Excitation-Contraction Coupling and Cardiac Contractile Force

EXCITATION-CONTRACTION COUPLING AND
CARDIAC CONTRACTILE FORCE

Excitation-Contraction
Coupling and Cardiac
Contractile Force
Second Edition
by
DONALD M. BERS
Professor and Chair Department of Physiology,
Loyola University Chicago,
Maywood, IL, U.S.A.
SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

A C.I.P. Catalogue record for this book is available from the Library of Congress.
ISBN 978-0-7923-7158-8 ISBN 978-94-010-0658-3 (eBook)

DOI 10.1007/978-94-010-0658-3
Printed an acid-tree paper
AII Rights Reserved

© 2001 Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers in 2001
Softcover reprint of the hardcover 2nd edition 2001
No part of the material protected by this copyright notice may be reproduced or
utilized in any form or by any means, electronic or mechanical,
includ ing photocopying, record ing or by any information storage and
retrieval system, withoutwritten permission from the copyright owner.
Vll
PREFACE TO FIRST EDITION
The main aim of this monograph is to provide an overview of calcium regulation in

cardiac muscle cells, particularly with respect to excitation-contraction coupling and the control
of cardiac contractile force. It is my hope that this book will be useful to students of the
cardiovascular system and muscle at all different levels and in different disciplines (such as
physiology, biochemistry, pharmacology and pathophysiology). I also hope that it will find use
for those studying developmental, comparative and disease processes as well as more integrative
phenomenon. I kept several goals in mind in writing this monograph. First, it should be easily
readable. Second, I chose to include numerous illustrations and tables to help integrate results
from numerous investigators in practical formats and also present key figures from important
papers. Thus, this monograph may serve as a resource of information for people working in the
areas described herein. Third, the presentation is a very personal one, and I have necessarily
drawn extensively on my personal experience in this field over the past 15 years. This, I think,
helps maintain a certain continuity of thought from chapter to chapter. Fourth, I have made
serious attempts to make each chapter "up to date", despite the breadth of topics covered. I have
also tried to be equitable in choosing references while not intending to be comprehensive or
exhaustive. Neither of these aims can be perfectly matched, and I apologize to the many
investigators whose papers I have not cited, but should have.
While I thank all of my colleagues who make this a stimulating area in which to work, I
would especially like to thank those who contributed by helpful discussions, providing original
figures, sending preprints of manuscripts, and by commenting on drafts of individual chapters.
These individuals include: S. Baudet, B.P. Bean, J.R. Berlin, J.H.B. Bridge, A. Fabiato, S.
Fleischer, J.S. Frank, C. Franzini-Armstrong, M.M. Hosey, L.V. Hryshko, N. Ikemoto, L.R.
Jones, W.J. Lederer, D.H. MacLennan, G. Meissner, M. Morad, K.D. Philipson, J.D. Potter, E.
Rios, R.J. Solaro, J.R. Sommer, J.G. Tidball, J. McD. Tormey, W.G. Wier, A. Williams, D.T.
Yue.
I also thank my many research collaborators over the years who have joined me in
making some contributions to this field, including: G.A. Langer, K.D. Philipson, D. Ellis, A.
Peskoff, J.L. Sutko, C.O. Malecot, K.T. MacLeod, J.H.B. Bridge, J.G. Tidball, M.J. Shattock,
S.M. Harrison, P. Hess, K.W. Spitzer, L.V. Hryshko, W.J. Lederer, J.R. Berlin.
Finally, a very special thanks are due to my wife, Kathryn E. Bers, whose combination of
patience and assistance have made this book possible.
ix
PREFACE TO THE SECOND EDITION
I have been delighted that so many people have found the I st edition of this book to be
nd
valuable. That has encouraged me to prepare a completely revised and entirely updated 2
edition. I have tried to maintain the flavor of the first edition, including some historical points
and classic observations, while integrating a large amount of new information into this frame-
work. The main aim is the same (to provide an overview of Ca regulation in cardiac muscle
cells, particularly with respect to excitation-contraction coupling and contraction). I have still
kept my initial four goals from the first edition in mind (readability, useful tables & illustrations,
up to date references and personal perspective). I have made every effort to integrate the wealth
of new data from various disciplines and perspectives from the past ten years with other work in
a seamless manner. I have added a tremendous amount of new material, but have really tried to
stay well focused, and limit the inevitable expansion of the volume. Nevertheless, this edition is
longer, but I hope to the true benefit of the reader. The new edition is organized in the same
overall sequence, but I have inserted one major new chapter on the action potential and ion
channels in the heart (Chapter 4, between Ca sources and sinks and Ca channels). I have also
included more discussion of heart failure and other pathophysiological issues. These aspects, I
felt, were lacking in the first edition and they make the book more complete. There are 178
figures, 27 tables and 2509 references in this edition. The number of references sounds like a lot,
but it was actually a challenge to keep it from getting longer. It is a tough trade-off in trying to
be equitable in choosing key references while not being exhaustive. I apologize again to the
many investigators whose excellent papers I have not cited, but well could have.
I hope that those people who embraced the first edition will appreciate the new edition
and the extensive rewriting. I hope this second edition will also be of value to new students of
the cardiovascular system at all levels, from medical/graduate students through the senior
investigators in the related fields which are discussed.
I want to thank all of my colleagues who continue to make this a stimulating field in
which to work. This is clearly a case where my own vision of how the heart works is built on the
shoulders and minds of a series of great scientists over the years. To those, too many to be
named, I thank you. I would especially like to thank those who contributed by helpful
discussions, providing original figures or data, sending preprints of manuscripts, by commenting
on drafts of individual chapters and collaborating with me to make some primary contributions in
this arena. These individuals are listed alphabetically on the next page.
I want to give very special thanks to a few coworkers who have helped me very directly
in preparing the final camera-ready manuscript. Ken Ginsburg, Tom Shannon and Lars Maier
patiently read and proofread every chapter and were constant companions. Teresa Carrillo, Lars
Maier and Klaus Schlotthauer did most of the painstaking work of preparing the extensive
reference list and checking it. Chris Weber prepared the very useful Index and Betty Weiss
helped in scanning some images from which I developed some of the figures. Finally, a very
special thanks is due to my wife, Kathryn E. Bers, whose tremendous patience, understanding
and encouragement have made this book possible.
x
ACKNOWLEDGEMENTS
These are the people who have really helped me learn about how the heart works. Thank you.
Julio Altamirano Larry Hryshko Burkert Pieske

Mike Artman Noriaki Ikemoto Steve Pogwizd
Jose Bassani Larry Jones Jim Potter
Rosana Bassani Hideki Katoh Pepe Puglisi
Stephane Baudet Arnie Katz MingQi
Bruce Bean Litsa Kranias John Reeves
Josh Berlin Glenn Langer Eduardo Rios
Lothar Blatter Jon Lederer Allen Samarel
Rolf Brandes LiLi Mike Sanguinetti
John Bridge Yanxia Li Hirosi Satoh
Joan Brown Steve Lipsius Martin Schneider
Mark Cannell David MacLennan Tom Shannon
Peace Cheng Ken MacLeod Mike Shattock
Simon Chu Claire Malecot Klaus Schlotthauer
Lea Delbridge Lars Maier Karin Sipido
Jaime DeSantiago Andy Marks John Solaro
Sanda Despa Abdul Matlib Jo Sommer
David Eisner Alicia Mattiazzi Ken Spitzer
David Ellis Eileen McCall John Sutko
Alex Fabiato Gerhard Meissner Jim Tidball
Mike Fill Rafael Mejia-Alvarez Andy Trafford
Sid Fleischer Ruben Mestril Dave Warshaw
Joy Frank Greg Mignery Chris Weber
Clara Franzini-Armstrong Martin Morad Gil Wier
Ken Ginsburg Rick Moss Alan Williams
Ana Gomez Mark Nelson PingXiao
Peter Haddock Jeanne Nerbonne Weilong Yuan
Simon Harrison Clive Orchard David Yue
Gerd Hasenfuss Ed Perez-Reyes ZhuanZhou
Marlene Hosey Art Peskoff MarkZiolo
Leif Hove-Madsen Ken Philipson
Steve Houser Valentino Piacentino
Finally, I thank the American Heart Association and National Institutes of Health, without whose
financial support this would not have been possible.
XI
INTRODUCTION TO THE FIRST EDITION
How is the heartbeat generated? What controls the strength of contraction of heart
muscle? What are the links between cardiac structure and function? How does our
understanding of movement in skeletal and smooth muscle and in non-muscle cells influence our
thinking about the development of force in heart muscle? Are there important species
differences in how contraction is regulated in the heart? While these important questions have
been asked many times, exciting results in many areas of mammalian biology have set the stage
for this refreshing new book on Excitation-Contraction Coupling and Cardiac Contractile
Force. This informative and quantitative book always remains readable. Don Bers explains how
contraction arises in heart and how it is controlled. Furthermore, he presents insightful and
stimulating discussions of apparently disparate results that will inform and delight both students
and "experts". In many ways, Don paints a modern "portrait" of how the heart works and in this
picture he shows a close-up of the structural, chemical and physiological links between excitation
and contraction.
The recent molecular investigations of excitation-contraction coupling in skeletal and
heart muscle have brought together cell physiologists, molecular biologists and physicians in
numerous research projects that form the background for this book. These new investigations
have led to the explosion of information that would challenge the individual who only seeks to
read the primary sources. Don simplifies our task by bringing much of this material together in a
single coherent presentation. Exciting questions abound and this book introduces and/or lays the
foundation for many of them. Some are stated explicitly by Don while others depend on Don's
presentation and the reader's background. For example, the five questions below are among the
ones that jump out at me. (1) In heart and skeletal muscle cells, the sarcolemma (SL) has been
reported to have many more dihydropyridine receptors than functional calcium channels. Is this
apparent excess real or an artifact? If it is real, what does this excess mean? (2) Another
question also centers on the dihydropyridine receptor (DHP-R) which is the L-type calcium
channel in heart and skeletal muscle. Recent cDNA sequence information along with
investigations of structure and function using DHP-R chimeras from heart and skeletal muscle
have suggested that a specific cytoplasmic domain or loop of the DHP-R can confer important
properties on this receptor/channel. With the skeletal muscle loop in place, E-C coupling in
skeletal muscle is "normal" (SL voltage-sensor-dependent calcium release) but when the cardiac
loop is in place, the E-C coupling resembles that normally seen in heart (calcium-induced
calcium-release). This raises the question of how this particular cytoplasmic loop normally
interacts with the SR calcium release channel (i.e. ryanodine receptor) in skeletal muscle. In
heart muscle the question is whether there is any interaction at all between the cytoplasmic loop
of the DHP-R and the SR calcium release channel. Furthermore, I must wonder if this interaction
(if any exists) changes during calcium overload or during maneuvers that change the inotropic
state of the heart muscle cell. (3) While calcium-induced calcium release (CICR) appears to be
the dominant factor in explaining the link between excitation and contraction in heart muscle, a
question lurks just below the surface. How is CICR modulated? What is the relationship
XII
between Ca influx and Ca release from the SR? It would appear that all elements of CICR can be
modified by intracellular calcium, drugs and neurohormones -- including calcium channels, Na-
Ca exchangers, Ca-ATPases, and calcium release channels. Furthermore the release process also
seems subject to modulation by intra-SR calcium and may also involve calsequestrin. (4) Do the
T-tubular membrane and the non-invaginated SL membrane participate in a similar manner in E-
C coupling? Do they have similar densities of DHP-Rs? Do they possess the same CICR
elements? (5) How can one make use of our knowledge of E-C coupling and Cellular Ca
regulation to develop improved inotropic agents? Many questions are raised by the book, and
each reader will undoubtedly focus on different ones. Although Don does not answer or directly
address all of our questions, he provides an improved vantage point for us to view the issues
important to each of us and to the field in general.
In his portrait of E-C coupling in heart, Don Bers presents many new findings
commingled with "established" truth and paints a new picture of how contraction arises and is
controlled in the heart. The picture is sharper and contains many new details. He assembles
important measurements in new and useful tables, presents figures from recent and more
classical publications and shows new figures to supplement his presentation. While integrating
new observations with traditional "facts", Don is able to retain both the excitement of discovery
and the inevitable controversy arising when important questions cannot be fully answered. This
book, written by an active research scientist, therefore provides a critical state-of-the-art report
on how the heart works as an electrically and chemically regulated contractile machine.
W.J. Lederer
Baltimore, Maryland
January 1991
XIII
INTRODUCTION TO THE SECOND EDITION

Since Excitation-Contraction Coupling and Cardiac Contractile Force was published IO years
ago, it has become a classic. The impact ofthe first edition was great. It was probably greatest on those of
us who work in the field because it has become an invaluable tool for us in our work. Furthermore, it has
become an important component in our teaching and research programs. The lofty "classic" status of the
first edition is well deserved but that presents a daunting challenge for a second edition. It is difficult for a
new version of any book to equal that kind of high achievement. The book had become so valuable to
those of us working in heart muscle, that we had it at hand during the writing of papers and grants and
during teaching. Students, postdoctoral fellows and visitors to our labs have come to expect to have copies
as departing gifts. Thus, when the word spread that the second edition was due soon, it was greeted with
excitement and trepidation. How can one best a classic? Would the new version be as useful as the first
edition? Would it be as comprehensive? How would current controversies be addressed? Would it be as
improved as were the updates of classic works by Bernard Katz (Nerve, Muscle and Synapse) or Bertil
Hille (Jonic Channels ofExcitable Membranes)?
The second edition of Excitation-Contraction Coupling and Cardiac Contractile Force is

stunning. It retains the zest and compact form of the first edition, contains the wealth of information and
analysis that has become the Bers' hallmark and it is well written. It is easy and delightful to read. While
the work does draw heavily from Don Bers' own work, it is appropriately and modestly done. The data
from all sources that is presented in the book is well chosen and new material is tightly integrated with the
old. The book presents important evolving topics, classic mateIial and issues that are now hotly debated.
All ofthis is done in a scholarly and evenhanded manner. The book does its best at integrating information
from multiple sources and providing thoughtful commentary and discussion. It is hard to pick a favorite
chapter because the weaker ones were significantly improved and all were made current. The ten chapters
cover the field ofEC coupling and cardiac contractility with the eye ofthe classicist but the viewpoint of a
modem biologist. I cmIDot help wondeIing if it should be called "Functional Genomics and Proteomics of
Heart Muscle" since novel molecular frndings are so well integrated with cellular and tissue function.
There is a theme that has been developing over the last 10 years that is of particular interest to me
and nicely treated by the second edition: local, subcellular, signaling. Virtually every presentation in the
second edition invokes, to some extent, "local control" as an element important to the overall signaling.
Such discussions include those on ea2+ sparks, mitochondrial signaling, extracellular matrix, channel
gating and modulation, transporter function, ryanodine receptor organization and signaling, behavior of the
sarcoplasmic reticulum, inotropic mechanisms, and many, many others.
On behalf of the many readers, contributors and colleagues of Donald M. Bers, I must end with a
note of appreciation and thanks. The revised version of Excitation-Contraction Coupling and Cardiac
Contractile Force is a magnificent work and will nicely replace our well-worn copies.
W.1. Lederer
Baltimore, Maryland
March, 2001
xv
TABLE OF CONTENTS
Chapter Page
1. Major cellular structures involved in E-C coupling................................................ 1
Sarcolemma and transverse tubules............................................................................... 2
Extracellular space......................................................................................................... 9
Sarcoplasmic reticulum............................... 10
Mitochondria 15
Myofilaments................................................................................................................. 16
Connections to the extracellular matrix......................................................................... 17
Other cellular constituents.................................................................. 18
2. Myofilaments: The end effector of E-C Coupling................................................... 19
Myofilament proteins 19
Mechanism by which Ca activates contraction............ 22
Acto-myosin ATPase............... 24
The length-tension relationship 27
The Ca sensitivity of the myofilaments 28
Force-pCa relation in intact cardiac muscle 31
Factors which influence the force-[Ca] relationship 32
Force velocity curves..................................................................................................... 35
3. Sources and sinks of activator calcium....................................................................... 39
General Scheme of Ca cycle in cardiac myocyte 39
Cellular volume conventions and Ca fluxes............................ 41
Ca buffering in the cytosol............................... 41
Ca requirements for activation of contraction 47
Ca dynamics during a twitch 48
Sources and sinks of Ca............ 50
Ca removal during relaxation 52
Ca influx vs SR Ca release in contractile activation............ 54
Mitochondrial Ca transport............................................................................................ 56
4. Cardiac action potentials and ion channels............................................................... 63
Action potential & heterogeneity...... 63
Resting Em Nernst potential & propagation................................................................... 64
Basic structure & function of ion channels 67
Permeation and selectivity....................... 68
Channel gating................................................................ 69
Em-dependent activation........... 70
Channel inactivation 71
Na channels........ 73
Ca channels.................................................................................................................... 76
K channels 76
K channel rectification............................................................................................ 76
IKI stablizes the resting Em....................................................................................... 77
Transient outward current (1\0) 78
Delayed rectifier K currents.................................................................................... 79
Other inward rectifier K channels........................................................................... 81
Na- and fatty acid-activated K channels......................... 83
CI channels............................................................................. 83
CFTR: cAMP-activated Cl channel........................................................................ 84
Ca-activated CI current l CI (Ca): A transient outward current........... 84
XVI
Swelling-activated CI current........................... 85
Stretch-activated channels................... 85
Non-selective channels 86
Pacemaker current Ir................................................................................................ 86
Ca-activated nonselective monovalent cation current Ins(Ca).................................... 87
Na/Ca exchange...................................................... 88
Na/K-ATPase 88
Currents during ventricular action potentiaL...... 91
Rapid upstroke of AP (Phase 0)........ 92
Early repolarization (Phase 1)................................................................................. 92
Plateau (Phase 2)............................................. 92
Late repolarization (Phase 3) 94
Pacemakers (AP Phase 4) 94
Early and delayed afterdepolarizations........ 97
Early afterdepolarizations (EADs).......................................................................... 97
Delayed afterdepolarizations (DADs)..................................................................... 97
Reentry of excitation..................................... 99
5. Ca influx via sarcolemmal Ca channels 101
Ca channel types 101
Molecular characterization of Ca channels 104
Ca channel selectivity and permeation 108
Numbers of Ca channels..... 114
Ca channel gating 114
Surface potential and activation 114
lea inactivation............ 116
Ca-dependent Ica facilitation or Ica staircase 119
Amount ofCa entry via Ca channels 120
Modulation ofI ca by agonists and antagonists 123
p-adrenergic modulation of cardiac Ca current.. 127
Other modulators of Ca current 130
6. Na/Ca exchange and the sarcolemmal Ca-pump 133
The sarcolemmal Ca-pump 133
Na/Ca exchange 135
Early characterizations in sarcolemmal vesicles 135
Isolation, cloning and structure 136
Na/Ca exchange current in myocytes and excised patches 138
Modulation ofNa/Ca exchange 142
Phosphorylation 145
V max vs. Ca requirements, site density and localization 146
Ca entry via Na/Ca exchange and contraction 147
Thermodynamic considerations 147
Competition among Na/Ca exchange, sarcolemmal Ca-pump
and SR Ca-pump during relaxation and at rest..... 152
Resting Ca efflux from cells 156
Transgenic mice and antisense knock-down of Na/Ca exchange 159
7. Sarcoplasmic reticulum Ca uptake, content and release 161
SR Ca-pump 161
Regulation of the cardiac SR Ca-pump by phospholamban 164
SR Ca-ATPase inhibitors 169
Regulation of the SR Ca-pump by Ca, pH, ATP and Mg 169
XVII
Calsequestrin 171
Thermodynamics and Ca-pump backflux 173
SR Ca content: Assessment in intact cardiac muscle and myocytes 177
Electron probe microanalysis 178
Caffeine-induced contractures, .0.[ Ca]i and fI Naic • 178
Rapid cooling contractures............................. 181
Cumulative extracellular Ca depletions.................. 183
Direct chemical and radiotracer techniques 185
SR Ca release channel or ryanodine receptor... 186
Molecular identity and structure of ryanodine receptors........ 187
Ca sparks: Fundamental cellular SR Ca release events.... 190
Quantitative aspects of SR Ca release flux............ 191
Regulation of SR Ca release 192
RyR adaptation or inactivation 194
Malignant hyperthermia, cyclic ADP ribose and toxins 196
Regulation by protein kinases, calmodulin and FKBP 198
Inositol 1,4,5 trisphosphate (lP 3) receptor. 200
Other SR channels related to Ca release 202
8. Excitation-contraction coupling 203
Voltage-dependent Ca release (VDCR) and skeletal muscle E-C coupling 205
Murine muscular dysgenesis: A model system 208
Skeletal muscle DHPR-RyR interaction 210
Cardiac muscle DHPR-RyR interaction? 212
Direct depolarization of the SR ? 213
Mg as a possible mediator ofVDCR in skeletal muscle 214
Ca-induced Ca-release (CICR) 215
Ca-induced Ca-release in skeletal muscle 215
Ca-induced Ca-release in mechanically skinned cardiac muscle 215
Ca-induced Ca-release: Support from intact cardiac myocytes 217
Repolarization induced tum-off of Ca-release in cardiac muscle? 219
Local control and CICR 221
Gain and fractional SR Ca release 224
Activation of SR Ca release and time course of Ca release flux 226
Termination of SR Ca release 227
Recovery from inactivation/adaptation 228
Spontaneous SR Ca release, cyclic contractions and Ca waves 230
Other E-C coupling mechanisms in heart 232
Alternative Ca triggers in cardiac CICR 232
Voltage-dependent Ca release (VDCR) in heart 235
Other E-C coupling proposals......... 236
IP3 -induced Ca release (lP3ICR) 237
Inositol (I,4,5)-trisphosphate induced Ca release in smooth muscle 237
Ca sparks in smooth muscle: SR Ca release can contribute to relaxation 240
Capacitative Ca entry in smooth muscle 240
IP 3 induced Ca release in skeletal muscle 241
IP 3 induced Ca release in cardiac muscle 241
Summary 243
9. Control of cardiac contraction by SR and sarcolemmal Ca fluxes 245
Species, regional and developmental differences 245
Ca removal fluxes 250
XVJll
Ca influx must match Ca efflux 253

Biphasic contractions 254
Rest decay and rest potentiation 255
Early electrical and mechanical restitution 256
Slow ryanodine receptor recovery and rest potentiation 256
Rest decay and SR Ca depletion 258
Ca influx and efflux in rabbit and rat ventricle 261
SR Ca refilling and post-rest recovery 265
Force-frequency relationships 268
Frequency-dependent acceleration of relaxation 270
10. Cardiac inotropy and Ca mismanagement 273
Cardiac inotropy 273
Hypothermic inotropy 273
~-adrenergic agents and cardiac inotropy 275
a-adrenergic agents and cardiac inotropy 282
Ca-Calmodulin dependent protein kinase (CaMKII) 285
Cardioactive steroids: Glycoside inotropy 286
Ca mismanagement and negative inotropy 294
Ca overload: Spontaneous SR Ca release 294
Afterdepolarizations and triggered arrhythmias 297
Acidosis 300
Hypoxia and ischemia 305
Reperfusion-Acute effects 308
Hibernation...................................... 309
Stunning 310
Preconditioning............................... 310
Hypertrophy 312
Heart failure (HF) 316
Twitch contraction, Ca transients and APD 317
Myofilament Ca sensitivity 317
Ca current 317
SR Ca-ATPase and phospholamban (PLB) 318
RyR and SR Ca content... 318
Na/Ca exchange 320
Na/K ATPase and [Na+]j 320
Other ion currents 321
Sites for induction of cardiac inotropy 323
Modulation of myofilament sensitivity 324
Phosphodiesterase inhibition.......... 327
Ca current modulation 328
Na/Ca exchange modulation 329
SR Ca uptake and release....................... 330
Conclusion 331
References 333
Index 407
XIX
TABLE OF FIGURES
Figure # Page
Chapter 1 Ultrastructure
I Schematic of skeletal muscle structure 2
2 Schematic of cardiac muscle structure 3
3 Extended junctional SR in bird heart 8
4 Surface coat and external lamina in mammalian heart 10
5 Extracellular space in mammalian heart II
6 Skeletal muscle triads ("feet") 12
7 Cardiac muscle triads ("feet") 12
8 Morphology offoot protein/ryanodine receptor 13
9 Schematic of T-tubule/SR junction 14
10 Diagram of skeletal vs.cardiac T-tubules/SR junctions 15
II Sarcomere organization 16
12 Schematic of actin cytoskeleton and extracellular matrix 18
Chapter 2 Myofilaments
13 Myofilament proteins 21
14 Ca-dependent regulation of acto-myosin (side view) 23
15 Ca-dependent regulation of acto-myosin (end-on view) 24
16 Cooperative activation of myosin 25
17 Crossbridge mechanical model 26
18 Crossbridge chemical cycle 26
19 Length-tension relationship (cardiac & skeletal) 27
20 Temperature effects on myofilament Ca sensitivity 29
21 Species differences in myofilament Ca sensitivity 30
22 Myofilament Ca sensitivity (in vivo vs."skinned") 32
23 Force-velocity and power-velocity in cardiac muscle 36
24 Quick release and re-stretch in cardiac muscle 38
Chapter 3 Sources & Sinks of Activator Ca

25 General scheme of Ca cycle 40
26 Cytosolic Ca buffering 43
27 Fast Ca buffering measurement 45
28 Total Ca requirements for myofilament activation 47
29 Ca changes during a twitch in rabbit myocytes 49
30 Selective inhibition of Ca transporters 53
31 Ca influx vs. SR Ca release during a twitch Ca transient 55
32 Mitochondrial Ca cycle (inner mitochondrial membrane) 57
33 Mitochondrial [Cal in intact cells 59
34 Mitochondrial NADH and regulation by Ca 61
Chapter 4 Cardiac Action Potential and Ion Channels

35 Regional variation in action potential configuration 64
36 Resting membrane potential and Nernst potentials 65
37 Propagation of depolarization in cardiac myocytes 66
38 Structure of some ion channels 69
39 K channel structure and gating 71
xx
40 Em-dependent gating of ion channels 73

41 K channel Em-dependence and rectification 78
42 Hyperpolarization-activated channel (Ir) 87
43 Na/Ca exchange current 89
44 Na/K ATPase mechanism 91
45 Rabbit ventricular AP and underlying currents 93
46 Pacemaker activity in nodal cells 95
47 Rabbit SA node AP and underlying currents 96
48 Afterdepolarizations in cardiac myocytes 98
49 Reentrant excitation in heart 100
Chapter 5 Sarcolemmal Ca Channels

50 Voltage dependence ofT & L type Ca channels 103
51 Schematic ofL-type Ca channel 107
52 Ca channel permeation model 110
53 Critical pore glutamates and Ca channel permeation III
54 Ca channel conductance and reversal potential 113
55 Surface potential and channel gating 115
56 Cardiac Ca current inactivation 117
57 Cardiac Ca current activation, availability and recovery 118
58 Ca current facilitation 119
59 Ic• during square pulse vs. action potential-clamp 121
60 Ca influx via Ica during an action potential. 122
61 Ca channel agonist Bay K 8644 and channel modes 123
62 Ca channel blockers (Em-dependence) 125
63 Ca channel antagonists and interaction sites 126
64 ~-adrenergic modulation ofCa channel 127
65 ~-adrenergic cascade (Ca channels and other systems) 129
Chapter 6 Na/Ca Exchange and Sarcolemmel Ca Pump

66 Na/Ca exchange in cardiac sarcolemmal vesicles 136
67 Schematic model of Na/Ca exchanger 137
68 Na/Ca exchange current in myocytes 139
69 Ca-dependent activation and Na-dependent inactivation ofNa/Ca exchange 140
70 Current-voltage relationships for INa/C 141
71 Ca entry and extrusion via Na/Ca exchange 142
72 ENa/c. and INa/C. changes during an action potential 148
73 Submembrane [Cal prediction 150
74 Local [Cali and [Na]; and INa/C. during action potential 151
75 Ca entry via Ica and efflux via INa/C 152
76 Paired RCC's: SR Ca-pump and Na/Ca exchange competition 154
77 Em-dependence of relaxation in myocytes 155
78 Na/Ca exchange and [Cali decline during voltage clamp 156
79 Rest decay of SR Ca content relies on Na/Ca exchange 157
80 Rest decay of SR in rat, rabbit and ferret.. 158
xxi
Chapter 7 SR Ca Uptake and Release

81 Schematic of SR Ca-ATPase structure 162
82 Structure of the skeletal muscle SR Ca-pump 163
83 SR Ca-pump transport scheme 164
84 Phospholamban structure and pentamerization 165
85 Influence of phospholamban on SR Ca transport 166
86 Model of phospholamban-SERCA2 interaction 168
87 SR Cat transport in intact and perrneabilized ventricular myocytes 171
88 Association of calsequestrin with junctional SR proteins 172
89 Energy in the trans-SR [Ca] gradient. 174
90 Unidirectional SR Ca-pump fluxes 175
91 Effect of Ca leak rate on SR Ca content. 176
92 SR Ca-pump fluxes during a cellular Ca transient. 177
93 Caffeine-induced Ca transient: SR Ca content and cytosolic buffering 180
94 Rapid cooling contracture in rabbit ventricular muscle 182
95 RCCs and Ca transients in guinea-pig ventricular myocyte 183
96 Ryanodine accelerates rest decay of SR Ca content. 184
97 Extracellular Ca depletions in rabbit ventricular muscle 185
98 Ryanodine effects on single cardiac SR Ca release channeL 187
99 Three dimensional structure of the skeletal ryanodine receptor. 189
100 Schematic of domains in cardiac ryanodine receptor sequence 190
101 Ca sparks in isolated mouse ventricular myocyte. . 191
102 Caffeine, Mg and [Ca]-dependence ofryanodine receptor gating 193
103 Luminal SR [Ca] and Adaptation shift cardiac RyR gating 195
104 IP 3 receptor channel: IP 3 - and Ca-dependence 201
Chapter 8 Excitation-Contraction Coupling

105 Contractions in zero Ca: Cardiac vs. skeletaL 203
106 Em-dependence of contraction and L'.[Ca]i in cardiac vs. skeletal muscle 204
107 Potential E-C coupling mechanisms in cardiac muscle 205
108 Intramembrane charge movement and SR Ca release 207
109 Spatial relationships of proteins at T -tubule/SR junction 209
110 Possible interaction sites between skeletal DHPR and RyRl 211
111 Bay K 8644 alters RyR2 function in a Ca-independent manner 213
112 Fabiato's CICR in mechanically skinned cardiac Purkinje fiber 216
113 Diagram ofCICR based on Fabiato's work 217
114 Ca release activated by Ca-"tail" current 218
115 Ca entry is required for Ca release in heart 219
116 Duration dependence of Caj transients in heart 220
117 Dependence of L'.[Ca]i on Ic• and E-C coupling gain 221
118 [Ca]i diffusion near the mouth of a Ca channel and in junctional cleft 222
119 Gain of E-C coupling and fractional SR Ca release in ventricular myocytes 225
120 SR Ca release flux measured by different approaches 227
121 Recovery ofE-C coupling in heart 229
122 Microscopic recovery of E-C coupling in a rat ventricular myocyte 231
123 [Na]i and Em-dependence of SR Ca release 233
124 Comparison ofIc• and INa/C. in triggering SR Ca release 234
125 IP3 -induced Ca release in smooth muscle 237
XXII
126 E-C coupling in smooth muscle 239

127 IPrinduced Ca release in cardiac muscle 242
128 E-C coupling in skeletal vs. cardiac muscle 243
Chapter 9 Control of Cardiac Contraction by SR & Sarcolemmal Ca Fluxes

129 Effects of caffeine and ryanodine on steady state twitch force 246
130 Post-rest recovery in different species (± ryanodine) 247
131 Spatial [Cali gradients in atrial myocytes 248
132 Spatial [Cali gradients in neonatal ventricular myocytes 249
133 Ca transients in rabbit ventricle with selective Ca transport block 251
134 rCa];-dependence of Ca transport in ventricular myocytes 252
135 Integrated Ca fluxes during twitch relaxation (rabbit vs. rat) 253
136 Biphasic contractions with milrinone 255
137 Rest decay and rest potentiation in rabbit, rat, ferret & human ventricle 257
138 Recovery of Ca spark frequency and rest potentiation in rat myocytes 258
139 Ca influx and efflux during twitches in rabbit and rat ventricle 262
140 Ca fluxes during action potentials 263
141 "Staircase" direction depends on pulse duration 263
142 Action potentials modified by Na/Ca exchange 264
143 Paired-pulses and sarcolemmal Ca fluxes 264
144 Refilling of the SR in rabbit ventricle 265
145 Ca fluxes and SR Ca content during refilling after depletion 266
146 Increasing SR Ca release enhances ,:\.[Ca]j only transiently 267
147 Frequency changes in rabbit ventricle 268
148 Force-frequency relationship in rabbit, rat, guinea-pig & human ventricle 269
149 Frequency-dependent acceleration of relaxation 271
150 Frequency-dependent acceleration of relaxation, post-rest twitches and mechanism 272
Chapter 10 Cardiac Inotropy and Ca Mismanagement

151 Hypothermic inotropy 274
152 Quick temperature switch and hypothermic inotropy 275
153 ~-adrenergic effects on [Ca]j and contraction 276
154 Isoproterenol effects on inotropy and lusitropy 277
155 ~-adrenergic receptor signaling in ventricular myocytes 281
156 <X- vs. ~-adrenergic effects on [Cali and contraction 283
157 <X- vs. ~-adrenergic effects on myofilament Ca sensitivity 284
158 <x-AR transduction pathway in ventricular myocytes 285
159 Twitch force vs. [Na]j in glycoside inotropy 288
160 Diastolic [Ca]j and glycoside inotropy 289
161 Na-pump inhibition effects on [Na];, twitch force & RCCs 290
162 Glycoside inotropy with SR Ca transport blocked 290
163 Ca influx via Na/Ca exchange can activate contraction 291
164 Extracellular Ca depletion is altered by Na-pump inhibition 292
165 Na-pump inhibition increases Ca influx and efflux 292
166 Ca overload induces aftercontractions and Ca transients 295
167 Spontaneous SR Ca release can trigger an action potential 297
168 Delayed afterdepolarizations: ':\'[Ca]j threshold for triggered AP 299
169 Acidosis decreases force, but increases Caj transients 301
XXJIl
170 Acidosis decreases myofilament Ca sensitivity 302

171 Acidosis increases [Na]; and partially restores contraction 303
172 Acid transporters involved in pHi regulation 305
173 Changes which occur during Ischemia and reperfusion 307
174 Hypertrophic signaling cascades 315
175 Contractile dysfunction and arrhythmogenesis in heart failure 322
176 Myofilament Ca sensitizer EMD-57033 on force and Ca transients 324
177 Phosphodiesterase inhibitor EMD-57439 on force and Ca transients 325
178 Myofilament Ca sensitization 326
xxiv
TABLE OF TABLES
Table # Page
1 Cellular elements (surface area/cell volume) .4
2 Electrophysiological surface: volume measures 6
3 Cellular elements (as % of cell volume) 7
4 Co-localization of key proteins in rat ventricular myocytes 15
5 Cardiac contractile proteins 20
6 Ca and Mg binding affinities to troponin C 22
7 Temperature alters myofilament Ca sensitivity 29
8 Factors that alter myofilament Ca sensitivity 35
9 Ca flux (or sites): conversion to /lmol/l cytosol. 42
10 Passive intracellular Ca buffering 46
11 Kinetic parameters for Ca buffering 50
12 Ca transporter contributions to relaxation 54
13 Energy requirements for cardiac ion transporters 62
14 Cardiac ion channels 75
15 Ca channel types 102
16 Properties of cardiac L- and T-type Ca channels 105
17 Selectivity of L-type Ca channel 109
18 Kinetic properties of the sarcolemmal Ca-pump 134
19 Factors that alter sarcolemmal NaiCa exchange 145
20 Ca transport during ventricular relaxation 153
21 SR Ca content measurements 179
22 Cardiac ryanodine receptor permeability and conductance 188
23 Factors which alter Ca release from the SR 197
24 Fraction of activator Ca from lea and SR Ca release 254
25 Hormone receptors and ion transporters in cardiac muscle 287
26 Heart failure: Alterations in expression & function 319
27 Cardiac phosphodiesterases and inhibitors 327
D.M. Bers.
Excitation·Contraction Coupling and Cardiac Contractile Force.
2nd Ed., Kluwer Academic Publishers, Dordrecht, 2001
CHAPTER 1
MAJOR CELLULAR STRUCTURES INVOLVED IN

EXCITATION-CONTRACTION COUPLING
Numerous cellular structures are involved with the process of excitation-contraction

coupling (E-C coupling) in cardiac muscle cells. This chapter serves to introduce some of these
components from an ultrastructural perspective, and key functional components will be discussed
in greater detail in subsequent chapters. Figures 1 and 2 are schematic drawings of the structure
of amphibian skeletal muscle (Fig 1) and mammalian ventricular muscle (Fig 2) from a classic
ultrastructural study by Fawcett & McNutt (1969; also based on the work of Peachey, 1965).
Despite important differences between skeletal and cardiac muscle which will be
discussed below, the general scheme of E-C coupling is similar. Electrical excitation of the
surface membrane leads to an action potential which propagates as a wave of depolarization
along the surface and along the transverse tubules (T-tubules). The depolarization of the T-
tubule (or sarcolemma) overlying the terminal cisternae (or subsarcolemmal cisternae) of the
sarcoplasmic reticulum (SR) induces the release of Ca from the SR. The details of how this
sarcolemmal depolarization is able to induce SR Ca release will be the subject of much ensuing
discussion. The Ca released from the SR then binds to the Ca-binding subunit of the thin
filament protein troponin which serves to activate contraction. Cellular Ca movement is also
complicated (especially in heart muscle) by the presence of Ca channels and transport systems in
the sarcolemmal membrane which may transport substantial quantities of Ca and may play an
important role in mediating or modulating Ca release from the SR.
There is a recurring theme that skeletal muscle contraction depends almost exclusively
on Ca released from the SR with quantitatively insignificant Ca entry across the sarcolemma
during a normal twitch. Cardiac muscle contraction, on the other hand, depends on both Ca entry
across the sarcolemma and Ca release from the SR (and the relative importance may vary, e.g.
Bers, 1985, 1991; see Chapter 9). While these conclusions are based mainly on physiological
experiments to be discussed later, it is notable that ultrastructural differences (Figs I and 2) are
consistent with this conclusion. That is, skeletal muscle has an extensive and well organized SR
network with large capacious terminal cisternae abutting the narrow T- tubules. In contrast,
cardiac muscle typically has a more sparse and less rigidly organized SR system with smaller
saccular enlargements at the cell surface and at junctions. with the much larger diameter T-
tubules (200 nm in heart vs. 30-40 nm in skeletal muscle). In addition, cardiac myocytes are
typically < 20 Ilm thick, whereas the diameter of skeletal muscle fibers can be many times larger
(up to 200 Ilm). The smaller diameter of heart cells makes diffusion from the extracellular space
(and T-tubule matrix) to the myocyte interior more plausible with respect to diffusional
limitations than would be the case for skeletal muscle. The larger volume:surface ratio in cardiac
2 D.M. Bers Cardiac E-C Coupling
Figure 1. Schematic diagram of T-tubules and SR associated with several myofibrils in frog skeletal
muscle. Each myofibril is surrounded by the meshwork of SR. The SR is greatly enlarged at the terminal
cisternae where it comes into close contact from both sides with the relatively narrow T-tubule forming the
triad at the Z-line. In mammalian skeletal muscle, T-tubules and triads are normally at the A-I band
junction. (From Fawcett & McNutt, 1969, by copyright permission of the Rockefeller University Press).
muscle T-tubules also means that a given ion flux across this membrane will produce smaller
depletions and accumulations of ions in the T-tubules. Thus, the structure of the cardiac myocyte
is consistent with a quantitatively more important role of transsarcolemmal Ca fluxes.
SARCOLEMMA AND TRANSVERSE TUBULES

The surface sarcolemma is physically continuous with the membrane of the T-tubule and
as such the two combine to form the permeability barrier between the inside of the cell and the
extracellular medium. Thus, from this functional perspective it is suitable to refer to the complex
simply as "the sarcolemma". As a point of semantic distinction it seems reasonable to use the
terms "surface sarcolemma" or "external sarcolemma" if one means to exclude the T-tubule when
referring to sarcolemma. Another semantic point is that the T-tubules are not strictly transverse,
but have many longitudinal and oblique components (Sommer & Waugh, 1976; Soeller &
Cannell, 1999), such that the term sarcolemmal tubule network would also be apt.
The ultrastructural organization of the cardiac sarcolemma is important for several
reasons. First, it is the site at which Ca enters (and leaves) the cell, so the localization of the
relevant transport systems is of functional importance. This is particularly the case because there
is differential distribution of ion channels, pumps and other membrane specializations. For
example, Almers & Stirling (1984) point out that the densities ofNa channels (Jaimovitch et al.,
1976), delayed rectifier K channels (Kirsch et al., 1977) and Na/K-ATPase pump sites (Venosa
& Horowicz, 1981) are considerably lower in skeletal muscle T-tubules than on the surface
sarcolemma, while the density of Ca channels is >4 times higher in T-tubules (Almers. et aI.,
1981). Indeed, vesicles isolated from skeletal muscle T-tubules have very high densities of
Chapter 1 Ultrastructure 3
Figure 2. Schematic diagram of T-tubules, SR and myofilaments in mammalian cardiac muscle.

Compared to skeletal muscle, cardiac T-tubules are much larger in diameter and the SR is more sparse, but
includes junctional couplings with the external sarcolemma as weJl as the T-tubules. Mitochondria are
plentiful and myofibrils are also more irregular in heart. (From Fawcett & McNutt, 1969, with permission
from Rockefeller University Press).
dihydropyridine receptors (DHPRs), which are Ca channels. Jorgensen et al. (1989) immuno-
localized skeletal muscle DHPRs in clusters, primarily in T-tubules. Brandt (1985) separated
SR-associated sarcolemmal vesicles from rabbit heart (a presumed T-tubular fraction). This
fraction had a high density of DHPRs (vs. muscarinic receptors), when compared to the non-SR
associated sarcolemmal fraction (i.e. presumed to be surface sarcolemma). Wibo et al. (1991)
also showed that DHPRs were 3 times more concentrated in a rat T-tubule vs. a surface
sarcolemmal fraction. Kawai et al. (1999) found that sealing off of rat ventricular T-tubules
reduced membrane capacitance by 26% (consistent with the 21-33% of sarcolemma being T-
tubular; Table 1) and that 75% of Ca channels were in T-tubules. This implies that Ca channel
density is -9 times higher in T-tubule vs. external sarcolemma. Doyle et al., (1986) found lower
density of DHPRs (vs. saxitoxin receptors) in putative T-tubular fractions than in surface
sarcolemma fractions from sheep or bovine hearts. Immunofluorescence studies by Carl et al.
(1995) indicated that in rabbit ventricle DHPRs co-localize with both ryanodine receptors and
triadin at dyads in T-tubules. In rabbit atrium and ventricle surface DHPRs were discretely
localized over junctional SR containing ryanodine receptors (see also pg 15). This is also the
case in chicken heart v here all dyads are superficial (Sun et al., 1995). Thus E-C coupling sites
where the SR comes into close contact with the sarcolemma are 1) almost exclusively in T-
tubules in skeletal muscle (Spray et al., 1974),3) mostly in T-tubules in mammalian ventricle or
2) primarily at the surface sarcolemma in mammalian atrium and avian heart.
Table I lists some quantitative ultrastructural data of surface areas of sarcolemma and
SR components in several cardiac and skeletal muscle preparations. Peachey (1965) reported
that in frog skeletal muscle the T-tubular area is -7 times the area of the external sarcolemma. In
mammalian skeletal muscle T-tubular area is somewhat less dominant, but still relatively large.
Table 1
2 3
Cellular Elements: Surface Area/Cell Volume (~m /~m )
Sarcolemma SR
External %ofSL Junet Junet Junet Free Tot
SL TT inTT ExtSL TT SR SR SR
Fineh ya 0.56 0 0 0.18 0.57 0.75
Mouse ya 0.28 0.22 44% 0.20 0.64 0.84
Mouse yb 0.324 0.34 51% 0.22 0.65 0.84
Mouse Y c 0.55 1.85
Mouse A C 0.26 2.69
Mouse LAb 0.65 0.10 13% 0.09 1.48 1.57
MouseRAb 0.62 0.08 11% 0.06 1.63 1.69
GPAd 0.014 0.08 t 2.21 2.29
GPyd 0.42 O.13 t 1.83 1.96
Ratye 0.27 0.07 21% 0.038 0.16 1.3 1.46
Ratyf 0.31 0.15 33% 0.023 0.069 0.19 1.0 1.19
Rabbit Y f 0.33 0.23 41% 0.010 0.042
Junet % of SL as dyad
SL Tot ExtSL TT
Rabbit Y g 0.35 0.25 42% 0.016 0.051 0.068 4.6% 21%
Ratyg 0.31 0.15 33% 0.023 0.069 0.093 7.7% 48%
Mouse Y g 0.31 0.17 37% 0.020 0.070 0.090 6.5% 40%
RatY h 0.24 0.44 64%
Frog yi 1.19 0 0 0.014 0.26 0.27
Frog A i 1.32 0 0 0.018 0.44 0.46
Lizard yi 1.14 0 0 0.045 0.46 0.50
Lizard Ai 1.25 0 0 0.056 0.89 0.94
Frog Sartoriusj 0.04 0.28 88% 1.4 4 5.4
Frog Sartorius k 0.064 0.22 77% 0.54 1.5 2.0
GP soleus' 0.116 0.064 36% 0.24 0.73 0.97
GP white vastus m 0.097 0.146 60% 0.41 0.91 1.32
GP red vastus m 0.097 0.148 60% 0.33 0.65 0.98
Sheep Purkinje n 0.037 0.423* 92%*
Dog Purkinje 0 0.096 0.041* 30%*
aBossen et al., 1978; ePage et al., 1971 ,rPage, 1978 k Mobley & Eisenberg, 1975
bSommer & Johnson, 1979 gPage & Surdyk-Droske, 1979 I Eisenberg et al., 1974
Bossen et af., 1981 . Soeller & Cannell, 1999 mEisenberg & Kuda, 1975, 1976
~Forbes & Sperelakis, 1995 I Bossen & Sommer, 1984 n Mobley & Page, 1972
Forbes & Van Nlel, 1988 J Peachey, 1965 o Eisenberg & Cohen, 1983
Ventricle (y), Atrium (A), Guinea Pig (GP), External sarcolemma (Ext SL), T-tubule (IT), Junctional
(Junct), Total (Tot). Mammalian skeletal muscle can be classified into slow (soleus), fast/glycolytic (white
vastus) and fast/oxidative/glycolytic (red vastus). *Intercellular clefts or folds for Purkinje fibers, which
lack TT. tExcluding corbular SR. In some cases right (R) and left (L) heart data were averaged.
There also seems to be a difference between fast (vastus) and slow (soleus) muscle which may
have a functional correlate (Eisenberg, 1983). In mammalian ventricle about 30-50% of the
sarcolemmal area is in the T-tubules and in mammalian atrium this fraction is < 15%. This may
be an upper limit since anecdotal reports often claim to see no T-tubules in mammalian atrium,
and Tidball et al. (1991) reported -20 times less T-tubular area in rabbit atrium vs. ventricle (and
also greater cell to cell variability in atrium). Bird, amphibian and reptilian hearts lack T-tubules
entirely. The ratio of external sarcolemma/cell volume is also inversely related to the cell
diameter (i.e. 2/radius if one assumes a cylindrical shape). The cell diameters in frog and lizard
hearts and mammalian atria are smaller than those of mammalian ventricular muscle. For these
smaller cells (with high surface to volume ratio), T-tubules are less important for inward spread
of activation (or Ca diffusion) than in mammalian ventricle or the even larger diameter cells in
skeletal muscle. Cardiac Purkinje fibers are cells which are specialized for electrical conduction,
and the large cross-sectional area decreases the longitudinal internal resistivity and hence
increases the rate of propagation of electrical impulses.
Soeller & Cannell (1999) used 2-photon fluorescence microscopy in intact rat ventricular
myocytes and found T-tubules to occupy 3.6% of the cell volume and a surface area of 0.44
flm 2/flm 3, vs. 0.24 flm 2/flm3 for the external sarcolemma (i.e. 64% of sarcolemma in T-tubules).
They also found that 60% of the T-tubular area is within 0.55 flm of the Z-line and includes
many non-transverse elements, leading them to suggest that T-tubules be renamed the
sarcolemmal Z-rete. Perhaps the term sarcolemmal tubule network is simpler and still de-
emphasizes the transverse component, but today T-tubule remains the standard term.
The complexity of the T-tubule network also means that when one changes extracellular
solutions around a myocyte there is a delay between bulk concentration changes and diffusion in
and out of the T-tubules (Blatter & Niggli, 1998). Shepherd & McDonnough (1998) measured
the functional time dependence of rapidly changing [Na]a or [Ca]a on Na or Ca current (INa or lea)
in guinea-pig myocytes. In atrial myocytes (which lack T-tubules) INa and lea changed with a
single fast time constant C't) of 20-30 msec (i.e. >90% complete in 85 ms, reflecting bulk solution
flow). For ventricular myocytes, 36% of the lea and INa also changed rapidly (reflecting surface
SL channels), but 64% changed more slowly (1:-200 msec) indicating that -64% of functioning
Na and Ca channels are in T-tubules (in agreement with data discussed on pg 3). If these
channels are concentrated in T-tubules, this value ought to be higher than the percent of
sarcolemmal area in T-tubules (21-51 % in Table I), but Soeller & Cannell (1999) remarkably got
the same number (64%). This T-tubule diffusional problem also requires >500 msec for an
abrupt change in surface [Ca]a or [Na]a to be fully transmitted to the T-tubules.
The surface:volume ratios in Table I are also valuable (see Chapter 3) in quantitative
evaluations of trans-sarcolemmal ion fluxes (e.g. Ca current) in terms of how they alter cellular
ion concentrations. Indeed, Satoh et al. (1996) measured cell volume and membrane capacitance
simultaneously in myocytes from several cardiac preparations and determined surface to volume
ratios (in pF/pL). These results in Table 2 are particularly useful for translating electrophysio-
logical measurements of ion flux (in pNpF or pmol/pF) to fluxes per unit cell volume (flM).
A major structural specialization of the sarcolemma is coupling with the SR (e.g. triads
and dyads). Figure I suggests that in skeletal muscle, most of the T-tubular membrane is
Table 2
Electrophysiological Surface: Volume measures
Rabbit Rat Ferret
3 mo. 6 mo.
Y Cell (pL) 30.4 30.9 36.8 30.9
CM (PF) 138 207 324 162
Length (J.!m) 143 124 140 138
Width (J.!m) 32 34 33 31
Depth (J.!m) 12 13 14 14
% Y cell Mitochondrial t 71 68 68 68
Surface:Yolume Ratios
CMN cell (pF/ pL cell) 4.58 6.76 8.88 5.39
CMN Cyt (pF/ pL cytosol) 6.44 9.94 13.0 7.96
J.!m2/J.!m 3 (as in Table I) 0.46 0.68 0.89 0.54
Data from Satoh et af. (I996) and t Barth et af. (I 992). Note that surface:volume ratio
in units of pF/pL are 10 times larger than in J.!m 2/lJm 3• Thus, using 1 IJF/cm2, 4.58
pF/pL would translate to 0.46 J.!m 2/lJm 3 in Table 1.
involved in junctional, triadic complexes with the SR (Peachey, 1965). The triad refers to the
coupling of two SR terminal cisternae to either side of aT-tubule. In cardiac muscle these
junctions are more apparent as dyads and can occur either at the surface sarcolemma or with T-
tubular membrane. In mammalian ventricular muscle Page & Surdyk-Droske (1979) found that
4-8% of the external sarcolemma is involved in junctional complexes whereas 20-50% of the T-
tubular membrane is so involved. They also reported that rabbit ventricle had a smaller
sarcolemmal fraction involved in SR junctions (4.6% of surface and 20.6% of T-tubule)
compared to rat or mouse ventricle (6.5-7.7% of surface and 40-48% of T-tubule). This
structural observation correlates with physiological data, indicating that ventricular contraction
in rat is more SR Ca-dependent than in rabbit, which is more SR Ca-dependent than frog (where
junctional couplings are sparse and mainly sub-sarcolemma; Fabiato & Fabiato, 1978; Bers et aJ.,
1981; Fabiato, 1982; Bers 1985 and see Chapter 9). That is, twitches in frog and to a lesser
degree rabbit (but not rat) can be supported largely by Ca entry from the extracellular space.
The sarcolemma also exhibits caveolae, which are flask-shaped invaginations (50-80 nm
in diameter) and contribute significantly (-10%) to the surface area of both surface and T-tubular
sarcolemma (Levin & Page, 1980; Anderson, 1993). Caveolae are probably neither pinocytotic
nor reservoirs of membrane for recruitment during physical stress (Sommer & Johnson, 1979;
Langer et aJ., 1982, Forbes & Sperelakis, 1995). They do seem to be the preferential location of
the scaffolding protein caveolin-3 and signaling molecules such as nitric oxide synthase and
protein kinase C (Song et aJ., 1996; Feron et aJ., 1996; Rybin et aJ., 1999).
The other major specialization of the sarcolemma is the region where cells are closely
apposed end to end, known as the intercalated disk. The ends of cardiac muscle cells generally
interdigitate (as in Fig 3) and the classic work of Sj6strand et aJ. (1958) described three
differentiations in this region known as 1) the nexus or gap junction, 2) fascia adherens or
intermediate junction and 3) macula adherens or desmosome. The fascia and macula adherens
appear to be of central importance in the mechanical connection of one cell to the next and the
macula adherens is sometimes likened to a spot-weld between cells. Intermediate and actin
Table 3
Cellular Elements: As % of Cell Volume
Junct Free SR
MF Mito Nucleus TT SR SR tot SR:TT
Finch va 57.3 34 0 0.21 0.62 0.83

Mouse va 54.3 37.5 0.8 0.22 0.65 0.87 0.75
Mouse Vb 54.3 37.5 0.95 0.22 0.65 0.87 0.92

Mouse L.Ab 53.0 20.3 0.25 0.10 1.62 1.73 6.92
Mouse R.Ab 52.6 19.8 0.20 0.06 1.69 1.76 8.80
Mouse VC 43.3 37.0 1.32 3.19 6.9 2.16
Mouse A C 44.6 25.7 2.1 1.52 12.3 8.09
Guinea Pig Ad 43.2 17.9 3.8 0.08 0.46 t 9.47 9.93 124
Guinea Pig V d 45.2 25.3 2.8 2.62 0.56 t 7.37 7.93 3.03
Guinea Pig A e 41.4 14 4 0.5 1.7 2.2
Guinea Pig V f 50 25 ~2
Rat V g 48.1 34 1.2 0.3 3.2 3.5 2.9

RatVh 46.7 36 2 0.3 3.2 3.5
Frog Vi 46.1 13.8 0 0.03 0.35 0.38
Frog Ai 42.4 12.0 0 0.03 0.53 0.56
Lizard Vi 50.1 25.8 0 0.09 0.6 0.69
Lizard A i 41.1 18.7 0 0.11 1.12 1.22
Frog vj 0 0.2 0.3 0.5
Frog Sartorius k 82.6 1.6 0.32 4.1 5 9.1 28.4
Guinea Pig
Soleus I 86.7 4.9 0.9 0.14 0.9 2.2 3.1 22.1
White Vastus m 82.0 1.9 0.15 0.27 1.6 3.0 4.6 17.0
Red Vastus" 80.3 8.2 0.9 0.28 1.2 2.0 3.3 11.8
Sheep Purkinje 0 23.4 10.3 1 0.23*
a Bossen et ai., 1978 h Page, 1978

b Sommer & Johnson, 1979 I Bossen & Sommer, 1984
Bossen et ai., 1981 J Page & Niedergerke, 1972
CForbes &Sperelakis, 1995 k Mobley & Eisenberg, 1975
d Forbes & Van Niel, 1988 1 Eisenberg et al., 1974
e Frank et ai., 1975 mEisenberg & Kuda, 1975
f Eisenberg, 1983 " Eisenberg & Kuda, 1976
g Page et ai., 1971 o Mobley & Page, 1972
Myofilaments (MF), Mitochondria (Mito), Nucleus (Nuc), Ventricle (V), Atrium (A), T-tubule (TT),
Junctional (June!). Manunalian skeletal muscle can be classified into slow (soleus), fast/glycolytic (white
vastus) and fast/oxidative/glycolytic (red vastus). *Intercellular clefts or folds for Purkinje fibers.
tExcluding corbular SR. The SR volumes estimated by Forbes & Van Niel were acknowledged to be
artifactually high due to a contrast effect. In some cases right (R) and left (L) heart data were averaged.
Figure 3. Finch cardiac muscle stained with colloidal lanthanum hydroxide as extracellular marker. Note
the interdigitation of cell ends and the lack ofT-tubules. Arrowheads indicate structures identified at higher
magnification as extended junctional SR. Bar = 2 /lm. (From Sommer & Waugh, 1976, with permission).
filaments also insert at the intercalated disk (Price & Sanger, 1983), emphasizing the mechanical
function of this cytoskeletal structure (see pg 17-18).
Gap junctions are predominantly on parts of the intercalated disk parallel to the long axis
of the cell (edges of interdigitations, Spray & Burt, 1990; Severs, 1990, 1997). Page & Shibata
(1981) estimated that gap junctions make up only 0.7-1 % of the total sarcolemmal area in rat and
rabbit ventricle (-3% in dog Purkinje Strand, Eisenberg & Cohen, 1983). Chen et a!., (1989),
however, suggested that these values in rat ventricle may underestimate the gap junctional area
by 3-4 fold (based on comparison of classical cross sections vs. en lace sections of the transverse
cell borders). These gap junctions serve as the low resistance electrical pathways that allow the
heart to function as an electrical syncytium. Revel & Kamovsky (1967) first demonstrated that
the membranes of the two cells did not fuse, but were separated by a narrow 2 nm gap at the
nexus and also observed hexagonal arrays, now known to be the functional units. A general
working model of the gap junction has evolved (e.g. Unwin & Zampighi, 1980; Makowski et al.,
1977) in which one hexameric unit with a central pore (or connexon) from each cell meet within
the gap and form a pore which allows direct connection between the cytoplasms of the two cells.
The molecular weight of the "connexin" monomers which make up the connexon differ in liver
(mw -28,000, Henderson et al., 1979; Hertzberg & Gilula, 1979) lens (mw = 26,000, Goode-
nough, 1979) and cardiac cells (mw = 43,000, known as connexin-43, Kensler & Goodenough,
1980; Manjunath et al., 1982; Beyer et al., 1987). The cardiac protein appears to have an extra
polypeptide on the cytoplasmic side which may contribute to functional differences in channel
properties (Manjunath & Page, 1985). Each connexin molecule has 4 transmembrane spanning
regions, an intracellular loop and a carboxy tail. Six connexins come together to form a
connexon with a central pore whose conductance can be regulated. While connexin-43 is the
dominant ventricular (and vascular smooth muscle) isoform, connexin-40 is abundant in the A-V
node, bundle branches, Purkinje fibers and some atrial cells. Connexin-45 is also expressed in
heart at much lower levels and connexin-37 and 40 are expressed in the vascular endothelium
(Beyer et al., 1989; Gourdie et al., 1993; Kanter et at., 1993; Severs, 1997; Spray, 1998). The
diameter of the pore has been estimated to be 1.6-2.0 nm by the cell-to-cell diffusion of
fluorescent tracers (Flagg-Newton et al., 1979). Notably, this allows ions such as Ca and K to
pass, as well as small signaling molecules like cyclic AMP and inositol phosphates. The single
channel conductance of connexin-43 (60 pS) is lower than that of connexin-40 (150-200 pS).
Cardiac gap junction permeability is decreased by high [Cali (DeMello, 1975; Weingart,
1977) and intracellular acidification (Reber & Weingart, 1982). This has the functional advan-
tage of uncoupling metabolically compromised cells. While the interaction of these effects is
complex and synergistic (Burt, 1987), Spray et al., (1982) concluded that the pH sensitivity
exceeds the Ca sensitivity. This may protect healthy cells from neighboring cells which have
either pathologically high [Cali or low pH; (or depolarization). The down-side may be that
diverting the normal electrical conduction pathway can also be pro-arrhythmic (see pg 99-100).
So far, sarcolemma has been used in reference to the phospholipid/cholesterol bilayer
with the integral proteins (e.g. ion channels and pumps) which are floating in it. The sarcolemma
as such has clear regional specialization (e.g. dyads & gap junctions), where the bilayer surfaces
are in close contact with special structures and where special proteins are located. The outer
surface of muscle sarcolemma is also invested with a layer abundant in acidic mucopoly-
saccharides (Bennett, 1963). Frank et al. (1977) divided this glycocalyx (or "sweet husk")
functionally into a surface coat (a less dense 20 nm layer adjacent to the sarcolemma) and an
extemallamina(amoredenseouter30nmlayerattheinterstitialinterface).This glycocalyx is
rich in sialic acid residues, which may account for fixed negative charges in this region and
explain the observation that pretreatment with neuraminidase decreases the labeling of the
glycocalyx with cationic electron-dense markers (lanthanum, ruthenium red and colloidal iron,
Frank et a!., 1977). Langer et al. (1976) found that such neuraminidase treatment greatly
increased cellular Ca exchange and suggested that sialic acid in the surface coat may importantly
regulate sarcolemmal Ca permeability. Exposure of cells to Ca-free solution causes the external
lamina to lift away from the surface coat (Fig 4, Frank et al., 1977). They attributed this to
break-down of sugar-Ca-sugar bridges and suggested that this might be involved in damage
associated with readmission of Ca (i.e. the Ca paradox described by Zimmerman & HUlsmann,
1966; see also Frank et al., 1982; Chapman & Tunstall, 1987; Bhojani & Chapman, 1990).
This separation of external lamina in Ca-free solution creates surface blebs which span
from T-tubule to T-tubule, such that the external lamina remains anchored by its extension into
the T-system. This also points to the fact that these surface layers remain associated with the
sarcolemma within T-tubules in cardiac muscle. This contrasts with skeletal muscle where the
glycocalyx does not appear to extend into the narrower T-tubules (Fawcett & McNutt, 1969).
EXTRACELLULAR SPACE
Knowledge of the contents of the extracellular space is important for understanding their
possible direct participation in cardiac function (above), but also for correcting measurements
made in intact preparations in terms of intracellular vs. extracellular concentrations. Frank &
Langer (1974) characterized the extracellular space in arterially perfused rabbit intraventricular
Figure 4. Rabbit ventricular muscle stained with colloidal iron. The muscle had been perfused in Ca-free
solution for 20 min. Note the separation of the extemallamina (EL) from the surface coat (SC) forming a
bleb (*). The EL is anchored where it penetrates into the T-tubule (TT). (From Frank et aI., 1977, by
permission of the American Heart Association, Inc.).
septum and also measured extracellular space volume by both morphological and chemical
means. They found that 59% of the extracellular space is vascular, 23% ground substance
(resembling the glycocalyx material described above), 7% connective tissue cells, 6% empty
space and 4% collagen. They also demonstrated that 36% of the cell circumference at its widest
point is within 200 nm of a capillary. This close proximity to capillaries is illustrated in Fig 5
and emphasizes the fact that in vascularly perfused ventricular muscle, the myocyte has rapid
access to the vascular contents (rather than requiring a strictly series model for diffusion through
a large intervening interstitial compartment).
Frank & Langer (1974) estimated tissue extracellular space morphometrically (27.6%
including T-tubules) and chemically (35.7% using 14C-sucrose and 36.2% using 35S-sulfate). Lee
& Fozzard (1975) reported a similar value (32.9%) for the 35S-sulfate space in superfused rabbit
papillary muscle. Bridge et al. (1982) measured a similar value for the extracellular space in
rabbit heart in vivo using 14C-sucrose (0.303 ml/g) and CoEDTA (0.294 ml/g), but found much
larger values in the isolated aqueous perfused intraventricular septum (0.51 ml/g for CoEDTA
and 0.46 ml/g for 14C-sucrose). While most extracellular space values for mammalian ventricle
are in the 25-35% range, the higher value reported by Bridge et al. (1982) for the intraventricular
septum may reflect tissue edema due to the low oncotic pressure of the aqueous vascular
perfusion. Thus, the extracellular space volumes used to correct tissue contents to intracellular
or extracellular contents should be measured under the same experimental conditions.
SARCOPLASMIC RETICULUM
The SR is an entirely intracellular, membrane bounded compartment which is not
continuous with the sarcolemma. The main function of this organelle in muscle appears to be
sequestration and release of Ca to the myoplasm. The volume of SR varies among cell types
Figure 5. Rabbit interventricular septal cell in transverse section. Note that -36% of the cell surface is
within 200 run of a capillary (cap), as indicated by the borders between arrowheads. Bar = 1 /lm. (From
Frank & Langer, 1974, by copyright permission of the Rockefeller University Press).
(e.g. Table 3) being most abundant in skeletal muscle, less abundant in mammalian heart and
least abundant in frog ventricle. This may reflect functional differences in the relative
importance of SR Ca in the activation of contraction (See Chapter 9). The SR in skeletal muscle
is very highly organized. This allowed Winegrad (1965) to perform his classical auto-
radiographic study in which he directly confirmed the terminal cisternae as the site of Ca release
from the SR. This was a very important conclusion because it indicated anatomical segregation
of transport functions within the SR. Except for the junctions between the SR and sarcolemma,
the SR membrane appears fairly homogeneous and contains mainly the SR Ca-ATPase pump
protein (Stewart & MacLennan, 1974; Katz et aI., 1986) which is manifest as intramembrane
particles ~8 nm in diameter and 3000-500011lm2 of SR membrane (Franzini-Armstrong, 1975).
These particles are also observed in isolated SR vesicles and the density may be slightly lower in
cardiac vs. skeletal muscle SR vesicles (Baskin & Deamer, 1969). Even the major part of the
terminal cisternae appear to have Ca-pump protein. Thus, the vast majority of the surface of the
SR is likely to function primarily to remove Ca from the cytoplasm. The relative area of the SR
surface involved in couplings with the sarcolemma also varies substantially from one muscle
type to another (see Junct SR in Table 1). Again, skeletal muscle has the greatest relative area of
junctional SR, with mammalian heart less, and frog heart the least.
The junctions of SR with sarcolemma (surface or T-tubule, see Fig 2) are highly
specialized and feature bridging structures or spanning proteins which have been called "feet" by
Franzini-Armstrong (1970, see Fig 6), and also pillars, spanning proteins, bridges and junctional
feet. Similar structures are seen in cardiac muscle at the junction of SR with either surface or T-
tubular sarcolemma (see Fig 7). Caldwell & Caswell (1982) showed biochemical evidence for a
high molecular weight protein which could be the junctional feet (see Chapter 7).
Figure 6. Tangential view of 3 triads from frog sartorius (arrowheads) showing periodic junctional "feet"
at the site where the SR and T-tubule come into close contact. Bar = 0.5 flm; inset is at 2.2x higher magn-
ification. (From Franzini-Armstrong, 1970, by copyright permission of the Rockefeller University Press).
Figure 7. Rat papillary muscle in a thin section electron micrograph (left) and freeze-etched electron
microscopy after ultra-rapid freezing without fixation (right). Junctional "feet" between the SR and T-
tubule (TT) can be seen to periodically span the gap. Bar=0.2 flm. (From Frank, 1990 with pemlission).
Based on their distinctive morphology and high affinity for the neutral plant alkaloid,
ryanodine, feet were purified and identified as the SR Ca-release channel in skeletal (lnui et af.,
1987a; Lai et af., 1987) and cardiac muscle (Lai et al., 1988a; Inui et aI., 1987b). This ryanodine
receptor protein is so large (560 kDa for the monomer; Takeshima et aI., 1989), that the
Figure 8. Morphology of the foot protein! ryanodine receptor/ Ca release channel of skeletal SR. Center)
Electron micrograph of negatively stained receptors showing the 4-fold symmetry, square shape (-27 nm/
side) and computer average of 240 images (bottom right). The foot protein extends -12 nm from the SR
surface of SR vesicles originating from terminal cisternae (top left) and the same square array can be seen in
tangential sections of these vesicles (top right, see also Fig 99). A model of the terminal cisternal
membrane (bottom left) shows a junctional face membrane with feet, and the remainder of the SR surface
covered largely by the Ca-pump protein. (From Fleischer & lnui, 1989, with permission).
functional tetramer can be seen at the electron microscopic level (Fig 8; Saito et al., 1988;
Wagenknecht et al., 1989, 1994, 1995; Seresheva et al., 1995, 1999; Sams6 et al., 1999). This
channel will be discussed in detail in Chapter 7 (see pg 189).
In addition Block et al. (1988) demonstrated that these feet are organized in a distinct
pattern on the SR underneath the T-tubular membrane in skeletal muscle and are matched by an
organized array of particles in the T-tubule membrane which are likely to be the sarcolemmal Ca
channel protein (or DHPR, see Fig 9). This arrangement is consistent with a stoichiometry of 1
ryanodine receptor tetramer (RyR) to 2 DHPR as measured in skeletal muscle, whereas in
mammalian ventricle the RyR:DHPR ratio is 4-10, depending on species (Bers & Stiffe), 1993;
Wibo et al., 1991, see Table 25, pg 287). That constrains the organization of the junctional
coupling as indicated in Fig 10 (and observed experimentally by Franzini-Armstrong & Protasi,
1997). Since the ryanodine receptors are clustered at these junctions, it may be important to
consider how many tetramers (or feet) there are in a typical array. Franzini-Armstrong et al.
(1999) estimated that these domains (referred to as couplons) in skeletal muscle contain from 17
feet (in guinea-pig slow soleus) to 38 feet (in fast rat extensor digitorum longus). In dog and
Figure 9. Three-dimensional reconstruction of the relative positions of key proteins at the skeletal muscle
triad. The SR is filled with calsequestrin and the non-junctional surface is covered with the Ca-pump
protein. The RyR feet are organized in two parallel rows and protrude from the SR. A similar array of
proteins (dihydropyridine receptor) exists in the T-tubular membrane, but the axis of fourfold symmetry is
rotated and they lie only over alternating foot structures (see also Fig 109). Based on toadfish swim-
bladder muscle (from Block et aI., 1988, by copyright permission of the Rockefeller University Press).
mouse ventricle the coupIon size was 90-128 feet for internal dyads, and 61-150 for surface
couplings (rat ventricle T-tubule couplons averaged 267 feet). Given the packing array, 100
ryanodine receptors would occupy a 60-100 nm diameter circle. So a typical cardiac couplon
may have about 100 RyR and 10-25 DHPR. They also measured the minimum transverse
distance between couplons (300-400 nm along mouse and rat T-tubules). The implications of
this arrangement for E-C coupling will be considered more specifically in Chapter 8. The SR
also contains calsequestrin, a low affinity, high capacity Ca binding protein (Ostwald &
MacLennan, 1974) which is especially concentrated in the terminal cisternae (Meissner, 1975).
Calsequestrin is crucial to the Ca buffering capacity of the SR (Chapter 7, pg 172).
Sommer and colleagues have also described what seems to be junctional SR (including
feet), which does not come into contact with any sarcolemma component. One specialized
region (extended junctional SR) is prominent in the interior of avian cardiac myocytes which
lack T-tubules (Jewett et al., 1971; Sommer & Johnson, 1979). This extended junctional SR
occurs along the Z-disk in bird ventricle where T-tubule/SR junctions would be prominent in
mammalian ventricle (see Table 1 and Fig 3). Corbular SR (Dolber & Sommer, 1984) is a
basket-like form of extended junctional SR. It exhibits the morphology of junctional SR, but it is
not in the vicinity of the sarcolemma, and is normally connected to the free or network SR only
at one point. Corbular SR has been described in mammalian ventricle (Dolber & Sommer,
1984), but is particularly apparent in atrium, Purkinje fibers (Sommer & Johnson, 1968; 1979)
and chicken cardiac muscle (Jewett & Leonard, 1973; Jorgenson & Campbell, 1984). Jorgensen
et al. (1985, 1993 & personal communication) found that up to 30-40 % of the calsequestrin and
ryanodine receptors in rat and dog ventricular myocytes are in this sort of non-junctional SR.
These "uncoupled" SR components with morphology like true junctional SR provide a functional
Chapter J Ultrastructure 15
A. Skeletal B. Cardiac
I~ SR
TT~ TT
tDCorbular ~~~~~
SR
~Corbular
V SR
Figure 10. Diagram comparing the organizational differences between skeletal and cardiac T-tubule
junctions. The upper diagrams are side views of the junction (trapezoids are ryanodine receptors, RyR and
filled ovals dihydropyridine receptors, DHPR). Lower panels are views from inside the T-tubule at the
junction. Note that DHPRs are sparse and less aligned in heart (modified from Bers & Stiffel, 1993).
challenge to models of E-C coupling. That is, what is the function of these SR components? Do
they participate in Ca release during the activation of a normal contraction? Is the mechanism of
release distinct from that of true junctional SR?
Scriven et al. (2000) examined the colocalization of several key proteins with respect to
E-C coupling (Table 4). While all of the DHPRs were near RyRs, some RyRs were not near
DHPRs (e.g. possibly in corbular SR). Notably, neither Na channels nor Na/Ca exchanger were
colocalized near RyRs, nor each other. Thus, both Na channels and Na/Ca exchange appear to be
excluded from the cardiac SR-sarcolemmal junctions. This may limit the role that these
transporters have in E-C coupling (see pg 205, 232-237).
Table 4
Co-localization of some key Proteins in Rat Ventricular Myocytes*
Co-Localization (%)
A (FITC Label) B (Texas Red) A with B B with A
DHPR RyR 56.7 ± 5.1 36.7 ± 4.8
Calsequestrin RyR 61.6 ± 7.2 55.8 ±.6.2
NaCaX RyR 5.8 ± 1.9 7.7 ± 2.3
Na Channel RyR. 2.9 ± 0.9 3.1 ± 1.2
NaCaX Na Channel 3.5 ± 1.5 3.6 ± 2.1
RyR RyR 64.7 ± 5.8 61.2 ± 5.7
*Ca channel is L-type Ca channel, NaCaX is Na/Ca exchange, RyR is ryanodine receptor. Data are
from Scriven et al. (2000) with one antibody labeled with FITC (fluorescein isothiocyanate) and the
other with Texas Red. Note that maximal colocalization (last line) is -65%.
MITOCHONDRIA
About 35% of the volume of mammalian and avian ventricular muscle cells is occupied
by mitochondria (see Fig 2, Table 2 & 3 and Barth et aI., 1992). The mitochondria are the site of
oxidative phosphorylation and the tricarboxylic acid cycle and the large mitochondrial content in
mammalian ventricle bespeaks the high demands on this organelle for energy supplied by aerobic
metabolism. The mitochondrial fraction of cell volume is lower in mammalian atrial muscle and
Purkinje fibers as well as amphibian and reptilian hearts and skeletal muscle (Table 3). Indeed,
there is substantial variation in mitochondrial volume among different types of skeletal muscles
that reflects differences in the oxidative capacity of those muscle types. The surface area of the
folded inner mitochondrial membrane in rat left ventricle has been estimated to be 20 )..lm 2/)..lm 3 of
cell volume (Page, 1978). This membrane is the site of control of metabolite and ion transport
(e.g. Ca and protons). The surface area is more than 10 times larger than that of the combined
sarcolemmal and SR membranes, so a modest Ca transport by mitochondria could have a large
impact on overall cellular Ca regulation (see Chapter 3, pg 56-62).
Cardiac mitochondria are usually cylindrical, but can flatten because of the tight packing
in cells. The cristae in cardiac mitochondria are more tightly packed than those in hepatocytes,
perhaps reflecting the continuous intensive energy demands of the myocardium (Sommer &
Johnson, 1979). A layer of mitochondria is often found just under the plasma membrane and
also between adjacent myofibrils. As mitochondria are squeezed in everywhere in cardiac cells,
it is not realistic to assign a specific functional role to the cellular mitochondrial distribution.
MYOFILAMENTS
The myofilaments occupy 45-60% of the cell volume in mammalian ventricle (Table 3,
Fig 2). This fraction is larger in skeletal muscle and smaller in atria and cells specialized for
electrical conduction (Purkinje fibers). Myofilaments are composed of the thick (or myosin) and
thin (or actin) filaments as well as associated contractile and cytoskeletal components.
Myofilament bundles or fibrils are less defined in cardiac vs. skeletal muscle due to branching.
The myofilaments are the contractile machinery of the cell and indeed they represent the
end effector responsible for transducing chemical energy into mechanical energy and work. The
sarcomere is the fundamental contractile unit in striated muscle and is bounded by the Z-Iine (Fig
11). The thin actin filaments (-10 nm thick) extend ~ 1 )..lm from the Z-Iine toward the center of
each sarcomere (for more detailed structure and function of the thin and thick filaments with
Z-Line M-Line Z-Line
L L I
oj. rTitin
Thick filament
.~! Thin filament
~
H H-Band
~ A-Band -------..j I-Band I
Figure 11. The organization of the sarcomere. The thin filaments meet at the Z-Iines and the center of the
thick filaments is known as the M-line. The I-band (or isotropic band) is the area where there are only thin
filaments and the A-band (or anisotropic band) is the length of the thick filaments. The region of the thick
filament where there is no overlap with thin filaments is known as the H-band (or H-zone).
respect to contraction see Chapter 2). Tropomodulin is localized at the tip of the thin filament
and may be involved in regulating the length of the fi lament (Sussman et al., 1994; Gregorio et
al., I 995a,b). At the Z-line a-actinin is also a crucial structural element. Connections from the
Z-line to the sarcolemma include actin filaments (F-actin), a-actinin, the 7-10 nm thick
intermediate filament protein desmin (Lazarides, 1980, 1982; Eriksson & Thornell, 1979) and
ankyrin. Ankyrin has also been found to associate with the sarcolemma and certain membrane
transporters, such as the Na/Ca exchanger (Li et al., 1993).
The thick myosin filaments are -1.6 /lm long (-15 nm thick). Myosin heads protrude
from the long axis every 14.3 nm, with the protrusion angle rotating 1200 at each point, such that
in one plane (as Fig II) the heads seem to be spaced 43 nm apart. These myosin heads create the
crossbridges that interact with actin to generate contraction. Myosin binding protein C
(sometimes called C-protein) appears to wrap around the thick filament and may be important in
stabilizing and modulating the thick filament (Offer, 1972; Freiburg & Gautel, 1996). C-protein
appears as 10 nm bands (near the crossbridge extension away from the filament axis) every 43
nm, within two 200 nm zones on either side of the M-line separated by a 400 nm bare zone.
Thick filaments are interconnected transversely at the M-line by M-protein and myomesin
(Obermann et al., 1995). Titin is an extremely long (3,000 kDa) structural protein that runs from
the M-line, through the thick filament and all the way to the Z-line (Gautel & Goulding, 1996).
Titin may be important in creating a scaffold for myosin deposition on the thick filament
(Trinick, 1994), and stabilizing it structurally in association with C-protein (Freiburg & Gautel,
1996). Titin is also extremely important in determining the passive stiffness of the heart (Brady,
1991). Regulation of myofilament force will be discussed in Chapter 2.
CONNECTIONS TO THE EXTRACELLULAR MATRIX

The myofilaments develop and bear the active force within the cardiac myocyte.
However, unlike skeletal muscle, cardiac myofilaments are not continuous in series from one cell
to another, but must transmit force across cell-to-cell junctions. In addition, the complex
mechanical stresses on cardiac myocytes require strong mechanical links between the
intracellular myofilaments and cytoskeleton and the extracellular matrix. The Z-lines appear to
be the anchor points where intermediate filaments of the cytoskeleton connect actin filaments to
the sarcolemma (Price & Sanger, 1983; Price, 1991). The points of attachment across the
sarcolemma occur at the intercalated disks (at fascia adherens and desmosomes discussed above)
and also on lateral surfaces aligned with Z-lines at costamere-like focal adhesions.
Figure 12 illustrates two of the major structural complexes involved in transsarcolemmal
mechanical connection to the extracellular matrix, integrins (left) and dystrophin complexes
(right). The right integrin complex emphasizes important proteins involved in the mechanical
connection at Z-lines (actin, a-actinin, vinculin, talin, paxillin, tensin, a- and ~-integrins, laminin
and collagen, Bloch, 1996; Schlaepfer et al., 1999). It is increasingly clear that these complexes
are also extremely important in the transduction of physical stretch of the myocardium to
intracellular signaling cascades (see pg 312-316). The left integrin complex emphasizes some of
the key signaling proteins so far implicated (including focal adhesion kinase or FAK, the small
GTP-binding protein Ras, and the protein kinase C 8 isoform with its anchoring protein RACK-
I). The right side of Fig 12 shows the dystrophin complex (Campbell, 1995) which links actin to
Figure 12. Schematic diagram indicating connections between the actin cytoskeleton and the extracellular
matrix via integrins (left) and the dystrophin complex (right). The two integrin complexes are drawn to
separately focus on the mechanically important proteins (right complex) and signaling proteins (left
complex), although the proteins and functions coexist there. The integrin side was adapted from an original
kindly supplied by A.M. Samarel and the dystrophin complex is based on Campbell (1995).
a complex of transmembrane proteins which link up to laminin and collagen in the extracellular
matrix. The -terminal of the large dystrophin molecule (400 kDa) binds to actin and a cysteine-
rich region near the C-terminal of dystrophin links it via ~-dystroglycan to the cluster of
transmembrane proteins. This cluster is composed of several dystrophin-associated proteins
(DAP) and glycoproteins (DAGs, & adhalin). On the outer sarcolemmal surface a-dystroglycan
binds to laminin in a Ca-dependent manner (Ervasti & Campbell, 1993).
OTHER CELLULAR CONSTITUENTS

Cardiac myocytes are typically mononucleate (although some are binucleate) and contain
Golgi apparatus, Iysosomes, lipofuscin granules and peroxisomes (Sommer & Johnson, 1979).
Lipid droplets and ~-glycogen granules are also present and are more abundant than in skeletal
muscle. Cardiac myocytes also have a well developed cytoskeleton, only briefly discussed
above, including microtubules and microfilaments. Microtubules have also been implicated in
altering cardiac mechanical properties in hypertrophy (Zile et al., 1999; Tagawa et al., 1998).
D.M. Bers. 19
CHAPTER 2
MYOFILAMENTS:
THE END EFFECTOR OF E-C COUPLING
When cytoplasmic [Cal rises, the myofilaments are activated in a [Cal-dependent

manner, thereby transducing the chemical signal and chemical energy (ATP) into mechanical
force or shortening. Under physiological conditions, skeletal muscle contractile force can be
varied by summation of contractions, tetanus and recruitment of additional fibers. Cardiac
muscle, on the other hand, functions as a syncytium such that each cell contracts at every beat.
The heart must also relax between contractions. Thus, there is neither the practical possibility of
recruitment of additional cells, nor summation, nor tetanization to alter the force of contraction to
meet altered demands. Therefore, in cardiac muscle, the force of contraction is varied in large
part by changes in the peak [Cali reached during systole (as well as sarcomere length).
For this reason I place considerable emphasis on the factors influencing the [Cali in
subsequent chapters, often with implicit assumptions about the consequent effects on myo-
filament activation. However, since contraction is the physiological function of cardiac muscle,
the fundamental characteristics of the contractile proteins and how they sense [Cal under
physiologically relevant conditions is important to consider.
MYOFILAMENT PROTEINS
Each thick filament is composed of -300 myosin molecules, but also contains other
proteins, such as titin and C-protein. Each myosin heavy chain (MW -450,000) has a long (~130
nm) a-helical tail and a globular head (Fig 13A). The tails of the myosin heavy chain form the
main axis of the thick filament. The heads form the crossbridges to actin on the thin filaments,
contain the site of ATP hydrolysis, and have two light chains associated with each head. The
myosin molecule is hexameric, composed of two heavy chains with their tails coiled around each
other and two myosin light chains per heavy chain. Based on susceptibility to specific proteases,
the myosin heavy chain has been broken down into light meromyosin (2/3 of the ISO nm tail) and
heavy meromyosin (HMM). HMM is further divided into subfragment 2 (S2, the residual 50 nm
tail) and subfragment 1 (Sl, 20 nm of neck and long globular head). The central region of the
thick filament, around the M-line (Fig II) is devoid of crossbridges, reflecting the tail-to-tail
abutment of myosin molecules there. The two myosin light chains (MLCI and MLC2) bind to
each myosin heavy chain at the base of the S I domain or in the neck region (Fig 13A). MLCI is
also referred to as the alkali or essential light chain (ELC). MLC2 is also called the phosphory-
latable or regulatory light chain (RLC). Both light chains confer physical stabilization of the
thick filament (along with C-protein and titin). However, the regulatory light chain (MLC2) may
also alter function in response to Ca binding or phosphorylation (see pg 34-35).
Table 5
Cardiac Contractile Proteins
Molecular Weight t
Myosin Heavy Chain 223,000
Myosin Light Chain I (essential) 21,000
Myosin Light Chain 2 (regulatory) 19,000
Actin 41,700
lX-Tropomyosin 67,000
Troponin T 38,000
Troponin I 23,500
Troponin C 18,400
t from Swynghedauw (1986) and Moss & Buck (2001) for mammalian ventricular muscle.
The proteins of the thin filament and their interactions (Fig 13B) have been reviewed in
detail (Zot and Potter, 1987; Solaro & Rarick, 1998; Solaro, 2000). The backbone of the thin
filament is composed of two chains of the globular protein G-actin, which form a helical double-
stranded F-actin polymer (Fig 14). Tropomyosin (Tm) is a long flexible protein which lies in the
groove between the actin strands and spans about 7 actin monomers (Fig 14). Tropomyosin is
also double-stranded and mostly lX-helical (coiled coil) and the two strands may be connected by
a disulfide bridge. The carboxy end also overlaps the amino end of the next tropomyosin by 5-10
amino acids, contributing to thin filament functional cooperativity that spans beyond a single 7-
actin span of one tropomyosin (Lehrer et al., 1997). At every seventh actin there is a troponin
complex attached to tropomyosin. The troponin complex is made up of three subunits: troponin
T (TnT, or the tropomyosin binding subunit), troponin C (TnC, or the Ca binding subunit), and
troponin I (Tnl, or the inhibitory subunit, which also binds to actin). The sites of interaction of
thin filament proteins are shown in Fig 13B, while the arrangement of these subunits in the thin
filament is indicated in Fig 14. TnT has a globular carboxy region and an elongated shape that
lies along Tm over about 3 actin monomers (Ohtsuki, 1979; Flicker et al., 1982). This
arrangement may allow TnT to better control the position of tropomyosin. Tnl (near its amino
terminal) interacts specifically with the carboxy ends of both TnT and TnC (Figs 13 & 14). The
strong binding of the amino end of Tnl to TnC depends on Ca or Mg binding to sites on the
carboxy end of TnC (physiologically almost always fully occupied; see pg 21 & 46). In the
resting state (low [Cali) the carboxy end of Tnl also binds specifically to actin, and this prevents
the myosin head from interacting with actin. When Ca binds to the amino end of TnC, at the
lower affinity physiological regulatory site, this part of TnC binds to the carboxy end of Tnl
causing Tnl dissociation from actin. This change in TnC-Tnl interaction is sensed by TnT and
causes movement of tropomyosin to allow myosin to interact with actin (see Fig 14-16).
Skeletal TnC has been extensively characterized and contains 4 Ca-binding sites, two
Ca-specific sites (Ki(Ca) = 200 nM in the troponin complex) and two sites at which Ca and Mg
bind competitively and with high affinity, known as Ca-Mg sites (Ki(Ca) = 2 nM; Kd(Mg) = 25 IlM
in the troponin complex). It is notable that the affinities of these sites on TnC are different when
the intact system is partly or wholly disassembled (e.g. in isolated TnC the Ca-specific site has
Chapter 2 Myojilaments 21
A. Myosin
~LMM (100 nm)

52 (50 nm)
51 (20 nm)
B. Thin Filament
Regulatory Proteins
SER-1
55 nm
Tnl
Figure 13. Myofilament proteins. A. The myosin molecule is -170 nm in overall length with two
globular heads (S I) and tails (including light meromyosin LMM + S2) which exist as a coiled coil. The two
light chains (LC) are indicated on the neck (Based on Warshaw, 1996). B. The thin filament regulatory
proteins interact extensively with one another (arrows); some of the interactions are Ca-dependent. Several
phosphorylation sites are indicated (Ser, Thr). (Based on a figure generously supplied by R.J. Solaro).
-10 times lower affinity for Ca than the whole troponin complex; see Table 6). The Ca-specific
sites are the sites which are responsible for the regulation of contraction (Zot and Potter, 1987).
Cardiac TnC differs significantly from skeletal TnC in that the cardiac isoform has only
one Ca-specific binding site at the amino end, but almost the same two Ca-Mg sites on the
carboxy end. With the affinities in Table 6, resting [Calj = 100 nM and [Mgl j = 1 mM, the
cardiac Ca-Mg sites would be -97% saturated (90% with Ca and 7% with Mg). Thus, these sites
would always be nearly saturated and this is probably important in the structural stability of the
TnC-Tnl complex (above). The Ca-specific site has a Kd -500 nM and thus would be expected
to respond to [Cali changes which occur physiologically (see below). Pan and Solaro (1987)
measured Ca binding in detergent treated canine ventricle which was otherwise intact. They
found a slightly lower affinity for the Ca-specific site of TnC during rigor compared to the
Table 6
Ca and Mg Binding Affinities of Skeletal and Cardiac Troponin
Ca Specific Sites Ca-Mg Sites
number K c• number K c•
of sites (M- I) of sites (M- I )
Skeletal TnC 2 3.2 X 105 2 2.lxI0 7 4 X 103
Skeletal TnC - Tnl 2 3.5 X 106 2 2.2 X 10 8 4 X 104
Skeletal Tn 2 4.9 X 106 2 5.3 X 10 8 4 X 104
Cardiac TnC I 2.5 X 105 2 1.4 X 10 7 7 X 102
Cardiac TnC . Tnl I 1.0 X 106 2 3.2 X 10 8 3 X 103
Cardiac Tn (reconstituted) I 2.5 X 106 2 3.7 X 10 8 3 X 103
Cardiac Tn (native) I 1.7 X 106 2 4.2 X 10 8
Values are from Holroyde et al. (1980) and Potter & Johnson (1982).
isolated TnC complex in Table 6 (1.2 x 10 6 M- I) and the affinity was further reduced ~3-4 fold
by adding ATP to dissociate the rigor complexes.
TnC can also bind other di- and trivalent cations (Fuchs, 1974). Kerrick et al. (1980)
demonstrated that cardiac myofilaments could be activated similarly by Ca or Sr, but that skeletal
muscle is much less sensitive to activation by Sr than Ca. Skeletal muscle can have a higher Ca-
sensitivity than cardiac muscle, partly due to lower pH sensitivity (see Fig 21D & pg 32).
Cardiac muscle also exhibits different contractile protein isoforms (e.g. troponins,
myosin and myosin light chains) than fast skeletal muscle and the expression of various cardiac
isoforms is modulated in vivo (Swynghedauw, 1986; Morkin, 1987). For example, there are two
main isoforms of the cardiac myosin heavy chain (a, ~), sometimes referred to as fast (a) and
slow (~) based on the myosin ATPase rate or muscle fiber shortening rate. Three different
dimers can form (aa, or VI, a~ or V2 and ~~ or V 3). There are species differences (e.g. rat ven-
tricle is mostly VI while rabbit and human ventricle are mostly V 3), adaptational differences (rat
ventricle shifts from a to ~ during hypertrophy) and thyroid hormone induces a switch from ~- to
a-myosin heavy chain production. There are also 4-5 isoforms of TnT in rabbit and human
ventricle (Anderson et aI., 1988, 1991, 1995) which confer different Ca sensitivities to the myo-
filaments. These change developmentally and also with hypertrophy or heart failure.
MECHANISM BY WHICH Ca ACTIVATES CONTRACTION

It is well established that the rise in cytoplasmic [Ca] is the event which activates the
myofilaments and a fairly clear picture (though not complete) of the molecular basis for this
regulation has developed, including mapping of peptide regions involved with changes in subunit
interactions (Zot and Potter, 1987; Solaro & Rarick, 1998). Figures 14 (side view) and IS (end-
on view) illustrate our current understanding of some of these interactions. At rest when rCa]; is
low, the Ca-specific sites of TnC are unoccupied. In this condition the interaction between Tnl
(carboxy-middle) and TnC (amino) is weak and this region of Tnl interacts more strongly with
actin. This favors the configuration where the troponin-tropomyosin complex is shifted
peripherally and more out of the axial groove of the actin filament. This position sterically
REST
Tropomyosin
MLC1
MLC2
+Ca 2 +
Figure 14. Side view of Ca-dependent regulation of acto-myosin interaction in cardiac muscle (based on
diagram supplied by R.J. Solaro and see also Rayment el aI., I993a,b). At rest the C-terminal domain of
Tnl is bound to actin, thereby aJ]choring the TnT-tropomyosin (Tm) complex and preventing the myosin
head (SI) from binding to actin. When Ca binds to the amino terminal ofTnC, this region binds strongly to
the carboxy terminal of TnI, which comes off actin, allowing the TnT-Tm complex to roll deeper into the
actin groove and exposing the sites on actin along the chain which interact with the myosin S I head.
hinders the binding of the myosin S I head to actin. When [Ca Ji rises, Ca binds to the Ca-specific
site of Tne. This strengthens the specific interaction of TnC with Tnl and effectively
destabilizes the interaction of TnI with actin. This favors the more axial location of the troponin-
tropomyosin complex, and removes the steric hindrance to myosin interaction with actin,
consequently allowing force production and/or shortening. The tight interaction between TnT
and tropomyosin is probably important in the transmission of this conformational change along
the thin filament to the actin monomers which do not have associated Tnl subunits.
The movement of tropomyosin deeper into the actin groove was first suggested by x-ray
diffraction studies (Haselgrove, 1973; Huxley, 1973) and the structural and biochemical results
generally support this steric hindrance model of myofilament regulation. However, Chalovich et
al. (198]) suggested that the regulatory proteins might directly block the acto-myosin ATPase
reaction in the absence of Ca, without preventing interaction between myosin heads and actin.
Thus, there may be weak binding interactions between actin and myosin that can still occur in the
sterically blocked (rest state), but for these to become strong or force-bearing and cycle through
the ATPase reaction, the interaction domains must become unblocked. Moreover, there is
evidence to suggest that when the myosin S I head binds to actin it pushes the tropomyosin
deeper into the actin groove than when Ca binding to TnC switches the filament into the open
state (Lorenz et aI., 1995). This is depicted in Fig 15A as 2 prospective Tm positions.
Figures ]4 & 16 illustrate how a series of 7 actin molecules can be cooperatively
activated by one troponin complex. The head to tail overlap of tropomyosin molecules in series
in cardiac muscle may also allow this cooperativity to spread to neighboring tropomyosin-actin
A. Relaxed B. Active
p
Myosin
81
+Ca
-Ca
p
\"'" Myosin blocked------'\

~.~ ~:
Figure 15. End-on view of Ca-dependent regulation of acto-myosin interac.tion in cardiac muscle (based on
diagram by Warber & Potter, 1986). In the absence of Ca (A), Tnl binds to actin thereby preventing
myosin from interacting with actin. This also draws Tm out of the groove between actins, thereby
extending the myosin blocking effect to adjacent actin monomers (which lack Tnl). In the presence of Ca
(B), Tnl binds more firmly to TnC and not to actin. In this case Tm is also not drawn out of the groove and
the interaction of myosin and actin can occur. The two dashed positions of Tm in A illustrate that Ca-
dependent shifts cause partial movement into the groove, but when myosin S 1 binds Tm shifts further.
units (Lehrer et al.,1997; Solaro, 2001). The binding of myosin heads to actin can also
contribute to this sort of long-range cooperativity along the thin filament (Fig 16). This is, in
part, a direct steric effect, but binding of myosin SI heads also increases the affinity of Ca
binding to TnC (Pan & Solaro, 1987; Fuchs, 1995). This would also contribute to cooperative
activation. In this manner the thin and thick filaments can be viewed as more dynamically
involved in their own state of activation, rather than as simply responding passively to the
ambient [Ca].
ACTO-MYOSIN ATPase
In the presence of sufficient Ca, myosin can interact with actin, which greatly increases
the ability of myosin ATPase to hydrolyze ATP and also allows transformation of chemical
energy stored in ATP to mechanical energy and work. A physical model for this transduction
known as the sliding filament theory, came from x-ray diffraction studies (Huxley, 1969) and
mechanical perturbation studies (Huxley and Simmons, 1971) and is illustrated schematically in
Fig 17. At rest the myosin heads (or crossbridges) extend from the thick filament perpendicular
to the filament axis. Upon activation the crossbridge can interact with the thin filament and
produce either force generation or relative filament movement by a rotation of the myosin head
(perhaps due to a series of stable states). Isometric force would be analogous to storing the
potential energy of this myosin head rotation temporarily in an elastic component of the myosin
Chapter 2 Myofilaments 25
Diastole
~~... Tm
~~"'\TnT
Tnl
Systole
Cooperative Activation l~
Figure 16. Cooperative spread of activation along the thin filament stimulated by myosin S I binding to
actin. When Ca binding to the left TnC complex allows myosin binding, this pushes tropomyosin further
from the blocking position along the actin filament. This increases the probability of additional downstream
myosin binding (right) and also increases Ca affinity at neighboring TnC molecules. (modified form of
figure generously supplied by R.J. Solaro).
molecule (Fig 17C). Alternatively, the rotational movement can produce relative motion of the
thick and thin filaments (i.e. shortening) if the muscle force exceeds the load (Fig 17D). The
amount of force developed by a single crossbridge is 0.5-1 pN and the physical movement or
filament translation from a single crossbridge cycle is 5-10 nm or 0.25-0.5% of sarcomere or
muscle length (Cooke, 1997; Molloy et al., 1995; Tyska et aI., 1999). Furthermore, Tyska et al.
(1999) showed that both myosin heads are required to generate maximal force and displacement
and nearly double both values (to 1.4 pN and 10 nm). The details of how the two heads work
synergistically are not yet clear.
The chemical steps involved in the crossbridge cycle have also been extensively
characterized and correlated with such physico-mechanical schemes (e.g. Goldman, 1987;
Brenner, 1987). The general chemical scheme is illustrated in Fig 18. At rest myosin (M) is
mostly complexed with ATP (M.ATP) or in the rapidly equilibrated M.ADP.P i where ATP is
technically hydrolyzed, but the energy has not been used. As [Cali rises M.ADP.P i can interact
with actin and phosphate is rapidly released. The acto-myosin passes through at least two
energetic states where ADP remains bound (A.M.ADP* and A.M.ADP) and these transitions
may encompass the so-called "power stroke" or myosin head rotation. The affinity of myosin for
actin increases along this series of steps and is strongest after ADP dissociates (i.e. A.M).
However, at normal [ATP]i, A.M binds ATP rapidly and this induces dissociation of actin and
M.ATP. The cycle can then continue until [Ca]i declines, thereby stopping myofilament
interaction (in the M.ADP.P i state) or until ATP is depleted. When ATP is depleted the cycle
stops in the A.M state (known as rigor) and crossbridges are firmly attached, creating the
A. Rest C. Force Development

Thick Filament Thick Filament
~
Thin Filamef1j
B. Attachment D. Shortening
Thick Filament Thick Filament
Figure 17. A mechanical model of active crossbridges based on the original diagram by Huxley and
Sinunons (1971). A. a detached crossbridge (e.g. M-ATP in Fig 18). B. an attached crossbridge prior to
developing force (e.g. A-M-ADP-Pi in Fig 18). C. an attached crossbridge developing force stored in the
elastic component (e.g. A-M-ADP* in Fig 18). D. a crossbridge rotated and translated so the filaments
slide relative to one another (e.g. A-M-ADP or A-M in Fig 18).
stiffness associated with rigor mortis). There may also be a small component of crossbridge
cycling that occurs in the absence of elevated rCa]; (Fabiato and Fabiato, 1975b; Goldman el aI.,
1984; Reuben el al., 1971; see also pg 32).
ATP Pi ADP
A -M~A-M-ATP---7A -M-ADP-P -4A -M-ADP*----.A-M-ADP-4. A-M
Ir !
M M-ATP ---+ M-ADP-P ~
f I
M-ADP* ---7
I
M-ADP ~ M
I
ATP Pi ADP
Figure 18. A chemical model of the steps in the crossbridge cycle (or acto-myosin ATPase) based on
Goldman & Brenner (1987). The top TOW shows the states in which actin (A) interacts with myosin (M).
The heavy solid lines indicate the normal reaction pathway, but all the reactions can occur and are reversible.
Two energetically different states of A-M-ADP are indicated by the inclusion of an asterisk on one.
100
><
III 80 Myofilament
:E overlap
~
~ 60
l:
0
'iii 40
l:
Ql
I-
20
0
1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8
Sarcomere Length (~m)
Figure 19. The length-tension relationship in frog skeletal muscle as described by Gordon et al. (1966) is
shown along with inset diagrams. The length-tension relationship for cat cardiac muscle (Allen et aI., 1974)
is also illustrated for the range of physiological sarcomere lengths (thick trace).
THE LENGTH-TENSION RELATIONSHIP

The relationship between sarcomere length and the maximum force which can be
developed is a central issue in the above physical models of muscle contraction. The relationship
was described in detail by Gordon et al. (1966) in frog skeletal muscle and their results are sum-
marized in Fig 19. The fundamental assumption is that the maximal force at any sarcomere
length is determined by the degree of overlap of the thick and thin filaments (i.e. how many
crossbridges can cycle). Thus, as the sarcomere is stretched from its optimal overlap (2 - 2.2 11m)
the force gradually declines until it is 0 at the point where there is no longer overlap (-3.6 11m).
At shorter sarcomere lengths (2.0 to 1.6 11m) the thin filaments cross over and may impede
effective crossbridge formation. Finally, when the thick filament collides with the Z-line (-1.65
11m) the resistance to shortening is greatly increased and externally developed force drops steeply
as the thick filament is compressed.
Cardiac muscle has a strong parallel elastic component which normally prevents cardiac
sarcomeres from reaching the "descending limb" of the length-tension curve (e.g. sarcomere
lengths >2.3 11m). This is of course a good thing for the heart since such long sarcomere length
could result in progressive decrease in contraction and failure of cardiac output. Thus the heart
normally functions along the "ascending limb" of the length-tension curve, so that one would
expect increased contraction with increase in sarcomere length (end diastolic volume or preload).
This increase in fiber overlap is undoubtedly involved in the classic Frank-Starling law of the
heart, whereby increased diastolic volume leads to increased systolic contraction. However, the
length-tension relationship for cardiac muscle is considerably steeper than that for skeletal
muscle at sarcomere lengths between 1.8 and 2.0 11m (Allen et al., 1974; see thick curve in Fig
19). Indeed, the changes in myofilament overlap may only explain ~20% of the classic Frank-
Starling effect (Moss & Buck, 2001).
Hibberd and Jewell (1982) and Kentish et at. (1986) demonstrated that the Ca sensitivity
of the myofilaments was significantly increased at longer sarcomere lengths which would
steepen the length-tension curve for a given [Ca]. Babu et at., (1988) suggested that this was due
to cardiac TnC, since when they reconstituted cardiac fibers with skeletal TnC they failed to see
the effect. However, this finding has been challenged by more recent results from three different
angles (Moss et at., 1991; Fuchs & Wang, 1991; McDonald et al., 1995a). Thus it cannot simply
be due to cardiac vs. skeletal Tne. Another important factor is geometric. That is, as sarcomere
length is increased the myocyte and sarcomere must become thinner to maintain constant
volume. This will cause the thick and thin filaments to come closer together in the myofilament
lattice, thereby enhancing the chance for crossbridges to bind to actin (see narrower interfilament
gap at long sarcomere length in Fig 19 insets). Indeed, osmotic compression of the myofilament
lattice can reverse the decreased Ca sensitivity and Ca binding seen at shorter sarcomere length
(Hoffman & Fuchs, 1988; Wang & Fuchs, 1994, 1995; McDonald & Moss, 1995; Fuchs &
Wang, 1996).
Allen & Kurihara (1982) provided evidence for this length-dependent Ca binding in
intact ferret cardiac muscle where a quick release and shortening of ferret ventricular muscle was
accompanied by a release of Ca from the myofilaments (measured using the Ca-sensitive
photoprotein aequorin). In addition, at longer steady state sarcomere lengths, SR Ca uptake and
release may be increased (Fabiato, 1980; Allen & Kurihara, 1982; Kentish & Wzosek, 1998).
Thus, there are multiple protective mechanisms which increase the heart's ability to contract as it
is stretched. Increased myofilament overlap, increased myofilament Ca sensitivity and increased
SR Ca release may all contribute to the Frank-Starling law of the heart.
THE Ca SENSITIVITY OF THE MYOFILAMENTS

Cardiac myofilaments are activated by Ca in a graded manner, so the relationship
between free [Cal and force development is of fundamental importance. Most of the
experimental results on this issue have been obtained in cardiac muscle preparations which have
had their sarcolemma removed or permeabilized and are called "skinned" preparations. Since
myofilaments are sensitive to submicromolar [Cal and contaminant [Cal is usually several
micromolar, Ca chelators such as EGTA are used to buffer [Cal in the range of interest. The
solutions used for these skinned fibers usually also contain ATP, Mg and some other solutes (e.g.
ATP regenerating systems, pH buffer and KCl). Computer programs (e.g. Fabiato, 1988; Bers et
at., 1994) are usually used to solve the multiple ion equilibria and prepare solutions of known
free [Ca].
The pCa (-log[Ca]) values reported for the threshold [Cal and [Cal at half maximal force
for mammalian cardiac muscle have been reported to be 5.9-7.0 and 4.7-6.0, respectively (Solaro
et at., 1974; Kerrick & Donaldson, 1975; Endo & Kitazawa, 1977; Fabiato & Fabiato, 1975a;
1978; McCLellen & Winegrad, 1978; Hibberd & Jewell, 1979; Fabiato, 1981a; Miller & Smith,
1984; Harrison & Bers, 1989a). While some portion of this large variance is due to species
differences and particular experimental conditions (i.e. ionic strength, temperature, pH etc.), it is
also possible that it reflects inaccuracies in calculated free [Cal. This issue has been critically
Temperature and Ca Sensitivity

~
u
0
~_--36
~_---29
-
N
N 100 ----22
I II
><
III
___- - - 1 5
E
~ ~---8
~ 50
c:
0
"iii 1
c:
Q)
I-
0+-.......,.~~;;;;=;=;;=Ff==-........,.=;::::;~;:;:;:--,---,-.-,.,...,-rT1
0.1 1 10 100
[Cal (~M)
Figure 20. The influence of temperature on the Ca sensitivity of chemically "skinned" rabbit ventricular
muscle (data from Harrison and Bers, 1989a have been redrawn). Both the Ca sensitivity and the maximum
force are reduced at lower temperatures.
Table 7
Temperature and the Ca Sensitivity of Rabbit Ventricular Myofilaments
Temperature pCal/2 Hill Maximum
(0C) Coefficient Force (%)
36 5.47 ± 0.07 1.75 ± 0.1 118.5 ± 10.0
29 5.49 ± 0.07 2.06 ± 0.1 108 ± 4.6
22 5.34 ± 0.05 2.15 ± 0.6 100
15 5.26 ± 0.09 2.49 ± 0.2 74.3 ± 6.0
8 4.93 ± 0.06 3.06 ± 0.5 57.2 ± 7.0
1 4.73 ± 0.04 2.94 ± 0.6 29.3 ± 5.4
Data (from Harrison and Bers, I989a) were fit to a Hill equation F=FmaJC I + {Kn/[CaJ} n).
addressed (Bers, 1982; Miller & Smith, 1984; Harrison & Bers, 1989b) and the errors in [Cal can
be greater than 3-fold (i.e. 0.5 pCa units). These inaccuracies can be attributed to a) incorrect
choice of (or use of) association constants, b) inappropriate modifications of the association
constants for differences in ionic strength and temperature, c) small systematic errors in pH and
d) overestimation ofEGTA purity.
This complication makes it more difficult to compare quantitative details of Ca-
sensitivity curves from one lab to another. While qualitative conclusions are probably valid, this
may not always be the case. For example, based on results from l2°C and 22°C in mechanically
skinned canine Purkinje cells, Fabiato (l985b) concluded that myofilament Ca sensitivity in
cardiac muscle may be increased by cooling, as is the case in skeletal muscle (Stephenson &
A. Fabiato, 1982 B. Harrison & Bers, 1989

100 100
..
><
E
80
Prepartum
Rat "-... 80
[:""
0~ 60 Rabbit 60
c Pigeon
0
'iii 40 Dog Atrium 40
cQ) & Ventricle
~ 20 20
'-Dog Purkinje
0 0
0.1 10 100 0.1 10 100
[Ca)(~M) [Ca)(~M)
C. Lues et aI., 1988 D. Solaro & Coworkers

100 100
><E 80 80
C 60 60
c
0 Monkey
'iii 40
cQ)
~ 20
Rabbit Ventricle (Adu~)
o 0+-~T"F':;::;""""-~~~"'-~~~"1
0.1 1 10 100 0.1 1 10 100
rCa) (~M) rCa) (~M)
Figure 21. Myofilament Ca sensitivity in cardiac muscle preparations. Data from A. Fabiato (1982), B.
Harrison & Bers (1989a), C. Lues et al. (1988) and D. Solaro et al. (1998) & Wattanapermpool et at.
(I 995a) have been redrawn (based on Hill equation fits, 100/( 1+ {Kn/[CaJ) n). All data were normalized to
the maximum force under the prevalent conditions and are from ventricle, unless noted. The conditions
including ionic strength (f) are slightly different in the three panels. The major variations are (in mM):
Temp. pH r [EGTA] [pH-buff] [Mg] [MgATP]
A. noc 7.1 160 10 30 3 3
B. 29°C 7.0 180 10 25 2.3 -5
C. 21°C 6.7 140 5 30 -2.5 -10
D. 23°C 7.0 160 10 60 2 -5
Williams, 1981; 1985; Godt & Lindley, 1982). However, a relatively minor adjustment of
Fabiato's solution calculations, based on our studies of the influence of ionic strength and
temperature on the affinity of EGTA for Ca (Harrison & Bers, 1987), greatly reduced the
apparent temperature-induced shift reported by Fabiato (Harrison & Bers, 1989b). Furthermore,
we found that over the temperature range 37°C to 1°C there was a progressive decrease in Ca
sensitivity of the myofilaments with cooling in chemically skinned rabbit ventricular muscle (Fig
20 and Table 7, Harrison & Bers, 1989a). Similar shifts were also reported for guinea-pig, rat
and frog ventricle (Harrison & Bers, 1990a). We also found that much of the difference between
the influence of temperature on cardiac and skeletal muscle could be attributed to the TnC type
in the muscle, based on experiments where rat ventricular TnC was extracted and replaced by
either rabbit skeletal TnC or bovine cardiac TnC (Harrison & Bers, 1990b). These results
indicate an interesting thermodynamic difference between cardiac and skeletal muscle regulation
at the level of Tne. That is, cooling decreases the Ca sensitivity of cardiac myofilaments, but
increases the sensitivity of skeletal myofilaments (largely due to the TnC type).
The foregoing indicates that minor oversights in the calculations of free [Ca] in the
complex solutions used in skinned fiber studies can lead to qualitative as well as quantitative
errors. It is therefore useful to obtain an independent check of the free [Ca] in the solutions used
whenever possible (e.g. with a Ca-selective electrode; Bers, 1982; Bers et al., 1994). With these
caveats in mind some factors which alter myofilament Ca sensitivity will be discussed below.
Skinned cardiac muscle fibers also exhibit hysteresis in the force-[Ca] relationship, such
that Ca sensitivity is apparently greater when [Ca] is decreasing than when [Ca] is increasing
(Harrison et al., 1988). This hysteresis diminishes with increasing sarcomere length and is
virtually eliminated at sarcomere lengths greater than 2.2 /lm. This implies that for full
relaxation to occur at shorter sarcomere lengths, the [Ca]i must be lower than would be supposed
from force-pCa relationships obtained in the activating direction. From a functional standpoint,
this may be an important consideration, since this may limit relaxation between contractions even
if [Ca]i has reached a stable diastolic level.
Figure 21 shows superimposed [Ca] vs. tension curves from skinned fibers from several
cardiac species. The data in Fig 21A are from Fabiato (1982). Fabiato & Fabiato (l978b)
reported comparative data under slightly different conditions (with 3 abscissa calibrations, illus-
trating the calibration problems above). In that report they found that the myofilament Ca sensi-
tivity of human atrium and ventricle were indistinguishable from those of rat and rabbit ventricle.
Figures 2IB-D are from other studies. The experimental conditions in the three panels are all
somewhat different. While neonatal ventricle and skeletal muscle myofilaments have somewhat
higher Ca affinity, the main point is that there are only minor differences in Ca sensitivity among
mammalian cardiac muscle preparations under the same conditions. The pCal/2 values for these
skinned fiber preparations are typically between 5.2 and 5.7 (2-6 /lM) with Hill coefficients 1.9-
2.5 at 22-29°C, at ionic strength ~160 mM, pH~7.0 with 3-5 mM Mg ATP and 2-3 roM free Mg.
These will serve as a base for the comparisons below.
FORCE-pC a RELAnON IN INTACT CARDIAC MUSCLE

All of the foregoing and much of what will come below is based on experiments in
muscles in which the sarcolemma has been disrupted so that the intracellular environment can be
controlled. However, this has the substantial disadvantage that some cellular constituents which
might affect myofilament Ca sensitivity may be lost. For example, Harrison et al. (1986) found
that carnosine and related compounds (imidazoles), which are abundant in skeletal muscle
(Crush, 1970) and may total as much as 10 roM in cardiac muscle (O'Dowd et aI., 1988)
significantly increase cardiac myofilament Ca sensitivity. Skinning also causes osmotic swelling
of the myofilament lattice in simple ionic solutions. This reduces apparent myofilament Ca
sensitivity in skinned fibers (see pg 28).
Yue et al. (1986) estimated the steady state force-[Ca]i relationship in intact ferret
ventricular muscle using aequorin to measure [Ca]i and ryanodine treatment to allow the heart
muscle to be effectively tetanized. They found a mean pCal/2 of 6.3 and a mean Hill coefficient
of 6. J. This is a remarkably steep curve and the half-maximum point is at a [Ca]i (500 nM),
considerably lower than almost any skinned fiber results. The myofilament force would go from
less than 5% at 300 nM [Ca]i to 95% of maximum at 800 nM [Ca]i. The specific factors
responsible for the disparity in Fig 22 are not entirely clear, but the intact cell myofilament Ca
100
><III 80 Yue et al,1986

E Ferret
~ (intact) Harrison &
~ 60
c: Bers, 1989
.2 40 Rabbit
VI
c: (skinned)
(I)
I- 20
0
0.1 10
rCa] (~M)
Figure 22. The myofilament Ca sensitivity in intact ferret ventricular muscle at 30°C (from Yue et aI.,
1986) and in chemically skinned rabbit ventricular muscle at 29°C (from Harrison and Bers, 1989a). The
curves are Hill curves based on the reported pCa 112 and n values.
sensitivity has been confirmed in subsequent studies using other Ca indicators (Gao et al., 1994;
Backx et al., 1995). Gao et al. (1994) found similar myofilament Ca sensitivity before and after
skinning when they used 0.5 ruM free [Mg] in the skinned fiber solutions (as they measured in
the intact muscle). Thus, 2 ruM free [Mg], used in many skinned fiber studies, may be too high,
but the difference in Fig 22 is not completely understood.
Sollott et at. (1996) demonstrated that even during diastole and very low [Ca);, there is
appreciable acto-myosin interaction contributing to resting force. They also showed that this
interaction was still Ca-dependent and correlated with very slow decline in rCa]; (even in cells
with Ca-free, EGTA containing solution). Stuyvers et at. (1997) also found that as diastolic rCa];
continues to decline and sarcomere length increases, muscle stiffness increases. This may be
indicative of dynamic structural changes which occur even in the apparent quiescent state.
FACTORS WHICH INFLUENCE THE FORCE-[Ca] RELATIONSHIP

A large number of factors are known to modify the relationship between [Ca] and the
force generated by cardiac myofilaments and these have been most extensively studied in
skinned cardiac muscle preparations (e.g. Table 8). As described above, both cooling and shorter
sarcomere length decrease the Ca sensitivity and also the maximum force generated by the
myofilaments (Hibberd & Jewell, 1982; Kentish et al., 1986; Harrison and Bers, 1989a).
Similarly, acidosis decreases both the Ca sensitivity and maximum force generated by the myo-
filaments (see Fig 170; Fabiato & Fabiato, 1978a, Blanchard & Solaro, 1984). This may be
particularly important during pathological conditions such as hypoxia or ischemia where
intracellular pH (pH;) is known to decline (Jacobus et al., 1982). An interesting note is that the
Ca sensitivity of myofilaments from perinatal hearts is much less affected by acidosis (Solaro et
al., 1986, 1988; Martin et at., 1991). The results suggested that the difference in pH sensitivity
was mainly due to a difference in Tnl (slow skeletal Tnl is expressed in the newborn heart).
However, it is not due to the unique N-terminal domain in cardiac Tnl (Guo et al., 1994), which
is crucial in modulation by phosphorylation (below). Skeletal muscle force is also less depressed
by acidosis, and both skeletal Tnl and TnC contribute to this effect (65 & 35% respectively,
Metzger et al., 1993; Ball et al., 1994; Ding et al., 1995).
It has been argued that the intracellular acidosis accompanying early ischemia is not
sufficient to explain the mechanical dysfunction (Jacobus et al., 1982; Weiss et aI., 1984; Allen
& Orchard, 1987) especially if one only considers the effect of pH; on the myofilaments.
Ischemia also results in decreases of [creatine phosphate] and [ATP] and increases inorganic
phosphate (P04 ) levels. While decreasing [ATP] (as MgATP) increases myofilament sensitivity,
it also decreases the maximum force (Best et aI., 1977). Kentish (1986) demonstrated that the
increase in P04 which accompanies the decline in high energy phosphates (ATP and creatine
phosphate) during ischemia can dramatically decrease both Ca sensitivity and maximum force.
Overall the increase in P04 and the decrease in pH; in ischemia combine to produce a dramatic
decrease in myofilament Ca sensitivity (with P0 4 being slightly more important). This
combination plays an important role in the early decline in mechanical function (see pg 307).
Increasing the free [Mg] also decreases myofilament Ca sensitivity (Fabiato & Fabiato,
1975b; Best et al., 1977). Increasing ionic strength (but not osmolality) decreases both Ca
sensitivity and maximum force of the myofilaments (Kentish, 1984). Imidazoles (e.g. carnosine
and N-acetyl histidine) which are naturally occurring in cardiac muscle cells (O'Dowd et aI.,
1988) increase myofilament Ca sensitivity (Harrison et al., 1986). These compounds are
chemically related to caffeine, which is often used in the study of cardiac muscle Ca metabolism
because it can deplete the SR of Ca. However, caffeine and other methylxanthines are potent
myofilament Ca sensitizers (Wendt & Stephenson, 1983; Fabiato, 198Ib). Interestingly, caffeine
exerts its potent sensitizing effect without affecting Ca binding to the myofilaments (Powers &
Solaro, 1995). This contrasts to other Ca sensitizers which exert their effect by increasing Ca
affinity (e.g. pimobendan and MCI-154, Fujino et al., 1988; Kitada et al., 1989). Caffeine and
other methylxanthines also inhibit phosphodiesterases, but the effects on the myofilaments are
not mediated by cyclic AMP.
A number of inotropic agents function in part via their ability to inhibit cardiac phospho-
diesterases and thereby elevate cyclic AMP (e.g. amrinone, milrinone, pimobendan and
sulmazole), while some increase myofilament Ca sensitivity (Endoh, 1998, Table 8, see also pg
324-328). Ca sensitizers include levosimendan (Edes et al., 1995), EMD 57033 (Solaro et al.,
1993; Vannier et al., 1997), pimobendan (or AR-L 115-BS, Fujino et al., 1988), sulmazole (or
UDCG-1I5-BS, Solaro & Ruegg, 1982), isomazole (Lues et al., 1988), BM 14.478 (Freund et
al., 1987), MCI-154 (Kitada et al., 1987; 1989), DPI 201-106 (Scholtysik et al., 1985) and
perhexiline & bepridil (Silver et aI., 1987), but not milrinone (Fujino et aI., 1988). Racemic
EMD-53998 has both phosphodiesterase inhibitory and Ca sensitizing actions, but these actions
are selectively due to either the (-) enantiomer (EMD-57439) or (+) enantiomer (EMD-57033)
respectively (see Figs 177 & 178; White et al., 1993; Solaro et al., 1993). Thus, myofilament
effects of some of these drugs contribute to their inotropic effects.
Ray and England (1976) and Solaro et al. (1976) demonstrated a cyclic AMP-dependent
phosphorylation of cardiac TnI in response to ~-adrenergic stimulation. This phosphorylation
decreases myofilament Ca sensitivity in intact ventricular muscle (Okazaki et al., 1990) and in
skinned fibers, a result which is mimicked by cyclic AMP (McClellan & Winegrad, 1978;
Holroyde et al., 1979; Mope et aI., 1980; Herzig et al., 1981). Phosphorylation of TnI (and the
shift in Ca sensitivity) can also be reversed by cyclic GMP or cholinergic agonists (Mope et al.,
1980; Horowits & Winegrad, 1983). Phosphorylation of Tnl at both Ser 23 & 24 appear to be
essential for this effect (Zhang et aI., 1995a). This region is in the N-terminal extension of TnI
that is not present in slow skeletal Tnl. Studies with mutant cardiac Tnl lacking this N-terminal
extension demonstrated that this phosphorylation was both necessary and sufficient to produce
the reduced myofilament Ca sensitivity seen with PKA (Guo et aI., 1994; Wattanapermpool et
aI., 1995b). Transgenic mice in which cardiac Tnl was quantitatively replaced with slow skeletal
Tnl exhibit higher myofilament Ca sensitivity, which is no longer affected by PKA phosphory-
lation (Fentzke et al., 1999). These data provide compelling evidence that Tnl phosphorylation
mediates the alterations in myofilament properties induced by PKA. While PKA also
phosphorylates C-protein, it is less clear wether this really alters myofilament function (Weisberg
& Winegrad, 1996; Winegrad, 1999; Solaro, 2001).
For ~-adrenergic stimulation to produce its well known inotropic effect, the amplitude of
the intracellular Ca transient must more than compensate for the reduced myofilament Ca
sensitivity (e.g. by enhancement of Ca-current and SR Ca release, see Chapters 5, 7 & 10). The
decline in myofilament Ca sensitivity induced by ~-adrenergic stimulation is accompanied by an
increased off-rate of Ca from TnC. In principle this could contribute to the faster relaxation of
contractions (or lusitropic effect) observed in the presence of ~-adrenergic agonists. Studies in
skinned cardiac muscle fibers using photolysis of a caged Ca chelator, diazo-2, have been
equivocal, finding either enhanced (Zhang et al., 1995b) or unchanged relaxation upon phospho-
rylation of cardiac Tnl (Johns et al., 1997). In intact fibers, McIvor et al. (1988) showed that
myofilament desensitization was not necessary for faster relaxation. That is, the acceleration of
relaxation could be attributed entirely to the other actions of isoproterenol (e.g. increased SR Ca
uptake rate, secondary to cyclic AMP dependent phosphorylation of phospholamban, Kirch-
berger et aI., 1974; Tada et al., 1974; Lindeman & Watanabe, 1985a). This could be true even
though phosphorylation of Tnl appears to occur at lower isoproterenol concentrations than for
phospholamban (Karczewski et aI., 1990). Indeed, Fentzke et al. (1999) found that even with
slow skeletal Tnl (lacking the phosphorylation sites), isoproterenol greatly accelerated relaxation
in myocytes. Li et al. (2000) also showed that in mice lacking phospholamban, PKA phosphory-
lation of Tnl produced no change in myocyte relaxation. However, with increasing force
generation in muscle (as opposed to unloaded myocyte shortening) there was a smalllusitropic
effect of isoproterenol in these phospholamban knockout mice. We concluded that all of the
lusitropic effect on unloaded myocyte relaxation was attributable to phospholamban phosphory-
lation, whereas with maximal force development the Tnl phosphorylation may contribute ~15%
to the lusitropic effect of isoproterenol.
One of the myosin light chains (MLC2 or regulatory light chain) can also be
phosphorylated by a Ca-calmodulin dependent protein kinase (Pires et al., 1974). Phosphory-
lation of this regulatory light chain by myosin light chain kinase is crucial in the activation of
smooth muscle contraction (Aksoy et aI., 1976; Walsh et aI., 1983). Yang et al. (1998) showed
that MLC2 phosphorylation in skeletal muscle could mimic the effect of reducing filament lattice
spacing (increasing the likelihood of myosin binding to actin). However, the physiological role
of this phosphorylation in cardiac muscle is not clear (England, 1984; Gevers, 1984; Solaro,
Table 8
Factors which alter Cardiac Myofilament Ca Sensitivity
Factor Sensitivity Maximum Force
Factor ref Change Direction L'.pCay, Direction L'.(%)
Temperature a 36~ noc dec 0.18 dec 16
2~ 8°C dec 0.20 dec 43
Sarcomere Length b 2.0~ 2.35 flm mc 0.21 mc 34
c 1.75~ 2.05 flm inc 0.25 mc 36
pH d 7.0~ 6.6 dec 0.34 dec 10
pH e 7.0~ 6.5 dec 0.29 dec 12
P04 (pH 7) f O~ 20mM dec 0.37 dec 69
MgATP (lmM Mg) g 0.03~ 4mM dec 0.3 dec 31
Mg (4mMMgATP) g 0.05~ I mM dec 0.4 mc 21
Mg (3mM MgATP) h 0.3~ 3 mM dec 0.33-
Ionic Strength (f) 0.1~ 0.2 M dec 0.3 dec 17
Tnl Phosphorylation j O~ -50% dec 0.45
Carnosine k O~ 15 mM inc 0.2 mc 3
Caffeine I O~ 20mM inc 0.32 dec 6
Sulmazole (AR-L115 BS) m O~ lmM mc 0.2 mc 7
Isomazole n O~ lmM inc 0.1 inc -15
Pimobendan (UDCG-115-BS) 0 O~ 50 flM mc -0.2 not.
MC1-154 p O~ JOflM inc -0.2 not.
Levosimendan q O~ 10flM mc 0.1 noll
EMD 57033 r O~ 10flM inc 0.3 inc 20
CGP-48506 s O~ 10flM mc 0.14 mc 13
Butanedione monoxime (BDM) t O~ 3mM dec 0.15 dec 30
a) Harrison & Bers, 1989a k) Harrison et aI., 1985

b) Hibberd & Jewell, 1982 1) Wendt & Stephenson, 1983
c) Kentish et al., 1986 m) Riiegg, 1986
d) Fabiato & Fabiato, 1978a n) Lues et aI., 1988
e) Blanchard & Solaro, 1984 0) Fujino et al., 1988
f) Kentish, 1986 p) Kitada et al., 1989
g) Best et aI., 1977 q) Edes et aI., 1995
h) Fabiato & Fabiato, 1975b r) Solaro el aI., 1993; Val1l1ier et al., 1997
i) Kentish, 1984 s) Wolska et al., J996a
j)Mopeetal.,1980 t) Gwathmey et aI., 1991
2001). Phosphorylation of this regulatory light chain by myosin light chain kinase increases the
Ca sensitivity of the myofilaments in skinned pig ventricular muscle (Morano et aI., 1985), and
increases the rate of force development (Morano et aI., 1995), but decreases myosin cross-bridge
cycling rate (Franks et al., 1984). Silver et at. (1986) showed a frequency dependent change in
the degree of phosphorylation of myosin light chains in rabbit ventricular muscle. Thus, while
phosphorylation of mynsin light chains occurs and may modify acto-myosin ATPase, the details
of this action and its physiological importance remain to be unequivocally established. Other
myofilament sites are also targets for phosphorylation (see Fig 13B and Solaro et al., 1987;
Solaro, 2001 for further discussion).
FORCE VELOCITY CURVES

So far most of the discussion has focused on the development of force under isometric
conditions (i.e. fixed sarcomere length) and at fixed resting force (or preload) and infinite
Force-Velocity Curve
2.0 .15 "tl
>.- 0
:E
...
;t:l/)
u-
o l/)
(!)
-~
1.5
-,,
"...0
Q)-
>tn .10
tnt: ',Power
.....
"•
t:~ 1.0 ..... .....
.
.- Q) ..... (!)
t:_ .....
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U
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.05
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~E 0.5
..... 0"
" .....
:s-"
CJ)- ;:::;:
""
0.0 .00
0.0 0.2 0.4 0.6 0.8 1.0
Force (fraction of maximum)
FiKllre 23. Force-velocity and force-power curves in cardiac muscle. The thick curve is at optimal sarco-
mere length for force development (Lm,x) and shortening velocity varies inversely with load (or force). At
shorter sarcomere length this curve shifts down and to the left (lower maximal force). Power (force x
velocity) is shown for the Lmax curve. Based on data and analysis in Moss & Buck (2001).
afterload. Indeed, ventricular filling, end diastolic pressure and volume are the preloads that set
the sarcomere length and position on the Starling curve (Fig 19) where contraction begins.
However, only the early part of ventricular contraction in situ is isometric (i.e. the isovolumic
phase when both the A-V valves and the aortic/pulmonic valves are closed). Once the force (or
ventricular pressure) development reaches the afterload (or aortic pressure), the aortic (or
pulmonic) valve opens and ejection begins. This is associated with shortening of individual
sarcomeres at a velocity that is related to the afterload (i.e. the force or pressure against which
the heart must perform work). Clearly physiological cardiac contraction is not isometric, nor is it
ideal to simply measure unloaded shortening (as often done with isolated cardiac myocytes).
Cardiac muscle normally shortens under a continually changing afterload (aortic pressure).
To understand the relationship between load (or force) and velocity of shortening,
muscles have traditionally been released from a certain sarcomere length bearing different loads
and the velocity of shortening measured (Moss & Buck, 2001). According to the classic work of
Hill (1938) one expects a hyperbolic relationship of the form (P+a)(V+b) = b(P max + a) (where P
and V are force and velocity, P max is maximal isometric P, and a and b are constants). As Figure
23 shows, this simply means that the maximum velocity (V max) occurs when there is no load and
that at P max the muscle cannot ~horten (V =0 = isometric). This is intuitive in that the greater the
load, the slower the shortening velocity. It also means that as the muscle shortens from L max and
maximal isometric force declines (Fig 19), the force-velocity relationship will fall along a lower
curve (lower solid curve in Fig 23, where P max is 62% of that at L max ). Thus as ventricular
ejection proceeds, the velocity of ejection decreases for a given load (or pressure, or force).
Thus higher initial sarcomere length (higher preload) partly explains why the early ejection phase
is the fastest. Of course the initial ventricular ejection phase is also the time when the aortic
pressure (afterload) is minimal (which would be relatively leftward in Fig 23).
The maximum shortening velocity (V max) occurs with no load and this reflects the
maximum turnover rate of the myosin ATPase cycle. That is, with truly zero load, a single
myosin head could (in principle) shorten the muscle at its maximal cycle rate. Moreover, the rate
limiting step is thought to be ADP release, which also governs the crossbridge detachment step
(Siemenkowski et at., 1985) Thus, situations which alter the isoform of myosin expressed can
alter Vmax (<x fast or ~ slow). The switch from <X- to ~-myosin in hypertrophic rats may be
adaptive in sustaining force or pressure longer during contraction, at the expense of V max' Since
slower myosin splits ATP more slowly, this may also conserve energy for a given force-time
integral. Figure 23 also shows that changes in sarcomere length typically produce greater
changes in maximal isometric force than in Vmax. This is because P max depends on the number of
crossbridges working in parallel, while V lllax is determined mainly by the single crossbridge
turnover rate. It should also be noted that even without an external load, the cardiac myocyte has
internal load due both to the mass of the cell and also internal elastic elements which resist
shortening (as well as lengthening). That is, even isolated myocytes spring back to a given
resting length after contraction and rCa]; decline (sarcomere length 1.85-1.95 f.lm; see Roos, 1997
for references and discussion). In the intact heart, where the extracellular matrix also contributes
to resting sarcomere length the values tend to be a bit longer (2-2.2 f.lm; Roos, 1997).
Figure 23 also shows how the power output of cardiac muscle changes with afterload.
Work is, of course, equal to force x distance and power (work/time) is then force x velocity. The
maximal power typically occurs at 20-40% of maximal isometric force, which coincides with the
typical degree of myofilament activation during a twitch (Harrison & Bers, I990a).
A large factor in the rate of activation of the myofilaments is the rate of rise of [Cali-
However, even at a constant level of Ca activation it takes a finite time for the crossbridges to
cycle and for development of force. Figure 24 shows an approach that has been used to assess
this fundamental activation characteristic of the myofilaments. The muscle is initially released
(by 10-20% of its initial length) such that force drops to zero. After 20-50 ms the muscle is
rapidly stretched back to the initial length, instantly breaking any attached crossbridges (note the
spike). The exponential rate constant of force redevelopment is indicative of the transition from
weak or unbound crossbridges to the strongly bound, force generating state (k'f> Brenner, 1986;
Moss & Buck, 2001). Cardiac ktr increases at higher steady rCa] (Wolff et al., 1995a; Palmer &
Kentish, 1998; but see also Hancock et aI., 1996). Swartz & Moss (1992) found that this effect
could be produced in skeletal muscle by either higher rCa] or N-ethylmaleimide Sl heads (which
mimic strongly bound crossbridges) and they inferred that this reflected Ca- or crossbridge-
dependent cooperative activation.
Brenner & Eisenberg (1986) proposed a crossbridge model where strong crossbridge
binding is regulated by the rate constant for formation ifapp) and dissociation (gapp) of the
complex, such that kr depends on bothfapp & gapp, but detachment depends only on gapp'
Palmer & Kentish (1998) showed that the rate constant of force development after flash
photolysis of caged Ca and ktr measured as in Fig 24 were almost identical, indicating that there
is little delay due to the local rise in [Ca]i> Ca binding and troponin-tropomyosin changes.
Clearly it is mostly due to crossbridges going from weak (or unbound states) to strong binding
states which develop force. They found that activation rate constants were much faster in rat
Quick Release and Re-stretch

1.0 r
-
length step CD
-
::J
40 Force <C
0.9
01
-::r
~
-
.§.. 30
III
Q.l 0
o 20 o'
-
(,)
Force redevelopment ::J

LL
0
10 F=Fss(1-e-ktrt) en
!E.
o
o 100 200 300 400
time (ms)
Figure 24. Redevelopment of force after release and rapid re-stretch in cardiac muscle. Force (F) drops
to zero with the release and shortening. A transient spike in force occurs upon re-stretch as crossbridge
attachments are stressed, then broken, allowing the re-attachment-force development to be monitored (based
on data in Moss & Buck, 2000).
than guinea-pig ventricle (14 vs. 2.7/s). They also flash photolyzed a caged Ca-trap (NP-EGTA)
to drop [Cal extremely fast and found a similar difference in relaxation rate constants (J6/s in rat
vs. 3/s in guinea-pig), despite similar Ca-dependence for steady state force (pCaJ/2 = 5.6). These
findings are consistent with the rat having 90% a-myosin (fast) and guinea-pig having 50-60% p-
myosin (slow) and also emphasize that intrinsic properties of the myofilaments can contribute
importantly in the kinetics of contraction and relaxation.
The control of the myofiJaments by [Cal and other factors is very complex. Furthermore,
the simplifying assumption often used (e.g. in subsequent chapters) that the myofiJaments
respond to a given [Cal; in a direct and simple black box manner is obviously incomplete.
However, this implicit assumption makes it considerably easier to discuss the equally compli-
cated area of [Cal; regulation in relation to cardiac muscle function. One must retain the
perspective that the relationship between [Cal; and contraction is not fixed and it is contraction
which is the physiological function of cardiac muscle cells. Indeed, even when cellular cardiac
physiologists talk about contraction, we usually mean quasi-isometric contractions in papillary
muscles (or muscle strips) or unloaded contractions in single isolated myocytes, rather than the
more complicated in vivo contractions. Again, the simplifications made in the name of
reductionist science should be borne in mind as we go forward.
D.M. Bers. 39
Excitation-Contraction Coupling and Cardiac Contractile Force.
2n~ Ed., Kluwer Academic Publishers, Dordrecht, 2001
CHAPTER 3
SOURCES AND SINKS OF

ACTIVATOR CALCIUM
GENERAL SCHEME OF Ca CYCLE IN CARDIAC MYOCYTE

Transsarcolemmal Ca influx and SR Ca release play dominant roles in the rise of rCa];
which activates contraction in the heart. It is useful at this point to present a simple working
model of cellular Ca fluxes which can serve as a background for further discussion (Fig 25).
During the cardiac action potential, Ca enters the cell via sarcolemmal Ca channels
(possibly more than 1 type) and there may also be some Ca entry via Na/Ca exchange. This Ca
which enters the cell can contribute directly to the activation of the myofilaments. Ca entry is
also involved in the activation of SR Ca release. This is the well known Ca-induced Ca-release
process described in detail in Chapter 8. Whether sarcolemmal Ca channels are, in fact, located
directly over the SR Ca-release channel has not yet been proven, but enough indirect evidence
(see Chapters 1, 7 & 8) consistent with this arrangement justifies inclusion in this working
model. The possibility of direct electro-mechanical coupling between the sarcolemmal Ca
channel protein and the SR Ca-release protein (as in skeletal muscle) is controversial and will be
discussed in Chapter 8. In any event, the myofilaments are activated by the combination of Ca
influx and SR Ca release. The degree of contractile activation depends on how much Ca is
delivered to the myofilaments (as well as how they respond, see Chapter 2). As rCa]; rises the
regulatory TnC binding site also has to compete with other cytosolic Ca buffers.
For relaxation to occur, Ca must be removed from the cytoplasm, lowering rCa]; such
that Ca will dissociate from TnC. Four Ca transport processes may be involved in removing Ca
from the cytoplasm (Bassani et al., 1992). Ca can be transported: I) into the SR by the SR Ca-
ATPase pump, 2) out of the cell by the sarcolemmal Ca-ATPase pump, 3) out of the cell by the
sarcolemmal NaiCa exchange or 4) into mitochondria via the Ca uniporter. These 4 Ca transport
systems are all in direct competition for cytoplasmic Ca (see below and Chapters 6 & 9).
Each of these transport systems will be discussed in considerable detail in later sections.
Ca influx via Ca current and Na/Ca exchange will be discussed in Chapters 5 and 6 respectively.
The SR Ca-ATPase and release channel will be the focus of Chapter 7, while Chapter 8 will
focus on the details of the E-C coupling mechanism itself. Ca extrusion from the cell via Na/Ca
exchange and the sarcolemmal Ca-ATPase will be discussed in Chapter 6. Mitochondrial Ca
transport will be discussed below (pg 56)_ Discussion of how these mechanisms interact
functionally will start below and recur throughout (especially in Chapters 9 & 10).
A small leak of Ca out of the SR into the cytoplasm must occur at rest. It is unimportant
here whether this leak occurs via the SR Ca-release channel (Ca sparks) or not. The point is that
3Na 2K Na
Sarcolemma
:1 ::::::::~a~
Ca 3Na H
~===:::---. '\
t=;-.....,)~
~
Ca .::::::: I :::::::. Ca
Myofil .. 'A
3Na
Ventricular Myocyte
Figure 25. General scheme of Ca cycle in a cardiac ventricular myocyte. Ca can enter via Ca channels
(Ie,) and NaiCa exchange (NaCaX). Ca influx controls SR Ca release by the ryanodine receptor (RyR). Ca
is removed from the myofilaments (Myofil) and cytosol by the SR Ca-ATPase pump (modulated by
phospholamban, PLB), sarcolemmal Ca-ATPase pump, Na/Ca exchange & mitochondrial (Mito) uniporter.
there must be some finite rate at which Ca leaks down its ~IO,OOO-fold concentration gradient
(-I mM inside SR: -100 nM in cytosol). This cytoplasmic Ca is then subject to the competing
Ca transport systems above. If all the Ca were re-accumulated by the SR there would be no net
loss of SR Ca. However, if some of that Ca leaked by the SR is extruded from the cell by Na/Ca
exchange or the sarcolemmal Ca-pump, it would represent a net loss of Ca from the SR and the
cell. This is the basis for the cardiac muscle phenomenon known as rest decay (Allen et al.,
1976; Bers, 1985), wherein most mammalian cardiac muscle preparations exhibit smaller post-
rest contractions after longer rest intervals (see Chapters 6, 7 and 9). Thus, the resting muscle
may not be at a true steady-state with respect to Ca fluxes for a rather long time.
A similar type of leak must exist at the sarcolemma, since the electrochemical gradient
between the extracellular space and cytoplasm is even greater than between the SR and
cytoplasm (due to the negative membrane potential). Again, it matters little whether this leak is
via sarcolemmal Ca channels or some less specific route. This Ca influx must be balanced by Ca
extrusion; otherwise such Ca entry could cause progressive gain or loss of cell (and SR) Ca.
Thus, the contribution of the sarcolemma to maintaining cellular Ca content within specific
limits is absolutely essential. The intracellular compartments can only buffer the changes in cell
Ca driven by the sarcolemma. In addition, the intracellular cytosolic Ca buffers are in a closed
system in quasi-equilibrium with free [Cali, and consequently are of finite capacity.
Chapter 3 Ca Sources& Sinks 41
This qualitative picture is useful, but it becomes apparent quickly that the actual
quantities of Ca movement are extremely important. It will therefore be critical to consider Ca
transport and cytosolic buffering much more quantitatively. This is the only way to address
questions such as: How much Ca enters the cell via Ca current vs. Na/Ca exchange? How much
SR Ca is released compared to Ca influx? How do the four Ca transport systems compete for Ca
extrusion from the cytosol during relaxation? Thus we must consider how Ca transport is
quantified and how much Ca is involved in activation of contraction.
CELLULAR VOLUME CONVENTIONS AND Ca FLUXES

A variety of different cardiac preparations and techniques have been used to measure Ca
transport (with different advantages and disadvantages). This information must all be brought
together in a common framework for overall quantitative consideration. We usually consider
free ion concentrations in mM or f!M. For the intracellular environment it makes sense then to
consider [Ca]; in nM or f!M and total cytosolic Ca in similar units (f!mol/L cytosol). A
fundamental convention must be developed with respect to the cytosolic volume of interest.
Fabiato (1983) suggested excluding mitochondrial volume as not readily accessible.
Since mitochondria occupy 30-35% of total cell volume in ventricular myocytes (see Tables 2 &
3), this constitutes a substantial decrement from total cell volume. Nuclear volume is about 2%
of cell volume in rat ventricular myocytes (Table 3), but Ca probably passes through large
nuclear pores such that this volume should probably not be excluded. The SR also occupies 1-
3.5% of cell volume (Table 3). While the SR is clearly a separate compartment, its absolute
volume is less accurately known. Partly to account for this we typically use the lower end of
non-mitochondrial cell volume (65%) for cytosolic volume. The myofilament space which
occupies almost 50% of the cell volume is considered to be part of the cytosol.
Ca fluxes into and out of the cytosol of ventricular myocytes have been measured in
several different units, depending on the type of preparation and measurement. For example, Ca
transport by SR or sarcolemmal vesicles may be reported in nmol Ca/mg SR protein, 45Ca fluxes
in heart tissue in f!mol/kg wet wt of whole tissue, while integrated Ca current may be reported in
pCoul/pF (or fmol/pF). Table 9 shows convenient conversion factors for some of the most
commonly used units and preparations. These values are not all from one species, but are
generally based on values from either rabbit or rat ventricle. My choice of common units for Ca
flux in Table I is somewhat arbitrary, but reflects the cellular focus here. Another value that
comes from these calculations is the protein concentration in a ventricular myocyte (109 mg cell
protein/ml cell volume) or 9.2 f!1 cell volume/mg protein or 5.95 f!1 cytosol per mg protein.
Ca BUFFERING IN THE CYTOSOL

Ca is very highly buffered in cardiac myocytes, as in all cells. Indeed, while the free
[Ca] transient in cardiac myocytes may rise from 100 nM at diastole to a peak near I J.!M, it takes
-100 f!M Ca added to the cytosol to produce this change (see below). Having established some
convention about cytosolic volume units, we can consider the cytosolic Ca binding sites in terms
of their concentrations (in f!mol/L cytosol or simply f!M) and in terms of their equilibrium
dissociation constants (K d , in f!M or nM).
Table 9
Ca nux (or sites): Conversion to /-lmol/L cytosol
Multiply by this factor
Preparation Measurement units for units umol/L cytosol
ventricular muscle flmol/kg wet weight 2.43 a
ventricular muscle flmol/kg dry weight 0.49 b
ventricular homogenate nmol/mg homog pn 292 c
ventricular myocytes nmol/mg cell pn 168 d
subcellular fractions (e.g. SR or SL) nmol/mg SR pn 29.2 e
f
ventricular myocytes fmol/pF 7046
ventricular myocytes flmol/L cell volume 1.55 a
a using 35% of cell volume occupied by mitochondria (Table 3), 1.06 g/ml ventricular density and 33%
extracellular space (Chapter I). [1.06 kg wet wt/L vent][1 L vent/0.67 L cell][1 L cell/0.65 L non-mito
volume]=2.43 kg wet wt/L cytosol, similar to 2.5 from Fabiato (1983) via a more complex calculation.
b similar to above, but including wet weight: dry weight = 5 (Langer et aI., 1990).
c using [120 mg homog pnlg wet wt][2.4 kg wet wt/L cytosol] = 292 g homog pniL cytosol.
d using the 1.83-fold and 1.66-fold purification of dihydropyridine and ryanodine receptors in isolated
myocytes (Bers & Stiffel, 1993; Hove-Madsen & Bers, 1993a) [0.574 mg cell pnlmg vent homog
pn][120 mg homog pnlg wet wt][2.43 kg wet wtlL cytosol]=168 g cell pniL cytosol.
assuming a lO-fold purification factor for SR or sarcolemma (SL). [I mg SR pnilO mg homog
protein)[120 mg homog pnlg wet wt][2.43 g wet wt/ml cytosol] = 29.2 mg SR proteinl ml cytosol).
More generically the conversion factor is 292/x, with x the purification factor for a given organelle.
Mitochondrial protein is about 40 mg/g wet wt (half that reported by Scarpa and Graziotti, 1973). This
limits potential purification of mitochondria to 3x (vs. 50-60x for SL or SR).
fusing 4.58 pF/pl for rabbit ventricular myocytes (see Table 2, pg 6; Satoh et aI., 1996) [4.58 pF/pl)[1 I
cell/0.65 I cytosol) = 7.046 pF/pl cytosol [xlOOO (pl/l)(flmol/fmol»). Satoh el af. (1996) found higher
values for ferret (5.39 pF/pl), young rats (6.76 pF/pl) and older rats (8.88 pF/pl) and these values are
2 3
slightly different than stereological measurements in Table 1 in rabbit and rat (0.6 and 0.46 flm /flm
respectively) which correspond to 6 and 4.6 pF/pl for 1 flF/cm 2
Ca which enters the cytosol may bind to any of numerous Ca-binding ligands in addition
to the Ca regulatory site on cardiac troponin C. Two general strategies have been used to
evaluate intracellular Ca buffering: I) calculations using the Ca binding properties of known
cellular constituents based on measurements in isolated systems (Fabiato, 1983) and 2) attempts
to directly measure the total Ca buffering properties in ventricular muscle or myocytes under
conditions approaching those in situ (Pierce et al., 1985; Hove-Madsen & Bers, 1993a; Berlin et
aI., 1994; Trafford et al., 1999). These approaches are described below and I will try to
synthesize them into a current "best guess" composite.
The first serious effort to estimate the total Ca requirements for contractile activation
was by Fabiato (1983). He collected data on known concentrations and Ca binding
characteristics of the following cardiac myocyte constituents: cardiac troponin C, the SR Ca-
ATPase, calmodulin, ATP, creatine phosphate and sarcolemmal sites (see curve #6 in Fig 26).
This was an extremely useful first approximation and indicated that a total of -57 flM total Ca
would be required to raise free [Ca]j from 100 nM to 1 flM (or to activate 70% of maximal
contraction). However, this estimated Ca buffering is likely to be low because there must be
additional Ca binding sites in the cell not on Fabiato's list, and K d values for Ca binding to
Cytosolic Ca Buffering
150 1.Calculated Total-New
2. Hove-Madsen-Equili b.
-
..... 0
~ ~ 100
3.Berlin-Fast + MF Ca/Mg
4.Trafford (Fast+)
..... >. 5. Fabiato/Bers-New Fast
16°
.... - 6.Fabiato-Orig Fast
~~ 7. Berlin-Fast
<l E 50
2:
0...l-C--'---'---'-5..L00-'---'--'--~10::-':0:-:0--'---'-~~~1500
Free [Cali (nM)
Figure 26. Ca buffering in cardiac myocytes. The data are: I) Calculated Total using values in Table 10,
2) measured by Hove-Madsen & Bers, 1993a, 3) fast buffering measured by Berlin et at. (1994) plus slow
myofilaments Ca/Mg binding as in Table 10, 4) from Trafford et al. (1999) based on integrated Ca efflux
during caffeine-induced contractures, 5) Fabiato's (1983) calculations updated as described in text, 6)
Fabiato's original values, 7) Fast buffering only from Berlin et at. (1994). The curves are all well fit by
hyperbolic functions of the form: L'l.Ca bound = (B max I {I+(K m I[Ca])} )+B",;n), where B",;n is the theoretical
Y-intercept at [Ca]=O (i.e. below diastolic [Cal,). The values below describe each curve. One can also
extract total rather than L'l.Ca binding, by using Bm,x -B",;n as B max in the classic binding hyperbola.
I.New 2. 3.Berlin 4. 5.Fabiato 6.Fabiato 7.Berlin
Calc. Eguilib. +Ca/Mg Trafford Update Orig. Fast
B max (~mol/L cytosol) 244 236 203 175 162 217 123
Km(Ca) (nM) 673 498 779 590 730 2307 960
B",;n (~mollL cytosol) -28 -39 -20 -25 -19 -8.8 -12
troponin C and the SR Ca-ATPase used by Fabiato (2 f!M and 1 f!M respectively) were probably
too high (see Table 10). Curve #5 in Fig 26 is an update to the Fabiato compilation. It includes
lower K! values (0.6 f!M) for both the SR Ca-ATPase and regulatory site on troponin C based on
various studies (e.g. Hove-Madsen & Bers, 1993b; Mattiazzi et al., 1994; Yue et al., 1986; Gao
et al., 1994). I have also updated the sarcolemmal Ca binding to include a slightly lower total,
based on more recent measurements of inner sarcolemmal sites from Post and Langer (1992;
B"",x=42 f!M, K d=13 f!M) and our own data at low [Ca] (Bers et al., 1986 and unpublished;
B"",x=15 f!M, K d=0.3 f!M). I also refer to this calculated total estimate as fast buffering since the
major contributors are expected to bind rapidly (see below concerning slow binding).
Direct Ca titration in whole ventricular homogenate (as well as particulate and soluble
fractions) was first done by Pierce et al. (1985). This study probably overestimated intracellular
Ca buffering because they include titration of sites on the external cell surface, disrupted
organelle sites and additional non-myocyte sites. However, they would also include
(appropriately) some intracellular sites not included by Fabiato (1983). This approach was
refined by Hove-Madsen & Bers (I 993a), who performed similar Ca titrations on isolated
ventricular myocytes which had been permeabilized by digitonin and where the SR and
mitochondrial Ca transport were blocked by thapsigargin and ruthenium red, respectively. The
equilibrium binding results of Hove-Madsen & Bers (1993a; see curve #2 in Fig 26) were lower
than reported by Pierce et al.(l985), but might still include some external sarcolemmal sites. On
the other hand, this overestimate may be minor at submicromolar [Cal because passive Ca
binding to the sarcolemma is asymmetric with the Ca binding phospholipids situated almost
exclusively on the inner sarcolemmal surface (Post et al., 1988). As Hove-Madsen & Bers
(1993a) pointed out, the SR Ca binding might also be underestimated in the presence of
thapsigargin, since this agent appears to lock the SR Ca-ATPase in a Ca-free E) state (Kijima et
al., 1991; Sagara & Inesi, 1991). The results of Hove-Madsen & Bers (l993a) are curve #2 in
Fig 26, and are labeled "Equilibrium" because the data were acquired on a very slow time scale
which would include both rapidly and slowly equilibrating sites.
Berlin et al. (1994) used a single cell intracellular titration strategy to assess cytosolic Ca
buffering using the fluorescent Ca indicator indo-I and voltage clamp. We blocked Ca transport
by the SR Ca-pump, Na/Ca exchange, sarcolemmal Ca-ATPase and mitochondria using
thapsigargin, Na-free solutions, high [Ca]o and a mitochondrial uncoupler. Then we measured
total Ca injected into the cytoplasm via lea (using voltage clamp) and the resulting change in free
[Cali (Fig 27A). Stepwise increases in [Cali accompany the lea traces. Closer inspection of these
[Cali steps (e.g. Fig 27B) reveals a rapid phase of[Ca]i increase followed by a slow decline. The
rapid increase, which was in phase with integrated Ca influx via lea (Fig 27B) is due to very rapid
Ca buffering. Note that entry of 9 IlM total Ca in Fig 27A only raises free [Cali by 0.1 IlM,
implying 90: 1 Ca buffering. Focusing on this rapid phase, Berlin et al. (1994) used only the
initial rapid Li[Cahound/Li[Ca]; component to find a mean fast cytosolic buffering with B max = 123
IlM and Kd = 960 nM (curve #7 in Fig 26). These results agree well with the fast buffering in
curve #6 calculated by Fabiato (1983). Trafford et al. (1999) used a related strategy, applying
caffeine to release SR Ca and measuring the rate of total Ca removal (via Na/Caexchange
current) at the same time as recording the Li[Ca]i (Fig 93). They found Bmax =175 IlM and K m=
590 nM. Notably their approach would measure both fast and some slow buffering.
The slow [Cali decline in Fig 27B could be due to either a slower phase of Ca buffering
or incomplete inhibition of Ca transport out of the cytosol. Berlin et al. (1994) set up and tested
conditions to ensure block of such transport, so residual transport is expected to be extremely
small. It is reasonable to expect slow Ca buffering by sites which are initially bound to Mg or
protons. This is because bound Mg or H must first dissociate before Ca can bind. This is why
EGTA is a slower Ca buffer than BAPTA (i.e. it exists mainly as H 2EGTA at neutral pH).
Troponin C and myosin both have divalent cation binding sites at which Ca and Mg compete
(CalMg sites, see Chapter 2, Table 6; Holroyde et al., 1979, 1980; Pan & Solaro, 1987). The
Ca/Mg sites on troponin C and myosin are expected to bind Ca slowly because they are nearly
saturated with either Ca or Mg at resting levels of [Cali (Robertson et al., 1981 and see Table
10). Simple inclusion of these sites along with the measured fast buffering sites in the cell in Fig
27B would cause [Cali to fall to the level indicated by the arrow (F+S) in the figure. Inclusion of
these myofilament CalMg sites would also raise the overall buffering curve up as indicated by
curve #5 in Fig 26 (Berlin-Fast + MF CalMg). This still doesn't explain all of the equilibrium
buffering measured by Hove-Madsen & Bers (I 993a) or all of the apparent slow buffering in Fig
27. For example, in Fig 27A the overall increase in [Cali expected from the total integrated Ca
A. 0.6
B.
-
1.2
10
-f
1.0 0
~
~ 0.8 (")
-
::L 1lI
~ 0.6 (1)
ro- ::l
S2. OA ':<!
n-
'-.
0.2
Il>
~'"ImJIDrr
'2
~
~.0.5 I OA-'-,---~---r---.---r---'
02468 10 0.2 OA 0.6 0.8 1.0
time (5) time (5)

Figure 27. Fast Ca buffering titration in a voltage clamped rat ventricular myocyte. Ca transport was
blocked by 10 11M thapsigargin (for SR Ca-ATPase), Na-free solutions inside and out (for Na/Ca
exchange), 10 mM [Ca]o (for sarcolemmal Ca-ATPase) and 1799 (for mitochondrial Ca uptake). A.
Voltage clamp pulses (-40 to 0 mV for 200 ms) injected lea to cause step-like increases in [Cali. B. The lea
integral from the fourth pulse in A (/ lea) follows the kinetics of the rise in [Cali. F+S indicates where [Cali
is expected to fall to based on curve #3 in Fig 26 (see text). Eq in panel A is the predicted final settling
points for [Cali if curve #2 in Fig 26 (equilibrium) is used (modified from Berlin et aI., 1994).
influx by the end of9 pulses would be to 1.75 11M considering only the measured fast sites (off
the scale of the graph). Inclusion of the calculated myofilament Ca/Mg sites would predict a
final [Cali of 1.15 11M, indicated by F+S in Fig 27A. Clearly free [Cali is falling below the F+S
level, but perhaps not all the way down to the level predicted by the equilibrium buffering (Eq in
Fig). There may well be additional slow Ca/Mg buffers such as Mg-sensitive Ca binding sites on
the inner sarcolemmal surface (Frankis & Lindenmayer; 1984; Bers et at., 1986), mitochondria
or other sites that contribute to equilibrium Ca binding (Hove-Madsen & Bers, 1993a).
To better marry the empirical titration results with our knowledge about the buffering
species, I have chosen the values in Table 10 (plotted as curve #1 in Fig 26). Specific values are
still provisional, but this provides a practical working model for the present Not surprisingly,
the proteins which show the largest change in bound Ca between 100 nM and I 11M are the regu-
latory Ca binding sites on troponin C and the SR Ca-pump (which are present at 70 and 47 11M in
the cytosol). However, these sites only account for about 50% of the total overall change in
bound Ca and 75% of the fast buffering. It can also be seen that even at the resting [Cali (100
nM) there is a substantial amount of Ca already bound (142 11M) with 83% of that at the CalMg
sites of troponin C. Furthermore, there are several constituents which don't make a large impact
by themselves (e.g. calmodulin, ATP and sarcolemma), but collectively make a buffering
contribution that is not negligible. These calculations are based on 0.5 mM [Mg]j at pH of 7-7.2
and mostly at room temperature. Obviously, changing conditions would change the buffering.
Extrapolation of data with these calculations depends on accurate knowledge of the Ca,
Hand Mg affinities of the various binding sites as well as what the conditions are in the cell. For
example, if the Mg affinity of the Ca/Mg sites is underestimated, then the "Berlin-Fast+ Ca/Mg"
Table 10
Passive Intracellular Ca Buffering a
Kd B l1lJlx Ca Bound
at 100 nM [Cali at I /lM [Cali Delta
(/lM) ----------------------- (/lmol CalL cytosol)b --------------------
Fast
Troponin C 0.6 70 10 43.9 33.9
SR Ca-pump 0.6 47 6.8 29.6 22.8
Calmodulin totar 0.1-1 24 0.45 3.57 3.1
ATP 200 5,000 0.35 3.46 3.1
Creatine phosphate 71,073 12,000 0.02 0.17 0.2
Sarcolemmad 13 42 0.32 3.0 2.7
Membrane/High+ e 0.3 15 3.7 11.5 7.8
Free [Cali -.ill. 1.0 0.9
Fast Total 21.7 96.2 74.5
Slow: Ca/Mg
Troponin C: Caf 0.013 140 117 137 20
Mg (Mg bound) 1111 Mg 7.1 Mg 0.8
g
Myosin: Ca 0.033 140 3 25 22
Mg (Mg bound) 3.64 Mg 136 Mg 114
Slow Total 120 162.2 ~
Total Ca 142 259 117
a these constants describe curve #1 in Fig 26. Values are mostly taken from Fabiato, 1983; Bers, 1991
and other sources noted in the text. Binding was calculated assuming [K] = 140 mM and [Mg] = 0.5
mM, where relevant (e.g. ATP, creatine phosphate, calmodulin, Ca/Mg sites).
b see Table 9 for unit conversion factors.
c calmodulin results are from four classes of binding sites which also exhibit specific affinities for H, Mg
and K and these characteristics were accounted for using the constants compiled by Fabiato (1983)
and Haiech et al. (1981).
d inner sarcolemmal binding measured by Post and Langer (1992).
e this is based on our earlier estimates of sarcolemmal Ca binding at low [Cal with K d -0.3 IlM. The
Bmax was reduced from our earlier estimates (Bers, 1991) because the moderate affinity sarcolemmal
sites from Post and Langer (1992) are now included. This modest B max value was also slightly
adjusted such that calculated total buffering matched the equilibrium measurements (curve #2 in Fig
26). These sites could also include some unaccounted for high affinity sites from other sources.
f Kct values are from Pan and Solaro (1987).
g values are trom Holroyde et al. (1979) and Robertson et al. () 981).
curve in Fig 26 could move up to curve #1 (Berlin et ai., 1994). Also, when Robertson et ai.
(1981) did detailed kinetic calculations of Ca binding to cardiac myofilaments, they assumed that
diastolic [Cali was 10 nM and free [Mg]i was 2 mM. This led them to predict that the Ca/Mg
sites on troponin C were 91 % saturated with Mg with little bound Ca at rest. Based on more
recent data, diastolic [Cali is probably between 100 and 150 nM and free [Mg]; is probably about
0.5 mM (Blatter & McGuigan, 1986; Murphy et ai., 1989a,b; Gao et ai., 1994). This means that
at diastole, the troponin C Ca/Mg sites would be 84% saturated with Ca and only 5% with Mg
(Table 10). Obviously the kinetics of binding will also be important to clarify further.
Table 10 reflects a Ca buffer capacity of 123 IlM/pCa unit (from 0.1 to 1.1 /lM [Cali),
much lower than cardiac cellular pH buffering (20-90 mM/pH unit, Ellis & Thomas, 1976;
Wallert & Frohlich, 1989; Bountra et aI., 1990). This is relevant because intracellular acidosis
can increase [Ca]; due to competition with Ca at intracellular binding sites (Bers & Ellis, 1982)
and changes in [Ca]j can lead to intracellular acidosis (Vaughan-Jones et al., 1983).
A subtle additional consideration is what constitutes accessible cytosolic volume. Our
standard cytosolic volume above only excludes the cell space occupied by mitochondria (~35%).
If we additionally exclude SR volume (3.5%, Table 3) and space occupied by the concentrated
cytosolic protein (-10-15%), all the curves in Fig 26 would be shifted up by another 15-20%.
Protein volume is based on cytosolic [protein] of 120 mg/ml cytosol and density of 1.2 g
protein/m\. This would bring Curve #3 close to Curve #1 in Fig 26 and to values in Table 10.
Thus, these estimates of cellular Ca buffering have caveats, but are meant as a best guess guide,
subject to further clarification.
Ca REQUIREMENTS FOR ACTIVATION OF CONTRACTION

Figure 28A illustrates the impact of cooperativity (assessed by the Hill coefficient) on
the [Ca] dependence of myofilament activation. As discussed in Chapter 2, measurements made
in chemically skinned cardiac muscle fibers are fit with Hill coefficients (n) of -2-3 (as for Ca
binding) and half-maximal force at K m -1-2 IlM. Measurements of myofilament Ca sensitivity in
intact ventricular muscle have given both a higher affinity (K m-0.6 IlM) and cooperativity (Hill
coefficient = 4-6, Yue et aI., 1986; Backx et al., 1995; Gao et aI., 1994). The result is a very
steep force dependence on rCa] between 300 and 800 nM (Fig 28A).
Figure 28B shows force as a function of the amount of Ca added to the cytosol from an
initial free [Ca] of 150 nM using the Ca buffering from Table 10 and curve #1 in Fig 26. It can
be seen that very little force is developed with the first 30 IlM Ca supplied. As the amount of Ca
increases the relationship is fairly steep, such that most of the force development occurs between
A. Free [Cali B. Total Activator Ca

100 100
Ky,=600 nM
Ky,=600 nM
80 n=4 .. ........ -
~ 80
---------~~ ~/~:::~:::-
..........
...........
60 ...... 60
/.·····K y,=1000 nM
40 40
..- //
n=2
~ .......- 1./ n =2
o
u.
20 ... 20 ··....·1
......... I
,--y~~~--,--~~~r--r--'--~"
0+-;""''''''':'''/-' O-F-'=:;=-.,--~~~~.--,~~-.-.~-,
o 500 1000 1500 2000 o ~ W 00 1W 1~
Free rCa) (nM) Activating Ll[Caltotal (jJmol/l cytosol)
Figure 28. Total Ca requirements for myofilament activation. A. Myofilament Ca sensitivity over the
range ofrelevant [Ca];, plot1ed as Force = Fm,xf(l + (Kn![Ca];Y), where n is the Hill coefficient, indicative
of cooperativity of both Ca binding and myofilament activation (see Chapter 2). B. The amount of added
total cytosolic Ca required to activate contractile force (from [Cali =150 nM), based on Table 10.
40 and 80 flM of added Ca. The steady state twitch in mammalian ventricular muscle is typically
sufficient to reach -40% of maximal force at 25-30°C (Harrison & Bers, 1989a). This would
require a free [Cal of -540 nM and about 62 flM of added total Ca. To reach 600 nM [Cali (the
Km for force in Fig 28A) would require between 31 and 74 flM for the seven curves in Fig 26
(from diastolic [Ca]j=150 nM). Thus, while some quantitative points could be known more
precisely, a reasonable current estimate of the Ca which must be added to the cytosol to activate
a normal ventricular twitch is -60 flM (i.e. flmol/L cytosol).
It should also be recognized that many factors can alter cytosolic Ca buffering and Ca
requirements for myofilament activation, such as pH and Mg. Under normal conditions
intracellular pH is 7.1-7.2 and free [Mg]j is probably -0.5 mM (Blatter & McGuigan, 1986;
Murphy et al., 1989a,b; Gao et aI., 1994). Both acidosis and increased [Mg]; can occur in
pathophysiological situations and would be expected to reduce cytosolic Ca buffering by
competition. In 10-15 min of ischemia [Mg]; can increase 3-fold as a consequence of decline in
[ATP], which normally exists mostly as Mg-ATP (Murphy et al., 1989b). Cytosolic Ca buffering
increases developmentally (Bassani et al., 1998) and there may also be temperature- and species-
dependent differences, which have not been systematically addressed.
Ca DYNAMICS DURING A TWITCH

In Figures 26 and 28 1 have dealt with cytosolic Ca buffering in a steady state manner,
without explicit consideration of kinetic aspects. Figure 29 shows a simplified kinetic model of a
myocyte Ca transient. The starting point is a generic free [Cali signal which starts at 100 nM,
rises to a peak of 744 nM at 70 ms and falls exponentially with a time constant 1: = 300 ms. This
[Cali transient is the driving function to determine Ca binding to each cytosolic ligand L in Table
II (using the general function d[Ca-L]/dt = kon[Ca]i [L] - kotJI:Ca-L]). Not all of the kinetic
parameters have been directly measured, so some approximations are used. Figure 29A shows
the time course of free [Cali as well as the change in total cytoplasmic [Cal (LlTotal Cacyt ) after
considering on- and off-rates for Ca (and Mg) binding to each cellular ligand (see Table II).
Figure 29B shows how Ca bound to different ligands varies dynamically during the Ca transient.
From these figures it can be appreciated that free [Cali peaks earlier than total cytosolic Ca and
also comes back to near initial value faster due to these intrinsic kinetics. Relatively slow
buffering can also create a ratcheting up of total cytosolic Ca (e.g. see CalMg sites). This would
be expected to be influenced by stimulation frequency. In Figure 29 total cytosolic Ca is 6.5 flM
higher at the end of 2 sec than before the pulse, with 5.3 flM of this still bound to TnC Ca/Mg
sites and 1.l flM still bound to myosin CalMg sites. At the end of 10 pulses at 0.5 Hz these
CalMg sites gain an additional 6 flM Ca (see also Shannon et aI., 2000a).
Figure 29C adds a simplified consideration of Ca fluxes which may underlie the Ca
transient in Fig 29A. This also serves as an introduction to the following chapters where these
fluxes will be addressed more directly. Fluxes in Fig 29C were calculated as described in Table
II. Ca current (Iea) peaked at 6.8 pAlpF with rising and falling time constants of 3 and 40 ms,
respectively (bringing in a total of 16 flM Ca; Yuan et al., 1996). The Ca transported by the SR
Ca-ATPase was calculated using a classic Michaelis-Menten relationship with a Vmax of 210
flM/sec, a Km of300 nM and a Hill coefficient of2 (Bassani et al., 1994a; Balke et al., 1994; see
also Chapter 7). The SR Ca leak was set to counterbalance the resting SR Ca-ATPase rate (21
o
700
A, ,.
~
E. ···.,.."Hotal Caeyt o
~ 500 '2~
~ $0<II
Q)
Q) 'C"l, "'"....•.••...•. g.
.t 300
20
()
Ol
100
0 200 400 600 800 1000
30 time (ms) 1200
B. C.
~
~ 1000
-=c: 20
"0
-="0
<II
800
SR Ca release
0
600
:l
>.
.
0
Cll () 400
lea
()
<l
10
craM
SL .
.5 200
() ..... -..
SR
.. _. __ Ca leak--
.............
, 0 --~._-_
<l
.. ' .:g.~.t)tg-- ........----.--.... ·200 SR Ca pump
0
0 200 400 600 800 1000 200 400 600 800 1000
time (ms) time (ms)
Figure 29. Dynamic Ca changes during a twitch in rabbit ventricular myocyte. A. Free [Cal; and change
in total cytosolic ([CaJc}'!). B. Associated changes in Ca bound to different cytosolic ligands. C. Ie" SR Ca
release flux, SR Ca-pump flux and SR Ca leak. See Tables 10 & II and text for details.
/lM/s) and was varied in direct proportion to the calculated intra-SR free [Cal ([CaJsR)' The
initial SR Ca content was set at 100 Ilmol/L cytosol (Bassani & Bers, 1995) with passive intra-
SR Ca buffering (B max = 180 IlmollL cytosol and Kd = 600 /lM; Shannon & Bers, 1997). The SR
Ca release flux in this particular model ends up being predicted as the amount of change in total
cytosolic [Cal required for the initial free [Cali waveform and not accounted for by either lea. SR
Ca-ATPase or SR Ca leak. Thus, the SR Ca release flux is the residual of the calculations, and is
similar to other estimates (e.g. Sipido & Wier, 1991; Wier et al., 1994; Shannon et al., 2000b).
It is not precisely known what normal resting, diastolic or peak [Cali really is in cardiac
muscle. This is largely due to questions about the in vivo calibration of the Ca, indicators which
have been used (e.g. Ca microelectrodes, aequorin, quin2, fura-2 and indo-I, Blinks, 1986). For
example, Ca microelectrodes can overestimate [Cali because of imperfect impalements, fura-2
and indo-I may underestimate [Cali because of binding of the indicator to intracellular
constituents (Konishi et al., 1988, Hove-Madsen & Bers, 1992; Bassani et al., 1995d; Harkins et
al., 1993). The situation is even more problematic when cells are loaded with the
acetoxymethylester (AM) forms of these indicators (rather than direct intracellular application of
the free acid form). In this case, the cellular signals are complicated by the fluorescence of
incompletely de-esterified forms of the indicator (Liickhoff, 1986) and intracellular
compartmentalization of the indicator (e.g. -50 % may be trapped in the mitochondria, Spurgeon
et aI., 1990). Bassani et at. (l995d) calibrated indo-l in situ in ferret using null-point titrations
and found that: a) the ~ for Ca-indo-l in the cell was 844 nM (vs. -250 nM in aqueous
solution), b) diastolic [Cali right after a train of beats at 0.5 Hz at 23°C was 294 nM, and c)
resting [Cali continues to decline very slowly with rest reaching -80 nM after 30 min. This
Table 11
3
Kinetic Parameters used for Figure 29
B ma • ken
(uM) (uM· 1sec' I) Reference
Troponin C 70 0.6 19.6 32.7 Robertson, Gao
Troponin C CalMg 140 0.0135 0.032 2.37 Pan, Robertson
(Mgsites) 1111 3.33 0.003 Pan, Robertson
Myosin CalMg 140 0.0333 0.46 13.8 Robertson
(Mg sites) 3.64 0.057 0.0157 Robertson
SR Ca-pump b 47 0.6 60 100 Diffusion
Calmodulin total 24 7 238 34 Haiech
Sarcolemma 42 13 1300 100 Post
Membrane/High IS 0.3 30 100 Bers
Flux Calculations
SR Ca-ATPase J SR = (210 )..lM/s)/(l+ {0.3 )..lM/[Ca]}2)
Ca current Ica = (9 pA/pF) exp(-t/40 ms)(l-exp(-t/3 ms))
SR Ca leak at t=O set at SR Ca-ATPase rate, changes proportional to [Ca]sR
SR Ca content initially 100 )..lM/L cytosol; [Ca]SR.bound = 180 )..lM/(l+0.6 mM/[Ca]sR)
SR volume taken as 3.5% of cell volume
SR Ca release L'.total cytosolic Ca (accounting for lea, SR Ca-ATPase and leak)
Bma • and Kd values are generally as in Table 10. In most cases values are only available for koo or kolT so
that the other was calculated using Kd = kow kon- If neither kon or kolT are available, diffusion limited
kon (100 flM·lsec·l) and Kd were used. References are Robertson et aI., 1981; Gao et aI., 1994; Pan
and Solam, 1987; Haiech el al., 1981; Post and Langer, 1992; Bers el al., 1986; Bers, I99l.
b
The 4 Ca-calmodulin binding sites were lumped as a single site. The Kd was artificially increased so that
the steady state Ca-calmodulin binding was well predicted over the relevant range of [Cali (0.1 to 3
flM) without requiring separate kinetic calculations of H, Mg and K competition at each of the 4 sites
(although these equilibrium interactions were included in Table 10 and Figs 26 and 28).
diastolic [Cali is probably on the upper limit of current estimates, but diastolic [Cali is probably
in the range 80-250 nM and peak [Cali during a normal twitch probably reaches 0.5-2 flM.
Another consideration relevant to the potential contribution of cellular sites to Ca fluxes
is the rates at which they can supply Ca to and remove Ca from the cytoplasm. The peak of the
Ca transient in mammalian cardiac muscle at ~30°C can be reached as early as 30 msec. Thus,
the rate of total Ca rise must be able to approach 2 mmol/L cytosol/sec (60 )..lmol/L cytosol/30
msec). The rate of Ca removal from the cytoplasm is about 10-fold slower, i.e. ~200 )..lmol/L
cytosol/sec (see Fig 29C). These rough estimates are given to indicate the general magnitudes
that might be expected of sources and sinks of Ca underlying contraction.
SOURCES AND SINKS OF Ca

Extracellular Space: The extracellular space (ECS~30% of the tissue volume, see Chapter I),
could supply more than enough Ca (2 mmol/L ECS x 0.55 L ECS/L cytosol = 1000 flmol/L
cytosol). This does not include low affinity Ca binding sites in the ECS which may at least
double this value (Bers & Langer, 1979; Philipson et aI., 1980). Clearly, this would be more
than enough Ca to activate contraction, but the critical question lies in the regulation of Ca
influx. The two main routes by which Ca is known to enter the cell are by voltage-dependent Ca
channels and the Na/Ca exchange system. Since there is a very large electrochemical gradient
favoring Ca entry at rest (Em -Eea~200 mY) any type ofleak could provide an additional route of
Ca entry. Ca current and Na/Ca exchange will be discussed in Chapters 5 & 6. There are also
two mechanisms which contribute to Ca efflux across the sarcolemma discussed in Chapter 6:
Na/Ca exchange and sarcolemmal ATP-dependent Ca-pump (or Ca-ATPase).
Inner Sarcolemmal Surface: The surface of the sarcolemmal membrane facing the cytoplasm can
also bind substantial Ca; and this may be as much as 60 flmol/L cytosol (Table 10, Bers et al.,
1986; Mansier & Bers, 1984; Post & Langer, 1992). This is a relevant range (although 20x less
than the ECS). A provocative hypothesis by Liillman & Peters (1977, 1979) was that Ca bound
to specific sites at the inner sarcolemmal surface could be released upon membrane
depolarization (due to locally induced acidification and altered binding affinity). This Ca
liberated upon depolarization could rebind during repolarization and relaxation. Negatively
charged phospholipids (phosphatidylserine and phosphatidylinositol) which comprise 7.2% of
the sarcolemmal phospholipids are exclusively on the inner sarcolenunal leaflet and would be
plausible sites (Post et al., 1988). Bers et al. (1986) measured a decrease in sarcolemmal Ca
binding at 300 nM [Ca] upon membrane depolarization from -80 to 0 mV (2 nmol/mg protein, 20
flmol/L cytosol). However, the Ca binding to these sites would also increase with lea and SR Ca
release. So this depolarization-dependent decrease in bound Ca was more than offset by a rise in
local [Cal to 1-5 flM. Thus, depolarization may simply decrease the ability of these sites to
buffer increases in local [Cal;. Physiological [Na]; (5-15 mM) and [Mg]; (0.5-3 mM) also
decrease sarcolemmal Ca binding (Frankis & Lindenmayer, 1984; Bers et at., 1986). Large rapid
changes in local [Na]i subsequent to Na channel current could also displace Ca from these local
sites, and may also enhance Ca entry via Na/Ca exchange (Akera et al., 1976; LeBlanc & Hume,
1990), but the role of these sarcolemmal sites as a source of activating Ca is not clear.
These are provocative ideas. However, intact cell data preclude any quantitative role for
these inner sarcolemmal Ca binding sites in E-C coupling. When extracellular Ca is removed
very quickly «1 sec) so that [Cal; and stores are not altered, depolarization does not produce
measurable contraction or [Cali rise under a wide array of conditions (Rich et al., 1988; Niibauer
et al., 1989, see Chapter 8). Thus, in the absence ofCa influx, depolarization alone does not lead
to a significant Ca release or [Cali rise. In conclusion, inner sarcolemmal sites are likely to serve
mainly as additional intracellular Ca buffering sites.
Sarcoplasmic Reticulum: The amount of Ca held in the SR is discussed in detail in Chapter 7
(Table 21, pg 179). The bulk of values under physiological conditions are in the range of 50-250
flmol/L cytosol (e.g. Solaro & Briggs, 1974; Hunter et at., 1981; Fabiato, 1983; Hove-Madsen &
Bers, 1993a; Varro et al., 1993; Delbridge et al., 1996). Thus, there is more than enough Ca in
the SR to support a single contraction. Again, the key issues are the regulation of SR Ca
transport. Chapter 7 will address both the SR Ca-ATPase and the SR Ca release channel.
Chapters 8 and 9 will focus on more integrated aspects of SR Ca release and Ca content.
Mitochondria: Mitochondria can accumulate massive amounts of Ca, especially when there is
sufficient inorganic phosphate, which can precipitate insoluble Ca-phosphate in mitochondria, a
process known as matrix loading (Lehninger et al., 1967; Carafoli & Lehninger, 1971; Carafoli,
1987). Indeed, isolated mitochondria can take up 100 nmol Ca /mg mitochondrial protein
(corresponding to 10,000 f!mol/L cytosol, assuming 40 mg mitochondrial protein/g wet weight)
and can store several times more (Carafoli, 1975). While this is a potentially enormous capacity,
it appears that under conditions anticipated in vivo, mitochondria are likely to contain very much
less (e.g. I nmol/mg, or 100 f!mol/L cytosol, Carafoli, 1987). Thus, mitochondria are a potential
source of activator Ca, but again the issue is whether the fluxes of Ca across the mitochondria on
the time frame of E-C coupling make significant contributions. This issue will be dealt with in
more detail in subsequent sections of this chapter.
It may be useful at this point to note that the same sites considered above as potential Ca
sources are also potential Ca sinks. The important question to be addressed from here is the
mechanism by which Ca is transported to and from these sources and sinks. First let us consider
where Ca goes during relaxation.
Ca REMOVAL DURING RELAXAnON

As mentioned earlier, four Ca transport systems can compete for cytoplasmic Ca during
relaxation in cardiac muscle: I) SR Ca-ATPase, 2) sarcolemmal Na/Ca exchange, 3) sarco-
lemmal Ca-ATPase and 4) mitochondrial Ca uniport system. Obviously, Ca entering the cytosol
from the extracellular space or SR is mostly bound to the various Ca buffers which were
discussed with respect to Figs 26-28 and Tables 10-11. Here I will consider how the four Ca
transport processes described above contribute to the Ca removal which allows relaxation to
proceed. In particular how do these Ca transporters compete dynamically during relaxation?
Bers & Bridge (1989) addressed this first by separately blocking SR Ca reuptake or Na/Ca
exchange during relaxation from rapid cooling contractures in rabbit ventricular muscle (see pg
152-155). Blocking Na/Ca exchange slowed relaxation by 30%, preventing SR Ca reuptake
slowed relaxation by 70%, while inhibiting both pathways slowed relaxation by > I000%. This
made it clear that the SR Ca-ATPase and to a lesser extent Na/Ca exchange could produce
relaxation, but that these were the main two relevant mechanisms. This work was extended in a
series of more detailed quantitative studies in isolated ventricular myocytes by Bassani et al.
(1992, 1993a; 1994a,b, 1995a; Puglisi et al., 1996) and others (see Chapter 9).
Bassani et al. (1992) initially evaluated this competition by using inhibition of each of
the four Ca transport systems and observing the impact on the rate of [Cali decline and relaxation
in rabbit ventricular myocytes. Figure 30 shows a summary of their results for relaxation, which
were in close agreement with data from [Cali decline. The normal twitch relaxes with a half-time
(ty,) of 170 ± 30 ms where all Ca removal systems are functional. Rapid and sustained
application of 10 mM caffeine causes abrupt SR Ca release via ryanodine receptors, and with
appropriate flow characteristics the rate of rise of rCa]; can be comparable to that during the
twitch. However, the sustained exposure to caffeine prevents net SR Ca reuptake, while the
other Ca removal systems can still function. When SR reuptake was inhibited this way (or by
thapsigargin, Bassani et al., I994a) relaxation was slowed by a factor of 3 (tv, = 540 ± 70 ms).
This result makes it clear that SR Ca uptake is important in relaxation, but also that a reasonable
rate of relaxation can be obtained by the other 3 systems. Then Na/Ca exchange was blocked at
the same time as SR Ca reuptake, by applying 10 mM caffeine in aNa-free, Ca-free solution
containing EGTA (Caff, ONa, OCa). Note that this blocks Ca flux via Na/Ca exchange in both
Relaxation of Rabbit Ventricular Myocytes

100 Block all 4
c
n...
0
-
80 Caff,ONa,10Ca (Mto only)
C'll 275
c Caff,ONa,OCa+FCCP
0 60
() (SLCa-ATPa5e only) 205
..Il:
C'll 40
<Il
....0
ll.
20
~
0
2 4 6 8 10 12
Time (s)
Figure 30. Relaxation in a rabbit ventricular myocyte with selective inhibition of Ca transporters.
Normalized cell relaxation is shown where: I) all Ca transporters function during a normal twitch (Tw), 2)
net SR Ca uptake is prevented during a contracture induced by 10 mM caffeine (Caff), 3) SR Ca transport
and Na/Ca exchange are prevented by caffeine in DNa, DCa solution (Caff,ONa,DCa), 4) only the
sarcolemmal Ca-ATPase is functional (Caff,DNa,OCa+FCCP), 5) only the mitochondrial Ca uniport is
functional (Caff,ONa,1 DCa) after pre-depletion of [Na]i, or 6) All four Ca removal systems were blocked by
combining the caffeine with ONa, IOCa and FCCP (after pre-depletion of [Na];). Relaxation traces are
based on mean ty, values (shown along traces) from Bassani et al. (1992).
directions. This maneuver slowed relaxation and [Cali decline by almost 20-fold (ty, =8.8 ± 1.0
s) compared to caffeine alone. Thus Na/Ca exchange is responsible for most of the relaxation
and [Cali decline during a caffeine-induced contracture (93%, Bassani et al., 1994a).
Even when both SR Ca-uptake and Na/Ca exchange are prevented, relaxation and [Cali
decline still proceed, albeit very slowly. This slow relaxation and [Cali decline, requiring tens of
seconds, could be due to Ca transport by the mitochondrial Ca uniporter or the sarcolemmal Ca-
ATPase. To inhibit mitochondrial Ca uptake in the intact myocy1e Bassani et al. (1992) used
application of FCCP and oligomycin (each at 1 JlM) a few seconds before exposure to caffeine,
ONa, OCa solution. FCCP is a protonophore which dissipates the mitochondrial proton and
potential gradient, thereby eliminating the driving force responsible for Ca influx into
mitochondria (oligomycin was included to minimize mitochondrial ATP consumption during the
brief exposure to FCCP). Inhibition of mitochondrial Ca uptake in this way slowed the mean
relaxation time by about 2-fold compared to caffeine, ONa, OCa (20 s vs. 8.8 s). Two different
strategies were used to inhibit the sarcolemmal Ca-ATPase (thermodynamic and
pharmacological). The thermodynamic approach used elevation of [Ca)o to 10-100 mM to
impede the sarcolemmal Ca-pump by steepening the [Cali I[Ca)o gradient. However, to do this
experiment in Na-free solution it is essential that the cells first be depleted of intracellular Na by
incubation in ONa, OCa. Otherwise extracellular Ca would enter in exchange for intracellular Na,
greatly complicating the interpretation. As seen in Fig 30, this slowed relaxation about 3-fold
with respect to caffeine, ONa, OCa (ty, went from 8.8 to 27 s). The second method employed
carboxyeosin, a potent inhibitor of the sarcolemmal Ca-pump (Gatto & Milanick, 1993) and
produced very similar results (Bassani et al., 1995a). That is, carboxyeosin slowed the ty, of
[Cali decline in rabbit ventricular myocytes during caffeine, ONa, OCa from 7.5 ± 0.5 s to 26.3 ±
2.1 s. When all four Ca transport systems were blocked, relaxation and [Cali decline were nearly
Table 12
Contributions of Different Ca Transporters to Relaxation
in Rabbit Ventricular Myocytes
Relaxation Relaxation Percent of Ca
Transporter analyzed rate A (S·I) removal Flux
SL-Ca-Pump Caff-ONa,OCa,FCCP 0.035 0.86%
Mito. Ca uniport Caff-ONa,+ lOCa 0.025 0.62%
Na/Ca exchange Cafft 1.22 30.0%
SR-Ca-Pump Twitcht 2.79 68.5%
All 4 systems Twitch 4.08 100.0%
SL is sarcolemmal, Mito. is mitochondrial and Caff is caffeine. Rate constants (A) are reciprocals of time
constants (In 2/tl/2) of relaxation. tThe A value for Caff is adjusted by subtracting those for the SL Ca-
ATPase + Mito (-0.06) which also function during a caffeine-induced contracture. tSimilarly ASR is
obtained by subtracting (ATw - ANaCaX - AM;to - ASL-Ca-pump). See pg 250-253 for more detailed analysis.
abolished (Bassani et al., 1992, 1995a). This indicates that these are the only four Ca removal
systems that need to be considered from any practical standpoint.
This series of studies allows a crude prediction of relative Ca extrusion rates in rabbit
ventricular myocytes (comparing ty, values). Compared to the Na/Ca exchanger, the SR Ca-
ATPase was 2-3 times faster and the sarcolemmal Ca-ATPase and mitochondrial Ca transport
were 37 and 50 times slower respectively (Bassani et aI., 1992).
Table 12 shows a more quantitative treatment of this data, considering each Ca transport
system to function independently with a rate constant (ASR, ANaCax, ASL-CaATP & AMito) that
contributes additively to the overall relaxation rate constant (A). This allows a breakdown of the
four individual values and their percent contribution to relaxation. We find that in rabbit
ventricle the relative values for SR Ca-ATPase and Na/Ca exchange are 68% and 30%, while the
sarcolemmal Ca-ATPase and mitochondrial Ca uniport contribute less than I% each. This is
consistent with both prior less direct estimates above (Bers & Bridge, 1989), as well as more
detailed quantitative analysis of rabbit ventricular myocyte Ca transients using integrated Ca
fluxes (where 70% of the transported Ca during relaxation was via the SR Ca-ATPase and 28%
via Na/Ca exchange; Bassani et al., 1994a; see pg 250-253). This balance of Ca fluxes differs
among species (e.g. SR Ca-ATPase contributes 92% in rat ventricle, Bassani et aI., 1994a;
Negretti et al., 1993) and in pathophysiological conditions (e.g. Na/Ca exchange contributes 50%
in failing rabbit heart (Pogwizd et aI., 1999). This will be further addressed in Chapters 9 & 10.
Ca INFLUX vs. SR Ca RELEASE IN CONTRACTILE ACTIVATION

The two main sources of Ca involved in the normal activation of cardiac muscle
contraction are Ca influx and SR Ca release. In the steady state the amount of Ca entry during
the cardiac cycle must be the same as the amount of Ca efflux. Otherwise the cell will gain or
lose Ca and not be in a steady state with respect to cellular Ca balance. The same is true for the
SR. Thus the quantitative analysis of the contributions of the SR Ca-ATPase and Na/Ca
exchange to relaxation should roughly reflect the fraction of activation by SR Ca release or Ca
SL..-:.40Jl'--O -70 E-"Cmc..:(_m-'-V)
1200
:?
.s Steady State
800
'iO Twitches
2-
400
0
2 sec
Superimposed lea -100
<-100
S
.9 -200 r. <
E::
3
)(
.
!:'
J INa/CaX ~ SR Ca load
-300
250 msec
-200 2 see
Figure 31. Ca transients, Ca influx via Ica and SR Ca content measured by integrating INalCa in a rabbit
ventricular myocyte (dialyzed with 50 IlM indo-I). The last 2 steady state Ca transients and superimposed
Ic, traces were during voltage clamp pulses to 0 mY (left) and rapid applications of 10 ruM caffeine at a
holding potential -70 mY caused SR Ca release and activation OflNalCa (right). Note different scales for lca
and INa/c, (from Delbridge et al., I996, with permission).
influx respectively. Thus, based on Fig 30 we might expect Ca influx and SR Ca release to
supply 25-30% and -70% of the activating Ca respectively in rabbit ventricle.
Delbridge et at. (1996) assessed this electrophysiologically, by measuring Ca influx via
Ic" the SR Ca content by caffeine-induced IN,/c" and using the fraction of this SR Ca content
released during a twitch (Bassani et a/., 1993b). Figure 31 shows steady state voltage clamp
pulses, Ca transients and Ic• (left). On the right, 10 mM caffeine is applied to release the SR Ca
content and prevent net reuptake. This causes a large and rapid Ca transient and an inward
current that decreases as rCa]; declines. This inward current is Na/Ca exchange current (INa/C.)
because it is abolished in the absence of extracellular Na (Li substituted) and in the absence of a
Ca transient (i.e. a second caffeine application did not cause a current or a rCa]; rise; Delbridge et
at., 1996). Furthermore, one sustained caffeine application was sufficient to empty the SR, since
a second caffeine exposure caused no further Ca release. The integral of Ic• allows direct
evaluation of Ca influx and the SR Ca content can be calculated from the integral of INa/c,. Since
Na/Ca exchange only removes 93% of the Ca during a caffeine-induced Ca transient in rabbit
(Bassani et at., 1994a), the SR Ca content is calculated by dividing the INa/c, integral by 0.93
(=87 11M). The other data required is the fraction of SR Ca content released during a twitch,
measured to be 43% in rabbit ventricular myocytes (Bassani et a/., 1993b). These Ca fluxes are
converted to I1mol/L cytosol using appropriate surface to volume ratios (Tables 2 & 9).
We then find that during the rabbit ventricular twitch Ic, brings in 9.7 ~IM Ca and the SR
Ca release is 37 11M (0.43x87 11M). Thus 23% of the Ca comes from Ic, and 77% from SR Ca
release. These numbers are in rather good agreement with the data in Fig 30, Table 12 and
Bassani et at. (1992, 1994a, 1995a), based on rCa]; decline during relaxation. This is remarkable
quantitative agreement between very different analytical methods, which have different intrinsic
limitations. Again, species-, development- and condition-dependent differences in this balance
of Ca fluxes will be discussed further in Chapter 9.
One may well ask whether Ca entry via Na/Ca exchange also contributes to Ca influx
during the cardiac action potential. This is certainly possible, but for various reasons I think that
the quantitative contribution of Ca entry via Na/Ca exchange is very small during the normal
action potential (:0;1 Ilmol/L cytosol) when compared to the Ca influx via lea. This issue will be
discussed in further detail in other chapters (e.g. 4, 6, 9 & 10). The amount of Ca influx via
Na/Ca exchange can vary dramatically under different conditions.
In conclusion, we now have a fairly clear quantitative picture about the number of Ca
ions involved in the activation of cardiac muscle contraction, including which transport pathways
are involved in bringing Ca into the cytosol, what the Ca ions bind to in the cytosol (and when)
and how Ca removal systems compete quantitatively during relaxation. For a typical 32 pL
myocyte the amount of Ca cycling during a twitch (~60 Ilmol/L cytosol) corresponds to 1.25
fmol Ca/cell or 750 million Ca ions/cell. Obviously, there is continual refinement of this
quantitative picture as more data become available, but we are probably much closer to reality
now than 10 years ago. There are also major species differences in the balance of Ca fluxes and
how these systems change during development, as well as under pharmacological and patho-
physiological situations. The snapshot described in this chapter may thus serve as a reference
point for much of the ensuing discussion throughout this book.
MITOCHONDRIAL Ca TRANSPORT
While the foregoing discussions indicate that mitochondrial Ca plays only a very minor
quantitative role in Ca fluxes associated with E-C coupling, mitochondrial Ca fluxes may still be
important with respect to mitochondrial function and energetics. Thus, mitochondrial Ca
transport will be considered in more detail here, rather than in a separate chapter.
As mentioned above (pg 52), mitochondria can accumulate large amounts of Ca, but
under physiological conditions the Ca content is probably on the order of 100 Ilmol/L cytosol.
Under conditions approaching those in vivo Fry et al. (l984a) showed that mitochondrial Ca
uptake was not appreciable in digitonin permeabilized cardiac myocytes until cytoplasmic [Cal
exceeded I 11M (where a Ca uptake rate of2-5 Ilmol/L cytosol/sec can be inferred).
Figure 32 illustrates the Ca cycle of mitochondria. Ca enters via a uniport system, down
a large electrochemical gradient (about -180 mY) set up by proton extrusion linked to the
passage of electrons down the cytochrome system in the respiratory chain. This Ca uniporter is
blocked competitively by physiological [Mg]; (Nicholls & Ackerman, 1982), lanthanides (Mela,
1969; Reed & Bygrave, 1975), and also potently by ruthenium red (Moore, 1971) and the novel
selective blocker Ru360 (Ying et al., 1991; Matlib et aI., 1998; Zhou et al., 1998). Ca entry via
the uniport pathway exhibits a sigmoid dependence on [Cal and under physiologic ionic
conditions has a Krn above 30 11M Ca. Thus, at the [Cali associated with the cardiac cycle (0.1-1
11M) the influx rate is expected to be quite low. In particular, Crompton (1985, 1990) developed
a model to describe Ca uptake by isolated mitochondria. At 0.1 and 111M [Cal mitochondrial Ca
uptake was 0.1 and 3.1 Ilmol/L cytosol/sec respectively (using 40 mg mitochondrial protein/ml
rca
Mitochondrial
Matrix
Ca
tOehydrogenases
!
tNADH-......
H+
Figure 32. The Ca cycle across the inner mitochondrial membrane. Ca enters via a uniport, down an
electrical gradient formed by H-pumping in the respiratory chain (Cyto). Ca is extruded by a Na/Ca
antiport and Na is extruded by NalH exchange thereby completing the cycle. Elevated cytoplasmic [Cal
can lead to elevated mitochondrial [Cal and increased activity of mitochondrial dehydrogenases.
cell and 0.65 L cytosollL cell). This is consistent with our data and analysis in intact myocytes at
[Cali between 0.1 and I flM, where mitochondrial Ca uptake was:S;1 flmol/L cytosol/sec (Bassani
et al., 1994a, I 995a). The ability of mitochondria to accumulate Ca led Lehninger (1974) and
Carafoli (1975) to speculate initially that it may contribute to cardiac relaxation by removing Ca
from the cytoplasm, but it seems now that the quantitative contribution is almost negligible.
Ca extrusion from the mitochondria is mainly via a Na/Ca antiporter, which may be
electroneutral (2: I), but might also be >2: I (Crompton et aI., 1976; Crompton, 1985; lung et al.,
1995). The [Na] dependence of this Na/Ca antiporter is sigmoidal with half-maximal Ca
extrusion at -5-8 mM Na, making this system quite sensitive to changes of [Na]i in the
physiological range (Crompton et al., 1976; Fry et aI., 1984b). While variations in bulk
cytoplasmic [Na] during the cardiac cycle are probably insufficient to cause rapid release of
mitochondrial Ca, large changes in [Na] can induce substantial mitochondrial Ca release in vitro
(Crompton et aI., 1976). There is also a Na-independent extrusion of Ca from mitochondria
which is less prominent in heart, but is more important in tissues where Na/Ca antiport activity is
low (e.g. liver & kidney; Crompton, 1985). The inner mitochondrial membrane has an active
Na/H exchange system (Mitchell & Moyle, 1967) which is the pathway for Na extrusion from the
matrix and also completes the cycle. In this way the energy for Ca extrusion via Na/Ca exchange
depends also on the proton movement during respiration and the consequently negative
intramitochondrial potential.
Under relatively physiological conditions there is probably only a small [Cal gradient
across the inner mitochondrial membrane, with intra-mitochondrial free [Cal ([Ca]M) being
slightly lower than [Cali (Moreno-Sanchez & Hansford, 1988; McCormack et aI., 1989; Miyata
et al., 1991; Zhou et aI., 1998, Fig 33). Based on the trans-mitochondrial potential (-180 mY)
[CalM would have to be 0.1-1 M to be at equilibrium. Ca is thus far from equilibrium and
considerable energy is required to extrude Ca from mitochondria up this electrochemical
gradient. While the Na electrochemical gradient may be the immediate source of energy, this
gradient is created by the proton gradient. Thus the true energy source is respiration and the
protonmotive force it generates. Figure 33 shows how [CalM responds to [Cali changes induced
by reduction in [Na]o, thereby promoting Ca entry via Na/Ca exchange, (Miyata et al., 1991). As
resting [Cali rises to 650 nM the value of [CalM stays below [Cali' However, as resting [Cali
becomes very high, mitochondrial Ca uptake appears to be activated and [CalM exceeds [Cali'
There is almost no detectable fluctuation in [CalM during an individual twitch in
ventricular myocytes (Miyata et al., 1991; Griffiths et al., 1997; Zhou et at., 1998). Chacon et
al. (1996) reported phasic [CalM transients in heart mitochondria which were kinetically identical
to the cytosolic signal, but these might be due to contamination of the [CalM signal by [Cali
(since the authors did not block mitochondrial Ca uptake or quench cytosolic indicator to confirm
their interpretation). Gunter et al. (1998; Sparagna et at., 1995) described a novel rapid Ca
uptake mode (RaM) in isolated liver mitochondria, which produces small amplitude, but very
rapid bursts of Ca uptake with Ca pulses. They found this to differ from Ca uniport flux in terms
of ruthenium red, Mg and spermine sensitivity, but RaM was also different in cardiac
mitochondria (Bunitas et al., 1997). This could produce up to 10 Ilmol/L cytosol Ca flux in 1-2
sec at physiological [Cal;, but the role in cardiac myocyte Ca regulation is not yet clear.
Crompton (1985) modeled the Ca transport of isolated cardiac mitochondria to phasic
changes in [Cali during the cardiac cycle. For a cytoplasmic [Cal change from -200 nM to -2
11M and back, [CalM increased by only -2% (vs. 1000% for the rise in [Cali)' On the other hand,
both this model and cellular data show that high stimulation rate (4 Hz) or strong cellular Ca
loading via the Na/Ca exchange cause a slow rise in [CalM over tens of seconds (Miyata et al.,
1991; Griffiths et al., 1997; Zhou et a/., 1998). Zhou et al. (1998) demonstrated that under these
conditions phasic increases of [CalM could be detected, but they were still slow and only
observed at diastolic [Cali > 400 nM. This is consistent with Fig 33 and the sigmoid dependence
of mitochondrial Ca uptake on [Cali (Crompton, 1976; Fry et al., 1984a,b). The 0.6% of twitch
[Cali decline attributed to mitochondrial uptake (Table 12) would be 0.36 Ilmol/L cytosol, or 0.7
IlmollL mitochondria. Assuming 100:1 Ca buffering of free [CalM, we expect mitochondrial
twitch Ca transients of -7 nM amplitude, with extremely damped kinetics compared to [Cali'
Mitochondrial Ca transport may be more consequential for Ca transient dynamics in
other cell types, such as neurons (Friel & Tsien, 1994) and adrenal chromaffin cells (Park et al.,
1996; Herrington et al., 1996; Babcock et at., 1997). These chromaffin cell studies showed that
a large bolus of Ca influx via lea produced large Ca transients which were substantially curtailed
by mitochondrial Ca uptake over 10-30 sec, allowing [Ca]; to recover to low levels much more
rapidly than when FCCP was present. Notably, this mitochondrial Ca uptake was only seen
when [Cali exceeded 500 nM for a relatively long time, consistent with cardiac data above. This
mitochondrial Ca in the chromaffin cell was then slowly extruded to the cytosol at a rate at which
the plasma membrane Ca-ATPase and Na/Ca exchange could remove it from the cell without
greatly elevating [Cali' Using a similar analysis to Table 12, relative contributions of SR/ER:
NaCaX: plasmalemmal-CaATP: mitochondrial Ca uniport were a) 68:30: 1: 1% in rabbit ventricle
b) 0:9:16:75% in chromaffin cells (Herrington et a/., 1996) and c) 7% plasmalemmal extrusion
Mitochondrial [Ca] in intact cells
2000
Miyata et aI., 1991
-
~ 1500
t:
::!!
'iii' 1000
~
500
0
0 200 400 600 800 '1000
[Cali (nM)
Figure 33. Mitochondrial free [Cal ([Ca]M) as a function of cytosolic [Cal ([Ca];). Increases of [Cali in
rat ventricular myocytes were induced by reducing [Na]o (i.e. via sarcolemmal NaiCa exchange). Mean
[Cali was measured using indo-I (loaded as the salt fonn) and [CalM was measured using indo-I (loaded as
the AM form) with Mn quench of cytosolic indo-I. Data are redrawn from Miyata et at. (1991) (without
error bars) and including a broken line corresponding to [CalM = [Cali (slope = I).
and 46% each for ER and mitochondrial Ca uptake in neurons (Friel & Tsien, 1994). The main
difference in heart is probably the extremely high density and Ca transport capacity of the SR
Ca-ATPase and Na/Ca exchanger. These systems cause rapid [Ca]i decline in heart which limits
the opportunity for the mitochondria to contribute to [Cali decline. In other words, if we were to
slow cardiac Na/Ca exchange and SR Ca-ATPase by 20-100-fold, the competition among the 4
Ca transporters would be more balanced. (e.g. see top traces in Fig 30).
Location is probably also important with respect to mitochondrial Ca transport. Rizzuto
et al. (1992, 1993, 1998) showed close proximity between mitochondria and IPrsensitive Ca
stores in non-cardiac cells. They also used mitochondrially-targeted recombinant Ca-sensitive
photoproteins to show that mitochondria near these Ca release sites sense a much higher local
[Cal (compared to bulk [Cali) and that [CalM is also higher than would be expected from bulk
[Cali' It is unclear how important this consideration is in cardiac myocytes. Parts of mito-
chondria are close to junctional SR in heart. However, this is partly a consequence of these two
important structures being physically excluded from the series arrangement of myofibers
necessary to transmit force. Local [Cali near both sarcolemmal and SR Ca release channels is
very high when the channels are open, and this is functionally important (see Fig 118).
Hunter & Haworth (1979) first described a large conductance pore in the inner
mitochondrial membrane known as the mitochondrial permeability transition pore (MTP; see
reviews by Zoratti & Szabo, 1995; Crompton, 1999). This pore allows passage of molecules up
to a molecular weight of 1500 Da, is activated by high [CalM and is blocked by cyclosporin A.
MTP is a complex of the voltage-dependent anion channel (VDAC) on the outer membrane, the
ADP/ATP translocase in the inner membrane and cyclophilin D (a matrix peptidyl-prolyl
cis/trans isomerase, which is also the receptor for cyclosporin A). The ADP/ATP translocase
60 D.M. Hers Cardiac E-C Coupling
may serve as the actual pore in the inner mitochondrial membrane, but many details of how this
complex works as the MTP are still to be worked out (Crompton, 1999). The MTP is opened by
high [CalM (K M-25 f..I.M, AI Nasser & Crompton, 1986) and oxidative stress. Openings of the
MTP cause abrupt dissipation of membrane potential in individual mitochondria and these can be
transient (Hiiser et al., 1998a). This depolarization allows Ca efflux to the cytosol, relieving the
mitochondrial Ca overload. However, this is costly because other mitochondrial contents are
lost, the mitochondrial FoFJ-ATP synthase consumes ATP (rather than making it) and the large
negative membrane potential must be reestablished by electron transport. Moreover, prolonged
MTP opening causes further dysregulation of Ca and energetic state leading to mitochondrial run
down and cell death.
Mitochondrial Ca fluxes might not be important quantitatively in E-C coupling, but the
small gradual changes in mitochondrial [Cal with heart rate changes or cellular Ca load may help
to regulate mitochondrial energy production. Three key mitochondrial matrix enzymes are
activated by low f..I.M [Cal (pyruvate dehydrogenase, a-ketoglutarate dehydrogenase and the
NAD-dependent isocitrate dehydrogenase, Denton & McCormack, 1980, 1985, 1990; Hansford,
1985, 1987). Thus, increases in mitochondrial Ca via the above mechanisms could occur when
cytosolic [Cal is relatively high and the energy demands are also high (i.e. when contractile
activation and Ca pumping are consuming ATP at high rates). In this way, the rise in
cytoplasmic (and mitochondrial) [Cal can increase oxidative metabolism and thereby increase
ATP production to meet increased demands. A potentially interesting twist on this is that
cellular Ca loading is often secondary to cellular Na loading, via sarcolemmal Na/Ca exchange
(e.g. when the Na-pump is inhibited by digitalis). In this case the increase of mitochondrial Ca
which could stimulate oxidative metabolism may be limited by the high [Na]j which would tend
to minimize the gain of mitochondrial Ca. Thus, energy supply may not go up to meet demands
and the cytoplasmic Ca load will be more severe. This might favor more force production, but
could also elevate diastolic [Cali and compromise cardiac relaxation and energy balance.
Brandes & Bers (1997) demonstrated a [Cali-dependent stimulation of mitochondrial
NADH production by measuring [NADH] in intact contracting ventricular muscle (Fig 34). With
a sudden increase in stimulation frequency or [Ca]o there was a transient decrease in [NADH],
consistent with NADH production not keeping up with the increased ATP and NADH
consumption. However, this [NADH] decline was followed by a recovery toward the initial
value. This recovery was entirely dependent on increased average [Cali' That is, a comparable
increase in work by increasing sarcomere length was associated with the same initial decline, but
no recovery (Fig 34C). We concluded that the increased average [Cali caused an increase in
[CalM and stimulation of dehydrogenases and NADH production. Indeed, when the elevation of
[Cali or frequency was terminated, there was an overshoot of [NADH] (again not seen when the
work increase was not due to higher average [Cali). The overshoot is probably due to slow
extrusion of [Ca]M and loss ofCa-dependent dehydrogenase stimulation. The time course of this
overshoot concurs with slow mitochondrial Ca efflux (20-40 sec, Bassani et al., 1993a).
In an extension of the studies described in Fig 30, Bassani et al. (1993a) evaluated the
fate of the Ca which was "forced" into mitochondria during caffeine-induced contractures with
Na/Ca exchange blocked. That is, under the Caff-ONa, OCa condition in Fig 30 -50% of the SR
Ca load is slowly taken up by the mitochondria over 20-30 sec (with the remainder transported
A. [Ca]o (mM) B. Frequency (Hz) C. Sarc. Length (IJm)

2 2.1
~ 30faAio.6
0.3 0.3 !~~l05 1 ~ 40
601 1.8 1.9
rll1L
1.7 1.7
515
u. t1:
20
o o
°
n
25 025 0.25 0.25
~:: ~ ~
",30 ~ 20 n
l
r-\
~ J \J l.
20
«
,; 10 ~10~ U
_ 5
MAX °
60
~65
60 ~60~""""""""""""1"""
l70
~ ~ ~ 55
z
I55
o
;:: 50
I i 50 MIN=SS
~ 45
~ 50'-r-_ _~ ~_ ~ 40 '-r--_~-~,-"-_~_ z e,,-_,-~,-~,-~,-
500 200 400 600 o 50 100 150 200
Time (sec) Time (sec) Time (sec)
Figure 34. Mitochondrial NADH fluorescence and force measured in rat ventricular muscle. Work was
increased by either elevating [Ca]o (A), increasing frequency (B) or increasing sarcomere length (C). Force
(in mN/mm 2 ) was converted to time-averaged force (middle traces) as an index of work level. Increasing
either [Ca]o or frequency increases average [Ca]i along with work, while increasing sarcomere length
increases work without substantial change in average rCa);. Increasing work in A & B causes NADH to
decline to a minimum (MIN) then recover during sustained high work load (SS) and finally overshoot
(MAX) upon return to original work level. Without [Ca]i change (C) there is no recovery and overshoot
phase (from Brandes & Bers, 1997, with permission).
out by the sarcolemmal Ca-ATPase). After removal of caffeine (in sustained ONa, OCa solution
with EGTA) this mitochondrial Ca was transported back to the SR with a time constant of 40 sec.
We concluded that this represented mainly the time constant of mitochondrial Ca efflux (with
relatively rapid reuptake into the SR, because [Cali remained low). Furthermore, the rate of Ca
redistribution from mitochondria back to SR was slowed when intracellular Na was removed
(consistent with the Na-dependence of mitochondrial Ca efflux in cardiac myocytes). Thus, it
seems clear that transient increases in mitochondrial Ca may accumulate gradually over a large
number of beats (e.g. during cytosolic Ca transients of higher frequency or amplitude) and [CalM
gradually dissipates over 1-3 min when the initial steady state is resumed.
It should be noted that mitochondria use the same pool of energy to phosphorylate ADP
to ATP as to drive Ca uptake (i.e. the protonmotive force). Energized, isolated mitochondria
have been shown to take up Ca at the expense of making ATP (Vercesi et al., 1978). This would
obviously be a dangerous situation in vivo, but it appears that at physiological [Mg]; the uniport
is inhibited strongly enough that mitochondrial energy is preferentially used to make ATP
(Sordahl, 1975). This may not be the case when the cell is exposed to chronically elevated [Cali,
where mitochondrial Ca uptake gets much more active.
The ability of heart mitochondria to accumulate massive amounts of Ca under
pathological conditions such as ischemia (Reimer & Jennings, 1992) may serve as an important
safety device for heart cells. Cellular Ca overload is a common early component of cell injury in
many cell types (Shanne et aI., 1979) and could quickly become disastrous in heart cells since
high cytosoJic [Cal would keep energy consumption by the myofiJaments and Ca-ATPases high
while mitochondrial Ca uptake could limit ATP synthesis. Sustained contracture due to rigor
crossbridges (induced by low ATP) could also worsen the situation by vascular compression
leading to decreased local blood flow. If the mitochondria can temporarily compensate for the
cellular Ca load by taking up large amounts of Ca, permanent cell damage might be avoided.
Unfortunately it is a double-edged sword, since Ca accumulation by the mitochondria diminishes
ATP production and eventually compromises the mitochondria. Thus the survival of the cell
might depend on whether the mitochondria can survive a given degree of transient Ca loading.
In conclusion, mitochondria playa minor role in Ca movements on a beat-to-beat basis.
However, with slower increases in "mean" [Ca]j mitochondrial Ca transport may be important in
increasing metabolism to meet metabolic demands. In more severe Ca overload, mitochondria
may provide a temporary Ca store to protect the cytoplasm from very high Ca levels.
It is worth considering the energy requirements for key ion transporters in heart. Table
13 shows that the Na/K-ATPase, sarcolemmal Ca-ATPase and SR Ca-ATPase all function at a
very high energetic efficiency (70-80%). The ~G required for the generation of normal ionic
concentration gradients is valuable to keep in mind, since anything that reduces ~GATP (i.e. lower
[ATP] or higher [ADP] or [PJ;) could also reduce the ionic gradients. The ATP required to
transport 60 JlM Ca into the SR, 15 JlM Ca out of the cell via Na/Ca exchange (which requires 3
Na, and 1 ATP/3 Na via Na/K-ATPase) and 15 JlM Na from INa out via Na/K-ATPase, would be
50 Jlmol ATP/L cytosol/beat or -300 JlL Oz/kg wet wtlbeat (or 1% of the cellular [ATP]lbeat).
At a heart rate of 1 Hz and cardiac Oz consumption of 100 ml Oz/kg wet wt/min Na and Ca
transport would require 15-20% of the total O2 consumption for the heart, with the myofilaments
accounting for most of the rest. Thus the myocyte uses -5-7% of its ATP for each beat.
Table 13
Energy Requirements for Cardiac Ion Transporters
Transport & [Xkow/[X]Hi Em ~Gx ~Gx Efficiency
Transporter Stoichiometry (roM) (mY) (mY) (kJ/mol) (%)
Na/K-A TPase 3Na out IONa/140Na o -80 461 44.5 76.4
+ 2K in 5KJ 120Ki
SL Ca-ATPase I Ca out 0.0001/2 -80 425 41.0 70.3
SR Ca-ATPase 2 Ca in 0.0001/1 0 492 47.5 81.5
FoF]-ATPsynthase 2.5Hint pH 7.1/8.1 -180 -604 -58.3 lOOt
L'.G x was calculated from L'.G x = RTln([XlLow/[X]Hi) - zFE m • tit is assumed that generation of I ATP
requires 2.5 protons to flow through the mitochondrial FoF]-ATP synthase, such that the energy available is
L'.G ATP = -58.3 kllmol. This is defined as 100% for comparison to energy use by the ATPases. However, it
may take 3 protons to generate this L'.G ATP making FoF]-ATP synthase efficiency 83%.
This chapter has provided a general framework and some quantitative values for the
processes involved in Ca regulation in cardiac myocytes. This sets the scene for more detailed
discussion of these key mechanisms and their interrelationships in the subsequent chapters.
D.M. Bers. 63
CHAPTER 4
CARDIAC ACTION POTENTIAL

AND ION CHANNELS
The initiating event in cardiac E-C coupling is the action potential (AP). The AP is the
membrane potential (Em) waveform that is determined by a complex interplay of many ion
channels and transporters, and the Ca transient itself. The AP is also the driving Em waveform
that influences ion channels and transporters, and results in the genesis of the Ca transient. The
AP is also responsible for the propagation of excitation information from cell to cell in the heart
and allows the heart to function as a syncytium (electrically and mechanically). The underlying
ionic currents also contribute to initiating pacemaker activity as well as arrhythmogenic delayed-
and early afterdepolarizations (DADs & EADs) and conditions for re-excitation and reentry.
This chapter is only a basic overview of cardiac ion channels and the cardiac AP. A
comprehensive treatment of this topic could readily fill another book of this type. For additional
depth and references I recommend some excellent recent reviews by Weiss (1997), Roden &
George (1997), Yellen (1998), Cmmeliet (1999) and Nerbonne (2000, 2001). The main focus
here will be on the ventricular myocyte AP and basic understanding of the ionic currents which
contribute to its shape and modulation.
ACTION POTENTIAL & HETEROGENEITY

Figure 35 demonstrates the classical phenotypes of APs in different regions of the heart.
The normal heartbeat initiates in the sino-atrial (SA) node since the cells in that region nonnally
have the fastest intrinsic pacemaker activity. The maximum diastolic polarization in these cells
is typically about -50 to -60 mV and a gradual pacemaker depolarization leads to an AP with a
slow rate of rise and consequently slow rate of local propagation. As will be clear from the
ensuing discussion, Na channels are almost entirely inactivated and don't participate in the rapid
depolarization phase (in these cells Ca channels serve this role). In the atrio-ventricular (AV)
node, the AP resembles that in the SA node. In atrial and ventricular muscle cells the resting Em
is near -80 mV and the AP has a very fast upstroke attributable to Na current, and overshoots 0
mV to reach a peak at +30-50 mV. Repolarization is much faster in atria than in ventricular
myocytes and Purkinje fibers. Thus in ventricular cells there is a more prominent plateau.
Moreover, the ventricular AP duration (APD) is shortest in epicardial cells, longer in endocardial
cells and longest in mid-myocardial cells (reflecting in part differential ion channel expression).
The longer endocardial AP also explains why T waves in the ECG are typically in the same
direction as the QRS complex (repolarization vector is in opposite direction of depolarization).
The long APD in ventricular myocytes serves two functions. First, it prevents electrical
re-excitation, by keeping the membrane depolarized (and thus Na and Ca channels inactivated).
This inhibits aberrant conduction pathway development. Second, it allows contraction to relax
Regional Variation in AP
Configuration
SANOd"[~
_--,4L....------- Atrial [
: - - - - - - ' < + - - - - - - - A V Nodal [ J"--

,-:-rn----Purkinje Fiber [ ~
Septum
--'~~::::::::f ~Apex Epicardial [ ~
Right Left
Ventricle '----'
Ventricle 200 msec
Figure 35. Regional variation in action potential configuration. The APs at right are representative of the
different shapes typically observed for the cardiac regions indicated. The position along the time axis for
AP upstrokes reflect the different delays from SA node firing. For example, the delay between atrial and
Purkinje fiber firing reflects slow transmission through the AV node (and the P-R interval in the ECG).
Atrial and ventricular myocyte AP upstrokes are responsible for the P wave and QRS complex in the ECG
(Fig kindly supplied by J. Nerbonne).
before the next beat (since the APD is almost as long as the Ca transient and contraction). This
also prevents tetanization of cardiac muscle, which would not be advantageous for cardiac
function in the way that it is for skeletal muscle function (i.e. The heart must fill between beats).
From the SA-node the wave passes to atrial muscle (fast propagation 0.1-1 m/s) and the
AV node, where conduction slows again progressively from atrial end (AN) to the central node
region (N; 0.01-0.05 m/sec), before slightly speeding through the last part of the node (NH). As
the wave gets through the His bundle, bundle branches and Purkinje fibers, propagation becomes
very rapid (2-4 m/sec) and it remains very fast through ventricular muscle (0.3-1 m/sec). At this
point I will consider the resting E"" how ionic channels influence Em and propagation.
RESTING Em, NERNST POTENTIAL & PROPAGAnON

The resting cardiac myocyte membrane is preferentially permeable to K (due mainly to
IK1 channels, see below). The Na/K-ATPase generates the ionic concentration gradients for Na
and K, but K-channels dictate the negative resting Em in myocytes (and virtually all cells). How
does this come about? Let's consider the simplest case of a generic cell (Fig 36) which has high
[K]j and low [K]o, but the membrane is impermeable with Em initially at 0 mY. If the membrane
is only permeable to K, then K will tend to flow down its concentration gradient, but as positive
charge leaves the cell a membrane potential is created (inside negative). This negative Em limits
further K efflux, so an electrical driving force opposes the driving force of the chemical gradient
([K]j I[K]o)' So at what point are these forces equal and opposite? This is easiest to consider
Chapter 4 Action Potential & Jon Channels 65
A. No Permeability B. K-Selective C. Nernst Em

+125 ECa
+70 ENa
140K+
0 OmV
5K+
=
At Em -89 5K+ -55 ECI
LiG Chem =LiG E1ec -82 Eres!
-89 EK
Figure 36. Resting membrane potential and Nernst potentials. The NaJK ATPase generates a [K] gradient
across the cell membrane even if there are no functional channels (A). The presence of only K-selective
channels (8) causes an Em to develop (E K) at which the K concentration gradient is exactly balanced
(thermodynamically) by a negative transmembrane Em (Nernst or equilibrium potential). Nernst potentials
for other ions (x) are also shown (C) according to (RT/zF) In([x]J[x];). The Goldman-Hodgkin-Katz
equation uses relative penneabilities for monovalent ions to estimate Em =(RTIF) In{([K]o+ PN,/PK [Na]o+
Pc/P K[Cl];) /([K];+ PN,IP K[Na]; + PCl/PK [Cl]o)}.
quantitatively with the free energy equation: e.G = RT In([K]J[K];) - zFE mwhere the two terms
reflect the energy in the chemical and electrical gradient respectively. When these are equal and
opposite e.G =0 and Em = (RT/zF) In([K]J[K];) or 61.5 10glo([K]J[K]j), which is the Nemst or
equilibrium potential for K (Ed. For [K]o = 5 mM and [K]; = 140 mM, EK = -89 mY. This
means that at an Em of -89 mY there will be no net K flux and the system will be stable.
Obviously this is too simple for a myocyte where there are many channels and even the
selectivity of K channels for Kover Na is not absolutely 100%. Similar Nemst potentials can be
calculated for Na, Ca and CI and are indicated in Fig 36C (+70, + 125 and -55 mY respectively).
So, if the membrane becomes more permeable to a given ion, the Em will move closer to the
Nemst potential for that ion (due to an increase in flux of that ion down its electrochemical
gradient). Thus, the opening of only a few Na selective channels (or finite Na permeability in K
or leak channels) will cause inward Na current and a more positive Em than -89 mY. The
relative permeability can be used to predict the actual Em using the Goldman-Hodgkin-Katz
equation or its more generalized forms (see Fig 36 legend; Hille, 1992; Campbell et aI., 1988).
To keep it simple, the resting Em in myocytes reflects only slight permeability to Na (~100 times
smaller than for K), such that resting Em is close to EK (-82 vs. -89 mY). In pacemaker cells,
which have less resting K permeability, other conductances move diastolic Em further from EK
and closer to EN. so that Em is considerably more positive (e.g. -50 to-60 mY).
To jump ahead a little in this simple permeability framework, when Na channels open Na
ions will come in (an inward current) causing Em to move toward EN.. The amount of current
flowing can be considered using Ohm's law: I = Ge.Y or INa =GNaCE m- EN.), where GNa is the Na
conductance. Of course as Em gets closer to E Na the driving force for current through the Na
( (\
Active R Passive
P~::~( iepol.
----++++++++++++++++++++++++++ C
++++-------------
40
Spatial Spread Charging time 20
Vx = Vo e-xll. ~ 0 ).. = ,}radius.Rm!(2.R,)

>
VI =V o(1- e- UT ) .§.
e
we 40
10
-------- w
.80./-------'---4-===9 01'-0- - ' - - - ' 5 - - - - ' 1 0

0
Distance (mm) time (msec)
Figure 37. Propagation of depolarization in cardiac myocytes. Inward ionic current (INa) in the active
patch at left causes passive spread of depolarization longitudinally through resistance (R; and Ro) which
includes gap junctions. The flow of positive charge (K ions) down the cell can charge the membrane
capacitance (em) and leak out via Rm(K channels). The degree and rate of spatial spread are dictated by the
length (or space) constant (Iv) and time constant (or) as indicated in insets. Technically Iv should use Ro+ R;
in place of R;, but Ro is usually small in comparison. This same cable model can be extended to the
transverse and depth dimensions in the real heart, and space constants differ in different directions. Insets
show expressions for passive spatial and temporal Em spread.
channel (Em - EN,) declines and the driving force for K current (Em - E K ) gets larger. At the
point where inward INa and outward IK are equal and opposite, net current is zero and this marks
the AP peak. As Na channels tum off (GN, approaches 0), outward K current repolarizes the cell
back to the initial diastolic Em.
The wave of AP depolarization proceeds via gap junctions and depolarizing current
spreads from one cell to the next, bringing Em in the latter cell beyond the threshold for an AP.
Figure 37 shows how electrotonic spread of current contributes to this activation. At the left a
patch of membrane where Na channels are open has a positive Em. This results in passive spread
of current through the cytoplasm and gap junctions (carried mainly by K ions), causing the
downstream regions to depolarize. Current flow in the reverse direction occurs in the
extracellular space (where Na and Cl carry the charge). This current charges up the membrane
capacitance to depolarize the downstream membrane electrotonically, without ions passing
through the membrane per se. This downstream membrane is thus being depolarized passively as
opposed to actively (as in the case where Na channels are open and ionic current causes
depolarization). This passive depolarization can bring the neighboring membrane closer to
threshold to trigger an AP at that point by opening Em-dependent Na channels (see below). This
passive current spread shows no refractoriness and conducts in both directions. An AP would
also propagate in both directions from an initial focal depolarization. However, ifNa channels in
the region to the left are refractory (due to recent inactivation) retrograde AP is prevented.
The ability of the membranes to propagate passive depolarization is extremely important
in determining the rate and fidelity of the wave of excitation that spreads through the heart. If
the active zone depolarizes faster and to more positive Em, this will spread faster and farther to
excite downstream regions with a faster conduction velocity. This is partly why atrial and
ventricular conduction are much faster than in the SA and AV node. The cellular geometry and
characteristics, especially resistance, also playa big role. As the passive current in Fig 37
proceeds down the cell and across gap junctions a limiting factor in how far it will reach is how
easily it flows longitudinally vs. across the membrane. Thus if the membrane resistance (Rm in
2
Q_cm ) is high compared to the longitudinal resistivity (Ri in Q-cm) more current will stay
intracellular and charge membrane capacitance further downstream. R; also gets smaller with
increasing cell diameter (like a larger diameter copper wire). The usual quantitative measure of
how well passive current spreads along a fiber is the space or length constant Ie = (radius x
Rn!(2R;))112 which is on the order of 2 mm in cardiac muscle. This means that at one point in
time it takes 2 mm for the impact of a local active depolarization to decay to 37% of its peak
value. More generally Em at a distance x from a point where it is Yo, is given by V x =Voe-x/l..
While external resistivity (R,) should also be added to R j in the OJ- equation, its value is usually
small in comparison to R i . Thus large diameter cells with many gap junctions and high R mwould
have the largest space constant and be best at high propagation velocity. Indeed, these factors
contribute directly to the slow propagation in AV nodal cells (small diameter) and the very high
rates of propagation in Pukinje fibers which have high diameters (large radius & small R j), no T-
tubules (hence higher R m) and many longitudinal gap junctions (lowering R;). The membrane
capacitance (C m) is also important in determining the time constant ("t=RmC m) for charging up the
membrane to the AP threshold. The equation describing this charging at time t is V,= Vo(l-e- II').
Thus, smaller 1: values will enhance propagation velocity.
The heart is normally well tuned in terms of its propagation network, so that APs are
activated in all cells in a progressive and synchronized manner. Local alterations in channel
function or passive properties (Rm, R j, Ie & 1:) can upset the normal activation pattern and
contribute to altered conduction patterns and reentrant arrhythmias.
BASIC STRUCTURE & FUNCTION OF ION CHANNELS

The overall structure of most of the ion channels discussed here (and in Fig 38) fall into
two main categories: I) Na, Ca and many K channels and 2) inward rectifier K channels. Na and
Ca channels have four homologous domains (I-IV), each of which has 6 transmembrane spans
(SI-S6). In each domain there is also a pore (or P) loop between Ss and S6 which dips back into
the membrane and is thought to line the actual permeation pore and be involved in channel
selectivity. The pore is created by one P-Ioop from each domain around the center with the
remaining transmembrane regions layered around this Ss-P-loop-S6 core. The S4 span in each
domain has a highly conserved stretch where every third amino acid is positively charged. These
S4 spans are thought to move within the electric field in response to changes in Em. That is, they
function as the Em-sensor of voltage sensitive channels (Yang et al., 1996; Mannuzzo et al.,
1996). Most Em-dependent K channels (and the pacemaker channel that produces If) have the
same overall structure except that each of the four domains is a separate protein, which
assembles into a tetramer that is analogous to the Na and Ca channel. Inward rectifier K
channels have a related structure, but are only ~400 amino acids long with only two trans-
membrane domains (M j and M 2) which are analogous to Ss and S6 above, including a pore loop
between them. These channels also function as tetramers with a central pore. These inward
rectifiers lack the S4 domain of Em-activated channels, and some are ligand-activated. The first
relevant ion channel crystal structure in Fig 39A (from Doyle et al., 1998) is from a bacterial K
channel that is similar in structure to these inward rectifier channels. They describe the shape as
an inverted teepee (more apparent in other structural views). The P loops converge to a very
narrow region at the outside mouth of the pore (and notably do not span the whole membrane
bilayer). This region contains the selectivity filter and can be occupied by 2 K ions in single file.
This pore is so narrow that these ions must shed all of their associated water molecules to fit.
After the selectivity filter there is an aqueous cavity which may serve to lower the electrostatic
barrier and ensure a low resistance and high throughput of ions. The inner mouth of the pore is
lined by the S6 or M 2 domain, and where they converge at the inner end may also be the site of
the activation gate (Yellen, 1998; Perozo et aI., 1999).
The activation gate for Em-dependent channels has long been expected to be at the inner
mouth of the channel (Armstrong, 1971, 1975; Yeh & Narahashi, 1977, Cahalan & Almers,
1979; Yellen, 1998). This is mainly because channel blockers can only enter the pore from the
inside when the channel is activated. Blockers can either inhibit closure of the activation gate
(foot in the door effect) or be trapped in the channel by activation gate closure (Yellen, 1998).
Permeation and Selectivity

Many ion channels are highly selective for one physiological ion and as such are called
Na, Ca or K channels. This selectivity is essential for their specific physiological function.
Selectivity is usually measured in a relative sense and PNa IPK for Na channels are -50-100: 1.
PK/PNa varies for different K channels, but are also in a similar range (Hille, 1992). Other
channels (e.g. If) show little discrimination among monovalent cations. Ca channels do not
discriminate very well among Ca, Ba and Sr, but PCa IPK and PCa IP Na are -1000 and 3000 (see
Table 17; Hess et at., 1986; Tsien et at., 1987). The channels gain selectivity because of binding
sites in the pore formed by the 4 P-Ioops coordinately binding one type of ion preferentially. For
K channels a signature 'GYG' sequence in the P loop seems to be essential. For Ca channels a
ring of four negatively charged amino acids (one glutamate from the P-Ioop of each domain)
forms the Ca-selective site (see Fig 53; Kim et at., 1993; Yang et aI., 1993). Na channels are
similar to Ca channels except that the analogous positions in domains III and IV are lysine and
alanine rather than glutamate. Heinemann et al. (1992) replaced either one or both of these
amino acids in the Na channel with glutamate and the resulting channel showed a remarkable
shift in selectivity toward that of a Ca channel (including block by 11M Ca). Thus, the P-Ioops
dictate the channel selectivity and probably do so by creating selective binding sites with
appropriate chemical coordination. These issues will be discussed more explicitly for Ca
channels in Chapter 5. Ion ch,mnels can pass _10 6 ionslsec and one may wonder how they can
attain such high throughput if i0ns bind with high affinity in the pore. One answer to this is that
many channels have multiple binding sites, such that a second charged ion entering the region
can help propel the first ion through the channel by electrostatic repulsion (i.e. destabilizing its
binding; see Chapter 5).
A. Na (or Ca) Channel

I P T11< resIStance
. II p
III IV
P P
,: Out
B. K Channel C. Inward Rectifier

D. Top View
Voltage Pore K Channel
Sensor
Na/K/Ca Channel
S1 S2 S3 S4 M1 M
~--~~'
NH 2 Inact NH 2 CO 2
Figure 38. Overall structure of some ion channels. A. The Na channel has four domains (I-IV) each of
which has 6 homologous repeating transmembrane regions (S\-S6) and a pore loop (P). Repeating
positively charged gating region in S4 is indicated by +. Other noted sites are I) the site responsible for low
cardiac TTX sensitivity (and high Cd sensitivity) compared to neuronal or skeletal muscle Na channels, 2)
PKA phosphorylation site (P), 3) site implicated in inactivation gate (IFM) and 4) sites mutated in
congenital long QT syndromes (LQT). The Ca channel has a similar topology (see Fig 51 Chapter 5). B.
Em-dependent K channels have similar overall structure, but 4 monomers (of SI-S6) are required to form the
channel. N-type inactivation domain and receptor region are indicated. C. The M],P-M 2 region in inward
rectifier K channels is analogous to just the Ss-P-S 6 region of Em,dependent K channels. D. A top view of
an Em-dependent channel showing a likely organization of transmembrane regions around the pore (+
indicates K ion in the selectivity filter; based on Durell et aI., 1998).
CHANNEL GATING
Ion channels can be activated and inactivated by changes in Em (e.g. depolarization or
hyperpolarization), binding of ligands (e.g. acetylcholine, ATP) or mechanical deformation (e.g.
cell swelling). At the single channel level, the opening and closing transitions of most channels
are very abrupt «< 1 ms), so that current through a channel changes in a square or seemingly
instantaneous manner between open and closed states (see Fig 40A). At the whole cell level one
measures a current which is the ensemble of all of the individual channel events and thus a
smoother function. This represents the average behavior of all Na channels and reflects
statistical or stochastic differences in latencies of opening, closing and reopening of individual
channels. Table 14 lists many of the most important cardiac ion channels, categorized by their
type of gating. At this point it is fair to say that we know the most about voltage-gated ion
channels in the heart, somewhat less about ligand-gated channels and least about mechano-
sensitive channels. Thus our appreciation of the different channels and their importance is still
evolving. Before discussing each individual channel it is useful to consider some general aspects
of channel gating, focused here on voltage-dependent channels.
Em-dependent Activation
Most Em-dependent channels are activated by depolarization, and this implies that there
is part of the channel which moves in response to a change in Em. The S4 region has been a
prime candidate since Noda et al. (1984) noted the repeating positive charge motif in the first
cloned Na channel. Depolarization would tend to move these charges outward across the
membrane electrical field, and this charge movement can be measured (Fig 40A) as a non-linear
component of capacitative current (Armstrong & Bezanilla, 1973). A key feature of such gating
current is that the total 'on-charge' which moves upon depolarization (outward current) must be
equal to the 'off-charge' moved upon repolarization (inward current). A complication in this
point is that some charge can become immobilized (or inactivated) and recover on a much slower
time frame, making it difficult to measure experimentally (when compared to the large phasic
component as depicted in Fig 40A). It should be noted that one usually has to block all ionic
currents across the membrane, in order to reliably detect gating currents (which are charge
movements confined within the membrane). Moreover, it is particularly difficult to measure
gating current attributable to a specific channel in cardiac myocytes because there are so many
channels with overlapping activation ranges (see Fig 40C).
The amount of charge moved per channel can be estimated in two ways. First, one can
measure the number of channels by either ligand binding experiments (e.g. JH-STX binding) or
by measuring whole cell current, divided by the single channel current and open probability
measured under the same conditions (N=V(ixxPo». Then the number of charges per channel is a
simple quotient. The steepness of the Em-dependence of conductance (current activation curves)
can also provide an estimate of charges moved per channel (but only the initial part or limiting
slope should be used; Hille, 1992). A steeper slope implies more charges/ channel. Activation
curves as in Fig 40 are normally described by a Boltzmann relation (l/{I+ exp((Eo5 - Em)/S)})
where Eo.5 is the Em for half-maximal activation and S is a slope factor (RT/zF), which gives the
slope in e-fold per S mY. Since RT/F is 25-26 mY, an S value of 4 would correspond to 6
charges moved across the membrane field. This is typical of many experimental estimates for
different channels. Values as high as 12-15 charges per channel (or 2.7-fold change in current in
2 mY) have been reported for Na, Ca and K channels (Schoppa et al., 1992; Hirschberg et al.,
1995; Noceti et aI., 1996). This could be due to 12 charges moving all the way through the
membrane electric field or 24 charges moving halfway etc. With 2':4 positive charges on each S4
region, there are> 16 of these candidate charges in Em-dependent channels. Indeed, mutations in
the S4 which neutralize or reverse charges produce reductions in charge movement consistent
with this idea (Aggarwal & MacKinnon, 1996).
The physical charge movement was initially considered to be a vertical outward
translation of S4 domains in Fig 38. However, more recent data suggests that the S4 may be tilted
and also interacts with amino acids on Sz and S3 (Papazian et ai., 1995; Tiwari-Woodruff et ai.,
1997; Durell et ai., 1998). The cartoon in Fig 39B shows how a modest degree of rotation ofS 4
along Sz may effectively move several positive charges from inside to outside, without dramatic
vertical movement (Yang et ai., 1996; Yellen, 1998). It is less clear how the gating charge
Chapter 4 Action Potential & Ion Channels 71
A. K Channel Structure B. Possible Gating Movement

EXTRACELLULAR
Selectivity
Filter
Pore
Helices
34A
Figure 39. Channel crystal structure and possible gating movement. A. Structure of the bacterial Ksca
channel which is analogous to inward rectifier K channels in heart (Doyle et aI., 1998). Four MJ-P-M J
regions fonn a narrow selectivity filter, internal aqueous cavity and inner narrowing which may be the
activation gate (reproduced from Doyle et aI., 1998, with pennission). B. Possible mechanism of charge
movement in Em-dependent channels (note that the K channel in A does not have SJ-S4 regions). Rather
than moving vertically through the membrane field, the charged S4 domains may rotate with respect to other
helices (e.g. SJ) to effectively move the charges from inside to outside during gating charge movement
(based closely on a figure by Yellen, 1998).
movement physically couples to the channel activation gate on S6 at the inner mouth.
Presumably this SJ-S4 movement causes movement of the SS-S6 to allow opening of the channel.
Figure 40B shows the Em-dependence of charge movement and current activation. It is
clear that charge movement occurs at significantly more negative Em values, and this is seen for
most channels. This is consistent with the channel having one or more Em-dependent transitions
prior to the channel opening step. Indeed, most state models of channel gating suggest that the
final closed to open transition is not intrinsically Em-dependent, and all of the Em-dependence
comes from transitions between closed states leading up to the opening step. This also makes
physical sense if the S4 (& S2) bears the intrinsic Em-dependence and this simply increases the
likelihood that the S6 (& Ss) will flip to the open state.
Figure 40C shows the Em-dependence of activation of several cardiac ion channels.
These channels all have relatively fast activation and it can be appreciated that with depolar-
ization one would progressively activate IN., ICa,T, ICa,L and Ito. Of course the activation kinetics
are not identical and the impact of individual currents also depends on the overall conductance
and the driving force (Em - Ex). The nature of these interactions will be discussed below.
Channel Inactivation
All Em-dependent channels exhibit deactivation, which is simply the reverse of activation
and would be in principle described by the same steady state activation curves as in Fig 40C.
Many Em-dependent channels also exhibit inactivation, which is a separate process from
72 D.M. Bers Cardiac E-C Coupiing
activation and this is an important semantic distinction. Inactivation of INa is illustrated in Fig
40A by the reduction in current amplitude under sustained depolarization. Inactivation rate is
generally found to be Em-dependent, getting faster with stronger depolarization. However, for Na
and some other channels, inactivation is not intrinsically very Em-dependent. The apparent E m-
dependence of inactivation derives from the strong Em-dependence of activation and the property
that the channel readily undergoes inactivation only from the activated state (Patlak, 1991). Thus
these processes are linked, but the linkage can be broken. Proteolytic enzymes applied
intracellularly or truncation/mutation of intracellular loops can remove inactivation (Armstrong
et ai., 1973; Hoshi et ai., 1990; McDonald et ai., 1994; StUmer et ai., 1989; West et ai., 1992).
Based on their early work Armstrong & Bezanilla (1977) proposed that inactivation was
mediated by a 'ball-and-chain' mechanism (see Fig 38B). Elegant confirmation of this
hypothesis comes from molecular studies with Shaker K channels (Hoshi et ai., 1990; Zagotta et
at., 1990) which are related to Ito in heart. They found that removal of the 46 N-terminal amino
acids (the ball) could prevent inactivation, and that this mutant channel could still be inactivated
by exogenous ball-peptide dialyzed into the cell from a patch pipette. This latter observation is
also important in showing that the process of inactivation depends on activation. That is, the
ball-peptide did not produce inactivation of channels until they were activated (but then it was
almost nonnal), implying that the activation process must present a target for the inactivation
ball. Isacoff et ai. (1991) demonstrated that the ball binds to the S4-SS region to block the
channel. MacKinnon et ai. (1993) also showed that the length of the chain connecting to the ball
dictates the speed of inactivation (shorter chain, faster inactivation) and also that binding of one
ball from one K channel subunit was sufficient to block the tetrameric K channel. The key
portion of the ball (20 amino acids) has II hydrophobic and 4 positively charged amino acids,
and both characteristics are probably functionally important. Notably, more positive charge
hastens inactivation (Murell-Lagnado & Aldrich, 1993) and this may confer some Em-dependence
to inactivation or recovery therefrom.
This mode of channel inactivation is referred to as N-type inactivation. Inactivation of
some components ofI,o in heart almost surely works this way (e.g. Kv1.4). There are also several
variations on this ball-and-chain scheme of inactivation. In some K channels a ~ channel subunit
appears to provide the ball (Rettig et at., 1994; Morales et ai., 1995) and in the Na channel it
appears to be more of a hinged lid formed by the intracellular loop between domain III and IV,
containing a critical 'IFM' sequence (lie, Phe, Met; Fig 38A, West et ai., 1992). There is also a
second general type of inactivation referred to as C-type, which again comes from work with
Shaker K channels (Hoshi et at., 1990, 1991). This type of inactivation appears to be due to a
constriction in the P-loop region of the pore and is linked to activation, but is generally a much
slower process than N-type inactivation, taking seconds, rather than msec (Yellen, 1998). The
rapid inactivation of cardiac I Kr (HERG) appears to be an unusually fast variant of C-type inacti-
vation (Smith et ai., 1996). Ca channels also show Ca-dependent inactivation which will be
discussed in Chapter 5 (see pg 116-119 & 122).
Once activated and inactivated, membrane repolarization is required for the channels to
recovery from inactivation. The rate of recovery from inactivation also depends on how negative
the Em is (faster at more negative Em). Figure 40B shows a Na channel availability curve
(sometimes referred to as a steady state inactivation curve). This is measured by holding Em at
A. INa and Gating Current B. INa Gating & Charge Movement
~ ~
0 "
~ ~ '" E'"
u
" '"
n~
on
Gating Current gE~
::l
0.5
0 ..
.. s:
zO
off O.O-l-----~"r_~~--.
·100 ·50
Em (mV)
C. Activation of Several Channels
1.0
"
.9
10
ICa •L
~
_ 0.5
"e
:;
o
·50 0 50
Em (mV)
Figure 40. Em-dependent gating of ion channels. A. Schematic diagram illustrating outward gating
charge movement upon depolarization, activation & inactivation of whole cell INa and 6 simulated single
channel sweeps (with their total, ensemble current). B. Steady state Em-dependence of INa activation,
gating charge movement and inactivation (or availability). C. Em-dependence of current activation in
several cardiac channel types.
different values to allow a state steady state condition and then depolarizing to test what fraction
of channels are available for activation. As Em becomes more positive Na channel availability
decreases. Thus if diastolic Em is more positive (e.g. due to membrane leak) fewer Na channels
are available and the rate of rise of the AP and the rate of propagation will be decreased.
INa activation and inactivation (or availability) curves in Fig 40B overlap such that there
is almost no availability when Em is held at values where activation is appreciable. This ensures
that activated Na channels inactivate nearly completely whenever activated by depolarization.
However, at about -52 mY there is a very small window where both channel availability and
activation are -3.5%. This would produce a steady state "window current" with a conductance
of about 0.1 % of maximum. Given the large INa in heart cells, small shifts in the activation and
inactivation properties could result in substantial sustained Na influx via window INa at relatively
negative Em. This same type of window current exists in Ca channels (especially from -25 to +5
mY for ICa,L) and also in K channels. For IKr where inactivation is fast compared to activation,
most of the IKr during the AP is this type of window current (see Fig 4IC-D).).
NaCHANNELS
The first channel cloned was the Na channel from the elctrical organ of electric eel
(Noda et al., 1984). The cardiac Na channel (Rogart et al.,1989; Gellens et al., 1992) and brain
and skeletal muscle channels are highly homologous. There are also 13 subunits associated with
Na channels, but it is somewhat controversial how these alter cardiac INa (Roden & George,
1997). The cardiac Na channel is much less sensitive to block by tetrodotoxin (TTX) and more
sensitive to Cd block than other Na channels. This has been attributed to a specific cysteine
(373) in the P-Ioop of domain I in heart vs. Phe or Tyr in other Na channels (Backx et aI., 1992;
Heinemann et at., 1992; Satin et aI., I992a). One must use> 10 flM TTX to block cardiac INa, vs.
10-50 nM for neuronal INa. Cardiac INa can also be blocked by type I antiarrhythmic agents (e.g.
IA: quinidine, procainamide; IB lidocaine, dilantin, mexiletine; I C flecanide, encainide,
propafenone, which prolong, shorten or don't change APD respectively). Some of these bind to
the S6 region of domain IV (Ragsdale et at., 1994). Most of these agents also exhibit Em- or use-
dependent block, meaning that they interact more strongly with open or inactivated channels.
This produces cumulative block of INa at high frequency. They thus block INa more effectively
during tachycardia and in depolarized tissue, conditions which can be pro-arrhythmic.
The function ofNa channels is to generate a large and very brief inward INa and cause the
rapid upstroke of the AP. This is accomplished by very brief channel openings with very short
latency, normally without reopenings (e.g. Fig 40A). That is, the channel inactivates quickly into
an absorbing state, requiring recovery at negative Em. However, cardiac Na channels can also
exhibit some persistent late openings (Saint et at., 1992; Zilberter et aI., 1994) and this may
contribute to a background Na leak current. An ultraslow component of INa inactivation (1: =600
ms) has also been reported in heart (Maltsev et at., 1998) which could contribute to EADs.
Hypoxia and lysophospholipids also cause persistent Na channel openings even at very negative
Em (Undrovinas et aI., 1992; Jue et aI., 1996).
A human congenital mutation (deletion of 3 amino acids, KPQ) in the cardiac Na
channel very close to the IFM region in the III-IV loop which is crucial in inactivation) results in
the long QT syndrome (Fig 38A; Bennett et aI., 1995). This is characterized by long ventricular
APD and may predispose these individuals to arrhythmias. The mutation does not greatly alter
macroscopic IN" but late bursting openings are seen at the single channel level, which could
cause the APD prolongation. Two other congenital long QT mutations in the Na channel gene
are in the S4-SS linker region in domains III and IV and produce similar INa effects (Wang et aI.,
1995a, 1996a; Dumaine et at., 1996). This site is analogous to the ball-receptor region involved
in N-type inactivation in K channels, as discussed above. The peak INa is very large (> I nNpF)
and even minor problems in inactivation can have significant electrophysiological consequences.
The amount of Na which enters the cell via INa is only 15 flM or so, but this is because INa is
normally so brief (Fig 45). If inactivation were only 99.5% complete, such that 0.5% of peak INa
flowed for most of the APD, this would easily double the amount of Na influx. This would
increase the burden on the Na/K-ATPase to extrude Na, but could also perturb Ca balance via the
Na/Ca exchange.
In the absence of [NaJo some Na channels can allow TTX-sensitive Ca influx (Cole et
aI., 1997; Aggarwal et at., 1997), but the amount of Ca current is several hundred times smaller
than for Na. The selectivity of cardiac Na channels for Na relative to Ca (PNa IP ca ) is >3000
(Nilius, 1988), such that it not clear how much Ca influx might occur under physiological
conditions. Santana et at. (1998; Cruz et at., 1999) showed evidence that isoproterenol and
digitalis glycosides cause cardiac Na channels to increase their Ca permeability such that PNalPCa
approaches I. This was based on shifts in reversal potential and also on the ability of a TTX-
sensitive current to activate SR Ca release. This provocative effect, which was dubbed 'slip
Table 14
Cardiac Ion Channels
Current Candidate Acti- Inacti- Role in Subunits? Blockers
Gene vation vation AP
Voltage gated Channels
INa SCN5A YYF YF Rapid Depol. ~ TTX,STX
I Ca.L alC, aID YF M Depol & Plat a2D, ~ DHP, ct>AA
I Ca•T alG, all-I YF F Depol-PMK ~ Mibefradil, Ni
lto,fast Kv4.2,4.3 YF F Early Repol ~ 4-AP,2,3-DAP
Ito,slow KvlA YF M Early Repol ~ 4-AP, 2,3-DAP
I Kr HERG M YF Plat-Repol MirPI Dofetilide, E-403]
I Ks KvLQTI YS x Plat-Repol MinK Chromanol 293
I Kur Kvl.5 F x Plat-Repol f1M4-AP
I Kp Kvl.5? F x Plat-Repol Ba
IK,slow Kv1.2 F VS Plat-Repol TEA
I K1 Kir2.1 (lRKI) YF x Rest Em Ba
If HCN2, HCN4 MS x PMK
Ligand Gated Channels
IK(Ach) Kir 3.1:3.4 ACh J.PMK
IK(ATP) Kir6.2 Pinacidil J.APD& PMK SUR Glibenclamide
IC1(Ca) ? [Ca]; Early Repol DIDS,nifJumate
ICI(cAMP) CFTR cAMP iRepol. 9-AC, DNDS
Mechanosensitive Channels
IC1(Swell) CIC-3 Swelling J.APD? Gd, DIDS
INS(str"ch) ? Stretch PMK? Gd
Abbreviations: F=fast, S=slow, M=moderate, Y=very and x=none. Depol=depolarization, Repol= repolari-
zation, Plat= plateau, PMK= pacemaker, TTX = tetrodotoxin, STX= saxitoxin, DHP= dihydropyridine,
ct>AA=phenylalkylamine, TEA= tetraethylammonium, 4-AP= 4-aminopyridine, 2,3-DAP = 2,3-diamino-
pyridine, DIDS= 4,4'-diisothiocyanatostilbene - 2,2'-disulphonic acid, DNDS= 4,4'-dinitrostilbene-2,2'-
disulphonic acid, 9-AC= 9-aminoacridine, ACh= acetylcholine. The nomenclature for Em-dependent K
channels (Kv) is based on homology to Drosophila gene families referred to as Shaker, Shab, Shaw and
Shal for Kvl.x, Kv2.x, Kv3.x and Kv4.x (Jan & Jan, 1992; Pongs, 1992).
mode conductance' has been challenged (Nuss & Marban, 1999; Balke et aI., 1999) and remains
controversial at present.
Na channels are modulated by cAMP-dependent protein kinase (PKA) and PKC (Cukier-
man, 1996). The site of phosphorylation is in the loop between domain I and II and results in an
increase of INa (Murphy et aI., 1993; Catterrall, 1995). However, phosphorylation results in a
shift of activation and availability to more negative Em, such that fewer Na channels may be
available for contribution to the AP. Activation of several receptors (e.g. muscarinic, a-
adrenergic, angiotensin II, and purinergic) can stimulate PKC activity and this can also modulate
IN., but the net effect on INa may depend on the specific method of activation (Ono et aI., 1993;
Cukierman, 1996).
Na channels require repolarization to recover from inactivation before another
ventricular AP can occur. The same is true for Ca channels which initiate APs in pacemaker
cells. Otherwise the cell is refractory to another electrical excitation. Recovery of INa requires
that the membrane be repolarized to near the diastolic level for a finite period of time (t ~ 10 ms
at -100 mY, 30 ms at -80 mY or 100 ms at -72 mY) before the cell is able to fire another AP.
Thus Na channels don't recover very rapidly until repolarization is nearly complete. In this way
the long AP contributes to limiting the ability of the cell to respond to an early depolarization.
Ca channels have the same property (but with longer recovery times, Fig 57) and are crucial in
recovery from refractoriness in SA and AV node cells
CaCHANNELS
There are two types ofCa channels in cardiac myocytes (T- and L-type). These will be
the subject of much more detailed discussion in Chapter 5 and will only be discussed briefly
here. ICa .L is the dominant lea and is in all myocytes. ICa,T is not detectable in most ventricular
myocytes, but is present to a variable extent in atrial and conduction cells (e.g. Purkinje fibers).
Both Ica,L and ICa,T activate rapidly upon depolarization (though not as fast as INa) and both show
inactivation (again, not as fast as INa)' Ica,T activates at more negative Em and inactivates more
rapidly than Ica,L (Fig 40C). ICa,T thus contributes only to the early phases of the AP. There is
some evidence to suggest that ICa,T can contribute to the late pacemaker depolarization seen in
some cells (Hagiwara et al., 1988; Zhou & Lipsius, 1994).
Ica,L inactivation is slower than ICa,T and is both E m- and Ca-dependent. The intrinsic E m-
dependent inactivation is relatively slow, so that with Ca influx and SR Ca release most of the
inactivation is Ca-dependent (see Chapter 5, pg 116-119). Indeed, with larger amplitude Ca
transients and lea the inactivation is greatly accelerated. This creates a negative feedback, such
that when Ca entry and release are large, less total Ca entry occurs during the AP (Puglisi et at.,
1999). ICa,L contributes inward depolarizing current to the cardiac AP. It may not contribute
much to the very rapid rising AP phase in myocytes (dictated by INa), but is the key depolarizing
current responsible for the slower rising AP in SA and AV node cells. Ica,L is also sustained
during the plateau phase of the AP and is the primary inward current at this time that must be
counterbalanced by K currents. lea is also stimulated by PKA, which increases both amplitude
and inactivation rate. Many of these points will be expanded upon in Chapter 5.
KCHANNELS
K channels in cardiac myocytes are the most diverse group and they serve numerous
important functions, but of course they all produce outward current at physiological Em values
and tend to drive Em toward E K (Fig 36C). They are structurally grouped into two classes (Fig
38B-C, Table 14). Em-gated K channels resemble Na and Ca channels and are activated by
depolarization. Some of these show inactivation and some do not (Table 14). The second group,
inward rectifiers, are generally ligand-gated and have fewer transmembrane spans (Fig 38C).
K Channel Rectification
Inward rectification means that the channel conducts inward current better than outward
current. Figure 41 A shows that without rectification K current would be a linear function of Em
with a reversal potential at EK. That is, the slope or conductance (gK) would be constant. With
weak inward rectification (as shown for IK(ATP») the outward current at Em > E K is less than
expected. For strong inward rectification (as for IK1 ), outward current can be completely blocked
at more positive Em. This inward rectification can be largely explained by open channel block of
outward current by intracellular Mg and polyamines, such as spermine and spermidine (Vanden-
berg, 1987; Mazzanti & DiFrancesco, 1989; Lopatin et al., 1994; Fickler et al., 1994). That is,
when outward current is favored (Em> E K) these positively charged species are driven into the
channel and prevent K from flowing out. For IKJ the concentrations of Mg and polyamines
required for 50% block are 11M and nM respectively, and normal [Mg]; and [polyamine]i are O. I-
I mM. This accounts for the strong inward rectification in this channel. Negatively charged
amino acids in the M 2 and carboxy tail appear critical for Mg and polyamine block (Yang et af.,
1995). After steps to more negative Em, inward IK1 shows an initial peak and slow decline, which
may also be due to block of inward IK1 mainly by extracellular Na (Biermans et af., 1987).
Inward rectification ofK channels in heart is functionally important, both in a permissive
sense for the long plateau phase of the AP, and also in limiting cellular K loss during activity
(which would have to be re-accumulated by the Na/K-ATPase). If K conductance did not
decrease during the AP there would be a huge outward K current at positive Em due to the large
electrochemical driving force (consider the extrapolation of the "no rectification" line in Fig 4lA
to positive Em). Thus, inward rectification allows a stable AP plateau with relatively small and
balanced inward and outward currents.
Some Em-dependent K channels exhibit outward or delayed rectification (e.g. see I Ks in
Fig 4IA), characterized by an increasingly positive slope at more positive Em. This increasing
slope upon depolarization constitutes apparent outward rectification. However, this conductance
increase is attributable to the Em-dependent gating of the channels rather than an intrinsic
permeation property. That is, the conductance increases with depolarization because more
channels are open, not because individual channels really exhibit rectification. It is referred to as
delayed rectification because these channels take a finite time to be activated (e.g. IKs activation
is very slow). Some K channels also exhibit negative slope regions at positive potentials due to
inactivation (see I Kr in Fig 4 I C). While the term is somewhat imprecise, these Em-dependent K
channels (IKr, IKs & IKur) are referred to as outward or delayed rectifier channels.
IKJ Stabilizes the Resting £11I

I KJ is the resting or background K channel responsible for stabilizing the resting Em near
E K. This current is thought to be carried by the Kir2.1 (IRKI, or other Kir2.x) channel protein
(Kubo et af., 1993). Figure 41B shows IK1 on an expanded scale. Conductance (gKb slope of the
curve) is very high around the resting Em and as such I KJ tends to bring small depolarizing or
hyperpolarizing influences back to rest. As Na channels are activated to bring Em near threshold,
gKJ declines allowing the positive feedback associated with the upstroke of the AP to occur.
Increasing [KJo (hyperkalemia) depolarizes Em due to a change in E K, and also increases gKJ (as
the square root of [K]o). While this moves the resting Em closer to a threshold for triggering an
AP, two factors cause hyperkalemia to actually reduce excitability. First, the increase in gKI
means that more inward INa would be required to overcome the I KJ which tends to keep Em near
E K. Second the depolarized Em reduces the number of Na channels available by increasing the
fraction in the inactivated state (Fig 40B). This reduces the amount of INa that can be mustered to
cause depolarization in the face of increased gKI' Conversely, hypokalemia causes increased
excitability, but the hyperpolarization is less than expected based on the change ofE K (since gK is
reduced, allowing other currents to move Em further away from E K).
A. Rectification
10
IK1 Em (mV)
strong 50
inward
-120 -60 -30 30

EK
C. IKr (steady state) D. IKr is a window current

Activation
slow
Availability
Inactivation
(v. fast) Window
/....~~-+ __ conductance
(Activ. x Inac!.)
-60 -40 40 80 -BO ·40 0 40

Em (mV) Em (mV)
Figure 41. K channel Em-dependence and rectification. A. A non-rectifying channel (dashed line) should
have an ohmic conductance (constant slope I-V curve, in symmetrical solutions) reversing at EK. Weak and
strong inwardly rectifying current (lK(ATP) & IKI respectively) show a gradual decline in slope at more
positive Em and may have a negative slope region. IKs exhibits outward rectification and is also time
dependent in activation (delayed). Currents are normalized in this panel. B. Raising [KJo makes EK more
positive and increases conductance ofl KI and IK, (C). C. Em-dependence of IKr at different [KJo. D. Very
rapid inactivation dictates IKr availability, so maximal conductance is proportional to the product of
fractional activation and fractional inactivation.
IKI density is generally very low in pacemaker cells and IKI may be absent in SA node
cells (lrisawa et aI., 1993). IKI is also lower in Purkinje fibers than in ventricular myocytes
(Cordeiro et aI., 1998). This facilitates the pacemaker induced APs in these cells. This is
because there is less outward I K1 to be overcome by INa or Ica , to cause the regenerative AP
depolarization. Indeed, these cells have much lower resting conductance (and capacitance since
they lack T-tubules). Thus a small inward pacemaker current can produce a large depolarization.
The low IKI also explains the more positive resting Em in these cells (further away from E K).
I KI also participates in the final stages of Em repolarization during the AP as the Em range
is reached where channels are not blocked (see Fig 45). Based on the strong inward rectification,
IKI has minimal involvement during the peak and plateau phases of the AP (despite the high
driving force for K efflux). This channel does not seem to be strongly modulated by PKA.
Transient Outward Current (I,,)

Transient outward K current (sometimes called 1,01) as well as a Ca-activated CI current
(or 1'02, see below) contribute to the early repolarization phase of the AP. These K channels are
Em-dependent and exhibit rapid activation and inactivation. There appear to be two main
components, one that inactivates faster than the other (l,o,faSt and lto,slow; Nerbonne, 2000, 2001).
Ito,faSt is mediated by Kv4.2 and/or Kv4.3, while lto,slow is attributable to Kv1.4 (Xu et al., 1999a;
Bou Abboud & Nerbonne, 1999). These two currents both activate rapidly (1: :s: 5 msec), but
lto,fast inactivates with 1: -100 msec vs. 200 msec for I,o,slow' Even more striking is the difference in
recovery from inactivation (1: = 30-50 msec for I,o,fast vs. 1 sec for lto,slow). This very slow
recovery OfI,o,slow would limit its contribution to the AP, especially at higher frequencies (due to
accumulating inactivation). Most mammalian myocytes exhibit I,o,fast. Guinea-pig ventricle
appears to lack appreciable I,D of either sort, and rabbit ventricular I,D is more like I'o,slow and
Kv1.4 (Fedida & Giles, 1991; Wang et al., 1999; Nerbonne, 2000, 2001). Slow recovery ofI,o in
rabbit limits the amount of APD shortening which occurs upon increasing frequency (due to
accumulation of inactivated channels). Both I'o,fast and I'o,slow are blocked by mM 4-
aminopyridine, but only I'o,fast is sensitive to Heteropodatoxins-2 and -3 (Xu et aI., 1999a). PKC
appears to attenuate I,o,fast and Kv 4.2 (Apkon & Nerbonne, 1988; Nakamura et aI., 1997) and can
also depress Kv1.4 induced currents in Xenopus oocytes (Murray et aI., 1994). CaMKII
phosphorylation ofKv1.4 slows inactivation, but accelerates recovery (Roeper et al., 1997).
There is also a transmural gradient of higher Ito amplitude with faster inactivation and
recovery from inactivation in epicardial vs. endocardial cells in ferret and human ventricle
(Niibauer et al., 1996; Brahmajothi et aI., 1999). There is also 5-6 times more I,o,fast in canine
epicardium vs. endocardium (Liu et aI., 1993), consistent with the more rapid repolarization and
shorter APD in epicardial cells (Fig 35). In mouse, there is I'o,slow in the septum but not in the left
ventricular apex (Xu et al., 1999a). It is possible that in ventricular muscle which is under more
mechanical stress, Kv4.213 (& I,o.fast) expression is lower, while Kv1.4 (& I,o,slow) is upregulated.
This would be consistent with the reduction of I,D which is a common finding in ventricular
hypertrophy and heart failure (Benitah et al., 1993; Beuckelmann et al., 1993; Tomita et al.,
1994; Niibauer & Kiiiib, 1998). Lower I,D could also contribute to the AP prolongation typical of
hypertrophy and heart failure. There is also a developmental increase in I,D as well as altered
isoform expression. Neonatal rat ventricle shows more of an I'o,slow phenotype which shifts to
lto,fast in adult (Wickenden et al., 1997). In rabbits as the density of I,D expressed increases
developmentally the reverse shift occurs from l to ,f.15' to I'o,slow (Sanchez-Chapula et al., 1994).
Delayed Rectifier K currents

There are at least three key delayed rectifier K channels in heart, IKr , IKs and IKur (for
rapid, slow and ultra-rapid). Sanguinetti & Jurkiewicz (1990, 1991) first distinguished two
different components of delayed rectifier K current in guinea-pig based on sensitivity to class III
antiarrhythmic drugs. IKr is blocked by dofetilide, E-4031, almokalant and sotalol, while IKs is
sensitive to clofilium, NE-10064, NE-10133, L-768,673 and HMR-1556 (Cameliet, 1993; Busch
et aI., 1994; Lerche et al., 2000; Xu et al., 2001). Both currents are present in many cardiac cells
types, including human ventricle with varying ratios (Beuckelmann et aI., 1993; Konarzewska et
aI., 1995; Salata et al., 1996). Some myocytes show only IKs (guinea-pig node, Anumonwo et al.,
1992), while others show only IKr (cat & rat ventricular cells, Follmer & Colatsky, 1990; Pond et
al.,2000). An even faster component called IKur was described in human and rat atrium (Boyle &
Nerbonne, 1991; Wang et al., 1993a) and a similar current (IKp for plateau) was described in
ventricular myocytes (Backx & Marban, 1993). I Kur and IKp may be the same current (Nerbonne,
2000, 2001) and will be considered such here. These channels are highly selective for K, but
their selectivity (PK/PNa ) is not as high as for IKh and consequently the Erev is slightly more
positive than E K(Carmeliet, 1999).
IKr (HERG). Sanguinetti et at. (1995, 1996a) and Trudeau et at. (1995) showed that IKr is
mediated by a K channel related to the mutant Drosophila gene ether-d-go-go (eag), called
human eag-related gene (or HERG). They also showed that mutations in HERG, which inhibit
IKr in a dominant negative manner, are responsible for congenital long Q-T syndrome (LQT2), a
reflection of increased ventricular APD. So, IKr is involved in ventricular repolarization. The
ventricular AP prolongation in long Q-T syndromes can lead to the clinical ECG observation
torsades de pointes (literally, turning of the points) and can reflect a cardiac substrate with
increased arrhythmogenic potential.
IKr activates slowly, but inactivates extremely rapidly. This is unusual behavior in that
inactivation is relatively divorced from, and faster than activation. It may be a consequence of a
very fast variant ofC-type vs. N-type inactivation (as mentioned above). The result is that the I-
V curve for IKr in Fig 41C is bell-shaped. Here the negative slope is due to inactivation at
positive Em, which creates unavailability of channels which would otherwise be activated at those
Em (i.e. this is not true inward rectification with respect to permeation). In this sense the IKr is
highly analogous to a window current (see Figs 40B & 41 C-D). Once activation reaches a steady
state (1 -50 msec at Em = 0 mY) IKr does not decline with time, so the time course doesn't look
like classic inactivation, as in INa or Ito.
Single channel conductance of IKr is -13 pS (Veldkamp et at., 1993). IKr amplitude is
increased by higher [K]o (like IKI ; as the square root of [K]o, see Fig 41C). This is not expected
thermodynamically, since raising [K]o would reduce the chemical driving force on K flux
through the channel. It must reflect allosteric regulation by [K]o. While HERG is sufficient to
produce IKr , the current is modulated by MirPl (minK related peptide I, where minK is
considered an important subunit ofI Ks (see below).
IKs (KvLOTl + minK). I Ks is a very slowly activating current which does not show appreciable
inactivation. The gene coding IKs was identified from positional cloning studies that identified
mutations in the most common congenital form of long Q-T syndrome (LQTl; Wang et at.,
1996b). Expression of both KvLQTI and minK (like a ~-subunit) is essential to fully
recapitulate cardiac IKs (Barhanin et at., 1996; Sanguinetti et at., I996b). KvLQTl has the usual
topology for Em-dependent K channels (Fig 38B) and minK is a 130 amino acid protein with a
single transmembrane domain, and was an early candidate gene by itself for IKs •
I Ks activates very slowly, over several seconds under sustained depolarization (1 -400
msec at Em = 0 mY). Thus activation is never complete during an AP. IKs activation also exhibits
remarkably shallow Em-dependence (Fig 41A) with a midpoint near 0 mV and e-fold increase for
15 mV (vs. 2-4 mV for INa)' Giv~n the major fundamental differences between IKr and IKs it is
rather surprising that the outward current carried by these channels looks similar superficially
during the AP (Fig 45). This results from very slow activation of IKs and the negative slope
conductance of IKr (i.e. gKr gets larger during repolarization (Fig 41D). IKs expression is two
times less in canine midmyocardium than either epi- or endocardium and this may contribute to
the very long APD which is characteristic of these cells (Sicouri & Antzelevitch, 1991; Liu &
Antzelevitch, 1995).
IKs is strongly regulated by PKA and PKC in an Em- and temperature-dependent manner
(Walsh & Kass, 1988, 1991). l3-adrenergic agonists increase IKs amplitude by 2-fold and shift
activation to more negative Em. PKC produced less increase in amplitude, but there was less
shift in Em-dependence. The PKA and PKC-mediated effects are additive, suggesting that they
may be mediated by phosphorylation at different sites. In contrast to these results in guinea-pig
ventricular myocytes, PKC can decrease IKs in rat and mouse myocytes, but this effect may be
due to phosphorylation of a different minK which contains a serine that is not in guinea-pig
minK (Varnum et at., 1993). PKA-dependent phosphorylation results in increased outward IKs
during the AP and contributes to the decrease in APD usually observed with sympathetic or 13-
adrenergic stimulation. The increase in IKs helps to offset the effect of PKA-induced increase of
Ica amplitude, which would tend to increase APD. IKs is also increased by elevated [Cali (Toshe
et al., 1987) which would speed repolarization during large Ca transients.
!Kur (Kv1.5). I Kur activates more rapidly (~=2-20 msec) than either IKr or IKs , does not inactivate
and is much more sensitive to block by 4-aminopyridine (i.e. 11M vs. mM required for IKr and IKs ;
Boyle & Nerbonne, 1991; Wang et al., 1993b). Heterologous expression of Kv1.5 produces
currents which resemble IKur and Kv1.5 message and protein is abundant in heart (see reviews by
Nerbonne, 2000, 2001). This led to the suggestion that Kv1.5 underlies cardiac IKur (Fedida et
al., ]993; Wang et al., 1993a; Barry et at., 1995). Feng et at. (1997) and Bou Abboud &
Nerbonne (1999) showed that antisense mRNA knock-down of Kv1.5 in human myocytes
specifically reduced IKur , clarifying that Kv1.5 does indeed underlie IKur . Similar tests have not
been reported yet to clarify whether IKp is indeed, also the product of Kv1.5. This may also be
the same current some have referred to as Iss (steady state) or Isus (sustained). IKur may be
particularly important in the early plateau phase where IKr and IKs are not yet activated (see Fig
45). Indeed, block ofI Kur in atrial myocytes greatly prolongs APD (Wang et al., 1993b).
Other Delayed Rectifiers. Nerbonne (2000, 2001) discusses some additional delayed rectifier K
currents, but these will only be mentioned briefly here. IK,slow is a rapidly activating and slowly
inactivating current in mouse. It is blocked by nanomolar concentrations of a-dendrotoxin and
appears to be attributable to Kv1.2 and/or Kv2.1 (Bou Abboud & Nerbonne, 1999; Xu et al.,
1999b,c). Iss is a slowly activating current that remains at the end of 10 sec long depolarizations,
and is relatively insensitive to 4-aminopyridine (Xu et al., I 999a,c), but its molecular identity is
not yet clear.
Other Inward Rectifier K Channels

!KiAChl (Kir3.1 & 3.4). IK(Ach) is a strong inward rectifier K channel which is activated by
muscarinic agonists such as acetylcholine (ACh). IK(Ach) is prominent in atrial myocytes and cells
of the SA and AV nodes, notably the sites where vagal innervation in the heart is highest. It is
also in ventricular myocytes at lower density. The parasympathetic activation of K current is
centrally important in mediating the physiological mechanism of heart rate slowing. That is, the
K current tends to stabilize Em near EK and increase the amount of inward current required to
depolarize cells to the threshold of AP activation. ACh secreted from parasympathetic neurons
binds to muscarinic M 2 -receptors which are members of the 7-transmembrane domain G-protein
coupled receptor family. This stimulates the GTPase activity of the heterotrimeric GTP binding
protein G j (with a, 13 & Y subunits) resulting in the dissociation of Gj,,-GTP from Gipy. While
there was initially some controversy as to whether the a or ~y subunit was responsible for
activating IK(Ach), it is now clear that Gipy is the species which binds to the carboxy terminal of the
IK(Ach) channel protein to cause activation (Reuveny et aI., 1994; Inanobe et aI., 1995; Wickman
& Clapham, 1995).
IK(Ach) is mediated by a heterotetrameric K channel composed of two Kir3.1 (or GIRKI)
and two Kir3.4 (GIRK4 or CIR, cardiac inward rectifier) molecules (Krapivinsky et aI., 1995).
The single channel conductance is high (~40 pS), and K selectivity is very high, as in IK1 .
Knockout of IK(Ach) by targeting the Kir3.4 gene, results in a mouse with depressed resting heart
rate variability and bradycardic response to vasoconstriction (Wickman et aI., 1998).
IK(Ach) can also be activated by some other agonists which couple to G-proteins, such as
adenosine, ATP (purinergic PI & P 2), endothelin, somatostatin, calcitonin gene related peptide,
and also by the generation of phosphoinositol 4,5-bisphosphate (PIP 2 ) via an ATP-dependent
lipid kinase (Berlardinelli & Isenberg, 1983; Lewis & Clapham, 1989; Kim, 1991a,b; Matsura &
Ehara, 1996; Huang et ai., 1998). IK(Ach) also demonstrates fade or desensitization, whereby
sustained exposure to ACh results in a gradual decline in current. A fast phase (within 30 sec)
may be due to reduced single channel open times, reflecting alteration in the G protein level
(Kim, 1993a), while the slower phase over -3 min may be due to receptor phosphorylation by a
receptor kinase (Zang et ai., 1993; Wang & Lipsius, 1995a).
!KfATP\ (Kir6.2 + SUR). A K current which is inhibited by intracellular ATP (IK(ATP) was first
described in heart cells by Noma (1983), but it is present and functionally important in many
other cell types (Ashcroft & Ashcroft, 1990). When [ATP]i falls during local ischemia these K
channels become activated and can shorten the AP and also prevent excitability (Nichols et ai.,
1991; Weiss et aI., 1992). This would be functionally important in conserving ATP by limiting
excitation and consequent contraction in cells where ATP is low.
The [Mg-ATP] required to half maximally inhibit IK(ATP) in excised patches is 15-50 ~M.
This is much lower than the levels of [ATP]i in the normal myocyte (5-10 roM), and even in the
early stages of ischemia where APD shortens and can be prevented (see pg 306). Intracellular
[ADP] is also a strong modulator of IK(ATP) and increases of [ADP]i from normal (::;20 ~M) to
above I00 ~M (as occurs relatively early during ischemia) can shift the apparent K m for ATP to
100 ~M. In this sense it is probably the [ADP]/[ATP] ratio which activates this channel (see pg
306-307). This makes sense teleologically, because this ratio is more directly related to the
energetic state of the cell. IK(ATP) may also be preferentially regulated by local glycolytic
enzymes which can regenerate ATP from local ADP (Weiss & Lamp, 1989).
These IK(ATP) channels are present at high density in cardiac myocytes and have a very
high single channel conductance (-40 pS). Thus, activation of<l% ofIK(ATP) (which can happen
with little reduction in [ATP]i) is sufficient to shorten APD by 50% (Nichols et ai., 1991; Weiss
et ai., 1992). Mg-ATP also prevents rundown of IK(ATP) possibly by preventing actin
depolymerization (Furukawa et ai., 1996) and also by stimulating PIP2 production by a lipid
kinase (Hilgemann & Ball, 1996). PIP 2 increases the [ATP] required to block IK(ATP)' Thus,
IK(ATP) channels are present in great functional excess with most fully inhibited, and only a small
fraction must be activated to protect the cell from depletion of ATP or increase of[ADP]/[ATP].
The down side of this is that local regions of cells which are not conducting the normal AP can
alter the normal conduction pathway and contribute to arrhythmogenesis. Even in cells which
retain APs the APD is shorter, reducing refractory period, which can be pro-arrhythmic.
To reconstitute cardiac IK(ATP) requires both the sulfonylurea receptor (type 2A, SUR2A)
plus the inward rectifier K channel Kir6.2 (Inagaki et at., 1995, 1996; Aguilar-Bryant et at.,
1998). This is a heteromultimer where the inner ring and pore is made up by 4 Kir6.2 molecules
and an outer ring includes 4 SUR2A molecules. SURs are members of the superfamily of ATP
Binding Cassette (ABC) proteins which include the cystic fibrosis transmembrane regulator
(CFTR) and multi drug resistance protein. These have 12-17 transmembrane segments and 2
characteristic nucleotide binding folds in intracellular loops. SUR2A gives the channel its
characteristic sensitivity to K-channel openers (cromakalim, lemakalim, pinacidil, nicorandil &
aprikalim) and blockers (glibenclamide, tolbutamide).
Work with IK(ATP) channel openers had suggested that IK(ATP) activation could be a key
cardioprotective mechanism induced by brief ischemic periods (preconditioning; Yao et at.,
1994; Gross, 1995; Grover et al., 1996). However, there is also a mitochondrial K(ATP) channel
(Garlid et a1.l996). Recent work with the selective mitochondrial IK(ATP) activator diazoxide and
blocker 5-hydroxydecanoate has shown that the mitochondrial IK(ATP) is crucial in mediating
cardioprotection in preconditioning, via a PKC-dependent pathway (see pg 311, Garlid et ai.,
1997; Liu et at., 1998; Sato et at., 1998a; Takashi et at., 1999). How opening mitochondrial
IK(ATP) channels would be protective is not known. The loss of mitochondrial membrane potential
would prevent mitochondrial Ca uptake (Hohnuhamedov et ai., 1999). On the other hand, this
would also limit ATP synthesis (see Chapter 3), so the protective mechanism remains unclear.
Na- and Fatty Acid-activated K Channels

A K channel activated by high [Na]i has been reported in cardiac muscle (Kameyama et
ai., 1984; Luk & Carmeliet, 1990; Lawrence & Rodrigo, 1999). This channel has high K
selectivity, but the K m for activation by [Na]i is very high (66 mM). The physiological role of
this current is unclear. Intracellular arachidonic acid, unsaturated fatty acids, phospholipids and
stretch can also activate K-selective channels in heart (Kim & Clapham, 1989; Kim, 1993b). The
specific physiological role of these currents is unclear, but they could serve the same function as
IK(ATP) to inhibit the AP under stressful cellular conditions.
CI CHANNELS
There are three types of CI currents in heart which will be discussed below (see Hume et
at., 2000 for review): a) Ca-activated CI current (ICI(Ca»), b) cAMP-activated Cl current (ICI(CAMP»)
and c) swelling-activated CI current (ICI(swell»). There is also a purinergic receptor activated CI
current (Matsura & Ehara, 1992; Levesque & Hume, 1995), but this will not be discussed further
here. These Cl currents are generally blocked by disulfonic stilbenes DIDS and SITS, except for
IC1(cAMP) which is not sensitive to these, but can be blocked by DNDS (see Table 14). The
reversal potential for CI is positive to resting Em in atrial and ventricular myocytes (Fig 36C)
which makes the functional role of CI channel opening less intrinsically obvious. Thus
activation of Cl currents may hasten AP repolarization, but also cause diastolic depolarization.
CFTR: cAMP-Activated Cl Channel.

The PKA activated IC1(CAMP» is present in ventricular myocytes, is less dense in atrial
myocytes, and is not present in rabbit SA node, canine myocardium or rat ventricle (Horowitz et
aI., 1993; Gadsby et aI., 1995). The current is carried by the cardiac variant of the cystic fibrosis
transmembrane regulator (CFTR). CFTR has two sets of 6 transmembrane domains (not
homologous to Na or K channels), separated by a large intracellular loop or regulatory domain
which contains one of the two nucleotide binding domain (NBD1, with NBD2 near the intra-
cellular C-terminal). Any pathway which activates PKA can cause phosphorylation in the
regulatory domain and activation of CFTR. Once phosphorylated, ATP binds at NBDI and is
hydrolyzed. While ADP remains bound at NBD 1 the channel is active and exhibits brief opening
bursts. When a second phosphorylation occurs and ATP is bound and cleaved at NBD2, ADP
bound at NBDI is stabilized and CFTR enters an active conformation characterized by long
openings (-1 sec) and brief closures (Ehara & Matsura, 1993; Gadsby et at., 1995). Dephos-
phorylation of CFTR or ADP dissociation from the NBDs causes channel deactivation.
IC1(CAMP) does not show appreciable Em-dependent gating and the current shows no
rectification in symmetrical CI solutions. However, since intracellular [Cl] is much lower than
[Cll o there is slight apparent outward rectification under physiological conditions. The
selectivity sequence for IC1(CAMP) is N0 3 > Br ~ CI ~ I (Overholt et al., 1993). When IC1(CAMP) is
activated it constitutes a static increase in CI conductance at the whole cell level. One
consequence is that the outward IC1(CAMP) during most of the AP will speed repolarization and thus
tend to shorten APD. This, along with PKA activation of IKs may offset the enhanced inward ICa
and contribute to the shorter APD seen with ~-adrenergic agonists and sympathetic stimulation.
Diastolic IC1(CAMP) would tend to depolarize Em somewhat toward ECI (about -55 mY). This effect
may be minor in ventricular myocytes where IK1 is relatively large, but could be larger in cells
with lower IK1 (such as pacemaker cells). However, pacemaker cells may have relatively little
IC1(CAMP) and since diastolic Em in these cells is already close to ECI> the impact may be small.
Ca-Activated CI Current IO(Cn;: a Transient Outward Current.

The Cal-dependent component of transient outward current which used to be referred to
as 1'02 is now clearly attributable to ICI(Ca), (Zygmunt & Gibbons, 1991,1992; Zygmunt, 1994;
Sipido et at., 1993; Kawano et at., 1995). This channel does not appear to have intrinsic E m-
dependence or rectification, but is activated by the rise in [Cal; during E-C coupling. IC1(Ca) has
been most extensively characterized in rabbit, ferret and dog myocytes, but is probably present in
many cardiac myocytes. Current density is higher in atrium. A high temperature-dependence
was indicated by the results of Puglisi et al. (1999) who saw almost no detectable IC1(Ca) in rabbit
ventricular myocytes at 25°C, despite prominent IC1(Ca) at 35°C. The kinetics of IC1(Ca) are much
faster than the global cellular Ca transient, as is true for INa/Ca and Ca-dependent inactivation of
ICa,L (Trafford et al., 1998; Puglisi et al., 1999). This is undoubtedly due to the much higher and
faster local Ca transient in the submembrane space (or junctional gap) due to both Ca influx and
SR Ca release, when compared to global [Cal; sensed by Ca indicators. In this way, Ca-
dependent currents (lCI(Ca), INa/Ca and Ica,d can serve as a valuable indicators of local [Cal;. This
also emphasizes that the local, rather than global [Cal;, is important in regulating these currents.
These 3 Ca-dependent currents can also have substantial impact on the AP. It is not clear how
much of the rapid early repolarization is due to IC1(Ca) vs. 1,0' but it is probably a significant
fraction (N.B. IC1(Ca) is not included in the Fig 45 model). At diastolic Em (below ECI) spon-
taneous SR Ca release can activate inward IC1(Ca), contributing to DADs (Zygmunt et aI., 1998).
Collier et al. (1996) reported that the single channel IC1(Ca) is small (-1 pS) and that the
apparent K m for [Cali activation was 150 IlM. This is relevant to the discussion above, because a
slow rise in global [Cali to 1 IlM would not activate appreciable Ic1(ca), whereas submembrane
[Cali may reach 50 IlM briefly at the peak of SR Ca release (Chapter 7) and this would activate
considerable IC1(Ca) with the short timecourse observed. IC1(Ca) can be blocked by mM DIDS, 50
IlM niflumate and glibenclamide (K m =65 IlM; Yamazaki & Hume, 1997).
Swelling-Activated CI Current
IC1(swell) was first reported in dog atrium and ventricle (Tseng, 1992; Sorota, 1992) and
seems to be more prominent in atrium than ventricle (Sorota, 1999). The likely candidate protein
for IC1(swell) is CIC-3 (Duan et aI., 1997a). CIC-3 is a member of a larger Cl channel family, and
these channels have 10-12 transmembrane spans, function as dimers and may have double
barreled pores (Jentsch et al., 1999). The current is activated slowly in response to an increase in
cell size (usually evoked by hypoosmotic superfusate or inflation via patch pipette). Activation
can be prevented by 50 IlM genestein, implicating a tyrosine kinase in the transduction pathway
(Sorota, 1995), and the extent of activation can be modulated by PKA and PKC (Sorota, 1999).
IC1(swell) is outwardly rectifying, even in symmetrical Cl solutions and has a large unitary
conductance, 49 pS (Vandenberg et aI., 1994; Duan et aI., 1997b). It can be blocked by I mM
DIDS, 100 IlM niflumate or 20 IlM tamoxifen, but there are no selective inhibitors. IC1(swell) can
be substantial (>50 pA) and contribute to depolarization of resting Em and shortening of APD
(Vandenberg et al., 1997). Clemo et al. (1999) found persistently activated IC1(swell) in dogs with
congestive heart failure and suggested that this contributes to electrophysiological dysfunction.
STRETCH-ACTIVATED CHANNELS
In addition to the IC1(swell) channels described above, there are stretch-activated channels
(SACs) in the heart, some of which are blocked by Gd, as reviewed by Hu & Sachs (1997). They
point out that mechanical stretch per se is different than cell swelling in response to osmotic or
hydrostatic pressure. If one wants to measure electrophysiological impact of the kind of
mechanical stretch which occurs in the heart this places challenging constraints on experimental
design. In rat atrial cells Kim & Fu (1993) reported a swelling-induced nonselective cation
current. Hu & Sachs (1996) found that swelling chick ventricular myocytes induced IC1(swell),
while mechanical strain activated a nonselective cation current. Clemo & Baumgarten (1997)
demonstrated a swelling-activated cationic current in rabbit ventricular myocytes. This current
was relatively non-selective (P0P Na =5.9), carried mainly inward Na flux at resting Em and was
blocked by Gd (Km = 0.5 IlM). Block of this current limited cell swelling in response to
hypotonic stress, suggesting that this channel along with IC1(swell) plays a role in volume regulation
and can cause diastolic depolarization. Stretch or swelling can also stimulate a K-selective
channel (Kim, 1992), IK(ATP) (Van Wagoner, 1993), increase Ica (Matsuda et al., 1996), and
inhibit IK(Ach) (Ii et al., 1998). The integrated electrophysiological role of these SACs is not
known, but nonselective cationic currents could contribute to arrhythmias.
NON-SELECTIVE CHANNELS
Pacemaker Current If
If was so named because it was a "funny" current, which activated slowly upon hyper-
polarization, in contrast to previously described cation currents activated by depolarization
(DiFrancesco, 1981 a,b, 1982, 1985, 1986, 1995). It is similar to the hyperpolarization-activated
current Ih in neurons. If is not selective between Na and K, but other monovalent cations (Li, Cs,
Rb) are almost imperrneant. The reversal potential is typically about -15 mV and unitary
conductance is small (-1 pS). Thus at negative Em net inward current is carried by Na and
contributes to diastolic depolarization. The time constant of If activation at -90 mV in SA node
cells is -500 ms (and ~2 sec at -70 mY). Thus If activates gradually at negative Em, and it does
not inactivate.
The protein responsible for If has recently been cloned (HCNI, 2 & 4; Ludwig et aI.,
1998, 1999; Gauss et al., 1998; Santoro et al., 1998; Shi et al., 1999) and is related to a class of
cyclic nucleotide gated cation channels. HCN stands for Hyperpolarization-activated Cyclic
Nucleotide-gated. These 700-900 amino acid channels appear to be homologous to the Em-
dependent K channels (Fig 38B) including 6 transmembrane domains, positive charges on every
third amino acid in the S4 domain and the GYG signature sequence in the P-loop. Differences in
amino acids neighboring GYG may result in lower selectivity for Kover Na (PK/P Na = 3-4;
Clapham, 1998). With these similarities it is not clear why this channel activates upon
hyperpolarization, rather than depolarization (as for Em-dependent K channels). One possibility
is that these channels have activation and inactivation like Ito, but are fully activated at all
relevant Em values. Then hyperpolarization may allow Em-dependent recovery from inactivation
(as for Em-dependent K, Na & Ca channels) such that If is really like a window current (see
Miller & Aldrich, 1996).
If is present in SA & AV node cells, Purkinje fibers, and also atrial & ventricular
myocytes, although myocytes do not readily display prominent pacemaker activity (DiFrancesco,
1995). The Em-dependence of activation differs dramatically in different tissues (Fig 42). In SA
node cells the Em-dependent activation range overlaps with the range of Em during pacemaker
activity (-40 to -60 mY) and the same is true for Purkinje fibers (-80 to -100 mY). While If
exists in ventricular myocytes (Yu et al., 1993, 1995) it is probably non-functional due to the
very negative Em range of activation (Fig 42). An exception could be if the Em-dependence
becomes more positive as a result of cAMP or some other potentially pathophysiological
modulatory effect. There also remains some controversy about the role of If in SA node cells
(see pg 94-97), based on relatively negative activation Em in some reports. This would certainly
be the case if one assumed that the Em-dependence of If in Purkinje fibers also was relevant to SA
node. Shi et al. reported that rabbit SA nodal cells expressed mainly HCN4, Purkinje fibers
express HCNI & 4 equally and ventricular myocytes express only HCN2. Thus there may be a
molecular correlate to the different Em-dependence of If in different cardiac tissues.
If Activation in Cardiac Cells

SA-node
0.5
0.0 +-,.-r-T"'""T..,......,--r-.,.;::::::;::::;=,......,.....,.--.;::::=r==r--,.-....."'T""'"'"T::::;::::p;::::;::=i~-..~
-160 -140 -120 -100 -80 -60 -40 -20 o
Em (mV)
Figure 42. Em-dependence of If activation during hyperpolarization. The rightmost 3 Boltzmann curves
are for SA node and have half-maximal Em (E o.s) at -78, -66 and -54 mV for acetylcholine (ACh), control
(Cd) and isoproterenol (Iso) respectively (slope values are J 0 mV/e-fold change). Values for Purkinje
fibers and ventricular myocytes are taken from Yu et al. (1995; Eo.s = -106 & -140 and slopes are 7 & 8.7
mV respectively).
Sympathetic stimulation or ~-adrenergic agonists (e.g. isoproterenol) and also para-

sympathetic stimulation by acetylcholine (ACh) release alter the Em-dependence of Ir, but not the
maximal current available (see Fig 42). The positive shift with isoproterenol means that at any
Em there will be more depolarizing inward current via Ir, and this would increase the rate of
depolarization and thereby the heart rate. ACh has the opposite effect, reducing Ir at any Em and
thereby slowing the rate of depolarization and heart rate. Thus, Ir and IK(Ach») contribute
significantly to the slowing of heart rate induced by parasympathetic stimulation of the heart.
Elevated [Cali also increases Ir(Hagiwara & lrisawa, 1989) and this would also tend to increase
heart rate.
The mechanism of sympathetic activation of Ir is mediated by the ~-adrenergic receptor
and production of cAMP by adenylyl cyclase. However, transduction does not depend upon
activation of PKA, but is due to a direct affect of cAMP on the Ir channel (DiFrancesco &
Tortora, 1991). This makes sense now that we know that this channel is related to a class of
cyclic nucleotide gated channels. Parasympathetic regulation is mediated via muscarinic
receptor and pertussis-sensitive G-protein (DiFrancesco & Tromba, 1988).
Ca-activated nonselective monovalent cation current Ins(Ca)-

Ins(Ca) was first described in heart by Colquhoun et al. (1981) and also characterized by
Ehara et al. (1988) in guinea-pig ventricular myocytes. Unitary currents (conductance =15 pS)
did not show any rectification and the channels were not selective among K, Na or Cs. Ins(Ca) was
half-maximally activated by 1.2 /-!M Ca with a Hill coefficient of 3 in excised patches. This
channel has the potential to contribute inward current upon spontaneous SR Ca release and
contribute to DADs along with IC1(Ca) and INa/Ca. The relative contribution of Ins(Ca) however is
small and it is not clear how functionally important this current is in ventricular myocytes.
Na/Ca EXCHANGE
Na/Ca exchange will be discussed in detail in Chapter 6, so I will only mention a few
salient characteristics here which are most relevant to Na/Ca exchange current (1Na/Co) during the
AP (Fig 45). The accepted stoichiometry of this reversible countertransport system is 3Na to
I Ca such that I positive charge is moved in the direction of Na transport. Thus Ca influx via
Na/Ca exchange produces outward INa/Co and Ca extrusion causes inward INa/co (Fig 43). The
direction and amplitude ofINa/co flow depends on Em as well as the intra- and extracellular Na and
Ca concentrations. INa/Co has a reversal potential (ENa/co) analogous to that of ion channels.
Under normal diastolic conditions ENa/co is typically about -40 mY, and inward INa/co occurs (Ca
extrusion) at resting Em -80 mY (A in Fig 43). However, since resting [Cali is low the amplitude
of diastolic inward INa/co is small (i.e. substrate concentration is limited). Upon depolarization to
the AP peak Em passes through the ENa/co and INa/co reverses and becomes outward (Ca influx, B
in Fig 43). Since [Cali rises rapidly due to Ico and SR Ca release, the ENa/co rapidly shifts to much
more positive potentials (note the shift in x-intercept in Fig 43, of the 1 flM vs. 150 nM curve).
This favors more inward INa/Co (Ca extrusion, 1 flM curve in Fig 43). The net INa/co current as the
AP proceeds through the plateau phase depends on both the time course of the Ca transient and
trajectory of the AP. As AP repolarization proceeds and [Cali remains elevated, INo/co becomes
strongly inward, as Na/Ca exchange extrudes Ca (from C to D to A in Fig 43). As [Cali declines
at diastolic Em, inward INa/co gradually declines (for more detail see pg 147-151).
When [Cali is elevated at resting Em during a spontaneous SR Ca release (E in Fig 43) a
large inward INa/co can be generated and this contributes to both a transient inward current (1,) as
well as the genesis of DADs. The relative contribution ofINa/c., Icl(co) and Ins(co) to DADs will be
discussed below (pg 97-99). Whatever Ca enters the cell via Ica at each beat, must be extruded
from the cell via Na/Ca exchange. Thus if 10 flM Ca enters via Ica 30 flM Na must enter to
extrude it. Interestingly, this Na influx is likely to be 2-3 times as much as enters via INo during
the AP. The Na ions which enter must in tum be extruded from the cell via the sarcolemmal
Na/K-ATPase.
NAlK-ATPase
The NaIK-ATPase transports 3 Na ions out and 2 K ions into the cell using the energy of
one ATP molecule, and thus moves one net charge out per cycle. This is the key transporter that
sets up the sarcolemmal ionic gradients for Na, K and Ca and consequently allows ion channels
to function. As indicated in Table 13 (pg 62) the Na/K-ATPase can generate a [Na]oI[Na]i
gradient of about 14 and a [K];I[K]o gradient of ~25 (based on L'.G ATP ). ATP is normally not rate
limiting for Na/K-ATPase, and local glycolysis may regenerate ATP, making the pump less
directly dependent on oxidative phosphorylation (Glitsch & Tappe, 1993). The main regulators
of Na/K-ATPase are the substrates [Na]i and [K]o. The activating K m for [Na]; is ~10 mM and
for [K]o is - 1.5 mM, so the pump is almost 80% saturated with respect to [K]o at normal [K]o of
5 mM. Normal [Na]; of -10 mM is right at the K m for [Na]j, assuring responsiveness to altered
[Na]; under physiological conditions. The precise K m for [NaJ may vary with different NalK-
ATPase isoforms and could cause resting [Na]; differences among species or regions (e.g. higher
[Na]; in rat than rabbit or guinea-pig ventricle; Shattock & Bers, 1989; Harrison el aI., 1992a).
Na/Ca Exchange Current (I NalCa )
Ca influx
80 J.~ _
-.--.,.--....-:==--r-o:;::::=-r-".----F.:;..-f!!.=--,..--r--r--r--r---r--r---, .. -_
EmlmV)
-1
Ca efflux
-2 'Na/Ca
(A/F)
Figure 43. Em-dependence of INa/Ca at different [Cali' INa/Ca was calculated using the equation and
constants in Luo & Rudy (I 994a) for [Na]i = 10 mM and [Ca]o = 2 mM. Inward currents (lNa/Ca <0) reflect
Ca efflux. As [Cali rises the reversal Em for INa/Ca (E NalCa ) becomes more positive. Abruptly increasing
[Cali at -80 mY as with caffeine increases inward INalC ' greatly (A to E). During the AP upstroke lNaiCa
reverses and becomes outward (A to B). As repolarization and [Cali decline INa/Ca becomes inward again (C
&D).
Like the Na/Ca exchanger, transport by Na/K-ATPase has a functional reversal potential
which depends on both intracellular and extracellular [Na] and [K] as well as the f,G ATP ' Under
normal conditions this has been estimated to be about -180 mV (Glitsch & Tappe, 1995), such
that Na/K-ATPase current (and Na transport) is outward over the whole physiological range.
However, this can change as f,G ATP declines during ischemia or energetic compromise, limiting
the Na/K-ATPase at negative Em (e.g. E rev shifts to -60 mV when f,G ATP falls from -58 to -39
kllmol). It is worth noting that at -80 mV almost all (95%) of the energy used by the Na/K-
ATPase goes into pumping Na out of the cell, because the electrochemical gradient for Na is so
much steeper than for K (150 m V vs. 8 mV). That is, at resting Em (close to E K) it takes little
energy to pump K in, but a great amount to pump Na out. Aside from this thermodynamic effect
Na/K-ATPase current is also weakly dependent on Em with a broad peak between -80 and 0 mV
(Gadsby et al., 1993; Bielen et aI., 1993). The result of these characteristics is that the Na/K-
ATPase contributes a relatively small outward (repolarizing) current throughout the AP (Fig 45).
The sustained outward Na/K-A TPase current thus also contributes slightly to the negative
diastolic Em. Abrupt block of the Na/K-ATPase normally only causes 2-4 mV depolarization
acutely (due to block of outward current), but of course in the long term would completely
dissipate Em and render cells inexcitable as the ion gradients run down.
Under typical experimental conditions the mean Na/K-ATPase current is 0.3 A/F over
the cardiac cycle (Fig 45). Since the Na/K-ATPase must extrude Na which enters the cell via INa
and Na/Ca exchange, it would be useful to know how well this current accounts for the known
Na influx. For 15 IlM Na entry via INa plus 36 IlM Na entry via INaiCa (to extrude 12 IlM Ca entry
via lea) per beat at I Hz would require extrusion of 17 IlM charge per second, or 0.27 A/F of
required Na/K-ATPase current. Thus there is good quantitative agreement between flux
estimates and Na/K-ATPase current measurements and >2/3 ofNa entry is via Na/Ca exchange.
The Na/K-ATPase is a member of the P-type ATPase pumps (like the SR and
sarcolemmal Ca-ATPases) and was first cloned by Shull et al. (1985) and Kawakami et al.,
1985). Three Na/K-ATPase isoforms exist in rat and human heart (al, a2 & a3, each -1000
amino acids or 110 kDa; Lucchesi & Sweadner, 1991; Wang et al., 1996c). These a subunits
exhibit all of the ion transport, ATPase activity and digitalis glycoside binding sites. The
associated ~ subunits (~I & ~2; 35 kDa) may playa role in ensuring appropriate processing and
insertion of the pump in the membrane, but only ~I is appreciable in human heart (McDonough
et al., 1990; Wang et al., 1996c). Expression of Na/K-ATPase is higher in ventricle than atrium.
McDonough et al. (1996) demonstrated that in rat ventricular myocytes the al isoform (lower
ouabain affinity) is preferentially in T-tubules, whereas a2 was homogeneously distributed.
Conversely, in rat vascular smooth muscle the a2 & a3 isoforms (high ouabain affinity) were
preferentially localized to junctions between SR and sarcolemma, while al was ubiquitous
(Juhaszova & Blaustein, 1997). This could reflect functionally important spatial compart-
mentalization. The a subunit has 10 transmembrane domains (with amino and carboxy tails
intracellular) and the 5th and 6th domains seem to be most important in ion binding and may form
the pore through which Na and K flow (Lingrel et al., 1997). External sites between domains 1-
2, 5-6 and 6-7 appear to be most important for specific binding of ouabain and other cardiac
glycosides. These compounds are specific inhibitors of Na/K-ATPase and have been used to
enhance cardiac contractility in the treatment of congestive heart failure for more than 200 years.
The inotropic mechanism of cardiac glycosides will be discussed in depth in Chapter 10. Suffice
it to say here that Na/K-ATPase inhibition would cause [Na]; to rise and this would shift the
balance of fluxes on the Na/Ca exchange to favor more Ca influx and less Ca efflux.
Figure 44 shows the accepted Na/K-ATPase transport mechanism as sequential in the
following sense (Liiuger, 1991; Rakowski et al., 1997). In the EI-ATP state of the enzyme the
cation binding sites face the cytosol, ready to bind Na. When 3 Na ions bind this causes ATP
hydrolysis and phosphorylation of the enzyme in a state where the Na ions are occluded
(inaccessible from either side). Then the pump shifts to the E2 conformation where the Na ions
are released to the outside and are replaced by 2 K ions. Binding of K ions stimulates dephos-
phorylation and K occlusion. Finally, intracellular ATP binds to the pump causing reversion to
the E, state where K is released to the cytosol and the pump is ready for another cycle. The
turnover rate is 75-100/sec (Nakao & Gadsby, 1986), -4 orders of magnitude slower than the
flux rate through a Na channel. The number of sarcolemmal Na/K-ATPase sites is much higher
than for Na channels (1200 vs. 3/J.lm2).
Na/K-ATPase in guinea-pig ventricle can be inhibited by ~-adrenergic agonists via a
cAMP/PKA pathway (Gao et al., 1996, 1998a) and stimulated by a-adrenergic activation (Wang
et al., 1998a). The ~-adrenergic-induced depression of Na/K-ATPase is still somewhat contro-
versial, since Ishizuka & Berlin (1993) found no effect on Na/K-ATPase current in rat myocytes
and increases in Na/K-ATPase were reported in rabbit, guinea-pig and sheep ventricle (Desilets
& Baumgarten, 1986; Glitsch et aI., 1989; Kockskiimper et aI., 2000). While there may be some
species-dependent differences, technical issues may also obscure the depressant effect of ~
adrenergic agonists (Gao et al., 1997c). The depressant effect also agrees with data in renal
cells, and there may also be isoform-specific differences (Blanco & Mercer, 1998). Reduction of
--,
Na Na
..3·~-O-·~~"
-----
--
p
-,
p/
-----,
Occluded
I
~ K
~~~~~-, .._~'=-;U2.
'- +
(I-~.-~
. - _ . _ -"
p .•- " /
ATP Occluded
Figure 44, Post-Albers scheme of Na/K-ATPase mechanism (see text; based on Liiuger, 1991).
Na/K-ATPase upon sympathetic stimulation could exacerbate the rise in [Na]j which occurs with
increased heart rate.
Different Na/K-ATPase isoforms have differing glycoside and [Na], sensitivity (Blanco
& Mercer, 1998). Thus, alteration in the balance of isoform expression or cellular localization
could be important in determining function and [Na]j. In failing human heart there is decreased
expression of the 2 main human Na pump isoforms (al and a3; Schwinger et aI., 1999b). This
could cause [Na]j to rise in heart failure and contribute to altered Ca homeostasis.
CURRENTS DURING VENTRICULAR ACTION POTENTIAL

Figure 45 shows a rabbit ventricular myocyte AP and the ionic currents which occur.
One practical way to evaluate the complex interacting currents during the AP is the following.
First one measures the Em- and time-dependence (& [Ca]j-dependence where relevant) of each
current, under well controlled voltage clamp conditions. Then the time-, Em- and [Cak
dependence of each current can be calculated at each point in time and used to calculate how the
integrated currents affect Em and [Ca]j during the next instant. These sort of numerical
integration models can be quite complicated, but some excellent versions have been developed
(e.g. Noble's Oxsoft Heart, Luo & Rudy, 1991, 1994a, Zeng et al., 1995; Jaffri et al., 1998;
Winslow et al., 1999; Nordin, 1993, Demir et al., 1994; Lindblad et aI., 1996). Figure 45 uses
the system of equations in the Luo-Rudy guinea-pig ventricular myocyte AP model (Luo &
Rudy, 1991, 1994a; Zeng et al., 1995) with some adjustments to make it more appropriate for
rabbit ventricular myocytes (e.g. Ito is included and IKr & IKs are adjusted; Salata et al., 1996). Dr.
J.L. Puglisi and I have mounted this on a user friendly platform (LabHeart) and we plan to make
it available in 2000. These models are certainly imperfect, but are being continually improved
and can be valuable adjuncts to guide appropriate experimental tests. One of the imperfections is
simply that the model can be no better than the precision and completeness of the data
concerning channel behavior, and one must always compromise with results from different
species and obtained under differing experimental conditions. An additional key issue is how to
deal with specialized spaces (SR-T-tubule junctions and subsarcolemmal space), where ions
and/or regulators may accumulate or deplete. With these caveats in mind let's consider which
currents contribute to each phase of the ventricular AP.
Rapid Upstroke ofAP (Phase 0)

Myocyte depolarization is normally initiated by passive current spread from a
neighboring active region (Fig 37), which can bring the cell to the threshold point where a
sufficient number of Na channels are activated that inward IN. exceeds outward IK . The rapid
upstroke of the AP in atrial and ventricular myocytes is primarily due to the regenerative
activation of IN•. That is, once threshold depolarization is reached for an AP, the opening ofNa
channels leads to further depolarization, which activates additional Na channels and depolari-
zation etc. This positive feedback creates a very rapid depolarization (200 V/sec) toward EN•.
Since there is always some finite K permeability, the Em never reaches EN. (+70 mY), but in
ventricular myocytes often reaches +35-50 mY. Depolarization also rapidly shuts off the
inwardly rectifying IKh limiting outward current during the AP upstroke (and later phases).
Depolarization stops, in part because Na channels inactivate, and this constitutes a built-in nega-
tive feedback or brake on IN.. Strictly speaking, the AP peak is where inward current (IN. + Ic.)
exactly equals outward current (carried mostly by K). With depolarization the driving force for
IN. (Em - EN.) gets smaller, while for K currents the driving force (Em - E K) increases. In
addition, the K conductance increase, rapidly for Ito & I Kur (more slowly for IKr & IKs). By the
peak of the AP I • is back down to -1 % of its peak value and Ic• is already at 43% of maximum
(see inset in Fig 45). Coincidentally, the absolute value of Ic•. L and IN. are both -4 A/F at this
point. Again, at the AP peak total inward and outward current are exactly balanced.
Early Repolarization (Phase 1)

Several factors contribute to the early repolarization phase, including additional IN.
inactivation and activation of outward Ito and 1cl (c.) (although the latter is not in the Fig 45
model). The same SR Ca release which contributes to the activation of Icl(c.) also inactivates Ic•
and that is responsible for the initial rapid decrease in IC.,L in Fig 45. I Kur also activates very
rapidly and may contribute to early repolarization. Probably 1'0 is the most dominant contributor
to the AP notch, and the decrease in the AP notch seen from epi- to endocardium follows that of
1'0 expression (see pg 78-79). Nevertheless, inhibition of SR Ca release can also diminish the
notch (due to effects on IcJ(c.) and Ic.). Atrial myocytes and rat and mouse ventricular myocytes
have much more 1'0, and consequently greater early repolarization. Moreover, in rat and mouse
ventricular myocytes there is almost no plateau phase, because early repolarization is so great.
IC.,r is not normally observed in rabbit ventricular myocytes, and was not included in the model
calculations. It is shown in Fig 45 simply to indicate the expected timecourse for an AP like this.
Plateau (Phase 2)
The long plateau phase of the AP can be relatively flat, domed, gradually declining or
some combination thereof. Its key characteristic is that Em does not change rapidly. This is
because inward and outward current are nearly balanced. During this time the inward current is
mostly carried by IC.,L and the outward current by delayed rectifier K currents (lKun I Kr & IKs). IKur
follows roughly the shape of the AP because its gating responds so rapidly to Em. The shape is
distorted with respect to the AP, mainly because currents are much larger at positive Em due to
Rabbit Ventricular AP
50 1500
1200
;;- n
.§. 900 ~
E :l
w -50 00
s:
300
-100
100
0 100 200 300
~I
100 msec
Em
~
INa
~ 12msec I
25x I ca
[.... o
<= -3
......
Ql
:;,
<.> -6
-9
o 100 200 300
9 h
II
II
II
/I
/I
II
: : Ito
1\
I I
1\
I I
I I
I \
I \
I \
a 10 20
\ \
\."\".-I..~~,~
\
100 200 300

time (msec)
Figure 45. Calculated Em, ionic currents and [Cal; during a rabbit ventricular AP. Calculations were done
using the LabHeart program (J.L. Puglisi & D.M. Bers) and the Luo & Rudy (1991, 1994a; Zeng et al.,
1995) equations and parameters for guinea-pig ventricle with some adjustments to better represent rabbit
ventricle (e.g. inclusion of 1'0, altered I K, and I K,; Salata et al., 1996). ICa,T was also not included, but it is
shown for general information. INalCa and Na/K-ATPase current are scaled up by a factor of 3 to make them
clearer. The time-expanded insets illustrate the temporal relationship of Em, INa and Ica,L in the first 9 msec
(upper) and K currents near the rapid AP upstroke at -8 msec (lower).
the higher driving force on K (Em - EK ). IKs activates more slowly and IKr increases as repolari-
zation progresses (Fig 41) and these two outward current sustain the latter plateau. There is a
delicate balance of currents during the plateau phase so rather modest differences in any of the
currents can alter plateau duration. For example, there is less IKs in midmyocardial cells and this
contributes to a very long APD in these cells and the V-wave on the ECG (Liu & Antzelevitch,
1995). PKA-dependent phosphorylation also alters IKs and I C.(cAMP) to shorten the plateau.
Moreover, mutations in HERG and KvLQTJ, the genes which code for the channels which carry
IKr and IKs cause congenital long QT syndrome in humans (Sanguinetti et al., 1995, 1996a,b) in
which ventricular APD is abnormally long. Alterations in other ionic currents can also perturb
the delicate balance and plateau duration.
Late Repolarization (Phase 3)

At some point repolarization accelerates greatly and Em goes from the plateau level near
o mV back toward the diastolic Em. There is a negative slope in the current-voltage relationship
of IKr and IKJ (Fig 4 IB,C), which means that as repolarization proceeds outward current increases
(causing further repolarization). This sort of positive feedback results in progressive acceleration
of repolarization. Since this occurs at more positive Em for IKr, that current is particularly
important in the early part of this accelerating phase of late repolarization. As repolarization
proceeds IKr deactivates at more negative Em. At Em below -30 mV IKJ increases with repolari-
zation, again accelerating repolarization. During repolarization inward INa/C. (Ca extrusion)
becomes more prominent because [Cali is still relatively high and the more negative Em causes a
stronger driving force favoring inward INa/C. (see Chapter 6).
PACEMAKERS (AP PHASE 4)

Many cells in the heart exhibit spontaneous pacemaker activity, e.g. SA node, AV node
and Purkinje fibers. Many different currents are involved in pacemaker activity and the relative
amounts vary in different regions and under different conditions (Irisawa et al., 1993;
Anumonwo & Jalife, 1995; Noma, 1996). This also depends on the Em range, [Cali and the
history. Figure 46 shows APs typical of SA node cells with some of the key players implicated
in pacemaker activity. Figure 47 is from the rabbit SA node model of Demir et al. (1994, 1999)
and includes more details and quantitative estimates. Notably, these cells have little or no IKh
and this is largely responsible for their relatively positive diastolic potential. As repolarization
proceeds in these cells delayed rectifier K currents are decreasing ('L -300 msec, Fig 47) and a
loss of outward current (even with an unchanging inward background current) will tend to move
the Em in a depolarizing direction. Thus turning off of K currents contributes to pacemaker
activity in a permissive sense (Noma, 1996). If is an inward current activated by hyper-
polarization and is often referred to as the pacemaker current. While there is little doubt that If
contributes substantially to pacemaker activity (especially in Purkinje fibers), there is significant
controversy about the predominance of If in SA node cells (DiFrancesco, 1995; Vassale, 1995;
Noma, 1996). The key aspects of debate are whether the Ir activation only occurs appreciably at
Em more negative than the pacemaker range, and also whether the activation is sufficiently fast
(especially at the positive end of their activation range). While If may be minor in true primary
pacemaker cells at the core of the SA node (with most negative Em = -60), it may be more
+40
+20
;;-
.s
E
w
-20
-40
Xc;aSpk ~ I
-60
INa/Ca Na/Ca
ICaT
Figure 46. Currents contributing to pacemaker activity in nodal cells (SA or A V). The schematic shows
that declining outward IK and inward Ir, INa/ca, Ica.T, ICa .L, and INa can all make contributions. Background
inward current and a sustained inward current activated by depolarization may also be involved (see text).
important in peripheral nodal cells and/or with the normal degree of sympathetic tone that the SA
node receives in vivo. Ir is also more generally accepted to play an important role in Purkinje
fiber pacemaking. Note that these subsidiary (slower) pacemakers are normally suppressed (or
overdriven) by the SA node. They only become the functional cardiac pacemaker when the more
primary pacemakers are non-functional (e.g. sinus block, AV block).
Nodal cells and Purkinje fibers also have much higher ICa.T density than do most
ventricular myocytes and the activation Em range for this channel is in the pacemaker range (Fig
40C). Thus ICa,T contributes inward current toward the pacemaker depolarization, and 40 f!M
[Ni] which blocks Ica,T rather selectively can slow pacemaker activity (Hagiwara et aI., 1988;
Zhou & Lipsius, 1994). Ica,L may also participate in pacemaker activity, but with the relatively
positive Em for activation this is probably only relevant in the latter part of pacemaking. On the
other hand, the regenerative activation of ICa,L is responsible for the upstroke of the AP in nodal
cells (just as INa is in myocytes and Purkinje cells). The slower activation of ICa,L and the lower
overall current density compared to myocyte INa are responsible for the much slower rate of rise
of nodal compared to ventricular AP (-5 vs. 200 V/sec).
As Em declines faster than [Ca]i, there is also an inward INa/ca component which may
contribute to early pacemaking. In subsidiary pacemaker cells Zhou & Lipsius (1993) showed
that there is a late component of diastolic depolarization which depends on both SR Ca release
and INa/Ca. Hiiser et al.(2000) provided evidence consistent with an intriguing local rCa];
signaling model (Fig 46). The activation OfICa,T may raise local rCa]; sufficiently to trigger local
SR Ca release events (Ca sparks, see Chapters 7 & 8). This local amplification of rCa]; increases
inward INa/ca(Ca efflux). This may occur late in diastolic depolarization because of recovery
times required for either ICa,T or the SR Ca release channel (see Chapters 5-9).
In nodal cells which show slowly rising APs, Ica (rather than INa) is the primary current
responsible for the upstroke of the AP. These cells do have Na channels (Muramatsu et al.,
40 Rabbit SA Node AP
20
~ 0
E -20
W
-40
-60
200
()
~
.eo ~
a
III
"E -200
~~ ~
() 6
~
-400
-600
0 0.00
()
~
.eo 0.05 !;
iil
"E -5 a
~
~
0.10
~
() ~
0.15
-10
0.20
50
.5
()
~
.eo .0 ~III
"E a
~~ ~
() -50 ~
Figure 47. Calculated Em and ionic currents during rabbit SA node APs, done using the Demir et al.
(1994, 1999) model. 18k is a composite background current, corresponding to Ca, Na & K leak currents.
1996), but since Em never gets very negative, they are mostly inactivated. The few Na channels
available, can contribute to the inward currents associated with diastolic depolarization (Fig 47).
However, based on pacemaker TTX sensitivity this may only be in perinodal cells with relatively
negative Em (Li et al., 1997a; Kodama et al., 1997). Figures 45 and 47 show that in SA nodal
cells IN. density is > 1000 times less than in ventricular myocytes and >50 times less than {C.,L.
Guo et al. (1995) also reported a sustained inward current which is activated by
depolarization in the range of the pacemaker potential. This may be distinct from lea or the
background inward current (IBk in Fig 47) which reverses at -21 mV (Hagiwara et aI., 1992;
Noma, 1996). This I Bk may be a relatively non-selective Na, Ca, K conductance (Demir et aI.,
1994). It is not entirely clear yet how these inward currents contribute to pacemaker activity.
In conclusion there are multiple, overlapping and redundant currents involved in cardiac
pacemaker activity. There is a hierarchy where the SA node is normally the primary or fastest
pacemaker, but if the SA node fails to activate the rest of the heart, there are other normally
slower pacemakers which can take over this role. These latent or subsidiary pacemakers exist in
regions near the SA node, in the atria, AV node (and valves) and in the His-Purkinje system.
Even ventricular myocytes can exhibit spontaneous pacemaker activity under unusual circum-
stances (particularly when sympathetic activity is very high). The differing balances of pace-
maker mechanisms in different regions also provides a different sort of fail-safe, because agents
which abolish SA node function may have little impact on other pacemakers.
EARLY AND DELAYED AFTERDEPOLARIZAnONS

Two AP aberrations that can be pro-arrhythmic are EADs and DADs (Cranefield &
Aronson, 1988). These are usually categorized by the take-off potential (see Fig 48). If Em has
already returned to the diastolic Em prior to the depolarization it is a DAD, whereas if it takes off
(net depolarization) from somewhere on the plateau or late repolarization phase it is an EAD.
These are also called triggered activities, because they depend on prior stimulation.
Early Ajterdepolarizations (EADs)

EADs are more prone to occur with prolonged APDs. This can happen with bradycardia
or partial inhibition of K channels. The latter can happen as a consequence of hypokalemia
(which reduces outward I K1 and IKr), pharmacological action (e.g. K channel blocker) or
congenital channel defects (long QT linked mutations which alter INa, IKr and IKs ).
Midmyocardial (or M) cells are particularly prone to EADs because of their long APD (Nuss et
al., 1999). EADs can occur at either the plateau range of Em or in late repolarization. It is likely
that those that occur at plateau Em are attributable to reactivation of Ca channels, which can
partially recover during very long APs, especially as [Cali declines (January & Riddle, 1989;
Shorofsky & January, 1992; Luo & Rudy, 1994b; Sipido et al., 1995a). That is, as Em falls into
the range of Ie. window current, Ca channels recover such that they can cause depolarization.
EADs which occur late in repolarization (e.g. below --40 mY) are not due to reactivation
of Ie. because the Ca channels which recovered from inactivation during the AP would not be
activated at this negative Em (Fig 40C). EADs occurring in this Em range may be due to Ca-
activated currents, and as such have more in common mechanistically with DADs discussed
below. Nuss et al. (1999) also reported spontaneousdepolarizations (especially in heart failure)
which were [Cali-independent, but of unknown basis.
Delayed Ajterdepolarizations (DADs)

DADs are most frequently observed under conditions that increase cellular and SR Ca
loading. The high SR Ca load and [Cali cause apparently spontaneous SR Ca release events
0-
mV
-80 SR Ca overload &

Spontaneous Release
INa/Ca' ICI(Ca)' l"sICa?
Figure 48. Afterdepolarizations in cardiac myocytes. This schematic shows fundamental differences in
early- and delayed afterdepolarizations (EADs & DADs) and the likely underlying mechanisms.
which elevate rCa]; and activate [Cali-dependent currents. I say "apparently spontaneous"
because the events are probably stochastic (making them seem random or spontaneous), but we
now know that elevated rCa]; and SR Ca load increase their probability. There is also a delay in
the availability of SR Ca release events after prior activation (Satoh et aI., 1997), explaining why
DADs typically occur after a finite time of repolarization. Digitalis glycosides, which block
Na/K-ATPase and cause cellular Na and thus Ca loading, are a common mode of induction of
DADs (Rosen et al., 1973a; Ferrier & Moe, 1973). ~-adrenergic agonists which stimulate both
ICa and SR Ca uptake can also increase the probability of DADs (Belardinelli & Isenberg, 1983a).
The elevation of rCa]; activates a transient inward current (I,;) in voltage clamp studies
(Lederer & Tsien, 1976; Kass et aI., 1978). Without voltage clamp this inward current would
depolarize the membrane transiently, causing a DAD. There are three potential candidates for
the Ca-activated inward I,;, namely INa/ca, ICI(Ca) and I"s(Ca). Most investigators find no evidence for
significant contribution of I"s(Ca) to Iii or DADs in ventricular myocytes, but there is evidence for
ICI(c,) and extensive data supporting a major role of INa/c, (Fedida et al.l 1987b; Laflamme &
Becker, 1996; Zygmunt et al., 1998; Schlotthauer & Bers, 2000). Kimura (1988) concluded that
85% of the It; was attributable to INa/Ca (at -80 mV and rCa]; =0.5 f.lM). Zygmunt et al. (1998)
attributed 60% of It; to INa/ca and 40% to Icl(c.) in dog ventricular myocytes, but in rabbit we find
Iii to be almost exclusively INa/C. (Schlotthauer & Bers, 2000; Schlotthauer et al., 2000).
It is important to consider not only the Iti , but the amount of depolarization that it
produces. E CI is typically -50 or -55 mY. Thus, while Icl(c.) is inward at -80 mY, it will become
smaller as depolarization occurs, because the driving force would be reduced by 50% by a 12 mV
depolarization. This will limit the ability of IC1(Ca) to produce robust DADs. At high [Ca]i the
value of ENa/ca is positive (Fig 43) so this argument is not as relevant for INa/Ca. In addition, we
find that the depolarization induced by rapid caffeine-induced SR Ca release can trigger an AP,
even when lCI (Ca) is blocked by niflumate (Schlotthauer & Bers, 2000). Moreover, blocking
Na/Ca exchange by replacing Na with Li completely blocks depolarization (although IC1(Ca) and
Ins(Ca) could still function). In conclusion, ventricular DADs are primarily due to INa/c" with IC1(Ca)
contributing somewhat less (especially near threshold) and Ins(c,) is probably unimportant.
In heart failure the Na/Ca exchanger is upregulated and IK1 is decreased (Studer et al.,
1994; Beuckelmann et aI., 1993; Kiiiib et al., 1996; Pogwizd et al., 1999). The greater Na/Ca
exchange increases the inward INa/ca for any given SR Ca release. The reduced IK1 destabilizes
the resting Em and allows a given inward current to more readily trigger an AP. This is the likely
basis for increased triggered arrhythmias in heart failure (see pg 299 & 322). Indeed, in the
human, triggered arrhythmias above (plus possibly abnonnal automaticity) are responsible for
initiation of almost all arrhythmias in non-ischemic heart failure and -50% of those in ischemic
heart failure (Pogwizd et aI., 1992, 1997, 1998). Thus nonreentrant mechanisms are likely to
account for initiation of most arrhythmias in patients with heart failure.
REENTRY OF EXCITATION
While reentry may only initiate a minority of arrhythmias, it is the main mechanism
responsible for the subsequent faulty propagation of the cardiac impulse leading to ventricular
tachycardia and fibrillation. I will not discuss the complexities of this issue in any detail here,
but simply indicate the classic picture of reentry and of spiral waves in the genesis and
propagation of reentrant arrhythmias.
Figure 49A shows a multicellular conduction pathway around an anatomical obstruction
(e.g. a blood vessel). The impulse goes around the ring in both directions. When the wavefronts
meet head-on, they annihilate each other because the previously activated tissue (well behind
both fronts) is refractory. This is a benefit of the long cardiac AP. This prevents propagation
from jumping ahead to re-excite tissue behind the opposing wavefront. Several pathophysio-
logical conditions set the scene for reentry in this situation (Fig 49B). There must be unidirectio-
nal conduction block, often due to decremental conduction approaching an inexcitable core from
one side. Reduced or heterogeneous refractory period (due to short APD) and slowed conduction
can also facilitate the appearance of reentry. Now as the same impulse approaches it can travel
down one branch but dies out going down the other (decremental conduction and inexcitable
core). One can think of this as a progressive decrease in space constant (Ie) such that propagation
is extremely weak near the inexcitable core. This time when the normal impulse reaches the
annihilation point (from Fig 49A) the impulse proceeds through. Now it can reach the damaged
region from the other side and can jump the inexcitable gap (unidirectional block) and re-excite
the tissue on the other side. Here one can consider that Ie is much longer when it gets to the
inexcitable gap from this side, and so can bring cells on the other side to threshold for an AP.
Since conduction to thd point has been slowed (and especially if refractory periods are short) the
impulse may propagate retrograde, and go around the ring again. This can produce a continuing
propagation around the ring (circus movement) and also branch off in other directions.
Spiral waves can be initiated by either a break in a propagating wavefront or crossing of
fronts in cells and multicellular tissues (Lechleiter et aI., 1991; Davidenko et aI., 1992; Lipp &
Niggli, 1993). Indeed, this is a general property of excitable media (Winfree, 1973). The break
creates an edge of very high convex curvature which slows conduction due to the relatively large
current sink to source ratio. This causes the wavefront to bend around this tip forming the
A. Normal
C. Spiral Waves
B. Reentry
Asymmetrical
Decremental Retrograde
Conduction & Re-excitalion
Unidirectional
Block
Retrograde
Jumps Gap
Figure 49. Reentrant excitation in heart. A-B. Classical model of reentry around an anatomical region.
Unidirectional conduction block, decremental conduction and reduced refractory period in a damaged
region contribute to increased probability of re-excitation. C. Spiral waves simulated in 2D cardiac tissue
(3 x 3 cm) using a modified Luo-Rudy AP model at 4 successive 12 ms intervals. White is depolarized and
the spiral period is 50 ms (B was generaously supplied by IN. Weiss).
curved ann of a spiral wave (Fig 49C). Further out along the arm the current sink-source
mismatch is less severe and the curvature decreases progressively, forming the increasing spiral
pattern. The exact form of this spiral, its stability and how it moves in the tissue depends on the
electrophysiological characteristics of the substrate, mass and geometry of the tissue (Weiss et
al., 1999). Indeed, the spirals can meander, break, up and form multiple spirals.
Thus alterations in myocyte properties as well as anatomical obstacles or architectural
features (which can change with cardiac ischemia and remodeling) can contribute to the genesis
and propagation of cardiac arrhythmias. It is important to extend the kind of understanding we
are developing for electrophysiological properties of ion channels and single cells to increasingly
complex tissues in approaching the real physiological situation. The focus in this chapter has
been more on the understanding of nonnal properties of cardiac ion channels and fundamental
electrophysiological properties of cardiac myocytes. It is important to keep in mind that the
balance of currents during the AP is delicate, and that any increase in inward current will tend to
prolong the APD and any increase in outward current will tend to shorten AP. Moreover, in
different regions of the heart the AP is probably tuned for optimal physiological function and the
AP characteristics are detennined by the ion channels expressed, the cell characteristics and also
the cells with which it is electrically connected. The infonnation here sets the scene for more
detailed discussion of Ca channels and transporters in the next few chapters.
D.M. Bers. !OI
2nd Ed" Kluwer Academic Publishers, Dordrecht, 2001
CHAPTER 5
Ca INFLUX VIA
SARCOLEMMAL Ca CHANNELS
Since the time of Ringer (1883) it has been known that extracellular [Ca] is important in
cardiac muscle contraction. Cardiac Ca currents were first characterized as "slow inward
current" or lsi, since several tens of msec were required for peak current to be achieved (e.g.
Rougier et al., 1969; Mascher & Peper, 1969; Beeler & Reuter, 1970; Ochi, 1970). Since the
advent of the patch-clamp and isolated myocyte techniques it is now clear that Ca current (Ica)
can reach a peak value in ~2-3 msec after a depolarization (e.g. see Fig 50). Thus the moniker of
slow inward current seems inappropriate, although Ica could be considered secondary, in
deference to INa which activates still faster than Ica (Fig 45). The inward Ica during the normal
cardiac action potential contributes to the AP plateau and is also involved in the activation of
contraction (directly and/or indirectly).
Ca CHANNEL TYPES
Hagiwara et al. (1975) were probably the first to demonstrate two classes ofCa channels
in a single cell type. Nowycky et al. (1985) characterized three types of Ca channels in dorsal
root ganglion cells, giving them names which have been adopted generally in the present
nomenclature. L-type Ca channels are characterized by a Large conductance (-25 pS in 110 roM
Ba), Long lasting openings (with Ba as the charge carrier), sensitivity to 1,4-dihydropyridines
(DHPs) and activation at Larger depolarizations (i.e. at more positive Em). T-type channels are
characterized by a Tiny conductance (~8 pS in 110 roM Ba), Transient openings, insensitivity to
DHPs, and activation at more negative Em. N-type Ca channels are Neither T nor L, are
predominantly found in Neurons, and are intermediate in conductance and voltage dependence.
There are also other Ca current types distinguished by electrophysiological and pharma-
cological phenotype (e.g. P/Q & R), which are more prominent in neurons and neuroendocrine
cells. The T-type Ca channels are the only low-voltage-activated (LVA) type, while all of the
other types are referred to as high-voltage-activated (HV A), although the Em-dependence is not
always as distinct as one would like. The ten different Em-dependent Ca channel genes are
indicated in Table 15, along with alternate names and properties.
This L, T, N classification is an oversimplification because there are large differences
even among L-type channels. For example, co-conotoxin can strongly inhibit neuronal L-type Ca
channels, but not cardiac or skeletal muscle L-type channels (McCleskey et aI., 1987). In
addition the activation and inactivation kinetics in skeletal muscle L-type channels are -IO-fold
slower than in cardiac muscle (Bean, 1989). Discussion here will focus on the Ca channel types
in heart, mainly L- and T-type (due to alC, aID, alG and aIH)'
Table 15
Ca channel Types
Isofonn Gene name Type Blockers Tissue
CACNAIA P/Q ro-agatoxin-IVA « 100 nM) Neurons
ro-conotoxin-MVIIC (> 100 nM)
CACNAIB N ro- conotoxin-GVIA «100 nM) Neurons
CACNAIC L DHP, AA, BTZ Heart, brain, lung
smooth muscle, endocrine
aID CACNAlD L DHP, AA, BTZ Neurons, heart
alE CACNAIE R 100 IlMNi Neurons, heart?
alF CACNAIF L? ?
als CACNAlS L DHP, AA, BTZ skeletal muscle
T-Type (LVA)
alG CACNAIG T Mibefradil, Ni (K j =250 IlM) Neurons, cardiac Purkinje
alH CACNAIH T Mibefradil, Ni (K j =12 IlM) Heart, kidney, liver
all CACNAII T Mibefradil, Ni (K j =216 IlM) Neurons
DHP is dihydropyridine, <1>AA is phenylalkylamine (e.g. D600) and BTZ is benzothiazepine (e.g.
diltiazem). Information in table is from various sources (e.g. Varadi et aI., 1999; Lee et al., I999b).
Cardiac muscle contains both L- and T-type Ca channels (e.g. see Fig 50 & Table 16),
but not N-type channels (Bean, 1985, 1989; Nilius et al., 1985). Iea,L is ubiquitous in cardiac
myocytes, whereas cardiac Iea,r was first described in atrial cells (Fig 50A, Bean, 1985), In
canine cardiac Purkinje cells there is robust Iea,r (Fig 50B, Hirano et al., 1989). For a test Em of
-20 mV Iea,L and Iea,r in Fig 50B are about equal, whereas at 0 mV peak Iea,L is 3 times larger
than Iea,r. L-type lea appears to be prominent in all cardiac myocytes, whereas T-type lea is much
more variable, Figure 50C shows total lea current-voltage relationships in four different cell
types, The extent of the hump at negative Em (about -40 mY) reflects the relative amount ofIea,r,
Purkinje cells seem to have the most, while pacemaker and some atrial myocytes also have
significant amounts (Hagiwara et al., 1988; Bean, 1989), but the amount of Iea,r in ventricular
myocytes is either modest in guinea-pig (Mitra & Morad, 1986) or undetectable in bullfrog, calf,
cat, rabbit, rat and ferret (Bean, 1989; Nuss and Houser, 1993; Yuan and Bers, 1994; Yuan et ai"
1996), Neonatal rat ventricular myocytes exhibit Iea,r (Wetzel et al., 1993; Gaughan et ai" 1998)
and significant Iea,r may reappear in ventricular myocytes during the development of ventricular
hypertrophy in cat (Nuss and Houser, 1993) and rat (Martinez et al., 1999),
Thus T-type current is typically small or absent in ventricular myocytes, but may be
more prominent during development and hypertrophy, The relative prominence of Iea.r in
pacemaker and conducting cells, and its activation at Em in the pacemaker range has led to
suggestions and evidence for a role of Iea,r in atrial pacemaking (see pg 94-96 and Hagiwara,
1988; Wu & Lipsius, 1990). Because Iea,r is relatively small and inactivates very rapidly the
total amount of Ca flux via Iea,r is small compared to that via Iea,L and negligible in most
ventricular myocytes, This may reflect different functional roles, where Iea,L is more involved in
triggering SR Ca release and refilling SR Ca stores (see below), rather than pacemaking,
Since Iea,L inactivation is Ca dependent (Kokubun & Irisawa, 1984; Lee et al.,1985; see
pg 116 -119) Iea,L is transient and the kinetics can be confused with T type lea' Therefore, L-type
Chapter 5 Sarcolemmal Ca Channels 103
A. Dog Atrium (115mM Sa; Bean, 1985)
-100 . 0
-ir-:
·500
- - - Difference Current
HP= -80 minus HP= -30 mV)
B. Dog Purkinje Fiber (2 mM Ca; Hirano et aI., 1989)
'50~
°lr
·90
300pA
C. Several Species (Total lea)

(mV)
-70 -10 10 50
./
0/
I,'
/
I
'
.'
•
I,'
Guinea- I,
I'
I,'
PigV. I,'
I'
I,'
I'
Dog At. I,'
I'
.!
I "
I'
--41
lea (Normalized)
Figure 50. Voltage-dependence of whole cell L- and T-type Ca channel currents. A. Ba currents (with
115 mM Ba) induced by Em steps to test potentials from holding potentials (HP) of-80 or -30 mY. The Em
protocol is shown in the top trace, currents in the middle trace, and the difference between these currents in
the bottom panel. The peak I Ba activated from -30 mV is attributed to L-type Ca channels (0) and the
additional transient difference current activated from -80 mV (e) is attributed to T-type Ca channels (from
Bean, 1985, with permission). B. Similar protocol to A, but in dog Purkinje cells and with 2 mM Ca as
charge carrier (from Hirano et al., 1989, with permission). The shifts in Em between A and B are due to
surface potential differences in 2 mM Ca VS. 115 mM Ba (see Fig 55, pg 115). C. Total lea from several
sources (HP = -80 to -100 mV), where the hump at --40 mV is due to T-type current and differs among the
tissues and species studied. Dog Purkinje (from Hirano et aI., 1989) and rabbit ventricle (OM Briggs &
DM Bers, unpublished) are in 2 mM Ca and dog atrium (from Bean, 1985) and guinea-pig ventricle (from
Mitra & Morad, 1986) are in 5 mM Ca, but are shifted -10mV to compensate for surface potential.
and T-type channels can be best distinguished when Ba is the charge carrier since L-channel
inactivation is then rather slow (e.g. Fig 50A). Where T-type current is prominent, it can also be
resolved by the difference in Em-dependence (Fig 50). In cells which have substantial ICa.T the
current-voltage relationship (from holding Em of -80 or -90 mV) also shows a prominent
"shoulder" at negative Em. While only T and L-type ICa is apparent in cardiac myocytes, N-, P/Q-
or R currents can be seen in intracardiac neurons (Jeong & Wurster, 1997).
The cardiac cell types in which ICa.T is more prominent also happen to be the cells which
have less extensive T-tubules (see Table I). It is not known explicitly whether T-type Ca
channels are excluded from T-tubules, but L-type Ca channels in cardiac and skeletal muscle
may be concentrated there (see Chapter I). In general, unless otherwise specified below, Ca
channels and Ica will refer to L-type Ca channels or ICa.L for cardiac myocytes.
MOLECULAR CHARACTERIZATION OF Ca CHANNELS

L type Ca channels are characteristically sensitive to dihydropyridines (DHPs). Most
DHPs act as Ca channel blockers or antagonists (e.g. nifedipine, nisoldipine and nitrendipine,
isradipine), while some DHPs act as Ca channel agonists (e.g. Bay K 8644, but only the (-) enan-
tiomer). The agonist agents appear to greatly prolong the open time of the channel (see Fig 61).
The DHP-sensitivity of L-type Ca channels and high density of DHP receptors in skeletal muscle
T-tubules were crucial to the initial extensive biochemical characterization and isolation of the L-
type channel proteins from skeletal muscle (Curtis & Catterall, 1984; Borsotto et aI., 1984;
Flockerzi et al., 1986). Five protein subunits were found to constitute the skeletal muscle Ca
channel (a" a2, ~, Yand 8) and all have been sequenced (Tanabe et al., 1987; Ellis et aI., 1988;
Ruth et al., 1989; Jay et aI., 1990; Bosse et al., 1990). The a, subunit of skeletal muscle (a,s) is
distinct from the cardiac channel clone (aiC Mikami et al., 1989). The a, subunit appears to bear
the main known functional characteristics of these Ca channels. That is, the al subunit bears the
channel, the DHP, phenylalkylamine and benzothiazepine receptors (Galizzi et al., 1986; Sharp
et aI., 1987; Sieber et al., 1987; Vaghy et aI., 1987; Striessnig et al., 1990a,b) and may contain
the sites phosphorylated by protein kinases A, CaMKII (Curtis & Catterall, 1985; Hosey et al.,
1986,1987; Imagawa et al., 1987a; Nastainczyk et al., 1987) and protein kinase C (O'Callahan et
aI., 1988). Transfection of DNA coding for the alC subunit into a cell line lacking the other
subunits was sufficient to produce Ern-dependent 1ca (Perez-Reyes et al., 1989). Moreover, the al
subunit structure is very much like the Em-dependent Na channel and tetrameric K channels in
Fig 38. That is, a,c has 4 homologous domains (I-IV), each of which has 6 transmembrane spans
(SI-S6, with charged S4) and pore loops between Ss and S6, and has a long carboxy tail (Fig 51).
There are alternate splice variants of alC (reviewed in Bers & Perez-Reyes, 1999). There is also
aID expressed in the heart (Takimoto et al., 1997; Wyatt et al., 1997), but it is not clear if there is
functional consequence to aID VS. a,c expression.
As for the Ern-dependent channels discussed in Chapter 4, Ca channels have the same
repeating positive charge at every third amino acid for a stretch of 10-15 amino acids in each S4
span. This is undoubtedly involved in the Ern-dependent gating of the,Ca channels. There are
differences in activation among different types of Ca channels, e.g. the skeletal muscle Ca
channel (a,s) activates very slowly in comparison to the cardiac aiC. In studies with chimeric Ca
channels Tanabe et al. (1991) found that differences in domain I between a,s and alC could
Table 16
Properties of Cardiac L- and T-type Ca Channels
L-type T-type
Activation range
5mMCa Positive to -30 mV Positive to -60 mV
110 mM Ba Positive to -10 mV Positive to -50 mV
Inactivation
Range Positive to -40 mV -90 to-60 mV
Ca dependent Yes No
Voltage dependent Slow Fast
Tail deactivation Fast Slow
Conductance
110 Ba 25 pS 8 pS
110 Ca 8 pS 8 pS
150 Na, EDTA 80 pS 50 pS
Mean open time Typically <1 ms Short, 1-2 ms
Kinetics (Ba) Multiple bursts/pulse 1 burst per pulse,
(inactivation)
Pharmacological sensitivity:
Dihydropyridines Yes No
Cd High Low
Ni Low High
Isoproterenol Yes No
Excised patch Loses activity Retains activity
This tabulation is from Hess (1988) with values mostly obtained at room temperature.
explain this activation difference, while Spaetgens & Zamponi (1999) found domains 11 and III
(& maybe IV) confer the more positive Em-dependent inactivation of alC VS. alE.
Ellis et at. (1988) cloned and expressed the a2 subunit and it is now clear that the a2 and
8 subunit are from the same a28 gene (Jay et al., 1991). However, the a28 protein gets cleaved
so that 2 different proteins were expected based on biochemical work. Normally the a28 con-
nection is by one or more disulfide links and the 8 portion anchors the complex in the membrane
(Fig 51). So far most work has been on one gene known to code a28, but alternative splicing in
different tissues gives rise to splice variants (Williams et al., 1992; Kim et al., 1992; Angelotti &
Hofmann, 1996). The U:1a is predominant in skeletal muscle and a2b in brain, whereas the a2c and
a2d variants are most common in heart (and are slightly shorter). Klugbauer et al. (1999) recently
found 2 more a 2 8 isoforms that are expressed primarily in brain.
When a28 is coexpressed with alC there is a ~2-fold increase in expression of DHPR
binding sites, gating currents, and ionic currents (Wei et al., 1995; Singer et al., 1991; Bangalore
et al., 1996). This may indicate that a28 plays a role in the formation of functional channels at
the plasma membrane surface. However, coexpression of a28 also increases the apparent affinity
of those channels for DHPs by 4-fold (Wei et al., 1995) and accelerates channel opening and
closing (Bangalore et al., 1996; see review by Felix, 1999). The a 2 8 subunit may also directly
bind drugs, such as the anticonvulsant, gabapentin (Gee et al., 1996), but the therapeutic impact
is unclear. Most of the 8 component seems merely to anchor the a28 subunit in the membrane
(since other transmembrane spanning domains can substitute), whereas the heavily glycosylated
extracellular (X2 domain seems to interact with the (XI subunit, at least in domain III (Gumett et
ai., 1997; Felix etai., 1997).
The y subunit appears to be restricted to the skeletal muscle Ca channel (Ruth et al.,
1989; Jay et al., 1990), but coexpression with (XIS can also increase DHP binding, and alter
allosteric action of pbenylalkylamines (Suh-Kim et al., 1996). Although there is no evidence for
a cardiac y subunit, additional y genes may be expressed in brain (Letts et al., 1998).
There are four ~ subunit genes (~1-~4), but the main cardiac isoform is ~2' There is
alternative splicing of ~2 exons near the amino terminus and in a central region near the (XI inter-
action domain (Perez-Reyes & Schneider, 1995; De Waard et al., 1994). Recent data suggests
that the ~2a isoform is only expressed in brain (Qin et al., 1998). ~3 and ~l variants have also
been detected in heart (Collin et ai., 1993; Hullin et al., 1992). Northern analysis of rat and
rabbit tissues indicates that ~2 mRNA is the most abundant ~ in heart, while ~3 is the most
abundant in lung. A ~2 specific antibody also was able to immunoprecipitate 80% of the DHP
binding sites from rabbit heart. Therefore ~2 is the predominant cardiac ~.
Coexpression of ~2 with (XIC causes a 10-fold increase in current, accelerates activation
and inactivation kinetics, shifts steady-state inactivation and greatly increases the number of high
affinity DHP binding sites (Perez-Reyes et al., 1992; Neely et al., 1993; Mitterdorfer et al.,
1994). Phosphorylation of the ~2 subunit may also be involved in the ~-adrenergic-induced
increased in lca (Haase et al., 1993; Biinemann et al., 1999). The ~ subunit may act in part like a
molecular chaperone, helping nascent (XIC molecules to fold properly and reach the plasma mem-
brane (Gao et al., 1999). However, the ~ subunit may also facilitate pore opening (Costantin et
ai., 1998; Yamaguchi et ai., 1998; Gerster et al., 1999) and stabilize a state of the (XIC which has
intrinsically higher DHP affinity (Mitterdorfer et al., 1994, 1998).
Specific interaction domains between (XI and ~ subunits have been elucidated (AID and
BID in Fig 51; De Waard et al., 1994; Pragnell et al., 1994). The site on (XI that interacts with ~
(AID) is in the loop between domain I and II. Splice variants of this loop may allow Ws to inter-
act differentially with the (XI variants. The site on ~ which interacts with (XI (BID) occurs just
after a region in ~2 that is highly spliced, again, indicating functional variations in (XI-~ subunit
interactions. Indeed, splice variants of both this site and the amino terminus (~2a vs. ~2b) can
alter channel kinetics when co-expressed with (XIE (Olcese et al., 1994; Qin et al., 1996). One
major difference between ~2a and ~2b is that only ~2a is palmitoylated. Mutation of the palmitoy-
lated residues alters its interaction with the (X subunit (Qin et al., 1998; Chien et ai., 1996).
Neuronal N- and P-type Ca channels are also strongly modulated by G-proteins binding
to the AID region of (XIA and (XIB, where they interact with ~ subunits (see Dolphin, 1998; Ikeda
& Dunlap, 1999 for .more detailed discussion and references). Stimulation of various 7 trans-
membrane spanning receptors activates heterotrimeric G-proteins which release their ~y subunit.
This G~r binds to the I-II loop of these Ca channel (XI subunits (at least partly displacing the
bound Ca channel ~ subunit) and causes depression of Ica activation rate and amplitude. This
depressant effect on lea (which may be tonic) can be relieved by strong depolarization, resulting
in an Em-dependent facilitation of neuronal Ica . This may enhance Ca influx in neurons firing at
higher frequencies. Large depolarizations appear to cause dissociation of G~r from the Ca
channel (Xl subunit, relieving the inhibition. G-protein dependent inhibition (and Em-dependent
A s-s linked
Repeats: Il III IV 8
extracellular
intracellular
EF-hand
PM
PO,
BTZ
IQ
PO,
RyR
Figure 51. A. Schematic diagram and subunit structure of the cardiac L-type Ca channel. A. Each ball
represents one amino acid. The main <XI subunit has the four domains (I-IV), each with 6 transmembrane
spans (SI-S6) and a pore loop (as in Fig 38). Ca channel antagonists dihydropyridines, phenylalkylamines
and benzothiazapines (DHP, PAA & BTZ) bind to the IIIS5, IIIS6 & IVS6 regions. Possible interaction
sites between the <XI subunit and the ryanodine receptor (RyR) are indicated, as well as a potentially
important phosphorylation site (P04), an EF-hand region and an IQ motif where calmodulin binds. The ~z
subunit is cytosol ie, can be phosphorylated (P04), has an apparent SH3 domain and interacts with <XI at the
<X- and ~-interaction domains (AID, BID). The <xzo subunit is coded by one gene, but then cleaved, but still
interconnected by sulfuydryl bridges. B. A cutaway view of how the <Xl subunits may encircle a central pore
(see also Fig 38 & 39; drawings kindly supplied by E. Perez-Reyes).
relieD does not occur appreciably in cardiac Ie" but this effect emphasizes the potential
modulatory roles for Ca channel subunits.
Other sites on the <Xle subunit shown in Fig 51 (to be discussed in greater detail later) are
possible interacting sites for the cardiac ryanodine receptor (II-III loop and carboxy tail),
phosphorylation sites on the carboxy tail (Ser-I928), EF-hand and IQ motifs (involved in Ca-
dependent inactivation) and sites where the 3 main classes of Ca channel blockers (DHPs,
phenylalkylamines and benzothiazepine) bind (IIIS s, IIIS 6 and IVS 6). In heart the alC, azo and ~z
subunits are about 200, 175 and 60 kDa respectively (with the az-o split at about 150 az, 30 0).
The three T-type Ca channels alG, alH and all have recently been cloned and expressed
(Perez-Reyes et aI., 1998; Cribbs et al., 1998; Lee et al., 1999a,b). These channels are
structurally very similar to L-type Ca channels (e.g. I-IV domains, SI-S6 transmembrane spans
and pore loop). The sequence is most highly conserved in the S4 and pore loop regions.
However, the overall sequence identity between T-type and any high voltage activated Ca
channel is low «15%). Notably, the pore loop in domain III has an aspartate rather than the
glutamate which is in all four domains of all other Ca channels forming the EEEE locus which is
critical for permeation (see pg 117) and all 3 T-type Ca channels have SKD in this spot rather
than TFE in ale). Two motifs present in alC that are not found in alH are the ~ subunit
interaction region (I-II loop) and the putative EF hand which might be involved in Ca-dependent
inactivation (below). The latter is not surprising because ICa,T does not exhibit Ca-dependent
inactivation. Mibefradil is a somewhat selective ICa,T inhibitor, with a K j -I f!M for all three ICa,T
types (about ten times lower than for ICa,L block, Cribbs et al., 1998). Ni also blocks ICa,T, but alH
is particularly sensitive (Table 15), whereas alG and all have Ni-sensitivity that is not much
different from that OflCa,L (Lee et al., 1999b). Since the ICa,T in cardiac pacemaker cells is highly
Ni-sensitive, it seems likely to be mediated by alH. On the other hand, ICa,T in Purkinje cells in
the heart is less Ni-sensitive, and may reflect aiG.
Messenger RNA for both alG and alH are found in human heart. Although alH was
cloned from an adult human heart library, much more mRNA is expressed in kidney and liver
(Cribbs et al., 1998). Future studies are required to determine which of these T channel subtypes
is expressed in pacemaker tissue and which is expressed in vascular smooth muscle. While ICa,T
seems to be recapitulated by expression of the al subunit alone, it is not yet known whether
auxiliary subunits contribute to ICa,T function (as seen for other Ca channels). While a small
amount of alE may be expressed in atria and contribute to ICa,T (Piedras-Renteria et aI., 1997), the
significance of this contribution to Ica in heart remains to be clarified.
Ca CHANNEL SELECTIVITY AND PERMEATION

McCleskey and Almers (1985) determined the narrowest point in the Ca channel pore to
be -6 A diameter since tetramethylammonium can permeate. Thus, the Ca channel must exert
selectivity by means other than exclusion by size. Hagiwara et al. (1974) observed that Ba
current through Ca channels was greater than Ca current, but that the Ba current was more
susceptible to block by Co. They concluded that this was due to competition at a binding site
within the channel, with a weaker affinity for Ba than for Ca, such that Co would prevent Ba
current more effectively. This also explains the higher control Ba vs. Ca current. Since Ba
doesn't "stick" as well in the channel, it can go through more quickly.
Hess et al. (1986) measured cardiac Ca channel selectivity based on current reversal
potentials in asymmetric salt solutions (see Table 17). They also reported single channel
conductances for several permeant ions. In the absence of divalent cations Ca channels carry
very large nonspecific monovalent cation current (INS, typically inward Na current). Na does not
Table 17
Selectivity of Cardiac L-type Ca Channel
Single-channel Unhydrated
Ion Pion/PCs Conductance (pS)! Radius (A)
Ca 4200 9 0.99
Sr 2800 9 1.13
Ba 1700 25 1.35
Li 9.9 45 0.60
Na 3.6 85 0.95
K 1.4 1.33
Cs 1.0 1.69
Mg ~O 0.65
*Selectivities as permeability ratios based on reversal potential measurements by Hess et al., 1986, with
values from Tsien et al., 1987. tFrom Hess et aI., 1986 with 110 mM divalent and 150 mM monovalent
(the latter is at pH 9.0 to limit proton block of the channel, Prod'hom et al., 1987).
compete very well for the Ca-selective site in the pore. Indeed, as [Ca]o is increased Na current
through cardiac and skeletal Ca channels is blocked by Ca with a K d ~I ~M [Ca] (see Fig 52A;
Almers & McCleskey, 1984; Almers et aI., 1984, Hess & Tsien, 1984). Mg also blocks INS (K j
-50 ~M) and it may be noted that one must chelate both extracellular Ca and Mg to measure
unblocked INS (e.g. with EDTA, rather than EGTA). As [Ca]o rises further in Fig 52A, ICa is not
measurable until nearly roM [Ca]o and saturates with a K d ~14 roM (Hess et al., 1986). This
disparity is consistent with more than one Ca binding site is in the Ca channel.
The anomalous mole fraction effect is a related phenomenon which is also consistent
with more than one site in the channel (Yue & Marb{m, 1987; Friel & Tsien, 1989). This effect
is where the current through a Ca channel is paradoxically smaller in mixtures of Ca and Ba (at
constant total [Ca]+[BaJ) than in either ion alone (Hess & Tsien, 1984).
These types of experiments led Almers & McCleskey (1984) and Hess & Tsien (1984) to
propose a model for Ca channel permeation which includes 2 Ca selective sites in the permeation
pathway (Fig 52C). The potential energy (LiG/RT) for a Ca or Na ion as it goes through an
unoccupied Ca channel are indicated by the solid (Ca) and dashed curves (Na). The two energy
wells correspond to 2 Ca binding sites. Ca can enter one site and change the energy profile (due
to its two positive charges). Ca can also move between these sites more easily than it can escape
the pore. When a Na ion enters the open site, it will not dwell long due to electrostatic repulsion
and lower affinity. Thus, Na current is blocked by ~M [Ca). Single occupancy by Ca would also
decrease the affinity for Ca at the other site from K d ~0.5 ~M to ~ 10 roM. If another Ca ion does
enter the other site, the repulsive forces are very strong, so there is a 50% chance that the first Ca
ion will rapidly leave the pore (even at Em=O). With the usual strong inwardly directed
electrochemical driving force for Ca entry (E ca - + 120 mY), this will result in a rapid inward Ca
current. In this way the strong repulsion of double occupancy overcomes the anticipated slow
off-rate expected of a high affinity site and allows both rapid ion flux through the channel and
high divalent cation selectivity. Indeed, the high Ca flux rates do not occur until roM [Ca],
where double occupancy of the channel is expected with this model (see Fig 52B). More than 2
binding sites in the pore might even help to explain some results (Yue & Marb{m, 1990).
A C.
1.7 250
C
~
::l 12 ~ Out In
0 150 ~
'""
0
~
0.8
100
§
1i
.t ~
0.' 50
~
~
(f)
B.
~
g
~
"~"O
0.8
06
__ •••Q....
--'"
I
0.8
0.6
8 O. \ 2 0' 0-5
Electic Distance
1.()
02 0.2
9 8 7 6 5 4 3 2 1
Exterral pCa
Figure 52. Ca channel permeation. A. Increasing [Ca]o blocks Na current through the Ca channel at Kd
-1 IlM. At higher [Ca]o (mM) the current is carried by Ca ions. B. fractional occupancy of the Ca channel
by none (0), I or 2 ions. Ca current increases when the channel is occupied by 2 Ca ions. C. Schematic
diagram of the Ca channel, showing two binding sites and the energy profile for a single Ca or Na ion as
they traverse the channel where the abscissa is the electrical distance across the membrane (from Almers &
McCleskey, 1984, with permission).
This strict sequential site model is heuristic, but similar characteristics can also be
obtained with a single selective site and repulsive interactions with an incoming Ca ion
(Armstrong & Neyton, 1992; Dang & McCleskey, 1998). Indeed, the key molecular site in the
selectivity filter is a ring of glutamate residues, one from each pore loop known as the EEEE
locus (El, Ell, EllI & ElY, Yang et al., 1993). The carboxyl groups of these glutamates could
wrap around Ca in a similar manner to the 4 carboxyl groups of EGT A. These glutamates were
individually changed to glutamines in heterologously expressed alC, neutralizing the negative
charges (Yang et al., 1993; Ellinor et aI., 1995). This resulted in different shifts in the [Ca]o
required to half-block INS carried by Li (Fig 53A). These 4 glutamates were not equivalent and
mutating two simultaneously resulted in an even more dramatic shifts in Ca block (nearly 1000-
fold). Since single point mutations alter Kd(Ca) there cannot be 2 discrete independent sites.
Additional mutagenesis studies of the EEEE locus (Ellinor et al., 1995; Chen et al., 1996a; Chen
& Tsien, 1997) have provided much additional detailed information and insight. Figure 53B
shows how the EEEE locus might provide coordinated Ca ion binding in the pore (Yaradi et al.,
1999). These 4 glutamates probably do not form two discrete binding sites, but rather a more
complex domain that can bind one Ca with high affinity (K d ~l IlM) or two Ca ions with low
affinity (Kd -10 mM; Yang et al., 1993; McCleskey, 1999). While the molecular details may be
getting a bit clearer, the functional behavior is not grossly different from that envisioned in the
two-site models. That is, a negatively charged region creates high affinity Ca binding, and
approach of another Ca ion (favored by other negative charges) stimulates the first Ca to move
Chapter 5 Sarcolemmal Ca Channels III
A
•
I+IV
.;;; Na+------.
1!o 0.5
il
~
u.
oL-_J-_-L-="";;:~'::='I..-i_
10.1 10" 10.5 10" 10.2

[Ca>+] (M)
B
Ca 2 + E. Ca Permeation [Ca)o >1 mM
Figure 53. Critical pore glutamates and Ca channel permeation. A. Shifts in the [CaJo-dependence of
block of Li current (lL;) through wild type (WT) rabbit ale Ca channel expressed in Xenopus oocytes.
Mutant channels were also expressed in which each of the P-Ioop glutamates in the EEEE locus (I, II, Ill,
IV) were substituted by glutamine as well as a simultaneous swap in I & IV (data from Yang et al., 1993,
with permission). B. Depiction of how the EEEE locus may coordinately bind Ca in the pore (based on
Yang et aI., 1993 and Varadi et aI., 1999). C-E. Schematic ofNa pemleation at low [CaJo, block by 1-500
f.lM [CaJo (one Ca in channel) and high Ca flux at mM [CaJo when the channel is doubly occupied.
on down the channel (Fig 53C-E). Moreover, Ca will be much more effective than Na in driving
off Ca (or blocking Na flux).
Nonner & Eisenberg (1998) proposed a continuous Poisson-Nernst-Planck (PNP) model
of Ca channel permeation, using a rigid electronegative tube, rather than the localized binding
sites and discrete steps of the other models above. Remarkably, this channel exhibited Ca-
selectivity and Ca block of Na current. While it could not readily explain the non-equivalent
effects of different glutamates in the EEEE locus (McCleskey, 1999), it is wise to consider these
challenging problems from multiple perspectives.
There is flickering block of Ca channels by protons (Prod'hom et al., 1987) and certain
multivalent cations (Lansman et al., 1986). If the blocking events are fast the single channel
conductance simply looks reduced, because the individual blocking events are not resolvable.
This is true at neutral pH for INs, and raising pH to 9 can allow the full 85 pS conductance to be
observed. This flickering block may reflect transient binding of these competing ions to the sites
in the pore and has allowed estimates of on- and off-rates in some of the studies cited above. The
block of Ca channels by other multivalent ions can also indicate the selectivity of the sites in the
channel. Certain ions may also block the channel, but still pass through. The voltage
dependence for the relief of block can indicate whether the ion can be "forced" through the
channel (e.g. at large negative Em), or must exit from the same side it entered (Lansman et aI.,
1986). It is interesting that Mg can readily permeate skeletal muscle Ca channels, but is almost
impermeant in the cardiac channel (Almers & Palade, 1981; Hess et al., 1986). This penneation
difference is particularly striking because the 4 P-Ioop cores are identical between alS, ale and
alD. There may also be more subtle species differences in cardiac Ca channel penneation which
cannot be attributed to P-Ioop differences (Yuan et al., 1996). Thus, there are likely to be more
factors involved in penneation than just the core P-loop regions. In symmetrical solutions the Ca
channel exhibits a very linear current-voltage relationship (Rosenberg et aI., 1986) indicating that
the penneation pathway is functionally symmetrical.
It is worth considering what the single Ca channel current and conductance are under
physiological conditions. We often consider ion channel conductance to be a relatively linear
function of penneant ion concentration. In the limit this is not the case for any channel, partly
due to limitations created by ion binding in the pore. However, it can also be due to access
resistance; i.e. as the current increases there is depletion of ions at one mouth of the channel and
accumulation at the other (Peskoff & Bers, 1988; Bers & Peskoff, 1991). This effect is slightly
mitigated by the local potentials created by the ion depletion and accumulation and any fixed
negative charges on or near the channel (or an applied field). That is, a local depletion of [Ca]o
outside a Ca channel will create a local negative potential which helps draw Ca toward the
channel mouth from the medium. The maximal flux through a channel would be 2nrD[X]0,
where X is the permeant ion, r is the channel radius (e.g. 0.3 nm) and D is the diffusion
coefficient (~1O-5 and ~3xlO-6 cm2 /s for Na and Ca respectively). For a narrow Na or K channel
(at 140 mM) maximal theoretical current would be ~50 pA. For a Ca channel at 2 and 110 mM
[Ca]o maximal current would be 0.4 and 24 pA respectively (Peskoff & Bers, 1988). Since single
channel lea of 0.2 pA occur physiologically in cardiac Ca channels, significant Ca depletion
occurs at the outer channel mouth and accumulation at the inner mouth (Bers & Peskoff, 1991;
see also Chapter 8, Fig 118, pg 222).
Having said this, Ca depletion/accumulation is not the nonnal limiting factor for Ie.,
especially at high [Ca]o' Single channel lea is nonnally measured at various Em to measure the
channel conductance (the slope in Fig 54A; Yue & Marban, 1990). Conductance measurements
at various [Ba]o indicate that the conductance saturates at high [Ba]o (although depletion would
be less severe). Figure 54B-C shows mean Ba and Ca conductance compiled by McDonald et al.
(1994) from 24 different studies of this sort (13 in cardiac and II in smooth muscle). Maximal
Ca and Ba conductance are 10 and 27 pS, with half-maximal values at 4 mM [Ca]o and 10 mM
[Ba]o. These Kill values may be related to the affinity of the binding site in the channel in the
doubly occupied state (Fig 53E). Notably, conductance for Na (which does not bind strongly) is
10 times higher than for Ca (or 20 times if we count ions rather than charge). Thus, the key
feature in limiting Ca (or Ba) flux through the channel is binding in the channel, or more
accurately the rate of Ca dissociation from the binding site(s) in the channel.
In discussing Ca fluxes it is common to infer Ca flux by integrating the Ca current. In

general this is reasonable when one has taken the care to block all other currents. The implicit
assumption is that all of the lea is carried by Ca ions. For much of the voltage range this is not a
bad assumption, especially as Pe';Pes is > 1000. Figure 54D shows currents carried by Ca and Cs
according to the Goldman-Hodgkin-Katz (GHK) current equation
A. Unitary Ba Currents B. Ca Channel Conductance

-40 Em (mV)
-120 -80 40
_ 25
en0.
Gmax = 27 pS
2.5 rr
'Sa Km =10 mM -:0-
-; 20
u
2.0 ~~
c !l.!.
~ 15 1.5
8fl
5mMBa
• -2.5 -g" 10
8max= 10 pS
1.0 ~ 0"
13 pS Km =4 mM
• 0 -
0
lea .5 3
<
400 mMBa iSa 0 0
0 20 40 60 80 100 120
• 35pS
-5.0 pA
[Divalentl (mM)
C. Ca Channel Conductance D. Ca Channel Current (GHK)

14 0.5 Cs efflux
(j) 12
0. Isa ~
8"10 90 120
iij 8
g
."
g
o
6
4
-0.5
t
Eea
-1.0
lea
(nA) -1.5
[Divalent) (mM)
Figure 54. Ca channel conductance and lea near E"y. A. Single channel Ba currents at different Em values
and [Ba]o (redrawn from Yue & MarMn, 1990), where linear regressions yield the indicated conductances.
B. Single channel Ca and Ba conductance as a function of [Ca]o and [Ba]o. The curves are fits to data
compiled by McDonald et al. (1994) from 24 different studies (using G=G ma ,l(1 +KnI[X]), where X is Ca or
Ba). The right ordinate assumes ohmic conductance and a driving force of 100 mY (as would be the case
for Em =0 mY and Eea = 100 mY). C. Same data as B, expanded to emphasize the physiological range. D.
Whole myocyte lea calculated from the GHK equation (see text) with Pe,=4xI0- 5 cm/s and Pe/P es = 1000
for a 150 pF cell. Ca influx and Cs efflux are shown separately along with total Ca channel current.
4 Pea ([CaL ~ rCa]; exp(-2kEm» Pes([CS]i - [Cs]o exp(-kEm) }

ICa= FkE m { + (I)
1 - exp(2kEm ) ) - exp(-kEm )
where k = 1/(25.7 mY) and Pea/Pes = 1000. The left term is the Ca flux and the right term is the
Cs flux. There will be little error in assuming that lea at +10 mV is all Ca influx (Ca influx
would be underestimated by only 4%). However, at the lea reversal potential (E"y=+52 mY) one
would assume zero Ca influx, whereas the actual Ca influx is 11 % of the value which occurs at
+ 10 mV. Erey is the Em where Ca influx is exactly balanced by Cs efflux through the channel.
This is distinct from the Ca equilibrium potential (Eea = + 125 mY) where there would be no net
Ca influx. Zhou & Bers (2000) demonstrated just this sort of Ca entry via lea at Em above Erey (by
measuring Ca channel influx with a fluorescent indicator while measuring lea by voltage clamp).
While this was an expected finding based on GHK theory, it should also be noted that the Ca
channel does not truly behave ideally with respect to GHK assumptions. This is due in part to
the binding sites in the pore and the ion-ion interactions which are known to exist. Ca influx
above the E rev for lea is important to keep in mind experimentally, since some investigators have
depolarized to E rev with the intention of stopping Ca influx (e.g. in E-C coupling studies).
NUMBERSOFCaCHANNELS
Schwartz et al. (1985) estimated that there are 30-50 times as many specific DHP
receptors in frog skeletal muscle as functional L-type Ca channels. In isolated mammalian
ventricular myocytes, the density of L-type Ca channels has been estimated from single channel
and whole cell lea measurements to be 3-5/f!m2 (McDonald et al., 1986; Tsien et at., 1983). Bers
& Stiffel (1993) measured specific DHP binding to isolated adult rabbit, rat, guinea-pig and
ferret ventricular myocytes to be -150 fmollmg protein (as did Green et al., 1985 and Kokubun
et al., 1986 in rat myocytes). In rabbit ventricular homogenate the receptor density is somewhat
lower (-90 fmol/mg homogenate protein), due to dilution with other proteins. Assuming 120 mg
protein/cm3 , 25% extracellular space and a surface to volume ratio of 0.6/f!m (including T-
tubules, Table 1), this would correspond to -20 DHP receptors/f!m2 . This is in the same range as
estimates of Ca channel density based on non-linear charge movement in cardiac myocytes
thought to represent gating of the Ca channel (3.7-5.5 nC/f!F, Field et al., 1988; Bean & Rios,
1989; Hadley & Lederer, 1989). Assuming that 6 elementary charges cross the membrane field
when a channel opens, these "gating currents" would correspond to 37-57 channels/f!m2 .
Ca channel density (N) can also be estimated based on whole cell and the single channel
current (lea & i ea) using the relationship N = Iea!Uea x Po). The calculated channel density
depends critically on the open channel probability (Po). The value of Po assumed from single
channel records must represent the true mean open probability of all functional Ca channels. The
relatively high Po (-0.8) used for the whole cell lea by McDonald et al. (1986) was probably far
too high, since whole cell lea can be increased several-fold in the presence of Bay K 8644 which
is known to increase po (Hess et al., 1984a; Bean & Rios, 1989). Lew et al. (1991) found mean
overall po = 0.03 at peak current (including those in relatively inactive modes) and with the
measured i Cll and lea the calculated N is 18 channels/f!m2, equal to the density of DHP receptors
in the same rabbit ventricular myocytes (l3-15/f!m2 ). Thus, the number of Ca channels and DHP
receptors are the same (-15/f!m2 or -300,000/cell) and only -3% of the Ca channels are open at
peak lea. Thus, there is no discrepancy between the density of cardiac DHP receptors and L-type
Ca channels. This may also be true for skeletal muscle Ca channels if the average po at the peak
of the whole cell lea is on the order of 0.03, rather than the value of 1.0 assumed by Schwartz et
al. (1985). Of course the Ca channels are not uniformly distributed, but clustered near SR
junctions (see Chapter I).
Ca CHANNEL GATING
Surface Potential and Activation
Cardiac lea is rapidly activated by depolarization, reaching a peak in -2-7 msec,
depending on the temperature and Em (see Figs 45 & 50). Ca channel activation seems to depend
primarily on Em, but as for most voltage sensitive channels, is also sensitive to changes in surface
Chapter 5 Sarcolemmal Ca Channels 1I5
Outside Membrane Inside
---------------------r--------
Wo =-30 mV , Plus
______t _
_ L
Em =---40mV
" Screening
,
,Wm =-88
"" Wi =-50
_ _ _ _ _ _ _ _ _ _ _ _ _ L_
Normal ,
Wm=-60mV ~
5nm ~---t"
Figure 55. Surface potential and channel gating. At higher extracellular divalent ion concentration,
external surface charge is screened thereby decreasing 'Vo and making trans-bilayer potential ('Vm) more
negative for the same Em. Thus greater Em depolarization is required to reach the same activating 'Vm.
potential (e.g. Wilson et ai., 1983; Hille, 1992). The surface potential arises from fixed negative
charges on the membrane surface (McLaughlin, 1977, 1989). Figure 55 illustrates the effect that
changing surface potential (\jIo and \jIi) can have on the electric field within the membrane (\jim),
which may be what the voltage sensors of the channel respond to. I have selected to consider the
situation near the threshold of Ica activation (Em= -40) in a physiological medium (2 mM Ca and
-150 mM ionic strength) and used the Gouy-Chapman theory of the diffuse double layer
(Grahame, 1947). The values for the surface potentials are somewhat arbitrarily chosen, but
would correspond to surface charge densities of -I elementary charge/ 100-300 A2 (which
roughly corresponds to expected surface densities of acidic phospholipids and sugars). This is
also similar to the value estimated by Kass & Krafte (1987) from divalent cation-induced shifts
of Ca channel gating in cardiac Purkinje fibers (i.e. 11250 N). The \jIi was assumed to be larger,
since negatively charged phospholipids are preferentially on the inner sarcolemmal leaflet in
cardiac muscle (Post et al., 1988) and the divalent cation concentration is lower inside the cell
than out. Divalent cations are especially effective in screening surface charge since they are
concentrated by the negatively charged surface and also bind to the negatively charged sites to
neutralize them (McLaughlin et al., 1981; Bers et at., 1985). Indeed, at I mM [Ca]o we estimate
surface [Ca]a and [Na]o to be 25 mM and 700 mM respectively (and surface [CI] is also reduced).
Given these conditions, the "normal" state at Em= -40 mV in Fig 55 would correspond to
a trans-bilayer potential of 'Vm= -60 mY. If extracellular divalent cation concentration is
increased such that the external surface potential ('Va) is nearly abolished then the trans-bilayer
potential is considerably more polarized ('Vm= -88 mY) for the same Em= -40. Thus a greater Em
depolarization will be required for the channel to "see" the same trans-bilayer field and hence
threshold for activation. This is why the current-voltage relationship with 115 mM Ba in Fig
50A is shifted to more depolarized Em compared with 2 mM Ca in Fig 50B (and Il's activation in
divalent-free solution is shifted -20 mV compared to lea). Ba does not shift activation as much
116 D.M. Bel's Cardiac E-C Coupling
as Ca does, probably because it does not bind as avidly to negative membrane surface sites (the
pure charge screening effect should be the same for all divalents). This effect applies to all E m-
dependent channels and applies equally to the Em-dependence of inactivation. It readily explains
the increased excitability of cells in low divalent cation concentrations. That is, low [Cal o shifts
the activation and inactivation of channels to more negative Em (closer to the resting Em)'
The effect of the surface potential decays exponentially over a few Debye lengths (one
Debye length -I nm). Surface potential clearly effects channel gating, but might also alter Ca
channel conductance, because of the surface Ca concentrating effect of the negative potential.
However, Coronado & Affolter (1986) demonstrated single channel conductance was relatively
insensitive to the surface potential. Thus, in contrast to the gating sensor, the opening into the
permeation pathway may be relatively shielded from \jIo effects (due to elevation above the
bilayer surface and/or a charge-free disc around the pore of about 2 nm).
As discussed in Chapter 4, most of the Em-dependence in Ca channel activation is
between closed states, with the final closed to open transition being relatively Em-independent.
Analogous to Fig 40B, the Em-dependence of Ca channel gating charge movement occurs at more
negative Em than that of lea (Bean & Rios, 1989).
lca lnaclivalion
Inactivation of Ca channels is time-, E m- and [Cali-dependent (Lee el aI., 1985; Kass &
Sanguinetti, 1984; Hadley & Hume, 1987). Figure 56A shows that inactivation of INs is very
slow (tl/2 >500 ms) and probably reflects purely Em-dependent inactivation which is very slow at
this test Em (-30 mY). As an aside, E m= -30 mV in the absence of divalent cations produces a
membrane field and 'l'm comparable to that at E m=-10 or 0 mV with normal [Cal o (due to surface
charge screening by Ca, see above). The divalent cation currents were recorded during pulses to
o mY. I should note that when Em is 40-50 mV more positive to this range the inactivation ofINs
increases (i.e. Em-dependent inactivation is more prominent, but still incomplete even at large
positive Em). The Ba current (IBa) in Fig 56A inactivates more rapidly (ty, = 161 ms) than INs, and
this may reflect a modest ability ofBa to mimic Ca-dependent inactivation (Ferreira el aI., 1997).
When Ca is the charge carrier, with 10 mM EGTA in the pipette to abolish cellular Ca transients
(and in this case SR Ca release), inactivation is faster still (ty, = 37 ms). This probably reflects
Ca-dependent inactivation due to Ca entering via the channel itself. EGTA is a slow Ca buffer
and thus cannot prevent a rise in local [Cali near the mouth of the Ca channel. High concen-
trations of faster Ca buffers (like BAPTA) can slow this Ca-dependent inactivation. During
normal E-C coupling there is also Ca released from the SR and this can further elevate local [Cali
near the L-type Ca channel. The top lea trace in Fig 56A is under conditions where a normal Ca
transient occurs in the cell (clamped using perforated patch mode). Ie. inactivates much faster
(ty, = 17 ms) and this emphasizes that SR Ca release also plays a major role in lea inactivation in
a physiological setting. Indeed, in action potential clamp experiments (Fig 60), normal SR Ca
release reduces the integrated Ca influx via Ie•. L by 50% (Puglisi et al., 1999).
Figure 56B shows the Em-dependence of inactivation at the end of a 500 ms pulse to the
indicated Em (Hadley & Hume, 1987). It can be seen that INS inactivation was incomplete after
500 ms, even at +60 mY. At strong positive Em (where little Ca enters), there is little difference
between lea and INS inactivation (i.e. it is more purely Em-dependent). The additional lea inacti-
A. Normalized Ca Channel Current B. Inactivation Em-dependence

1.0
lea (perforated) with [Ca]; transients 1: 0.8

~
lea (ruptured & EGTA) <3 0.6
.~
I Sa (EGTA) ~ 0.4
G>
0:: 0.2
00 -'-....,.....~-r~--"F-.:r--....,.....--'H,!>a"'d"fleL.&'4-'H"'ump'e
-40 -20 0 20 40 60
50 ms Pre-pulse Em (mV)
Figure 56. Cardiac Ca channel inactivation. A. Normalized Ca, Ba and Na currents (Ica. lea & INS) meas-
ured at 0 mY (except INS at -30 mY to obtain comparable activation state). Ica was recorded under both
perforated patch conditions (where normal SR Ca release and Ca transients occur) and in ruptured patch
with cells dialyzed with ] 0 mM EGTA (to prevent global Ca transients). lea was also recorded with
ruptured patch (with 10 mM EGTA in the pipette). Extracellular [Ca] and [Ba] were both 2 mM and INS
was measured in divalent-free conditions (10 mM EDTA inside and out) with [Na]oat 20 mM and [Na]; at
10 mM. Peak currents were 1370, 808, 780 and 5200 pA and were attained at 5, 7, 10 and 14 ms for ICa
(perforated), ICa (ruptured), lea and INS respectively, with tv, of current decline of 17, 37, 161 and >500 ms
respectively. B. Amplitude of INS and ICa through Ca channels (at -10 mY) after 500 msec pulses to the
indicated Ern in guinea-pig ventricular myocytes. INS inactivation is Ern-dependent, and the additional
inactivation with Ca is attributed to Ca-dependent inactivation (redrawn from Hadley & Hume, 1987).
vation at intermediate Em has the same Em-dependence as inward ICa amplitude (e.g. maximal at
-OmY, see Fig 50C). This fast Ca-dependent inactivation is the overwhelmingly dominant
inactivation on the time scale of an AP (or voltage clamp pulse in Fig 56A).
The Ca-dependent inactivation ofI ca appears to depend on rCa]; and as such may provide
a sort of feedback control to limit further Ca entry. A remarkable feature of the Cal-dependent
inactivation is that it is still readily apparent even when rCa]; is fairly heavily buffered by EGTA
or BAPTA, which abolishes contraction. This suggested that Ca entering via ICa must exert the
inactivating effect locally, and perhaps directly at the channel, prior to mixing with bulk [Ca];.
HOfer et al. (1997) estimated a K; for [Ca]i of 4 flM based on single Ca channel currents.
Novel insight into the molecular mechanism was provided by de Leon et al. (1995) in
studies of a putative Ca-binding EF-hand region present in the cardiac alC (Fig 51) that is not in
alE (a neuronal Ca channel which does not show Ca-dependent inactivation). By donating this
EF-hand region of alC into alE in chimeric Ca channels they could confer Ca-dependent inacti-
vation upon alE. However, a study disrupting the Ca binding site by mutagenesis did not confirm
this (Zhou et al., 1997). More recently three independent groups have demonstrated that
calmodulin may mediate Ca-dependent inactivation of alC, converging on the following model
(Zulkhe & Reuter, 1998; Peterson et al., 1999; Qin et al., 1999; Zulkhe et al., 1999). Calmodulin
may already be bound to the Ca channel at rest. This may require I Ca bound to calmodulin-alc
with relatively high affinity (Kd =29 nM) in the carboxy terminal of alC near the IQ motif (amino
acids 1624-5 in Fig 51) prior to Ca channel activation. When the channel opens and local [Cal
rises, more Ca binds to calmodulin (which has 4 Ca binding sites) causing a stronger interaction
with the IQ motif of the Ca channel and consequent inactivation. Some aspects of this model
A. Activation & Availability B. Recovery from Inactivation

Activation -90 mV
1.0
Q)
u
c: Q)
III :0
ti .!l!
:::l
"tl .~
c: III
<3 0.5 'E 0.5
.~ ~
:::l
1;j
U
Qi
~
RabbitV.
O.O+-~~,....,~~~,..,~~....,~~---.-,
-40 -20 o 20 o 500 1000 1500 2000
Em (mV) Recovery time (msec)
Figure 57. Cardiac Ca channel activation, availability and recovery from inactivation. A. lea availability
is measured by depolarizing from -90 mY to the indicated Em for 2 sec and then testing the remaining
available lea at Em =0 mY ([Ca]o=2 mM). The result is referred to as a steady state inactivation curve.
Activation is measured by dividing the peak current by the apparent driving force (Em -E rev) according to
Ohm's law (G=I/,6.Y). Both curves are described by a Boltzmann relation (pg 70). Thick curves are for
rabbit and broken curves for rat (data from Yuan et al., 1996). B. Time and Em dependence of recovery
from inactivation of rabbit ventricular lea. After a 2 sec pulse to + 10mY to induce complete inactivation,
Em is held at the values indicated for different times before testing lea available with a pulse to 0 mY. Data
are composite from several studies in our lab at 2 mM [Ca]o and 23°e.
require further clarification, including whether the Ca-calmodulin-IQ region functions like a "ball
and chain" as in N-type inactivation (pg 72).
If voltage clamp pulses (or presumably action potentials) are very long, lea can be largely
inactivated during the cytosolic Ca transient, but then partly recover as [Cali declines (Sipido et
aI., 1995a). This sort of reactivation of L-type Ca channels during long action potentials may
contribute to arrhythmogenic early afterdepolarizations (EADs; January & Riddle, 1989).
Figure 57A shows typical steady state activation and availability (or inactivation) curves
for lea in rat and rabbit ventricular myocytes, where SR Ca load and transients were prevented by
high intracellular EGTA (from Yuan et aI., 1996). The Ca channel activation variable (d~) starts
increasing at --40 mY and is maximal by 0 mY. The inactivation variable (j~) begins decreasing
at --45 and is nearly 0 at -IS mY. Rat vs. rabbit lea activates at more negative Em, inactivates at
more positive E illi has slower Ca-dependent inactivation and shows a larger window current. In
both cases the maximal steady state window lea occurs at about -28 mY and the conductance at
that point is 1% of maximum for rabbit, but 5% for rat (d= x!=). This could provide significant
sustained Ca influx at Em from --45 to -IS mY. Josephson et al. (1984) reported that guinea-pig
ventricle shows a larger window current than rat. Cohen & Lederer (1988) showed a develop-
mental decrease in window lea after birth in rats, but in their case there was SR Ca release and
they attributed this difference to maturation of SR Ca release and cross-signaling.
Once the Ca channel is inactivated, recovery from inactivation is also [Ca]i- and E m-
dependent. Figure 57B shows that recovery is much slower at Em = -50 than at -90 mY. This
means that Ca channel recovery only becomes very fast as AP repolarization is nearly complete.
At -50 mY one can appreciate that frequency-dependent accumulation of inactivated channels
may occur (e.g. at I Hz). Moreover, drugs like the protein kinase inhibitors H-89, KN-62 and
A. B.
o mV I L « 900
-...
-90 mV
Co
~
c:
QI
...
<3800
Cll
U
200
pA
L 1 3 5
2 min rest, 0.5 Hz
700...L..,r-r-,..-,-r-",,-,-,,--T"""''---r-r-r--.--,
7 9 11 13 15
10 ms Post-Rest Pulse Number
Figure 58. Ca-dependent facilitation ofI ea or lea staircase. lea at 0.5 Hz (from -90 to 0 mY) after a 2 min
rest period in a ferret ventricular myocyte. The first post-rest pulse (PR) and the second, third and steady
state (SS) pulses are shown in A and the whole post-rest lea "staircase" in B. It may be noted that when the
holding Em is -40 mY a declining lea "staircase" is seen (redrawn from Hryshko & Bers, 1990).
KN-93 can slow recovery at -90 mY, so that it resembles that seen at Em = -50 mV (Yuan &
Bers, 1994; Li et al., 1997b). Clearly, this can reduce apparent lea in a protocol dependent way.
Ca-dependent ICa Facilitation or lea Staircase

Increasing the frequency of voltage clamp pulses from a holding potential of about -40
mV typically results in a progressive decline in lea amplitude (Tseng, 1988; Hryshko & Bers,
1990). This negative lea staircase probably reflects insufficient time for Ca channels to recover
from the inactivated state between pulses (at -40 mY). In contrast at suitably physiological
holding potentials (-80 mY), Fig 58 shows that there is a pulse-dependent progressive increase in
lea amplitude and a prominent slowing of inactivation (Mitra & Morad, 1986; Lee, 1987; Argibay
et al., 1988; Boyett, & Fedida, 1988; Fedida et al.,1988a,b; Gurney et al.,1989; Hryshko & Bers,
1990; Tseng, 1988; Zygmunt, & Maylie, 1990). This positive lea staircase is Ca-dependent (not
apparent with Ba as the charge carrier), still occurs in the absence of SR Ca release, and three
groups independently demonstrated that Ca-dependent lea facilitation is mediated by CaMKIl-
dependent phosphorylation (Yuan & Bers, 1994; Anderson et al., 1994a; Xiao et aI., I 994a;
Dzhura et al., 2000). Of course SR Ca release (a much larger local Ca flux than lea) can amplify
the effect, explaining some disparate results (Delgado et aI., 1999). Since this Ca-dependent
facilitation is still observed when cells are dialyzed with 10 mM EGTA (but is abrogated by 20
mM BAPTA), the Ca-dependent activation of CaMKIl must be highly localized near the L-type
Ca channel (Hryshko & Bers, 1990). This facilitatory effect of Ca entry on subsequent lea is
distinct from, but coexists with the Ca;-dependent inactivation described above. Indeed, biphasic
effects of [Ca]i on unitary lea have been reported (Hirano & Hiraoki, 1994).
Zuhlke et al. (1999) found that point mutations in the isoleucine in the IQ domain which
abolish Ca-calmodulin dependent inactivation could either enhance (De to Ala) or abolish (De to
Glu) Ca-dependent Ica facilitation. Thus the calmodulin involved in activating CaMKII and Ica
may be the same tethered calmodulin which is involved in Ca-dependent inactivation. I further
speculate that the slowing of Ica inactivation during this positive Ica staircase (Fig 58) might
result from some degree of functional shift of calmodulin from the "inactivation target" to
CaMKII as a target. The physiological impact of this Ca-dependent facilitation is not entirely
clear. However, it may partly offset direct Ca-dependent inactivation and create an additional Ca
channel memory that has a time scale of at least a few seconds.
A voltage-dependent facilitation of Ica has also been described in chromaffin cells
(Artelejo et al., 1990, 1992), skeletal (Sculptoreanu et aI., 1993a) and cardiac Ca channels
(Pietrobon & Hess, 1990; Sculptoreanu et al., 1993b; Xiao et al., 1994a; Kamp et al., 2000). In
cardiac Ca channels this effect seems to be mediated by cAMP dependent protein kinase and may
require coexpression of Ca channel ~ subunits (but is distinct from the G-protein mediated
modulation of neuronal Ca channels discussed on pg 106). This facilitation occurs with pulses to
very positive potentials (e.g. >+ 100 mV) and also dissipates extremely rapidly upon repolari-
zation to physiological membrane potentials in ventricular myocytes. For example, if one
repolarizes from +100 to -60 mV for more than ~IO ms the facilitation is almost abolished (and
even faster at more negative, diastolic Em). Thus this voltage-mediated Ica facilitation is probably
not physiologically important in the normal cardiac myocyte.
AMOUNT OF Ca ENTRY VIA Ca CHANNELS

The amount of Ca entry via Ica is functionally important with respect to the Ca
requirements for myofilament activation. During a square voltage clamp pulse to 0 mV the Ica
waveform can be integrated to infer a Ca influx of ~ 10 flmol/L cytosol (see pg 55; this is
equivalent to peak Ica = 1 nA, triangular shape 120 ms long for 30 pL myocyte cytosol).
However, Ca influx during the AP may differ significantly from the usual square pulse. Figure
59 compares square pulses and AP waveforms used as command potentials in voltage clamp (i.e.
AP clamp; Doerr et aI., 1990; Arreola et al., 1991; Yuan et al., 1996; Grantham & Cannell, 1996;
Linz & Meyer, 1998). Peak Ic• during the AP clamp is lower and occurs later than for the square
pulse. This is because at the AP peak (+50 mY) Ca channels activate rapidly, but the driving
force for Ca is initially low because Em is close to the reversal potential for Ic• (~+60 mY). As
Em falls the driving force apparently increases faster than the channels inactivate, producing a
larger current at later times during the AP. In rabbit ventricle Ic• is also more sustained during
the AP than a square pulse. The bottom panels in Fig 59 show running integrals of Ca influx. In
rat ventricular myocytes the amount of Ca influx for the same 200 ms square pulse is higher than
in rabbit (Yuan et aI., 1996). This is due to the difference in Ic• activation and inactivation.
However, the rat ventricular AP is very brief and with species-appropriate AP waveforms the Ca
influx is much higher in rabbit vs. rat ventricular myocyte (21 vs. 14 flmol/L cytosol).
The data shown in Fig 59 overestimate physiological Ca influx because these measure-
ments were in cells dialyzed with EGTA (such that there was less Ca-dependent inactivation of
Ic.). We also recently measured integrated Ic• during AP clamp in rabbit ventricular myocyte at
25 and 35°C where normal SR Ca release occurred, but all other currents, including INa/C. and
Icl(c.) were blocked (Puglisi et al., 1999). Figure 60A shows that for steady state AP clamps (and
contractions) Ic• peaks earlier and higher at 35°C than at 25°C, but the integrated Ca influx is
80 Rat 80 Rabbit
40 40 AP
>
E- o 0
E Step
w -40 -40
-80 -80
0 0
LL
:< -5 -3
'"
.2
-10 -6
-15 -9
Ste 20.6 ± 1.8
~3 3 ~mol/l cytosol
<5 AP
E 2 2
~ AP Step
<3 1 13.8 ± 1.3
..=, ~ mo III cytosol
0 0
o 100 200 0 100 200 300
time (msec) time (msec)
Figure 59. lea during square pulse and AP-clamp. Rat and rabbit ventricular myocytes (25°C) were
voltage clamped with either a square voltage step or an AP waveform (measured from 5 other cells under
physiological conditions). All other currents were blocked e.g. by replacement of K with Cs and Na with
TEA (inside and out) and cells were dialyzed with 10 mM EGTA to prevent Ca transients. Running
integrals ofCa influx during AP-clamp are shown along with mean values (data from Yuan et al., 1996).
almost the same. Figure 60B shows how lea changes during APs as the SR is refilled (after prior
depletion) and contractions recover to steady state (at 25°C). As the contractions and SR Ca
release get larger lea inactivates more rapidly and completely during the AP. This is an obvious
consequence of more profound Ca-dependent inactivation of lea. Considering that there is no SR
Ca at the first pulse (and peak lea hardly changes), the decline in integrated lea from pulse 1 to
steady state (pulse 10) indicates how SR Ca release limits Ca influx. Figure 60C shows that at
both 25 and 35°C integrated lea decreases from 12 to 6 llmol/L cytosol. This indicates that lea
inactivation due to SR Ca release decreases net Ca influx by -50% at both temperatures.
Quantitatively similar conclusions were found in square voltage clamp pulse experiments in rat,
ferret and guinea-pig myocytes (Adachi-Akahani et al., 1996; Sham et al., 1995a; Trafford et al.,
1997; Terraciano & MacLeod, 1997; Linz & Meyer, 1998).
The kinetics of this SR Ca release-induced lea inactivation can also provide information
about the timing of SR Ca release (Puglisi et al., 1999; Linz & Meyer, 1998). We took the
60 12
AP-Clamp
A B
30
> 0>
E 0
"Q)
'c
6
E t::
l1J -30 0
.c:
(j)
-60 0
-90
750 1000
0
~ 25°C
8 ~
ro -500 8
.C?
3-500
-1000
0 50 100 150 200 250 ·1000
0 50 100 150
time (ms) time (ms)
C
~ 0.4
D 15
[>
LL
~=(i)
C3
c.
12 Q .'?<i
8 0.3 § "C c 10
ro '2 -;~
.:l
'0 0.2 3
0 j e
.$
f ~
01
35'C
;S
C'l
'$.
.
~:E 5
o~
~
0
en a:
SR Ca Reloading_ 0 rn
0.0 -= 10 50
2 4 6 8 10 20 30 40
Pulse Number time (ms)
Figure 60. Ca influx during AP at 25 and 35°C in rabbit ventricular myocyte. lea during AP-c1amp. A.
AP waveforms and lea during steady state AP-c1amps. The AP waveform was recorded under physiological
ionic conditions (another cell) and used as the command Em in this cell, where all other ionic currents were
blocked (Cs and TEA replacing K and Na). B. After SR Ca was depleted by a brief caffeine-application
(with Na), a series of AP-c1amps were given, and the SR and contraction recover to steady state over 10
sequential pulses. C. Integrated lea for each pulse dUling SR Ca reloading in experiments like panel B at 25
and 35°C. D. The difference current between pulse I in panel B and pulses 3, 5, 7 & 10 (Jd!O: taken as an
index oflocal [Cal due to SR Ca release) was differentiated (dIJW/dt) to provide an index of SR Ca release
rate as sensed by the L-type Ca chaJ1J1el (data are from Puglisi et al., 1999).
difference in lea traces between pulse #1 and the other pulses in Fig 60B (lDilf) as an index of the
local [Cali produced by SR Ca release, as sensed by the L-type Ca channel. The rate at which
this local [Cali changes (dIDw/dt) may thus be the locally sensed rate of SR Ca release. Figure
60D shows that the local SR Ca release flux sensed by the Ca channel reaches a peak in 5 ms,
regardless of the amplitude of release (or 2.5 ms at 35°C). This is much faster than the rate of
rise of the global cellular Ca transient, but is not surprising given our expectation that Ca release
via ryanodine receptors occurs very near the L-type Ca channel. It also emphasizes that Ca-
sensitive ionic currents can be excellent sensors oflocal [Cali in the cell.
A~
CONTROL
L
BAY K8644
L.:. 70mv
RP -40mv
mode 2
"'I~~~
~""I'""""
B '~';U ",~.~ .... r~ -,
~~""""".~ 0 ~.......-
k' k'
~
." flo
-.., I' . 11 I' ~l

C,~C.~o
mode 1 /" Higresl
~ Affnity,: r Dff'
[~
Caag:m sis
-"4~\ I,l.~-..r--
k, k
....-1' I. ,',. Ti' • ..,.,
~ C,=C• .;;bO
J k'l k·t
~
~ rnode~
.. ,
r
I' I , _~..,~
t I •• "',.",..· .... ~
.j.'~
......... "III~ill.t
LI
HlgreslAffinty for
1pAL
20 ms
DHP ca
artagonisls
C .'\wi
, .. ,.... --L'"
.5
20ms
Figure 61. Effect of the Ca channel agonist Bay K 8644 on Ca channel gating from single channel patch
clamp recordings. A. Voltage clamp protocol referenced to the resting potential (RP) of about -60 mV.
B. Single sweeps in the absence (left) and presence (right) of 5 11M Bay K 8644. C. Average current from
all single sweeps. D. Model of Ca channel gating modes proposed by Hess et al. (1984). The transitions
between modes is slow compared to the gating within a mode (indicated as C], C2 and 0 for two closed and
one open state). (All panels are from Hess et al., 1984, with permission).
MODULATION OF lea BY AGONISTS AND ANTAGONISTS

A hallmark of L-type Ca channels is their sensitivity to DHPs (e.g. nifedipine,
nitrendipine, nimodipine, nisoldipine, isradipine or PN200-11O, Bay K 8644, azidopine and
iodipine). Indeed, the specific binding of DHPs to the (X) subunit of the L-type Ca channel was
impoltant in the isolation of the protein. Most of these DHPs decrease lea and are known as Ca
channel blockers or Ca channel antagonists. Some DHPs, notably (-) Bay K 8644, (+) S-202-
791 and CGP 28392 (known as Ca channel agonists), greatly increase lea by increasing the
duration of single Ca channel openings, without appreciably altering single channel conductance
(Brown et al., 1984; Hess et al., 1984a; Kokubun & Reuter, 1984; Kokubun et al., 1986). Bay K
8644 can increase channel open times from -0.6 msec to -20 msec and Hess et al. (1984a)
suggested that Bay K 8644 binding to the Ca channel stabilized a state of the channel, called
"mode 2" in which long stable openings occur (see Fig 61). In the presence of Bay K 8644 the
channel can still switch to the normal state (mode 1) where the openings are shorter and
indistinguishable from the control conditions. The mode 2 type openings can also be seen under
control conditions, but they are quite rare (Hess et al., 1984a; Yue et al., 1990). Thus, Ca
agonists can greatly increase the likelihood of these long lasting mode 2 openings. Ca antagonist
DHPs, on the other hand, inhibit lea apparently by favoring a mode of channel gating (mode 0)
characterized by the channel being unavailable to open. Switching between modes occurs on a
slower time scale than bursts of activity (several seconds). Indeed, normal Ca channels can be
quiescent for many seconds of depolarizing pulses (mode 0), can then have very infrequent and
brief opening of 0.15 ms (mode 0.; Yue et aI., 1990), switch to a mode where occasional bursts
of 0.5-1 ms occur (mode I) and rarely make excursions to mode 2 (where openings of 10-20 ms
are observed). While this modal model of Ca channel gating is widely used, its applicability has
also been challenged (Lacerda & Brown, 1989).
The long openings induced by Bay K 8644 would be expected to lead to slower Ic•
inactivation during a voltage clamp pulse. This is not usually observed for Ic• because the larger
Ca influx also produces greater Ca-dependent inactivation. Interestingly, when Bay K 8644 and
~-adrenergic agonists (which also enhance mode 2) are applied together there is a dramatic
slowing ofI c• inactivation (Tsien et aI., 1986; Tiaho et al., 1990). A prominent feature ofI c• in
the presence of Bay K 8644 is large, long-lasting "tail" currents. Tail currents are due to the
rapid increase in Ca driving force when repolarization occurs with Ca channels still open (before
they deactivate at negative Em). The last sweep in Fig 618 with Bay K 8644 shows a single
channel tail Ca current. Bay K 8644 also shifts activation and inactivation to more negative Em
(by 10 to 20 mV). The non-DHP Ca channel agonist FPL-64176 also shifts activation and inact-
ivation Em negative and produces even more dramatic tail Ic• than Bay K 8644, but it does not
compete with DHPs at their binding site (Rampe & Lacerda, 1991; Kunze & Rampe, 1992). Bay
Y 5959 is a new DHP Ca channel agonist (Bechem et al., 1997) which also produces very
prolonged tail Ic• and slows Ic• activation (both like FPL-64176).
Early experiments indicated that the affinity of cardiac microsomes for nitrendipine was
1000 times higher than the concentrations required to block Ic• (Bellemann et aI., 1981; Lee &
Tsien, 1983). This was explained by the voltage dependence of DHP binding to Ca channels.
For example, Bean (1984) showed that depolarization of Em from -80 to -10 mV in ventriculaJ
myocytes decreased the apparent K d for Ic• inhibition by nitrendipine by > 1OOOx, from -500 nM
to 0.36 nM (Fig 62). Similar results were found by Sanguinetti & Kass (1984) and the}
concluded that DHPs bind preferentially to the inactivated state of the channel. This conclusion
was supported by 3H-DHP binding experiments in isolated sarcolemmal vesicles (Schilling &
Drewe, 1986) and myocytes (Green et aI., 1985; Kokubun et al., 1986). These results fit well
with the modulated receptor hypothesis described by Hondeghem & Katzung (1977) and Hille
(1977) to explain the voltage- and use-dependent block ofNa channels by local anesthetics.
Sanguinetti & Kass (1984) also compared DHPs (and verapamil) which are neutral at
physiological pH (nitrendipine and nisoldipine) with ones that bear a net charge at pH = 7.4
(verapamil and to an intennediate degree, nicardipine). The charged ligands appeared to block
the channel only when it was in the open state (i.e. requiring voltage pulses) and as such are
strictly use-dependent. These charged ligands may need for the channel to open to gain access to
the receptor site. The neutral ligands, on the other hand appear able to block Ic• whether the
channel is in the open or inactivated state (i.e. at depolarized holding potentials without requiring
pulses) and as such are more strictly voltage-dependent than use-dependent. The more
hydrophobic nature of these ligands may allow them to gain access to the receptor site even when
the channel is inactivated. It is also possible that uncharged lipophilic DHPs act by first
partitioning into the membrane bilayer and approaching DHP receptors by lateral diffusion
(Herbette et aI., 1989; Valdivia & Coronado, 1988). There is good evidence to support the idea
1.0-.---__
III
..1i =
V h -80
CIl
.2: Kd =430 nM
10 0.5
Qj
a:::
O.O+-----.---------'lf-------r------,
-12 -10 -8 -6 -4
log [Nitrendipine] (M)
Figure 62. Different Ica blocking effectiveness of nifedipine in canine cardiac myocytes depends on
holding potential (V h)' The apparent K d for ICa block decreased by 1200-fold when V h was depolarized
from -80 to -10mV (curves are fit to original data taken from Bean, 1984). Incomplete Ica block occurred
when Vh = -80 may be due to a fraction of ICa which is not through L-type Ca channels.
that DHPs gain access to the receptor from the external side of the membrane (and external
protons can get access whether the channel is open or not; Kass & Arena, 1989; Kass et al.,
1991; Strubing et aI., 1993). This would be consistent with the receptor being at the external end
of the channel. Benzothiazepine Ca channel blockers (e.g. diltiazem) also appear to gain access
from the extracellular side (Hering et aI., 1993a,b). Access for phenylalkylamines (another class
of Ca channel blockers, e.g. verapamil) appears to be at the inner sarcolemmal surface since
impermeant ligands are relatively ineffective from the outside (Hescheler et al., 1982; LeBlanc &
Hume, 1989).
The voltage-dependence of the DHPs compared to the use-dependence of verapamil may
explain the greater efficacy of the DHPs as vasodilators and the relative lack of effect on cardiac
muscle at therapeutic levels. That is, since resting vascular smooth muscle is usually at more
depolarized levels of Em and can undergo long further depolarizations, DHPs will interact
preferentially with these smooth muscle Ca channels rather than cardiac Ca channels which do
not spend long enough times at depolarized enough potentials to be blocked by therapeutic
concentrations of DHPs. Of course this also explains why the cardiac effects observed with Ca
antagonists are more pronounced in pacemaker cells which have relatively depolarized diastolic
Em level. This effect also contributes to the antiarrhythmic effect of Ca channel blockers.
At least three classes of drugs interact specifically with the L-type Ca channel: I) DHPs
(above), 2) phenylalkylamines (AAs, such as verapamil, D600, D888 and D890) and 3)
benzothiazepines (BTZs e.g. diltiazem), and these sites in tum interact allosterically (Fig 63A,
Glossmann et aI., 1984, 1985). DHP and benzothiazepine binding reciprocally stimulate binding
at the other site and AA binding reciprocally inhibits DHP binding and benzothiazepine
binding. Ca and other divalent cations stimulate DHP binding, but depresses AA and
benzothiazepine binding. It has also been argued that there are two distinct DHP receptors, one
126 D.M Bers Cardiac E-C Coupling
B.
A.
Figure 63. Ca antagonist binding to Ca channel. A. Functional interrelationship between dihydropyridine

(l,4 DHP), phenylalkylamine (<1>AA) and benzothiazepine (BTX) receptor sites on the L-type Ca channel
(e.g. DHP occupancy increases BTX and Ca binding, but inhibits <1>AA binding; after G10ssmann el al.,
1984, 1985). B. Sites on the alC channel IJIS s, IlIS 6 and IVS 6 transmembrane domains which are thought
to be critical in binding DHP (d), <1>AA (<1» and BTZ (b) (based on review by Mitterdorfer el aI., 1998).
for Ca antagonists and one for Ca agonists (Kokubun et al., 1986; Brown el aI., 1986), but in
general these DHPs exhibit competitive binding. The stereoisomers of two well known Ca
agonists appear to act as pure Ca antagonists (-)-R-202-791 and (+) Bay K 8644; Williams et
aI., 1985; Franckowiak et aI., 1985; Kokubun el al., 1986) A further complication is that one
stereoisomer which is a Ca agonist (+)-S-202-791), switches from an Ica agonist at negative test
potentials to an Ica antagonist at Em -0 mY (along with a change from allosteric enhancement of
PN200-11O binding to competitive inhibition; Kokubun et al., 1986; Kamp et al., 1989). Even
nitrendipine, a DHP known as a Ca antagonist, can increase Ica activated from negative holding
potentials (Brown et al., 1986). Thus, while the precise nature of the DHP receptor(s) is not
entirely clear, these compounds are extremely valuable tools in understanding and modulating Ca
channels. Diphenylbutylpiperidine neuroleptics (e.g. fluspiriline and pimozide) and the benzoyl-
pyrrole Ca agonist FPL-64176 may also bind to the Ca channel at an independent receptor
(Gould et al., 1983; Galizzi et aI., 1986; Kunze & Rampe, 1992).
Substantial information has been obtained about the sites of interaction of DHPs, <l>AAs
and benzothiazepines with the Ca channel alC subunit (reviewed in Hockerman et al., 1997;
Mitterdorfer et al., 1998). Studies using photoaffinity labeling with reactive ligands such as 3H_
azidopine indicated that sites in IIIS 6 and IYS 6 interact with DHPs and BTZs (Striessnig et al.,
1990a, 1991; Nakayama et al., 1991; Kraus et al., 1996). Similar studies with <l>AAs only
identified sites on IVS 6 (Striessnig et aI., 1990b). Mutational "gain of function" analysis has
identified alC amino acids that when substituted into non-DHP-sensitive channels (e.g. alA) can
endow them with DHP sensitivity (Grabner et al., 1996). Sinnegger et al. (1997) found 9 non-
conserved amino acids on alC (in IIIS s, IIIS 6 and IVS 6) which could confer full DHP sensitivity
A. PKA and ICa I-V B. Activation & Availability

-60 -40 -20 0 20 40 60 1.0 <p--....a-;O--<:l....
Q)
...c":
U
.g 0.5
c:
o
()
-60 -40 -20 o 20

-24
Em (mV)
C. PKA slows Normalized IBa Decline D. PKA slows ICa Decline for Similar Amplitude
+Forskolin 31~30~0~P~A-.;;;!.~~~,:_-
I 50 ms
ICa
Control
Perforated [Cala
Control 2 mM
647 pA
Forskolin
+Forskolin
0.5 mM [Cal o
100 ms 1200 pA
Figure 64. 13-adrenergic agonist effects on lea. Forskolin (I flM, a direct activator of adenylyl cyclase)
was applied for 5-10 min to increase cAMP and activate PKA (see Fig 65). A. Forskolin increases Ie, at all
Em values, but especially at negative Em (200 ms pulses from Em = -90 mY to indicated Em). B. Activation
and inactivation curves after forskolin are both shifted to more negative Em values (methods as in Fig 57).
CoD. Effect of forskolin on normalized lea and loa decline measured in perforated patch mode and lea in
ruptured patch with 10 mM EGTA in the pipette. lea and IBa were recorded by Yuan & Bers (1995) in ferret
ventricular myocytes at 23°e in perforated (A, e & D) and ruptured patch modes (B & C) with all other
ionic currents blocked.
to alA (Figs 51A and 63B). The converse "Ioss-of-function" approach substituting ale residues
with either alanine or alE/B residues provided complementary data about 13 sites which
contribute to DHP binding on ale (2 on IIIS s, 7 on IIIS 6 and 4 on IVS 6; Peterson et al., 1996,
1997; Schuster et al., 1996). Similar studies with AAs and BTZs identified key sites on IIIS 6
and IVS 6 only (Fig 63B; Hockerman et al., 1997; Hering et al., 1996). Notably, there is signifi-
cant overlap of the sites involved in antagonist binding, despite a lack of classical competition
among these drug classes. Additionally, this SS-S6 region is likely to form part of the pore lining
and activation gate regions of these channels though not the selectivity filter (see Figs 38 & 39).
~-ADRENERGIC MODULAnON OF CARDIAC Ca CURRENT

The activation of lea by 13-adrenergic agonists in cardiac muscle is a classic observation
(e.g. Reuter, 1967). This occurs mainly through PKA, causes a 2-4 fold increase in basal Ie, in
ventricular myocyte and shifts the voltage-dependence of activation and inactivation to more
negative Em (Fig 64A-B; Tsien et al.1986; Hartzell, 1988; McDonald et al., 1994). The shift in
activation gating causes the lea increase to be most prominent at negative Em and maximal Ie, to
be at more negative Em. At the single channel level PKA has no effect on unitary conductance,
but there are fewer blank sweeps (without any openings) and an apparent increase in open times
which may reflect a shift toward mode 2 gating (Cachelin, 1983; Vue et aI., 1990).
The ~-adrenergic cascade also interacts with other signaling pathways in modulating lea.
For example, acetylcholine (ACh) has no effect on basal lea in ventricular myocytes, but strongly
antagonizes the catecholamine- or forskolin-stimulated lea (Fischmeister & Hartzell, 1986;
Hescheler et aI., 1986). Part of this effect of ACh may be mediated by the muscarinic receptor-
mediated activation of G j which inhibits adenylyl cyclase and thus net cAMP production
(Lindeman & Watanabe, 1989). In addition, ACh increases cGMP which can either decrease
cAMP by stimulating a cGMP-activated phosphodiesterase (POE-II) in frog (Fischmeister &
Hartzell, 1987) or increase cAMP in mammalian heart by a cGMP-inhibited POE-III (Ono &
Trautwein, 1991; McDonald et al., 1994). Indeed, in atrial latent pacemaker cells, where ACh
depresses basal lea (due to higher basal cAMP than in ventricular myocytes), withdrawal of ACh
causes a dramatic overshoot of lea amplitude above control (Wang & Lipsius, 1996). They
attributed this to further elevation of cAMP, and concluded that this rebound increase in lea was
responsible for transient post-vagal tachycardia. Increased cGMP can also activate cGMP-
dependent protein kinase (PKG), which does not appear to alter basal Ie., but can (like ACh and
cGMP) antagonize cAMP-mediated increases in lea (Ono & Trautwein, 1991). Nitric oxide also
stimulates cGMP production in cardiac myocytes (Balligand et al., 1993) and this can mediate Ie.
regulation as above (Mery et aI., 1993; Kirstein et al., 1995; Han et al., 1994a). Indeed, there is
evidence to suggest that nitric oxide is required for the ACh-mediated antagonism of cAMP-
activated lea in cardiac myocytes (Han et aI., 1994a; Wang & Lipsius, 1995b). ACh has also
been shown to stimulate protein phosphatase activity, which would also reverse PKA mediated
phosphorylation (Herzig et aI., 1995).
Figure 65 indicates the ~-adrenergic agonist pathway. Occupation of the ~-adrenergic
receptor by an agonist activates a GTP binding protein (G s) and the a subunit (Gsa) dissociates
and activates adenylyl cyclase, producing cAi\1P. The increase in cAMP leads to the dissociation
of the regulatory and catalytic subunits of the cyclic AMP-dependent protein kinase (PKA). The
PKA catalytic subunit phosphorylates several proteins including the L-type Ca channel and also
the ryanodine receptor, phospholamban and troponin I.
Support for this ~-adrenergic pathway for lea comes from experiments showing that the
effect can be mimicked by intracellular cAMP, cAMP analogs and phosphodiesterase inhibitors
(Tsien et aI., 1972; Tsien, 1973; Vogel & Sperelakis, 1981; Cachelin et al., 1983; Nargeot et aI.,
1983; Kameyama et al., 1985), direct activation of adenylyl cyclase by forskolin (Wahler &
Sperelakis, 1985; Hescheler et al., 1986) or non-hydrolyzable GTP analogs (Josephson &
Sperelakis, 1978) and intracellular application ofPKA catalytic subunit (Osterrieder et aI., 1982;
BlUm et aI., 1983). Dephosphorylation of the Ca channel by protein phosphatases (I and 2A)
also abolishes the increase in lea seen with isoprenaline (Hescheler et al., 1987a), but does not
decrease basal lea. The latter finding also indicates that phosphorylation of the Ca channel is not
required for channel activity in ventricular myocytes.
PKA is probably anchored in the vicinity of the Ca channel by an "A kinase anchoring
protein" AKAP-79 (Gao et al., 1997a; Fraser et aI., 1998). This crucial localization may have
caused difficulties in demonstrating PKA-dependent activation of lea in heterologous expression
~-Adrenergic
Activation
Ca ----tt==}+1
_ _ _~--
_ _ Ca
"
,---~ rmillT in ,
-
.::::::: I :::::::'
'::::::: I i!i!!!:: I
Ca Myofilaments
Figure 65. Dual pathways for activation of Ca channels by ~-adrenergic stimulation. The classic pathway
is via stimulation ofadenylyl cyclase (AC, via activation of the GTP binding protein Gs), increased [cAMP]
and phosphorylation of the Ca channel by the catalytic subunit of the cAMP-dependent protein kinase
(PKA, where Reg is the regulatory subunit of PKA). Another minor pathway is via a direct effect of the
activated CI. subunit of Gs on the Ca channel (~-Rec= ~ adrenergic receptor, Epi= epinephrine, Norepi=
norepinephrine, MrRec= Mrmuscarinic receptor, AKAP=PKA anchoring protein, PLB= phospholamban,
ACh = acetylcholine.
systems (Zong et al., 1995). Forskolin, which increases cAMP (by stimulating adenylyl cyclase)
activates PKA and strongly activates Ica in heart cells (Fig 64), fails to effect the heterologously
expressed alC, although the PKA inhibitor H-89 could decrease Isa (Perez-Reyes et al., 1994).
Despite this issue, the cloned alC and ~2A subunits have been shown to be substrates for PKA and
PKC (Haase et al., 1993; Puri et al., 1997). The phosphorylation site on alC (Ser-1928) can
increase ICa in an AKAP-dependent manner, but in heart cells Ser-1928 and the distal part of the
C-terminus might be cleaved off in some fraction of Ca channels (Gao et al., 1997b). Two non-
consensus PKA sites on the ~2A subunit (Ser-478 & 479) can be phosphorylated by PKA and
increase ICa (Biinemann et al., 1999).
A more rapid direct activation of Ica by GSa was suggested to be physiologically
important (Yatani et al., 1987; Yatani & Brown, 1989), but this effect (when observed) is a very
small fraction of the overall ~-adrenergic response (Pelzer et al., 1990; MacDonald et al., 1994).
How is Ca channel gating modified by ~-agonists? Here let us consider the relationship
between whole cell Ica (I) and single channel current (i): I = Nf x Po x i, where Nf is the number
of functional channels in the cell and Po is the probability that the channel will be open. Since
single channel conductance is unaltered, i is not changed (Reuter et al., 1982). Single channel
recording indicated that Po was increased by ~-agonists or dibutyryl-cAMP (Cachelin et aI.,
1983; Brum et al., 1984). There may also be some increase in N r , but truly "new" channels do
not appear in membrane patches with ~-stimulation (as would be expected if dormant channels
become functional; Bean et aI., 1984). The apparent increase in Nf could also be due to an
apparent shift toward greater mode 1 and mode 2 gating (vs. mode 0, Oa and mode 1; Yue et aI.,
1990; Cachelin et al., 1983; Brum et al., 1984). Thus, the increase in lea induced by ~-adrenergic
stimulation (and channel phosphorylation) is probably entirely due to an increase in po of the
channel. This increased Po is mediated either by a change in the Em dependence of activation or a
shift toward modes of channel gating where longer openings are favored.
As shown in Fig 64, PKA causes greater increase of lea at more negative potentials and
this is due to negative shifts in the Em dependence of both activation and inactivation. This shift
brings the Em-dependence of lea activation closer to the Em-dependence of Ca channel gating
current (which is not much shifted by ~-stimulation; Bean, 1990). Bean suggested that ~
stimulation may increase lea by making the coupling between the charge movement and opening
of the Ca channel more efficient. It is of interest to note that the negative Em shift in gating is the
opposite direction that one would expect for simple surface potential effects of adding negative
P04 groups to the membrane or channel surface (see Fig 55). Thus the phosphorylation must
change the channel sufficiently to overcome this effect.
PKA typically accelerates lea inactivation, but this is probably due in large part to the
greater Ca-dependent inactivation secondary to larger lea amplitude. Figure 64C-D shows that Isa
inactivation is slowed by PKA-dependent phosphorylation, and that if [Ca]o is lowered in the
presence of forskolin (to limit the increase in lea) lea inactivation is also slowed by PKA. Thus
Ca channel phosphorylation slows both Ca- and Em-dependent inactivation, but higher Ca influx
and SR Ca release may functionally reverse this effect in the normal cellular environment.
Both ~l and ~2-adrenergic agonists couple to Gs and can produce inotropic effects in
heart, but they may couple differentially to L-type Ca channels vs. other cellular targets (Xiao &
Lakatta, 1993; Xiao et al., 1994b; Hool & Harvey, 1997). Additionally the ~radrenergic
receptor may also couple to a G j transduction system (Zhou et aI., 1999), complicating the
resulting effects and interpretations. Intracellular Mg can block lea, but Mg-ATP also stimulates
lea independent of phosphorylation (O'Rourke et al., 1992). At physiological intracellular Mg-
ATP this regulatory effect is probably maximal.
OTHER MODULATORS OF Ca CURRENT

Other hormones are known to modify cardiac Ca currents, although for many of these
results are conflicting or the effects are relatively small (McDonald et aI., 1994). For example,
most results with a-adrenergic agonists indicate no affect on lea (Hescheler et aI., 1988;
Hartmann et al., 1988; Ertl et al., 1991). However, phenylephrine induced a small transient
decrease followed by a substantial increase in lea only when using perforated patch recording
(Zhang et aI., 1998; Liu & Kennedy, 1998). This variation of whole cell voltage clamp limits
rundown of lea and prevents the washout of normal intracellular constituents which occur in
conventional ruptured-patch recording and can alter cellular responses. Histamine, acting at H 2
receptors, can also increase cardiac lea by activating adenylyl cyclase and the cAMP cascade
(Hescheler et aI., 1987b; Levi & Alloatti, 1988). Atrial natriuretic peptide (ANP) can reduce lea,
but this may depend on intracellular GTP and cAMP levels (Anand-Srivastava & Cantin, 1986;
Cramb et al., 1987, Gisbert & Fischmeister, 1988; LeGrand et aI., 1992). ANP may activate Gj
and prevent cAMP dependent enhancement of cardiac lea in a similar manner to acetylcholine.
ANP may also activate guanylyl cyclase and cGMP production, which can modulate lea via PKG
as well as stimulate breakdown of cAMP by phosphodiesterase type n. As mentioned earlier,
cGMP is not generally found to alter basal ICa (Ono & Trautwein, 1991), but some investigators
contend that PKG can directly decrease cardiac lea (Wahler et at., 1990; Sperelakis et at., 1996).
Endothelin increases lea in smooth muscle (Inoue et at., 1990), but in cardiac myocytes it either
produces a small reduction of Ie" particularly without pipette GTP, but could enhance lea with
GTP in the pipette (Tohse et al., 1990; Lauer et al., 1992). In perforated patch measurements
endothelin had no effect on basal ICa, but reversed p-adrenergic agonist stimulation of lea via the
ETA receptor and pertussis toxin-sensitive G j (Thomas et al., 1997).
Adenosine, acting at PI-purinergic receptors can activate G j and prevent lea activation by
p-adrenergic agonists, but does not alter basal Ie" analogous to ACh above (Belardinelli &
Isenberg, 1983b; West et al., 1986). Activation of P 2 -purinergic receptors by adenosine can
increase lea via stimulation of Gs (Scamps et al., 1992), but inhibitory effects have also been
reported (e.g. Qu et al., 1993). Angiotensin n can increase Ie" possibly via stimulation of
protein kinase C (PKC) rather than PKA (Allen et al., 1988; D6semeci et al., 1988; LeGrand et
al., 1991). Phorbol esters directly stimulate protein kinase C (PKC) and in cardiac myocytes
were shown to increase (D6semeci et al., 1988; Lacerda et al., 1988), decrease (Tseng &
Boyden, 1991; Scamps et al., 1992; Zhang et at., 1997a), have biphasic effects (Lacerda et al.,
1988) or not change lea (Walsh & Kass, 1988). While this leaves the issue somewhat unclear,
Zhang et at. (I997a) have shown that the inhibitory effect of the phorbol ester PMA that they
observe is attributable to a C2-containing PKC isoform (PKC-a, -P or -y).
Protein tyrosine kinase (PTK) inhibitors (e.g. genestein) decrease lea in ruptured patch
experiments (Katsube et al., 1998; Hool et al., 1998; Ogura et al., 1999), but also have a
coexistent larger and slower stimulatory effect that is apparent in perforated patch experiments
(Wang & Lipsius, 1998). This may mean that a membrane associated PTK stimulates basal Ie"
while a cytosolic PTK is normally inhibitory. Blocking PTKs with genestein also increases the
sensitivity to p-adrenergic agonists with respect to stimulation ofl ea and IK (Hool et at., 1998).
Thus tyrosine kinases may depress responsiveness to adrenergic signaling and reflects a cross-
talk between these kinase cascades. Arachadonic acid and some of its metabolites (epoxy-
eicosatrienoic acids, EETs) can also inhibit cardiac ICa (Petit-Jaques & Hartzell, 1996; Chen et
at., 1999a). While arachadonate may act by stimulating a phosphatase, EETs seem to directly
accelerate inactivation and decrease single channel po and conductance.
Regulation of lea by these other pathways is indeed potentially confusing given the
number of conflicting results. However, most of these seem to work via G" Gj (and possibly
other G-proteins) and modulation of cyclic nucleotides (cAMP & cGMP) and protein kinases
(PKA, PKC, PTK and PKG). Clearly additional work will be required to clarify the details of
these intermingling pathways for many of these important modulatory mechanisms.
In conclusion, I would like to emphasize that L-type lea is the main route of Ca entry into
the cell (vs. leak, Na/Ca exchange or Iea,T) and that lea plays a central role in cardiac E-C
coupling and overall Ca regulation and contraction. The kinetics and amplitude of the lea during
the action potential are critical factors in controlling the amount of Ca released by the SR (see
Chapter 8). Ca which enters as lea may also contribute directly to the activation of the
myofilaments as well as to the replenishment of SR Ca stores (see Chapter 9). The amount of Ca
influx via Ic• must be extruded from the cell during the same cardiac cycle (e.g. via Na/Ca
exchange) for a steady state to exist. Any uncompensated Ca influx could constitute a
progressive Ca load for the cell. Due to the high conductance of these ion channels, a relatively
small number of Ca channels which fail to inactivate could lead to substantial Ca gain (e.g.
during a window Ica , especially at depolarized Em). This, of course, can compromise relaxation
and contraction and even be arrhythmogenic (see Chapters 4, 6 & 10).
D.M. Bers. 133
Excitation·Contraction CouplinQ and Cardiac Contractile Force.
CHAPTER 6
Na/Ca EXCHANGE AND

THE SARCOLEMMAL Ca-PUMP
THE SARCOLEMMAL Ca-PUMP

The two known mechanisms responsible for extrusion of Ca from cardiac myocytes are
the sarcolemmal Ca-ATPase pump and Na/Ca exchange. A plasma membrane Ca-pump was first
reported in erythrocytes (Schatzmann, 1966), but is ubiquitous (Schatzmann, 1982, 1989;
Carafoli & Stauffer, 1993; Guerini et al., 1998). The red cell plasma membrane Ca-pump has
been most extensively characterized and appears closely related to that in other tissues. Two key
features of the Ca-pump are its stimulation by Ca-calmodulin and by PKA-dependent phos-
phorylation. The plasma membrane Ca-pump is a P-type ATPase (like the Na/K-ATPase, H/K-
ATPase and the SR Ca-ATPase). That is, it transfers the energy of ATP to a high energy
phosphorylated intermediate (aspartyl residue), energy which is then used in the ion transport
step (e.g. Figs 44 & 83). The purified protein (138 kDa) is no more similar to the SR Ca-pump
protein than it is to the Na/K-ATPase or H/K-ATPase (Niggli et al., 1981a; Verma et al., 1988).
The plasma membrane Ca-ATPase was first cloned by Shull & Greeb (1988) and there are four
human isogenes (PMCAI-4) and also numerous splice variants (Carafoli, 1994). A central
stretch of ~80 kDa is all that is required for Ca transport (James et at., 1988). The overall
structure is similar to the related, and better characterized SRiER Ca-ATPase (SERCA; see
Chapter 7, Fig 81-82). There are probably 10 transmembrane domains (TMI-I0) which make up
-20% of the protein, while the cytosolic domains make up ~80%. The data are much more
compelling for the SR Ca-ATPase, but TM4, 5 and 6 may be involved in Ca translocation. The
TM2-3 loop is analogous to the hinge domain of SERCA which is thought to couple ATP
hydrolysis to Ca transport. The even larger TM4-5 loop contains the site which binds ATP (and
fluorescein isothiocyanate or FITC) and also the aspartate which is phosphorylated at the active
site of the enzyme. A third important cytosolic domain is the carboxy terminal tail where a 30
amino acid stretch contains the regulatory calmodulin binding domain and also has regulatory
sites which are phosphorylated by PKA and PKC (James et al., 1988, 1989; Wang et al., 1991).
This carboxy terminal is thought to interact with the other 2 cytosolic domains in an
autoinhibitory manner, such that Ca-calmodulin binding and phosphorylation relieve this
inhibition (Carafoli, 1994). One Ca ion seems to be transported per ATP hydrolyzed (Rega &
Garrahan, 1986) and Ca extrusion by this pump appears coupled to proton influx (I Ca: I H,
Kuwayama, 1988). The turnover rate of plasma membrane Ca-pumps may approach -20/sec
with Km(Ca) -I J.lM (Schatzmann, 1989).
The cardiac sarcolemmal Ca-pump was first described in vesicle studies by Caroni &
Carafoli (1980). They also demonstrated a stimulatory effect of cAMP-dependent phos-
phorylation (-3-fold) and calmodulin on the pump (Caroni & Carafoli 198Ia,b). They found a
K m(ATP)-30 flM, K m(Ca)=0.3 flM and V max =31 nmol/mg protein/min in the presence of
endogenous calmodulin vs. Km(Ca)=11 flM and Vmax=IO nmol/mg protein/min in calmodulin
depleted preparations. Dixon & Haynes (1989) measured the cardiac sarcolemma Ca-ATPase
activity stimulation by calmodulin, cAMP-dependent protein kinase or both (Table 18).
Calmodulin profoundly effects Km(Ca) and VmaX> while smaller effects were observed with PKA.
Table 18
Kinetic Properties of the Cardiac Sarcolemmal Ca-Pump
Vrnax Vrnax * Km(Ca) n (Hill)
nmol/mg pn/min flmol/L cytosol/sec nM
Basal 1.7 ± 0.3 0.28 1800 ± 100 1.6 ± 0.1
+PKA 3.1 ±0.5 0.50 1100± 100 1.7±0.1
+Calmodulin 15.0±2.5 2.43 64± 1.4 3.7±0.2
+PKA+calmodulin 36.0± 6.5 5.83 63 ± 1.7 3.7 ± 0.1
Data are taken from Dixon & Haynes (1989). 'assuming 30-fold enrichment of sarcolemma, 120
mg protein/g wet wt and 2.43 g wet wt/ml cytosol (see Table 9, pg 42).
The maximum rate of Ca extrusion via the sarcolemmal Ca-pump from cardiac myocytes
based on these V max values is -5.8 flmollL cytosol/sec. Bassani et al. (1995a) found a very
similar Vmax in intact ventricular myocytes (2 flmol/L cytosol/sec in rabbit and 10 flM/sec in
ferret), but the apparent Km(Ca) = 0.3 flM was intermediate and closer to the Caroni & Carafoli
value above. This might reflect a partial calmodulin activated state in the intact cell. The
affinity of the sarcolemmal Ca-pump for Ca-calmodulin is high (K m -I nM; Graf & Penniston,
1981), but direct information about dynamic regulation in myocytes is lacking. The activating
effect of calmodulin on the Ca-ATPase can also be mimicked by acidic phospholipids, and in the
normal cellular environment this may produce 50% maximal activity (Niggli el aI., 1981 b).
There are no highly selective inhibitors of the sarcolemmal Ca-ATPase. However, eosin
and other fluorescein analogues can potently inhibit plasma membrane Ca-ATPase (Gatto &
Milanick, 1993). Eosin and carboxyeosin inhibit the pump with K j values of 50 and 20 nM
respectively, without altering Na/Ca exchange (Gatto el al., 1995). In ventricular myocytes 15
flM carboxyeosin blocked sarcolemmal Ca-ATPase function, but may also inhibit the SR Ca-
ATPase by -20% (Bassani el al., 1995a).
During normal cellular Ca transients in rabbit ventricular myocytes the transport rate by
the sarcolemmal Ca-ATPase is probably <I flM/sec and also it would take -60 sec to produce
relaxation by itself (see Fig 30, pg 53). This rate of Ca transport is quite slow compared to that
by sarcolemmal Ca channels (300 flM/sec), SR Ca release (1000 flM/sec), SR Ca-ATPase (200
flM/sec) or Na/Ca exchange (30 flM/sec); see Figs 29 and 45. Thus, while the sarcolemmal Ca-
pump can have a high affinity for [Ca];, the transport rate is too slow for it to be a major
contributor to Ca fluxes during the cardiac cycle. The sarcolemmal Ca-ATPase appears to be
nearly 5 times stronger in ferret than in rabbit or rat ventricular myocytes (Bassani el al., 1994b,
1995a). Even in the absence of SR Ca uptake, Choi & Eisner (1999) showed that Ca removal by
this system in Wistar rats is only -25% of that by the Na/Ca exchange, whereas our estimates are
<13%, 6% and 14% in in Sprague Dawley rats, rabbits and ferrets, respectively (Bassani el al.,
Chapter 6 Sarcolemmal Na/Ca exchange and Ca-pump 135
1994a, 1995a). Thus, even with species differences the sarcolemmal Ca-ATPase is only a 'minor
Ca transporter on a beat-to-beat basis. It might, however be more important in slow longer term
extrusion of Ca by the cell (but see Lamont & Eisner, 1996, and the end of this chapter).
Na/Ca EXCHANGE
Von Wilbrandt & Koller (1948) proposed that the site of action of Ca on the heart was
likely to be at the cell membrane and that contractions depended on the ratio [Cal/[Na]o.
Liittgau & Niedergerke (1958) suggested that Na and Ca compete for an anionic site (R2")
responsible for bringing Ca into the cell, consistent with a CaINa 2 model. Repke (1964) and
Langer (1964) suggested that intracellular Na might be linked to Ca influx in some way. This
helped explain the basis of digitalis inotropy and the "staircase" or positive force-frequency
relationship in cardiac muscle (where increasing frequency leads to increased contractile force).
Reuter & Seitz (1968) in heart and Baker et al. (1969) in squid giant axon were the first to
document the presence of a Na/Ca exchange countertransport system. In the ensuing 30 years
Na/Ca exchange has been characterized and its role in cardiac Ca regulation clarified (see
reviews by Khananshvili, 1998; Reeves, 1998; Blaustein & Lederer, 1999; Egger & Niggli, 1999;
Philipson & Nicoll, 2000; Hryshko, 2001).
Early Characterizations in Sarcolemmal Vesicles

Much important seminal work on sarcolemmal Na/Ca exchange came from studies of
isolated cardiac sarcolemmal vesicles (SLV). The general strategy for studying Na/Ca exchange
in SLV is illustrated in Figure 66. Reeves & Sutko (1979) were the first to demonstrate Na/Ca
exchange in SLV by measuring 45Ca uptake into a relatively crude preparation of SLV preloaded
passively with Na and diluted into a Na-free medium. Pitts (1979) made early estimates that the
Na/Ca exchange stoichiometry was 3:1 (Na:Ca). Studies showed that Na/Ca exchange is both
sensitive to Em (since more positive intravesicular potential increased Ca uptake) and can
generate a voltage gradient (Bers et aI., 1980; Reeves & Sutko, 1980; Philipson & Nishimoto,
1980; Caroni et al., 1980). These results were consistent with an electrogenic Na/Ca exchange
(i.e. >2Na:lCa), but the stoichiometry was most clearly demonstrated by Reeves & Hale (1984)
using a thermodynamic approach. Their measurements of the [Na] gradient required to prevent
net Ca transport at various membrane potentials indicated a stoichiometry of 3Na: 1Ca. This
stoichiometry is completely consistent with a wide array of other studies and is now generally
accepted as the true stoichiometry of Na/Ca exchange in cardiac muscle (e.g. Philipson & Nicoll,
2000). Some intriguing new data indicate a higher stoichiometry of ~4: 1 (Fujioka et al., 2000) or
a loss of electrogenicity at very acidic external pH (Egger & Niggli, 2000). It is probably
premature to dismiss the enormous body of work consistent with the 3: 1 stoichiometry, but it
may not be quite as rigidly fixed as we have thought for the past 15 years. Thus, in most of the
ensuing discussion I will retain the 3:1 stoichiometry, unless otherwise noted.
It was surprisingly difficult to separate inside-out and right-side out sarcolemmal vesicles
to study the symmetry of the Na/Ca exchange. Philipson & Nishimoto (1982a) and Philipson
(1985) circumvented this limitation by comparing Na/Ca exchange in vesicles which were loaded
with Na only by the action of the sarcolemmal Na/K-ATPase pump (i.e. inside-out vesicles) with
that in the whole population ofSLV. They found a similar Km(Ca) (-25 ~M) and Na-dependence
20
Load
Ca uptake -------- _ Na
c:
'iii 15
'0
Q.
W
+- 10
NCO
o
~c: 5 Na o-dependent ---------- . 45Ca
Ca release •••.•
"'~~ Release Dilute in
---. 50 mM Na
Binding blan
O+-~~__,_~~~~~__,_~_;...~.....--,
o 4 8 12 16
time (min) Na
Figure 66. Na/Ca exchange in isolated cardiac sarcolemmal vesicles. Vesicles are pre-equilibrated with
140 mM Na and then diluted 50x into a solution with 45Ca and 140 mM KCI or, in the case at left, 280 mM
sucrose + 50 IlM CaC1 2 . Parallel samples diluted without changing [Na] serve as binding blanks. Samples
are filtered or quenched with a La containing solution. After uptake has occurred, addition of 50 mM NaCl
induced a rapid efflux of Ca, indicative of the reversibility of the Na/Ca exchange system (left panel is
reproduced from Bers et al., 1980, with permission).
of Ca efflux (Km~30 mM) in both populations, suggesting a relatively symmetrical exchanger,

although their later work clearly showed asymmetry (see below; Li et al., 1991). In SLV the
Km(Na) is generally 20-30 mM, while values for Km(Ca) vary widely (2-200 f.tM), although most
reports give 20-40 f.tM (Reeves & Philipson, 1989). Electrophysiological measurement described
below, where the sidedness is more clearly defined, have provided more direct concentration
dependence and symmetry information.
Isolation, Cloning and Structure

Philipson et al. (1988) identified 70 and 120 kDa proteins as the Na/Ca exchanger and
suggested that the smaller protein may be a proteolytic fragment of the larger. Nicoll et al.
(1990) cloned the cardiac Na/Ca exchange (NCXI) and found it to consists of 970 amino acids
(MW = 108 kDa) including 12 putative transmembrane domains and one very large cytoplasmic
hydrophilic domain. The first 32 amino acids and first transmembrane span constitute a signal
peptide which is cleaved off during processing (Durkin et al., 1991; Hryshko et al., 1993), such
that the mature protein is 938 amino acids long and the amino terminal is g1ycosy1ated and
extracellular. Earlier models suggested 1I transmembrane domains. However, new topological
data indicate only 9 transmembrane spans (Fig 67) based on access of epitope-specific antibodies
and of sulfhydryl reagents to substituted cysteine residues (Nicoll et al., 1999; Iwamoto et al.,
1999; Doering et aI., 1998). In the current model there are 5 transmembrane domains on the
amino part of the molecule, a large cytoplasmic or f-loop (550 amino acids or 59% of the whole
protein) and 4 additional transmembrane domains on the carboxy end plus a putative P-Ioop like
domain. Deletion of the large intracellular f-loop (Matsuoka et al., 1993) showed that the loop is
not essential for transport ofNa or Ca, but it does abolish allosteric regulation by rCa]; and [Na]j
Na/Ca Exchanger
____----8-8
Outside
Inside
eOOH
f-Loop;-;c!!IIallll!lbl!lin·d!l!ljn·g-"1A~ltlil!. s~p"lic~e~~~
680-685
Figure 67. Working model of the Na/Ca exchanger based on the work of Nicoll et al. (1999) indicating 9
membrane spanning segments, glycosylation (CHzO), a-repeats and alternative splicing domain (Alt.
splice). The original I" transmembrane domain (TM) is cleaved during maturation and not shown. Former
TM6 (0Id-TM6) is now thought to be in the cytosolic f-Ioop. Former TM9 is now modeled as a P-like loop
(as in ion channels) between TM7 & TM8. The figure is based on Philipson & Nicoll (2000).
(see below). Similarly, chymotrypsin treatment of the intracellular side of Na/Ca exchange in
excised membrane patches also abolishes these modes of regulation (Hilgemann, 1989). There
are also two 23 amino acid regions of intramolecular homology (a repeats a-I & a-2) which face
opposite sides of the membrane. There is also a region that is somewhat similar to a region of
the NaIK-ATPase and SR Ca-ATPase that is thought to be involved in Ca transport (Clarke et al.,
1989b; Nicoll et al., 1996). The second transmembrane domain and the a-repeats may contribute
to the ion translocation pathway (Nicoll et aI., 1996), but additional clarification is required.
There is also a charged segment of 20 residues (219-238) at the amino end of the f-Ioop (XIP)
which resembled a calmodulin binding domain, and may be autoinhibitory. Exogenous XIP
(exchange inhibitory peptide) can inhibit Na/Ca exchange from the cytosolic side in SLY,
excised sarcolemmal patches and when dialyzed into whole myocytes (Li et al., 1991; Chin et
al., 1993).
In addition to the gene which generates the cardiac Na/Ca exchanger (NCXI) there are
two other genes (NCX2 & NCX3) which are mainly found in brain and skeletal muscle (Li et al.,
1994; Nicoll et al., 1996). All 3 isoforms have similar, but not identical functional properties
(Linck et aI., 1998). NCX 1 is present in heart (one of the richest sources of Na/Ca exchange)
and the gene is subject to alternative splicing in the f-Ioop. Six exons (A-F) participate and all
NCXI splice variants have either exon A or B, but not both. Exon A is more common in
excitable cells (e.g. heart, neurons and skeletal muscle) while exon B is more commonly found in
kidney, liver, lungs and astrocytes (Quednau et al., 1997; He et al., 1998). Na/Ca exchange
activity in cardiac muscle is > IO-fold higher than in smooth and skeletal muscle and neuronal
tissue (Slaughter et al., 1989; Donoso & Hidalgo, 1989; Blaustein, 1989; Barzilai et al., 1984,
1987). Cardiac NCXl.l expresses exons ACDEF (Philipson & Nicoll, 2000; Kim et al., 1998).
Another Na/Ca exchanger family (NCKX) which exchanges 4Na for 1 Ca plus IK is prominent
and functionally important in rod cells in the retina (Schnetkamp et al., 1989; Re'ilander et al.,
1992). Further discussion will concentrate on cardiac NCX I.
The Na/Ca exchanger is reversible and carries out ion transport in a consecutive or
"ping-pong" reaction mechanism (Khananshvili, 1990; Hilgemann et al., 1991) similar to the
Na/K-ATPase in Fig 44 (pg 91). For example, 3 Na ions bind with the exchanger open toward
the outside and are then transported by a conformational change allowing dissociation of 3Na
inside and binding of an intracellular Ca ion. The conformation then switches back to face
outside and the Ca can be released to the extracellular space, completing a cycle. This would
result in movement of one net charge inward per cycle and thus Ca extrusion constitutes an
inward INa/Ca. This electrogenicity also indicates that Na/Ca exchange is sensitive to Em as well
as intracellular and extracellular [Cal and [Na]. The Na/Ca exchanger can also operate in Na/Na
exchange or Ca/Ca exchange modes (Reeves & Sutko, 1979; Philipson & Nishimoto, 1981;
Slaughter et aI., 1983). These modes are most evident in the absence of Ca or Na respectively,
but do not produce net ion movement. Thus, these fluxes are not functionally important.
Na/Ca Exchange Current in Myocytes and Excised Patches

Cardiac ionic currents attributed to Na/Ca exchange were first reported by Horackova &
Vassort (1979), but acceptance was slow because of voltage clamp limitations in multicellular
preparations and lack of clear expectations for the measured current. The developments of whole
cell voltage clamp, ventricular myocyte isolation techniques and SLV data on Na/Ca exchange
allowed faster progress. Nao-dependent inward currents were observed in single myocytes during
caffeine-induced or spontaneous SR Ca release (Clusin et al., 1983; Mechmann & Pot!, 1986)
and were attributed to Na/Ca exchange. These currents are likely responsible for the transient
inward currents (ItiS) and delayed afterdepolarizations (DADs) discussed on page 98. Another
manifestation ofINa/ca is the so called "creep" currents described by Eisner & Lederer (1979) and
Hume & Uehara (l986a,b). Depolarizing voltage clamp pulses in Nai loaded cells are associated
with a declining outward "creep" current (likely due to declining Ca entry via Na/Ca exchange).
Repolarization also produces an inward tail current which slowly declines as Ca is extruded via
Na/Ca exchange current. These creep currents were [Na] and [Cal-dependent and could be
suppressed by La and dichlorobenzamil (Bielefeld et al., 1986; Hume, 1987).
Kimura et at. (1986, 1987; Miura & Kimura, 1989) provided key compelling electro-
physiological characterizations of Na/Ca exchange in intact ventricular myocytes. They blocked
all other known ionic currents (including Na/K-ATPase) and dialyzed the cell with known [Na]
and [Cal solutions. Figure 68 shows that with [Cali near zero, there was no current activated by
140 mM Na o. However, when [Cali was raised to 430 nM, application of 140 mM Na o activated
an Em-dependent current (Fig 68B-D) which was inward at Em = -90 mV. Thus this inward
current was dependent on [Na]o, [Cali and Em. They also demonstrated an Em-dependent outward
current which depended on [Na]; and [Ca]o. The outward INa/ca (Na efflux and Ca influx) also
appeared to require a certain amount of[Ca]j acting as an allosteric regulator (KI/2 -22 nM [Cali,
Miura & Kimura, 1989). Stimulation by Caj was also suggested in sarcolemmal vesicle studies
(Reeves & Poronnik, 1987).
1 min
A [ 60mV
I\Iii II \II I [ -30
-120
140 mM·Na+ Internal Ca" 140 mM·Na+
1+ I : I I I I ! i
#H+@+ rn-rrrrrT ¥.V"::;:r- iilr [
0.2
'h' OnA
a b t Ie ,n,
de;g
B 0'5nA c 0·5nA
c
-=:~~::::==t::::::~a, b
° 50mV c
e e
Figure 68. Na/Ca exchange current recording in a dialyzed and voltage-clamped guinea-pig ventricular
myocyle, A) Slow recording of Em (top) and INa /Ca where the spikes are from ramp depolarizations used to
generate the current voltage relationships in B, C and D at the times indicated (a-h). The bars in A refer to
changing the extracellular solution from 140 mM LiC] to 140 mM NaCl ([Ca]o was I mM throughout). At
the arrow [Cali was increased from nominally Ca free to 430 nM with 42 mM EGTA and 140 CsCl
throughout. Ouabain, Ba, Cs, D600 and tetraethylammonium were used to inhibit other ionic currents,
Application of Na o stimulated INa/C, only after [Cali was raised, The gradual decline in IN,/c, (d-g) was
supposed to be due to local Ca depletions (from Kimura et al., 1987, with permission).
Hilgemann (1990) developed the giant excised patch technique to study cardiac Na/Ca
exchange under more controlled conditions in patches detached from the cell. By dialyzing patch
pipettes and rapidly changing bath solution around the intracellular surface of the Na/Ca
exchanger (Fig 69A), Hilgemann and colleagues comprehensively characterized cardiac Na/Ca
exchange (Hilgemann, 1990; Hilgemann et ai" I 992a,b; Matsuoka and Hilgemann, 1992;
Hilgemann & Collins, 1992; Collins et al., 1992), Figure 69 shows the method and some
characteristics of INa/c, measured this way. Regulatory rCa]; is required (K m - 0.3 JlM) for INa/ca
activity, even when Na; is the transport substrate on the inside. With high [Ca] in the pipette
([Ca]o) application of 100 mM Na to the inside surface activates outward INa/ca (Fig 69B).
Sustained exposure to high [Na]; results in a Na-dependent inactivation of INa/Ca' Notably this
Na;-dependent inactivation is prominent only at high [Na]; (e.g. >30 mM), levels which may not
be attained under normal physiological conditions. In addition, Fig 69C shows that when rCa]; is
high, Na;-dependent inactivation does not occur. However, removal of rCa]; still deactivates
outward INa/Ca. Reported kinetics of Ca;-dependent activation and deactivation vary (Fig 69C,
Hilgemann et aI., 1992a; Kappl & Hartung, 1996; Weber et al., 2001). We find that outward
INa/Ca in intact ventricular myocytes is activated during SR Ca release just as fast as rCa]; rises
(-50 ms), and that INa/ca Ca;-activation state can vary dynamically during Ca transients over the
physiological range with K m=125 nM, Weber et al., 2001), Thus, Ca;-dependent activation may
have a short-term memory of [Ca];.
Levitsky et al. (1994) found high affinity Ca binding to two neighboring acidic regions
in NCXl (446-454 & 498-509). Mutations of the three sequential aspartate residues in these
regions disrupt both Ca binding and allosteric regulation of INa/ca by rCa]; (Matsuoka et al.,
A
Reversal of
Catdependent
c activation
25pA L
B 10 sec
Na; (mM)~ 100 o 100 lJL

15
Figure 69. Na/Ca exchange current in giant excised patch showing secondary regulation by Caj and Naj.
A) inside-out patch conditions (-10 ~m diameter tip) with external surface bathed in high [Cal, but without
Na. B. Application of 100 mM Naj (at 1 ~M Ca) causes activation and then inactivation of INa/c,. C. when
bath [Cal is elevated to 15 ~M application of 100 mM Naj activates outward INa/c" but inactivation is not
seen. Deactivation is observed upon removal of regulatory [Ca]j. The patch here is from a Xenopus oocyte
expressing NCX 1. (modified from Philipson & Nicoll, 2000, with permission).
1995). In the Drosophila Na/Ca exchanger (Calx), Ca binding to the analogous region decreases
I a/C, and this effect is prevented by similar mutations (Hryshko et aI., 1996; Dyck et aI., 1998).
This raises the intriguing possibility that Ca binding to the same site produces opposite effects in
NCXI vs. Calx. A deletion mutation (Li680-685; see Fig 67) in the NCXI f-loop abolishes
allosteric regulation by both Caj and Naj (Maxwell et al., 1999: Weber et aI., 2001), so this
region is also crucial in regulation. The XIP region of NCXI is involved in Naj-dependent
inactivation since mutations in this region alter the process (Matsuoka et al., 1997).
The physiological functions of the Caj-dependent activation and Naj-dependent
inactivation in intact cells are still not clear. However, the Caj-dependent activation could
stimulate Ca extrusion by INa/c, when [Ca]j is relatively high and turn the exchanger off as [Ca]j
falls to diastolic levels (thereby limiting how low [Ca]j goes). This Caj-dependent activation
could also stimulate greater Ca influx via Na/Ca exchange when conditions favor this direction
of Ca flux. The Naj-dependent inactivation could prevent excess Ca influx and cellular Ca
overload under conditions of high [Na]j where net Ca influx might be strongly favored.
However, the very high [Na]j required to observe Na-dependent inactivation of INalCa may render
this regulatory mechanism physiologically unimportant.
Figure 70 shows the E m- and [Nakdependence ofI Na/C , at two [Ca]j which reflect resting
and peak systolic [Ca]j (Matsuoka & Hilgemann, 1992). These data were obtained at
physiological [Na]o (140 mM) and [Ca]o (2 mM) and after treatment with chymotrypsin to
remove allosteric regulation by Naj and Caj. The complex dependence of the INa/C. on Na, Ca and
Em can be seen and is further addressed below (see Thermodynamics, pg 147).
Ca and Na affinities as substrates for ion transport have also been measured by several
groups under a variety of conditions and there is functional competition between Ca and Na at
No: (mM) No: (mM)

100
A. 1 IJM [Cali 60 pA '~g 50
._!.;!;~;~: ::
30
20
15
0_ ""., ••• ;':f 10
5
150 mV
·200 100 200 m'
-40
Figure 70. INa/c, Current-voltage relationships from excised giant patches from guinea-pig ventricular
myocytes. Patches were treated with chymotrypsin to remove secondary regulation and all other known
currents were blocked. A. With constant [Ca]o (2 mM), [Na]o (150 mM) and [Cali (l flM), [Na]i was
varied from 5 to 100 mM. The lower 3-4 curves probably reflect the INa/c, expected in intact cells when
[Cali is high (e.g. peak systole). B. The same conditions as A, except [Cali is reduced to 100 oM,
comparable to diastolic [Cali (trom Matsuoka & Hilgemann, 1992, with permission).
both sides. There is general agreement that the Km(Na;) of the Na/Ca exchanger is 20-30 roM,
with a somewhat higher value of Km(Nao) 50-70 roM. Moreover, the [Na]-dependence exhibits a
Hill coefficient of 2-3, consistent with the stoichiometry of 3Na per transport cycle (Miura &
Kimura, 1989). Miura & Kimura (1989) estimated Km(Cai) to be 0.6 flM, while values of 1-4 flM
were found by Hilgemann et al. (1991; Hilgemann, 1996). Some Km(Caj) estimates may also be
complicated by the coexistence of Caj-dependent allosteric regulation. Values for Km(Cao) have
ranged from 0.1 to 1.5 roM, but most estimates are near the 1.2 roM value reported by Kimura et
al. (1987). It should be borne in mind that the experimentally measured Km(Ca) can also be
affected by other conditions such as [Na], Em and pH.
Figure 71 shows simultaneous measurement of INa/c, and Ca transients in a guinea-pig
ventricular myocyte where other channels and transporters including SR were blocked
(Barcenas-Ruiz et al., 1987). During long depolarizing voltage clamp steps the Na/Ca exchanger
can bring in enough Ca to produce high [Ca]j (>1 flM). Then on repolarization INa/C. also
produces an inward tail INa/C. (lower traces in Fig 71A) as well as rCa]; decline. The plot ofINa/c.
as a function of rCa]; is fairly linear, suggesting that Ca extrusion via Na/Ca exchange is not
saturated even at relatively high physiological [Ca];. This result suggests that the functional
Km(Ca;) for the Na/Ca exchange is probably higher than 1 IlM.
Many di- and trivalent cations block Na/Ca exchange activity (e.g. Cd 2+ > La 3+ > y3+ >
Mn2+ > C02+ > Mg2+; Bers et aI., 1980). Trosper & Philipson (1983) found that trivalent cations
were more effective than divalents and that Ba 2+ and Sr2+ also inhibited Na/Ca exchange (Table
19). Tibbits & Philipson (1985) also showed that Sr2+ and to a lesser extent Ba2+ could also
substitute for Ca2+ and be transported in exchange for Na. Egger et al. (1999) also showed that
Ni can be transported weakly by Na/Ca exchange, probably by an electroneutral Ni/Ca exchange,
thus explaining the abolition ofINa/c. typically observed with roM Ni.
1.6
B 0
1.2
0
~
3 0.8 0
elf a
£ 0.4 a
0
0'~80::-----4:':0---:0"------'4C:-0
V (mV)
~~:1 ~
r ~C' 0.00 0.40 0.80
[CaJ;(~M)
1.20 1.60
Figure 71. Ca influx and extrusion by INa/c, in a guinea-pig ventricular myocyte. A. Pipette [Na] was 7.5
mM. Outward currents during depolarization were off-scale. Band C show the Em dependence of the Ca;
transient and the [Cali-dependence of the "tail" current observed upon repolarization to -80 mY. Other
ionic currents were blocked by Cs, tetraethylammonium, verapamil and ryanodine and [Cali was assessed
by fura-2 fluorescence (from Barcenas-Ruiz et al., 1987, with permission).
Charge movements by the Na/Ca exchanger have been used to measure "half-reaction
cycles" (Hilgemann et at., 1991; Niggli & Lipp, 1994). The idea is to start with all of the
exchangers facing inside (e.g. due to high [Na]a without Ca anywhere) and then suddenly apply
substrate (e.g. [Na];) to the inside such that neither the full NaiCa exchange cycle nor net ion
movement can occur. In this case the added [Na]; will bind and be translocated through a half-
reaction such that the exchanger will move to face outside. Hilgemann et at. (1991) found that
charge moved across the membrane when Na was carried, but not when Ca was carried.
Furthermore, their results suggested that one positive charge moves per Na-half cycle and that
this occurs near the extracellular end of the ion pathway. This would be consistent with the
"unloaded" exchanger having two negative charges. The number of charges moved then
provided an estimate of the surface density of Na/Ca exchanger in the membrane (400//lm 2) and
with a V rnax for INa/c, (20-30 A/F) they calculated a maximal molecular turnover rate (SOOO/sec).
Niggli & Lipp (1994) used flash photolysis to rapidly raise rCa]; and found that some charge
moves with both the Na and Ca transporting half reaction. Interestingly, Hilgemann's group
found that squid Na/Ca exchanger (NCX-SQI) gave the opposite result from canine or guinea-
pig heart NCXI, in that all of the charge moved in the Ca translocation step and none in the Na
translocation step (He et at., 1998).
Modulation ofNa/Ca exchange

Table 19 lists some factors which alter cardiac sarcolemmal Na/Ca exchange. Early
evidence, partly from SLV, suggested stimulatory effects of ATP on NaiCa exchange and Ca
affinity (Caroni & Carafoli, 1983; DiPolo & Beauge, 1988; Hilgemann, 1990). MgATP can
reactivate inactivated INa/c, in giant excised patches (K m ~3 mM) and it was suggested that this
might be due to activation of phospholipid translocase or flippase reorienting phosphatidylserine
to the inner surface (Collins et al., 1992; Hilgemann & Collins, 1992), consistent with activation
by acidic phospholipids (Philipson, 1984). Further studies showed that ATP could activate a
lipid kinase causing phosphorylation of phosphatidylinositol (PI) to form PIP z (Hilgemann &
Ball, 1996). Apparently PIP z stimulates INa/C. by preventing Nai-dependent inactivation and
might interact with the positively charged XIP domain (He et al., 2000; Philipson & Nicoll,
2000). Under physiological conditions ([ATP]i > 5 mM) it would be expected that this PIP z
pathway would always be fully active. This would again imply that Nai-dependent inactivation
does not occur, except under pathophysiological conditions where [ATP]i falls appreciably.
Na/Ca exchange is remarkably sensitive to alteration of the lipid bilayer. Exchange is
increased by a) reconstitution with acidic phospholipids, b) addition of exogenous negatively
charged amphiphiles or c) phospholipase cleavage of native phospholipids to yield negatively
charged membrane lipids. Certain anionic head groups are more stimulatory for Na/Ca exchange
than others and several cationic amphiphiles are inhibitory (Philipson, 1984). The effects of
charged amphiphiles and phospholipids does not appear to be simply due to surface charge
effects (e.g. concentrating Ca near the exchanger). Indeed, Na/Ca exchange is virtually
unaffected by changes in surface charge or surface [Cal (Bers et al., 1985). The organic divalent
cation dimethonium screens surface charge and thereby reduces surface [Cal and Ca binding.
But even when dimethonium strongly decreased Ca binding to SLV, it did not affect Na/Ca
exchange. Thus, the negatively charged amphiphiles may enhance Na/Ca exchange via a more
specific interaction with a site on the Na/Ca exchanger.
The stimulatory effect of negatively charged amphiphiles on exchange activity is
enhanced if the lipophilic portion of the molecule also disorders the bilayer, especially toward
the center of the bilayer (e.g. by unsaturated bonds or inclusion of doxyl- groups; Philipson &
Ward, 1987). Optimal reconstitution of exchange activity requires acidic phospholipids and
-20% cholesterol with other related sterols unable to substitute (Vemuri & Philipson, 1988a).
Low pH inhibits NaiCa exchange in SLV, while alkalosis enhances exchange (Philipson
et al., 1982). Giant patch recordings of outward INa/c. confinued this, but also showed that
chymotrypsin almost abolished pHi-dependence (Doering & Lederer, 1993, 1994). Possibly the
pH regulation (like that of regulation by Cai and Nai) is mediated by the large cytosolic loop f.
Egger & Niggli (2000) evaluated the effect the effect of extracellular pH (pH o) on cardiac INa/C•.
They found maximal INa/c. at pH o = 7.6 and 50% of that value at pH o of 10 and 5.5. At very low
pHo «6) inward INa/c. declined much more than Ca transported by Na/Ca exchange, suggesting a
progressive reduction of the electrogenic nature of Ca transported by Na/Ca exchange.
The pathophysiological consequences of the pH and lipid effects above are not yet clear.
However, during cardiac ischemia, the declining pH would be expected to decrease Na/Ca
exchange, production of fatty acids (e.g. arachidonate, see Chien et aI., 1984; Philipson & Ward,
1985) would increase NaiCa exchange and redox stimulation might also stimulate Na/Ca
exchange. These latter effects might limit the depression of Na/Ca exchange during ischemia
and help minimize cardiac Ca overload. On the other hand SLV from ischemic myocardium
exhibit reduced NaiCa exchange (Bersohn et al., 1982) and fatty acids also increase passive Ca
penneability in SLV (Philipson & Ward, 1985) so that the net result in ischemia is still unclear.
Ca chelators, such as EGTA stimulate Na/Ca exchange at a given [Ca], by reducing

Km(Ca) (e.g. from -20 to -I f1M, Trosper & Philipson, 1984) and similar effects have been
reported for the plasma membrane Ca-pump (Schatzmann, 1973; Sarkadi et al., 1979). This
might partly account for the lower Km(Ca) estimated in intact cells perfused with EGTA buffers
than in SLV where Km(Ca) determinations were often made without Ca chelators. It is not yet
known whether the Ca affinity of the exchanger is also increased by the related Ca chelators
which are used as fluorescent Ca indicators in intact cells (e.g. indo-I and fura-2). This could
complicate the use of these indicators in the study of Na/Ca exchange.
The Na/Ca exchanger can be inhibited by a number of drugs (Table 19), but most of
these agents are neither very potent nor very selective. That is, they inhibit other ion transport
systems and channels at even lower concentrations than those required to inhibit Na/Ca exchange
(Kaczorowski et al., 1989). For example, dichlorobenzamil inhibits sarcolemmal Ca channels,
and related amiloride derivatives are potent inhibitors of Na/H exchange, Na-coupled sugar and
amino acid transport, Na, K & Ca channels, and cholinergic and adrenergic receptors This lack
of a specific inhibitor (ligand) of Na/Ca exchange has been a serious limitation, both to the
isolation of the protein and characterization of the physiological action of the Na/Ca exchanger.
XIP inhibits Na/Ca exchange non-competitively at low IJM concentrations. However, it
is of quite limited use in physiological experiments because the peptide must be applied
intracellularly and because XIP also potently inhibits both the sarcolemmal and SR Ca-ATPase
(Enyedi & Penniston, 1993). Side effects are not entirely surprising since XIP resembles a
calmodulin binding domain and 8 of the 20 amino acids are positively charged. Thus while XIP
may be useful in studying isolated INa/ca in excised patches, it is less help in clarifying the role of
Na/Ca exchange in the complex cellular environment. FMRF-amide is a tetrapeptide which can
inhibit Na/Ca exchange non-competitively with a K j -I IJM (DiPolo & Beauge, 1994; Khanan-
shvili et al., 1993). An analogous cyclic hexapeptide (FRCRCF-amide) was also a potent Na/Ca
exchange inhibitor with K j between 2-10 f1M in SLV (Khananshvili et al., 1995, 1996) and in
patch clamped myocytes an apparent K i of -20 nM (Hobai et al., 1997a).
KB-R7943, an isothiourea derivative, has been reported to be a relatively selective
inhibitor of Na/Ca exchange (Watano et al., 1996; Iwamoto et al., 1996a; Watano & Kimura,
1998; Iwamoto & Shigekawa, 1998). KB-R7943 blocked outward INa/ca in myocytes with a Ki =
0.32 IJM, while much higher concentrations were required to block inward INa/ca (K i = 17 IJM;
Watano et al., 1996). This apparently selective block of outward INa/Ca (Ca influx) with low [KB-
R7943] has been confirmed in cellular [Ca]i measurements (Satoh et al., 2000), where 5 IJM KB-
R7943 blocked Ca influx via Na/Ca exchange upon abrupt [Na]a removal, but had no effect on
[Ca]i decline during relaxation when the SR Ca-pump was not functional. They also showed that
KB-R7943 could prevent Ca overload and spontaneous arrhythmias induced by strophanthidin-
induced block of the Na/K-ATPase, without preventing the inotropic effects. While this
intriguing compound may be useful, it also inhibits INa (K i = 14 IJM), Ica (K j = 8 IJM) and inward
rectifying IK (K i = 7 IJM; Watano et al., 1996). It is also unclear from a mechanistic standpoint
how it seems to produce relatively direction-specific block (Kimura et at., 1999). Thus, KB-
R7943 should be used cautiously and with appropriate controls.
Table 19
Factors That Alter Cardiac Sarcolemmal Na/Ca Exchange
Enhancers of Na/Ca Exchange Reference
Em Depolarization ~ i Ca entry a
pH Alkalosis b
ATP roM ATP (1 Km(Ca» c,d
PIP 2 via ATP-stimulated lipid kinase e
[Cali 40-125 nM ~ i I NaiCa (regulatory) d,f
Proteinase Trypsin, Chymotrypsin, Pronase, Papain, Ficin g
Phospholipase Phospholipase C & D ~ i Exchange h
Anionic Amphiphiles SDS, Lauric acid ~ i Exchange (JK m) i,j
Redox Modification FeS04 & DTT, Glutathione k
Ca Chelator EGTA, EDTA, CDTA (1Km) I
Lipids Cholesterol, Unsaturatedldoxyllipids m
Inhibitors of Na/Ca Exchani;e
pH pH ~ 1 Exchange b
Inorganic Cations La 3+>Nd 3+>Tm 3+~y3+>Cd2+> n,a
Sr2+>Ba 2+~Mn2+ >Mg2+
2
Cd 2+, Ni + Ki(Cd)= 320 flM; Km(Ni) = 200 flM o
XIP K i = 0.1-1 flM p
FMRF-amide K i = 1.5 flM q
Cationic Amphiphiles Dodecylamine ~1 Exchange (1V max) J
Adriamycin (doxorubicin) r
Chlorpromazine r
Tetracaine, Dibucaine, Ethanol s
Verapamil, D600 Ki(Verapamil)= 50-200 flM; KiCD600)= 22 flM t
Quinidine u
Polymyxin B g
Quinacrine K i = 10-50 flM v,w
Bepridil K i =30 flM x
Dichlorobenzamil K i =4-17flM y,w
Harmaline K i =250 flM z
Methylation a
KB-R7943 Ki(lNalCa)= 17 flM inward, 0.3 flM outward ~
a) Bers et al., 1980; Caroni et al., 1980, Philipson & Nishimoto, 1980, b) Philipson et al., 1982, c) Caroni
& Carafoli, 1983; DiPolo & Beauge, 1987, d) DiPolo & Beauge, 1988, Hilgemann, J990, e) Hilgemann &
Ball, 1996, I) Miura & Kimura, 1989; Weber et al., 2001, g) Philipson & Nishimoto, 1982b, h) Philipson &
Nishimoto, 1984, i) Philipson, 1984, j) Philipson et al., 1985, k) Reeves et al., 1986, I) Trosper &
Philipson, 1984; m) Philipson & Ward. 1985; Philipson & Ward, 1987; Vemuri & Philipson, 1987, n)
Trosper & Philipson, 1983,0) Hobai et aI., I997a, p) Li et aI., 1991, q) DiPolo & Beauge, 1994; Khanan-
shvili et al., 1993, r) Caroni et al., 1981, s) Michaelis et al., 1987; Michaelis & Michaelis, 1983, t) Van
Amsterdam & Zaagzma, 1986; Kosnev et al., 1989; Kimura et aI., 1987, u) Ledvora & Hegvary, 1983;
Mentrard et al., 1984, v) De la Pena & Reeves, 1987, w) Bielefeld et al., 1986, x) Garcia et al., 1988, y)
Kaczarowski et al. 1985, z) Su1eiman & Reeves, 1987, a) Vemuri & Philipson, 1988b, ~)Watano et al.
1996.
Phosphorylation
Evidence that Na/Ca exchange can be stimulated by phosphorylation in squid giant axon
(see DiPolo & Beauge, 1991) has generally not extended to cardiac Na/Ca exchange despite
attempts to evaluate this sort of effect (Philipson & Nicoll, 2000). There are a couple of
exceptions to the predominantly negative results with respect to phosphorylation. Iwamoto et aI.
(1995, 1996b, 1998) detected effects ofPKC on cardiac and smooth muscle Na/Ca exchange that
were initially attributed to phosphorylation, but the functional effects were modest and the latter
work indicated that NCXI phosphorylation was not required. Stimulatory effects reported for
Phenylephrine, angiotensin 11 and endothelin-I may be via PKC (Ballard & Schaffer, 1996). Fan
et al. (1996) found that PKA activation can inhibit Na/Ca exchange activity in frog heart, an
effect which might be due to a unique exon present in the frog, but not mammalian Na/Ca
exchanger (Shuba et al., 1998).
VmaT VS. Ca Requirements, Site Density and Localization
To appreciate the contribution of Na/Ca exchange to cellular Ca fluxes, let us consider
the rate at which this system transports Ca. In SLY, Ca transport rates of ~25 nmol/mg
protein/sec are typically observed (Reeves & Philipson, 1989). Assuming 30-fold purification of
SLV and 120 mg tissue proteinig wet wt tissue this would be -240 ftmol/L cytosol/sec. INalCa of
-300 pA (or 3 A/F) are also typical in mammalian ventricular myocytes (e.g. Miura & Kimura,
1989), corresponding to 200 ftmol/L cytosol/sec. Based on the rate of Ca extrusion during
caffeine-induced contractures maximal physiologicalCa efflux is -50 ftmol/L cytosol/sec
(Bassani et al., 1994a). Thus Ca transport rates under physiological conditions probably are
<100 Ilmol/L cytosol (1 A/F). However, when outward INa/ca is driven by saturating
concentrations of substrate (100 mM [Na]; & >10 mM [Ca]o) and large positive Em the maximal
I a/Ca can be 30 A/F or 10-20 times higher (Li & Kimura, 1990; Hilgemann, 1990). It is under
these real V max conditions that the turnover rate of 5000/sec was estimated (Hilgemann et aI.,
1991). This is much higher than most membrane transporters such as Na/K-ATPase, which has a
maximum turnover rate of 60-200/sec (Freidrich et al., 1996). The site density for Na/Ca
exchangers is 200-400/ftm2 (Hilgemann et aI., 1991). This is lower than the value of 1000/ftm2
which can be inferred for the Na/K-ATPase (Colvin et al., 1985), but 10-20 times higher than the
density of DHPR and functional Ca channels (see pg 114). There is some controversy about the
spatial distribution of Na/Ca exchange molecules in the ventricular sarcolemma. Frank et al.
(1992) indicated that Na/Ca exchangers are located preferentially in T-tubules, while Kieval et
al. (1992) found a more uniform sarcolemmal distribution.
Under physiological conditions the Na/Ca exchanger probably only reaches a turnover
rate of -300-500/sec (because of non-V max conditions). While this is still fast flux for an ion
pump, it is more than 1000 times smaller than an L-type Ca channel (or RyR). A single Ca
channel current of 0.3 pA at 2 mM [Ca]o corresponds to 106 Ca ions/sec (-3000 times larger).
Thus, while the normal cellular peak ICa (6 A/F) and INa/ca (I A/F) may differ by only a factor of 6
(and for Ca flux by only a factor of 3), it will take -1000 times as many Na/Ca exchange
molecules to carry out this Ca transport. This has important implications with respect to spatial
aspects of Ca signaling which are important in E-C coupling. For example, the opening of a
single Ca channel in the junctional cleft will bring in 1000 Ca ions/ms in a membrane area of
0.0003 ftm 2, whereas -3000 Na/Ca exchange molecules would be required to produce the same
Ca influx. These Na/Ca exchange molecules (even at 400/ftm2) would occupy -811m2 of
membrane or a disc of 2.5 11m diameter, which is more than 10 times larger that the whole
junctional region and more on the scale of an entire sarcomere. Thus, Ca entry via Ica is 25,000
times more "focused" than INa/ca, making it a much more effective way to elevate local rCa]; to
relatively high levels. Na/Ca exchange is still an important quantitative mode of Ca transport
during the cardiac cycle, it is simply less capable of producing local rCa]; spikes than is Ica .
The ability of Na/Ca exchange to extrude Ca in ventricular myocytes varies in different
species. Sham et al. (I 995b) found the amount of inward INa/C. to vary with hamster >guinea-pig
> rat ventricle. I would extend this provisionally based on our data on Na/Ca exchange-
dependent rCa]; decline of caffeine-induced contractures in several papers to be hamster>guinea-
pig >rabbit >ferret >cat >dog >mouse ~ rat. Su et al. (1999) found an almost inverted sequence
when measuring outward INa/Ca induced by abrupt Na o withdrawal (mouse >rat > rabbit >dog >
human). It is not clear why these observations differ, but the former data are for forward mode
(Ca extrusion) whereas the latter are for Ca-influx mode. It is possible that this discrepancy is
partially attributable to higher [Na]; in rat and mouse, which favors outward INa/Ca'
Ca Entry via Na/Ca Exchange and Contraction

Rapid reduction of [Na]o around intact cardiac muscle cells causes large contractions
attributed to Ca influx via Na/Ca exchange (e.g. Chapman & Tunstall, 1980). However, this
doesn't address whether Ca entry via Na/Ca exchange can contribute appreciably to force
development under more physiological conditions. Many results indicate that Ca entry via
Na/Ca exchange can contribute quantitatively to the direct activation of the myofilaments, since
contractions and Ca transients can be activated by action potentials and long depolarizing voltage
clamp pulses even when both sarcolemmal Ca channels and SR Ca release are inhibited (e.g.
Eisner et al., 1983; Cannell et aI., 1986; Hume & Uehara, 1986b; Barcenas-Ruiz et al., 1987;
Bers et aI., 1988). However, with [Na]; of only ~ 7 mM very large or very long dep01arizations
are required (Fig 71).
In intact rabbit ventricular muscle, we showed that action potential activated contractions
can be nearly abolished by blocking Ca channels with nifedipine (Bers et aI., 1988). However, if
[Na]; is elevated (e.g. to 15-20 mM), large twitch contractions can still be elicited in the presence
ofnifedipine (see Fig 124). We concluded that Ca entry via Na/Ca exchange does not normally
contribute significantly to the activation of contraction, but can if [Na]; is elevated by inhibition
of the Na-pump. There was also indirect evidence to suggest that, with elevated [Na];, Ca entry
via Na/Ca exchange could trigger Ca release from the SR. While Cannell et al. (1987) indicated
that this may not occur when [Na]; is only ~ 7 mM, Leblanc & Hume (1990) showed that Ca entry
via Na/Ca exchange may induce SR Ca release. In the latter case, they suggest that tetrodotoxin-
sensitive Na entry may increase the local subsarcolemmal [Na]; thereby activating Ca entry and
SR Ca release. The possible role of Na/Ca exchange in SR Ca release will be discussed further
in Chapters 8 & 10, but this is probably only relevant when [Na]; is high.
Thermodynamic Considerations
Before further discussion of cellular Ca flux via Na/Ca exchange it is worth considering
the thermodynamic basis that governs net transport. If there is more energy in the inwardly
directed electrochemical gradient for 3 Na ions than for one Ca ion, Ca extrusion via this coupled
transporter is thermodynamically favored (Mullins, 1979). That is
(6.1)
30
[Na];= 8.9 mM
peak [Cali = 1 IJM
0
........~
~ -30
2.0
~
15
W
E
'0
.!!!.
-60 1.0
;:
.5 ~
-90
..
--;;
(.)
z
U;I 20
40
0
,
100 200
time (ms)
300 400 o 100 200
time (ms)
300 400
.5
E •'\ tCainflux
!!:!.
.
0 - .......... tCa efflux
Cl>
-20 " --'
,,
(.) I
0
I
' ' ' \ , INa/Ca 0.5 ~
u. -40 2!
OJ --'
c: -60 \ ~------------
1.0
:~ --- Em -ENa/Ca
-80
0
Figure 72. Changes in ENa/ca and INa/c, during an action potential in rabbit ventricle. When Em> ENa/Ca,
Ca influx via the Na/Ca exchanger is thermodynamically favored (shaded areas). When Em < E Na/C" Ca
extrusion is favored. Resting [Ca];=150 nM, [Ca]o=2 mM and [Na]o =140 mM for all traces, with peak
[Cali (at 40 ms) indicated. Na activity coefficient of 0.786 is assumed (i.e. [Na]; of8.9 & 12.7 implies aNa;
of? & 10 mM). ENa/c, is from Eq 6.2 and current uses the Luo & RudX (1991) equation:
IN,/c,= kNa/ca {e~vk[Na]i3[Ca]o - e(~-I)Vk[Na]o3[Ca];} }I {(K mN / + [Na]o3)(K mea +[Ca]o)(1 +ksate(~-I)Vk)},
with kNa/ca= 2000 NF, K m.N,=87.5 mM, Km.c,=1.38 mM, ksat = 0.25, 11=0.35 and k=F/RT=I/(26 mY).
where n is the coupling ratio and E ca and EN, are the equilibrium potentials for Ca and Na (Ex =
(RTIzF) log ([X]J[X]i)' Then for n=3, the potential at which the gradients are equal (ENa/ca ) is
the INa/co reversal potential or the Em at which the INa/co is zero (exactly analogous to the reversal
potential of an ion channel). Hence,
ENa/Ca = 3ENa - 2E ca (6.2)

Thus, whenever Em is more positive than ENa/co Ca entry via the exchanger is favored and when
Em is negative to ENa/ca Ca extrusion is favored.
Figure 72 illustrates how ENa/co may be expected to change during the action potential in
ventricular muscle under normal conditions. The shape of the ENa/co curve is dictated here by the
[Cali transient (which alters E ca). For typical diastolic values of [Na]o and [Na]; (140 and 8.9
mM respectively; E Na = +73 mY) and [Ca]o and [Cali (2 mM and 150 nM, respectively; E ca =
+125.8 mY) ENa/co would be -32.6 mV. Thus at a resting Em of -80 mV Ca extrusion and inward
INa/co would be thermodynamically favored (ENa/ca >E m). During the action potential, there is a
very brief period where Ca influx via the exchanger is favored (Em>ENa/ca, shading in Fig 72). It
can be appreciated that the precise length of this period is rather sensitive to changes in peak
[Cali, the time course of the Ca transient, [Na]; and the shape of the action potential. The lower
panels show how the thermodynamic driving force (Em - ENa/ca) and INa/ca changes during the AP.
This illustrates the dynamic, yet delicate balance of Ca fluxes mediated by the NaiCa exchanger.
Indeed, increasing intracellular Na activity from 7 to 10 mM (8.9 to 12.7 mM [Na];) in the right
panel prolongs the time when Ca influx would be favored, even though peak [Cal; is much higher
(which by itself would favor Ca efflux).
While the simple thermodynamic consideration above can be sufficient to predict the
direction of Ca transport by Na/Ca exchange and the driving force, the INa/ca amplitude is also
subject to kinetic limitations (depending on substrate concentrations). Indeed, as [Cali declines,
the inward INa/ca in Fig 72 is not as large as might be expected from the driving force. That is, the
driving force for Ca extrusion via Na/Ca exchange during diastole is large, but the net Ca
extrusion is limited by the low diastolic [Cali' Several quantitative models ofINa/ca have included
these considerations (e.g. DiFrancesco & Noble, 1985; Hilgemann, 1989; Beuckelmann & Wier,
1989; Matsuoka & Hilgemann, 1992; Luo & Rudy, 1994a). Figure 72 uses the equation ofLuo
& Rudy (I 994a), but I now prefer a more complete version:
(6.3)
where K mAJlo is the K m for allosteric Ca;-activation (125 nM). Other constants are transport Kms
for Na & Ca inside and out (in mM): K mcao (1.4), K mca ; (0.0036), K mNao (88), K mNa ; (12). The ks",
(0.27) assures saturation at very negative Em, 11 (0.35) is the energy barrier position controlling
Em-dependence of INa/ca and k=F/RT (V max ~ 10-20 A1F). We developed this equation during
studies of allosteric regulation (first factor in numerator) OfINa/Ca by [Cal; (Weber et al., 2000).
The INa/ca analysis in Fig 72 is simplified because it assumes that [Cal; is uniform during
the Ca transient. However, during the twitch there are likely to be spatial gradients of [Cal near
the membrane and sites of SR Ca release (Langer & Peskoff, 1996). Trafford et al. (1995) used
INa/ca to demonstrate that the local subsarcolemmal [Cali ([Ca]sm) sensed by the Na/Ca exchanger
differed from the global [Cal; during caffeine-induced Ca transients. Figure 73 shows this type
of experimental analysis. Figure 73B shows [Cal; and INa/ca plotted against each other for each
time point in Fig 73A. During the rising phase of the Ca transient (Activation) INa/ca amplitude is
much larger than for the same [Cali during relaxation. Using INa/ca as a bioassay for local [Cali
and assuming that [Cali = [Ca]sm during the latter part of [Cal; decline (regression line), we can
infer [Ca]sm from INa/ca for any point. Figure 73C shows that [Ca]sm rises earlier and to a much
higher peak value than [Cali sensed by the bulk cytosolic Ca indicator. This emphasizes the
utility of electrophysiological indicators of local [Cal. It also indicates that large spatial
gradients of [Cal; exist during what appear to be relatively homogeneous signals from fluorescent
Ca indicators (see also Fig 60, pg 122, where lea was used in a similar manner).
During a normal twitch these gradients could be even faster and steeper due to the rapid
and highly synchronized twitch SR Ca release compared to that induced by caffeine. Indeed,
Egan et al. (1989) interrupted the action potential by repolarization and used tail INa/ca to predict
the twitch Ca transient sensed by the Na/Ca exchanger. Figure 74 combines these considerations
into the context of Fig 72 concerning INa/ca during the cardiac action potential. The Ca transient
in Fig 74A produces a change in ENa/Ca that is between the two cases shown in Fig 72, and the
ISO D.M. Bers Cardiac E-C Coupling
;,~tJ~I\
3000
2000
~500 ~ ~
:!:
.s
a 7;
-.eo tpicaffeme
l)
« -100 _ I ~ 1000
~ -200 I 2 sec I
!J. -300
B-100
~~~
~Z
! -200
----.-,.-----------.- ..... --- .. --.--_ ..
-300
__ L~r~~.i~t~I}R!<:)CC!I.Lc;C!l.l _. _
: from INa/Ca :
f ;
o 500 1000 1500 2000 2500 3000

[Cali (nM)
Figure 73. Submembrane [Cal predicted from INa/c,. A. caffeine-induced Ca transient and INa/c, in a rabbit
ventricular myocyte. B. INa/c, plotted as a function of global rCa]; for the traces in A (note that during
activation INalC, is larger in amplitude than for the same [Cali during [Cali decline). During [Cali decline the
rCa]; is assumed to directly control INa/c., such that a linear regression (between resting [Cali and [Cali
decline below 800 nM) predicts local submembrane [Cali ([Ca]snJ' Dotted lines indicate [Ca]sm sensed by
the Na/Ca exchanger at 3 different points before the steady state (SS) relation is assumed. C. Complete plot
of [Ca]sm and [Cali for this caffeine-induced Ca transient (data acquired by K.S. Ginsburg).
INaiC • in Fig 74C (driven by global [Ca]i and [Na];) is similar to that in Fig 72. However, if we
instead allow the [Ca]sm in Fig 74B to drive I alC, the initial outward current only lasts 0.3 ms
instead of 75 ms (and reduces total Ca influx from 0.3 to 0.01 flmol/L cytosol). Thus the high
local rCa]; in the junctional cleft (produced by lc. and SR Ca release) can shift Na/Ca exchange
rapidly into the Ca efflux mode. Thus high local rCa]; may limit any net Ca entry via Na/Ca
exchange during the AP. On the other hand, there may also be a transient increase in
subsarcolemmal [Na]; ([Na]sm, Fig 74B; Leblanc & Hume, 1990). The high [Na]sm favors more
outward INa/C. and in the example in Fig 74C this increases the peak and prolongs the outward
IN,/C. duration to 3 ms (with total integrated Ca influx of 0.08 flmol/L cytosol). While this by
itself does not preclude a potential triggering role for outward INa/ca, the total Ca influx via Na/Ca
exchange is negligible with respect to total cellular Ca fluxes.
It seems probable that under normal conditions the Na/Ca exchanger serves mainly as a
means of Ca extrusion and that Ca current is the main means of Ca entry into cardiac myocytes.
Figure 75 shows the quantitative functional relationship of Ic• and I alC. when SR function was
suppressed by 10 mM caffeine (Bridge et al., 1990). A guinea-pig ventricular myocyte was
voltage clamped using a dialyzing pipette containing Na-free, 130 mM CsCI solution to prevent
Ca entry via Na/Ca exchange and K currents, respectively. Depolarization in the absence ofNao-
Chapter 6 Sarcolemmal Na/Ca exchange and Ca-pump lSI
A 30 Em 1.0
,~-~ .8 '0
:;- I /- . . .
E.. I .......... ~
:c
--- ---
w
E ·30 I ..... .....[Ca]; .6
I ..... $
I .......... .4
,
·60
I
I .2
·90
o 200 300 400

B
~
15
~
E..
~ 10
o
~
[Ca]sm
(Cal;
f\
100 200 300 400
c !DriVen by [Cal sm & (Nal sm ICal,m & INal,m
ICa); & (Na);

" \" Doven by (Ca); &.(Na);. "
.
~
3- -1
\,
-2 Driven by {Cal sm
& INa);
100 300 400 0 10 20 30 40
time (ms)
Figure 74. Local [Cali and [Na]; effects INa/C. during cardiac action potential (AP). A. Bulk [Cali and
ENa/c. during a normal AP in rabbit ventricular myocyte. B. Submembrane [Cali ([Ca]sm) and [Na]; ([Na]sm)
predicted using exponential time constants of rise (2 and I ms respectively) and fall (30 and 8 ms,
respectively) where the [Ca]sm was added to the global [Cali (which rises and falls with time constants of30
and 200 ms respectively). C. INa/C. calculated from Eq 6.3 using the different [Cal & [Na] values as
indicated and the same parameters as Fig 72, except ENa/c. =700 NF and ksat = 0.1 (expanded time scale at
right). D. Cartoon showing how local [Cal and [Na] in the junctional region are interdependent.
activated inward Ic • and cell contraction. Repolarization by itself did not induce relaxation until
Na o was added using a quick solution switch. This relaxation was associated with an inward
INa/C. producing Ca extrusion and relaxation. Integration of the nifedipine-sensitive Ic • and the
I Na/C • (Fig 7SB) showed twice as much Ic • as INa/Co> consistent with all Ca entry via Ic • (2 charges/
Ca) and all extrusion via a 3Na: 1Ca Na/Ca exchange (I charge/Ca). This demonstrates the
ability of Ca influx via Ic• to support contraction and INa/C. to extrude Ca and produce relaxation.
Keep in mind that under normal twitch conditions (with functional SR) the Na/Ca exchanger
removes only 8-30% of the total activator Ca during relaxation. As stressed in Chapter 3 the
amount of Ca extrusion from the cell must be matched by the Ca influx per beat for the cell to
remain in steady state Ca balance. Since Ca influx via Na/Ca exchange is likely to be very small
(Fig 74), Ica provides almost all of the Ca influx.
145 111M
[Naja
A B
'60
•
I'·'
0-
,A
n=4
'20
~F
lea W
too n=3
~
z 40
...:::.
1,·0
microna
00 120 '00
!ICa'dl(nA.mS)
.v
-~ -40
~
Figure 75. Ca entry via lea and Ca efflux via I NaiCa . A. A voltage clamped guinea-pig ventricular
myocyte was equilibrated with 10 mM caffeine to prevent SR Ca uptake and release. The ICa associated
with the 2 sec depolarizing pulse in Na-free 2.7 mM Ca solution activated a contraction. Relaxion was
induced when [Na]o was rapidly returned, activating Ca extrusion via l'Jaica. B. The integral of lNaiCa is
plotted as a function of the integrated lCa . The jlc. is roughly twice the lNaiCa as would be expected for a
Na/Ca exchange coupling ratio ofn=3. (from Bridge et al., 1990, with permission).
COMPETITION AMONG Na/Ca EXCHANGE, SARCOLEMMAL

Ca-PUMP AND SR Ca-PUMP DURING RELAXATION AND AT REST
As discussed in Chapter 3 (pg 52-54), during relaxation there is dynamic competition
among Na/Ca exchange and the SR Ca-pump, sarcolemmal Ca-pump and mitochondrial Ca
uniporter. During relaxation the SR Ca-pump is generally dominant over the Na/Ca exchanger in
mammalian ventricle, but this dominance varies between 2-3:1 in rabbit and guinea-pig to >10:1
in rat and mouse. Table 20 lists estimates of the quantitative contribution ofNa/Ca exchange vs.
other Ca transporters to relaxation and [Ca]; decline during twitches and also during caffeine-
induced contractures (where net SR Ca uptake is blocked). During a twitch, the Na/Ca exchange
contributes a variable amount toward relaxation or [Ca]; decline (3 - 47%) depending on species
and condition. Almost all of the remaining Ca removal is due to the SR Ca-pump (50-96%), with
the combined action of the sarcolemmal Ca-pump plus mitochondrial uniport never being as
much as 10%. During caffeine-induced contractures or twitches when the SR Ca-pump is
blocked with thapsigargin the Na/Ca exchange is by far dominant (75-93%). This suggests that
Na/Ca exchange is several times more effective in mediating Ca efflux from the cell than is the
sarcolemmal Ca-ATPase (see also below).
Figure 76 shows paired rapid cooling contractures (RCCs), a complementary approach to
that shown in Fig 30 for evaluating the competition between the SR Ca-pump and Na/Ca
exchange during relaxation (Hryshko et ai., 1989c; Bers et at., 1989). Rapid cooling of cardiac
Table 20
Ca Transport During Ventricular Myocyte and Muscle Relaxation
Percent of Relaxation/ [Ca]j decline due to:
Species Temp --Twitch--- --Caffeine/Thapsigargin--
ref (0C) NCX SR Slow NCX SL-pump Mito
Rabbit [Cali a 22 28 70 2 93 3 3
b 35 27 70 3
b 25 23 74 3
g 22 91 6 3
Rat [Ca]i a 22 7 92 I 87 -13-
c 27 9 87 4 68 -32-
d 23 8 87 5 80 20 0
Mouse [Ca]j e 23 9 90 I
(PLB-KO) e 23 3 96 <1
Rat neonate rCa]; f 22 46 50 4
Ferret [Ca]i g 22 29 65 6 75-85 14-20 4
Rabbit Relax b 25 28 70 2
b 35 21 76 3
Ferret Relax b 25 30 63 7
b 35 28 67 5
Cat Relax b 25 47 51 2
b 35 32 66 2
Dog rCa]; I 37 27 73
Failing rCa]; I 37 56 44
Paired RCCs
Rabbit Relax h 29 23 76
Rabbit Relax h 29 27 73
Guinea-pig [Ca]i 30 36 64
Guinea-pig rCa]; J 22 30 67 3
Human Normal k 37 37 63
Human Failing k 37 42 58
PLB-KO, phospholamban knock out, NCX=Na/Ca exchange, SR=SR Ca-pump. Slow=combined sarco-
lemmal Ca-ATPase (SL-pump)+mitochondrial uniporter (Mito). Caffeine/thapsigargin=relaxation with non-
functional SR. In some cases, the Slow sytems were not assessed, so all is assumed to be SR+NCX. a)
Bassani et al. 1992, 1994a, b) Puglisi et al. 1996, c) Negretti et al. 1993, d) Choi & Eisner, 1999, e) Li et
al. 1998, I) Bassani et al., 1994c, g) Bassani et al. I 994b, 1995a, h) Hryshko et al. 1989, i) Bers et al.
1989, j) Terracciano & MacLeod, 1994, 1997, k) Pieske et al. 1999. I) O'Rourke et al., 1999.
muscle to ~0-1 °C in <I sec causes release of all SR Ca while simultaneously inhibiting Ca
transport mechanisms (see also details in Chapter 7). This [Ca]j results in slowly activating
contractures at this temperature and the RCC amplitude is a useful index of the amount of Ca
which was in the SR at the time of cooling. Rewarming the muscle re-activates Ca transport
systems (e.g. SR and sarcolemmal Ca pumps and Na/Ca exchange) allowing relaxation to occur.
In Fig 76 we lIsed paired RCCs such that the second RCC (RCC2) is used to assess the
fraction of Ca released at the first RCC (RCC l ) which was resequestered by the SR during
relaxation of RCC I . For example, if all of the Ca released from the SR at RCC I were
resequestered during relaxation, RCC 2 should be the same amplitude as RCC I (RCCiRCC I
=100%). This is essentially the case in Fig 76B where the rabbit myocyte was bathed in Na-free,
n
A. NT B.ONa,OCa
'~'e" ~
uRft
c
o
n 10
uM
• Ul
1
~
co
o
55 NT I 0 Na, 0 Ca
C o
NT ONa,OCa
=====-==:::-:======1 °C
[Cali ~
h. " RCC RCC, RCC3
~CC~C~
Figure 76. Paired rapid cooling contractures (RCCs) used to assess the resequestration of Ca by the SR.
A) Paired RCCs induced after a train of electrically evoked twitches in a rabbit ventricular myocyte. RCC l
is smaller because some of the Ca released during RCC I is extruded by Na/Ca exchange during relaxation.
B) When Na/Ca exchange is prevented during rewarming (by Na-free, Ca-free solution), the SR can
resequester all of the Ca released at RCC, (so RCCl/RCC, is I). C and D) A similar protocol in a guinea-
pig ventricular myocyte except that [Cali was measured (using indo-I) and three RCCs were used instead of
two (from Hryshko et al., 1989c (top) and Bers et al., 1989 (bottom), with permission).
Ca-free solution during cooling and rewanning (to prevent Na/Ca exchange). However if Na/Ca
exchange was allowed to compete with the SR (Fig 76A), RCCiRCC j ~75%. This again implies
that Na/Ca exchange extrudes an amount of Ca responsible for ~25% of developed tension in the
presence of an intact SR. Fig 76C-D show similar data for rCa]; during triple RCCs in guinea-pig
ventricular myocytes. In this case Na/Ca exchange reduced the amplitude of the second and third
Cal transients by 36% and 51 % (with respect to the preceding RCC).
Figure 77 shows the voltage-dependence of relaxation mediated by the SR and Na/Ca
exchange (Bers & Bridge, 1989). When relaxation was mediated by Na/Ca exchange (in the
presence of caffeine) relaxation was highly voltage-dependent (voltage clamp data are also
shown from Bridge et at., 1988). This is expected on thermodynamic grounds, since Ca
extmsion via Na/Ca exchange is favored by more negative Em (see above). A slight acceleration
of relaxation by nifedipine at depolarized Em raises the possibility of a small window ICa at these
potentials which could slow relaxation. In contrast to the results with Na/Ca exchange,
relaxation mediated by the SR (in Na-free, Ca-free solution) was entirely independent of voltage.
Crespo et at. (1990) reached similar conclusions, by comparing the slow phase of [Ca]; decline in
ryanodine-treated guinea-pig ventricular myocytes (attributed to Na/Ca exchange) with the rapid
rCa]; decline in rat ventricular myocytes (attributed primarily to SR Ca uptake).
Em-dependence of Relaxation in
Ventricular Myocytes ,"!" 1,0
o
-....
'5":::0
CD
3~
+ Nifed
N"--o'
III III
~ ::::l
-
DO .5-:::0
* III
CD
SR-dependent (ONa)
II •
-80 -60 -40 -20 o

Em (mV)
Figure 77. Voltage dependence of relaxation when Na/Ca exchange is prevented. RCCs and rewanning
of rabbit ventricular muscle were done in solution which either prevented SR Ca reuptake by 10 mM
caffeine (eo) or blocked Na/Ca exchange by ONa,OCa solution (_0, Em was varied by altering [KJo during
the RCC and rewanning). Parallel data were obtained with 10 11M nifedipine to block lea (0 0, data from
Bers & Bridge, 1989). Also shown are voltage clamp data (* from Bridge et aI., 1988) from guinea-pig
ventricular myocytes where relaxation rate was measured in the steady presence of 10 mM caffeine.
Figure 78 shows an experiment in which the Nao-dependent component of relaxation was

examined in a voltage clamped rat ventricular myocyte where [Cali was measured using indo-l
(Bers et ai" 1990). The cell was depolarized from -50 to + 15 mV for 50 msec (top) or 1 sec
(bottom) in the presence (left) or absence (right) of 145 mM Na o. After repolarization to -50 mV
the decline of [Cali for both short and long pulses was monoexponential and the time constant ('t)
was slowed by ~20% by blocking Na/Ca exchange (O[Na]o). During the long pulses at +15 mV
[Cali also declined along a monoexponential, but was relatively insensitive to [Na]o' This is
consistent with the voltage dependence expected of Na/Ca exchange, where Ca extrusion is less
favored at more positive Em (such that Ca is preferentially pumped into the SR).
Figures 76-78 demonstrate that the Na/Ca exchange can compete with the SR Ca-pump
during the relaxation of cardiac contraction with the Na/Ca exchanger being responsible for -20 -
30% of the decline in [Cali or force during cardiac relaxation. Despite the general similarity of
the numbers it should also be appreciated that this fraction will vary depending on certain
variables such as the action potential configuration, the resting rCa]; and [Na];, peak [Ca];, the
timecourse of the Ca; transient and the state of the SR and the Na/Ca exchanger itself. For
example, the long depolarization at +15 mY in Fig 78 (bottom left) biases the competition
between the SR Ca-pump and the Na/Ca exchange in favor of the SR, Indeed, the next
contraction and Ca transient after that pulse was larger, reflecting the enhanced SR Ca load. On
the other hand, if the cell and SR are already heavily Ca loaded, the decline of [Cali can still be
accelerated by Na o at + 15 mV (see Fig 10 in Bers et al., 1990),
0.7 145 mM [Naja o [Naja

:J-j' -50mV Em
:2 0.5
2:
<il'
~ 0.3
...JI---"- _
:2 0.5
::l.
<il'
~ 0.3
0.1 1.0 s 1.0 s
~ ~fr:::===:J--
1
0
c -1.0
.s
~ r
<3 -2.0
Figure 78. The effect of Na o on the decline of [Cali in a rat ventricular myocyte with depolarizing pulses
from -50 mY to +15 mY for 50 msec (top) or 1 sec (bottom). The dialyzing patch-clamp pipette contained
<0.5 mM Na to prevent Ca influx via Na/Ca exchange and 70 IlM indo-l to assess [Cak During the long
pulses and after repolarization, the [Cali was fit with a monoexponential decline (curves superimposed and
time constants 1: indicated). During the long depolarizations there may be a small residual Ca influx via Ca
channels (see current records at bottom). (from Bers et at., 1990, with permission).
Resting Ca efJlux from Cells

Finally, let us consider the relative roles of Na/Ca exchange and sarcolemmal Ca-pump
in diastolic efflux of Ca. Indeed, without energy-dependent Ca extrusion the resting Em would
drive [Ca]j to nearly 1 M at equilibrium! This is emphasized by the work of Choi & Eisner
(1999). They showed that when Na/Ca exchange and sarcolemmal Ca-pump are both blocked
(by ONa o and carboxyeosin) raising [Ca]o from 0 to 0.1 mM caused large [Ca]j increases.
Moreover the rate of diastolic Ca influx into cells has been estimated to be 0.5-3 /lmol/L
cytosol/sec (Diaz et a/., 1997a; Trafford et at., 1997). This would rapidly load the cell if the Ca
extrusion mechanisms were not able to counterbalance this Ca influx at resting [Ca];.
Philipson & Ward (1986) extrapolated SLY results and concluded that Na/Ca exchange
and the sarcolemmal Ca-ATPase pump were likely to extrude Ca from the cell at comparable
rates. Barry and Smith (1984) suggested that a Nao-independent mechanism was responsible for
the major fraction of 45Ca efflux from cultured chick heart cells. These initial studies were
complicated by undetermined Ca-Ca exchange and Barry et at. (1986) later concluded that this
Nao-independent component of 45Ca efflux was only ~20% of the Ca efflux via Na/Ca exchange.
Rest Decay of SR Ca content
U 100
U
ONa,OCa
....III ....III
0:::: Q)
....1/1
Ii
....
U "0
>.
-
III 50
...0 ....
<l Q)
1/1
....
.c
en ~
0
c: ~
..9! ~Iength
Ryanodine
<l
a
a 1 234 5
Rest Duration (min)
Figure 79. Rest decay of SR Ca relies on Na/Ca exchange. After steady state 0.5 Hz stimulation guinea-
pig ventricular myocytes were rested in either normal Tyrode's or aNa, OCa solution (to block Na/Ca
exchange). RCCs were used to assess SR Ca content as either tl[CaJj (indo-I) or cell shortening (L\length).
Ryanodine (I f.lM, 20 min,.) did not abolish RCCs, but greatly accelerated the rate of rest decay (see
Chapter 7) (data from Bers et al., 1989).
We have generally found that blocking either Na/Ca exchange or the sarcolemmal Ca-
ATPase does not produce a significant rise in resting [Ca]j, although blocking either one may
slow the decline in rCa]; during repetitive stimulation (since Ca enters with each pulse). Most
would concede that the Na/Ca exchange is dominant over the sarcolemmal Ca-pump during rCa];
decline. However, some posit that the sarcolemmal Ca-pump may be especially important in
governing diastolic Ca efflux because it can have a higher affinity (Km(Ca)~60 nM, see Table 18)
than the Na/Ca exchange (Km(Ca) ~I f.lM).
We have examined which Ca transport system functions during prolonged rest, when
[Ca]j remains low (Bers et aI., 1989; Bassani et al., 1994b; Bassani & Bers, 1994). During rest
there is some finite rate of Ca leak from the SR lumen to the cytoplasm. Ca in the cytoplasm is
then subject to either extrusion from the cell (via Na/Ca exchange or the sarcolemmal Ca-pump)
or resequestration by the SR (for the moment ignoring mitochondrial uptake). lf all of this Ca is
resequestered by the SR, then the Ca content of the SR would stay constant at rest. lf some of
this Ca is extruded from the cell, then the SR Ca content would progressively decline during rest.
Thus the rate of decline of SR Ca content during rest provides an index of how well the trans-
sarcolemmal Ca transporters compete with the SR Ca-ATPase (reflecting cellular Ca efflux by
the Na/Ca exchange or sarcolemmal Ca-pump).
Figure 79 illustrates the process of rest decay in guinea-pig ventricular myocytes. RCCs
were induced at various times after the last stimulated twitch and Ca transients and myocyte
contraction were recorded (Bers et al., 1989). With longer rest intervals in normal Tyrode's (NT)
the amplitude of the RCC and accompanying Ca transient are progressively smaller. When the
rest period is in Na-free, Ca-free solution (ONa,OCa ) there is almost no decline in the amplitude
Rest Decay of SR Ca in
Rat, Rabbit & Ferret
...
01
::J .-.
----- Rat--NT
-I-
~Z 100 , -D- Rat-DNa,DCa
-n.__!"
c1Q
... 01
, , -0- Rabbit-ONa,OCa
0_
urn 90
" '~
01>-
" \
t:......\
c'O
.- III 80 ~\..... 'n-- _
~2 \ ----c:. -f::,- Ferret-DNa,DCa
Illrn
u_
-- --- -- ....
\
_0 70
.............
~~
... 0
III -.- Ferret-NT
-Ill 60
I
~-
Q.
---..;:"....,...- ~ Rabbit NT
-+-- Rat-DCa, 14DNa
50
0 234 5
Rest Duration (min)
Figure 80. Rest decay of SR Ca in rabbit, rat and ferret. After steady state stimulation myocytes were
rested in either normal Tyrode's (NT .... e), ONa,OCa solution (to block Na/Ca exchange oo,s) or ONa,
140 mM Na o (after depleting Nai, to enhance Ca extrusion by Na/Ca exchange, T). Caffeine-induced
contractures were used to assess SR Ca content. (data from Bassani et aI., I994b, Bassani & Bers, 1994).
of the post-rest RCCs and Ca transients even after 5 min of rest. This is consistent with Na/Ca
exchange being the main means by which Ca is extruded from the resting cell. Indeed, the Na-
free solution which is used to prevent Na/Ca exchange is also Ca-free to prevent Ca gain via
Na/Ca exchange in the absence ofNa o . Under these conditions the sarcolemmal Ca-pump should
be even better able to extrude Ca into the Ca-free solution. Even so, there is practically no rest
decay in the absence of Na/Ca exchange. This result demonstrates that in intact guinea-pig
ventricular myocytes, Ca efflux during rest (as well as during relaxation) is mainly via Na/Ca
exchange with the sarcolemmal Ca-pump making at most a very small contribution.
Figure 80 shows that the same results are obtained in rabbit ventricular myocytes (in this
case using caffeine-induced contractures to assess SR Ca content after rest. That is, DNa, DCa
during rest completely abolishes resting loss of SR Ca in rabbit (0 vs. e). In rat ventricular
myocytes SR Ca content is stable during rest and usually does not decline (., Bassani & Bers,
1994). This may be due to the relatively high [Na]j reported in rat (16 mM or aNaj =12.7 mM)
which would place E NaiCa at or below resting Em (-80 mY), such that Ca extrusion via Na/Ca
exchange is not thennodynamically favored (Shattock & Bers, 1989; see also Chapter 9, Fig
140). In this case blocking Na/Ca exchange in rat by DNa,DCa solution (D) does not change
things much, despite the fact that the sarcolemmal Ca-pump should be unimpeded. On the other
hand, if rat myocytes are pre-depleted of Naj (by exposure to DNa,DCa) and Ca extrusion by
Na/Ca exchange is stimulated in OCao, 140 mM Na o solution (T) rat myocyte SR loses Ca during
rest as fast as rabbit. So again it seems that the Ca transport across the sarcolemma is dictated by
the Na/Ca exchanger, not the sarcolemmal Ca-ATPase. Finally, we tum to ferret ventricular
myocytes, where rest decay of SR Ca content occurs in NT as well as in ONa, OCa solution. The
inability of ONa, OCa to completely block resting SR Ca loss suggests that the sarcolemmal Ca-
ATPase does indeed contribute to resting cellular Ca efflux (perhaps about equally with the
Na/Ca exchanger). This is completely consistent with the relatively powerful sarcolemmal Ca-
ATPase reported in ferret ventricular myocytes (i.e. 5 fold higher Y max than in rabbit; Bassani et
al., 1994a,b, 1995a). Indeed, in ferret vs. rabbit we find that the sarcolemmal Ca-ATPase
contributes more significantly to twitch relaxation (6 vs. 0.5%) and to [Cali decline during
caffeine exposure (14-20% vs. 3% see Table 20). Thus, one should be careful about
generalizations and there are significant species differences in how Na/Ca exchange and
sarcolemmal Ca-ATPase interact.
Transgenic Mice and Antisense Knock-down ofNa/Ca exchange

Philipson's group generated transgenic mice overexpressing canine NCXI and myocytes
from these mice have been studied (Adachi-Akahane et al., 1997; Yao et al., 1998; Terraciano et
al., 1998; Weber et al., 2001). Exchanger activity was increased 2-3 fold based on INa/C. or [Cali
decline during caffeine exposure, whereas other Ca transport systems (e.g. Ic• and SR Ca-
ATPase) appeared unaltered. One could imagine that higher Na/Ca exchanger expression might
reduce SR Ca content, based on the foregoing discussion about the dominant role of this system
in Ca extrusion from the cell and its minor role in Ca influx. The first two groups above found
no change in either twitch amplitude or SR Ca content (based on caffeine-induced Li[Ca]; and
integrated INa/C.). This could be explicable if (as described above for the rat) EN • /C • is poised right
at the resting Em. Indeed, Yao et al. (1998) found [Na]; to be -16 mM in both wild type and
transgenic mice, giving an ENa/c. = -85 mY for [Cali = 125 nM or -80 for [Cali = 150 nM. Thus,
it is plausible that the Na/Ca exchange reaches equilibrium during diastole and doesn't then
support substantial diastolic Ca flux. Terraciano et al. (1998) found a large increase in SR Ca
content and consequently larger twitch size in the mice overexpressing NCX1. The difference is
unexplained, but even a small systematic difference in myocyte [Na]; could have a large impact.
Moreover, Yao et al. (1998) claimed the additional Na/Ca exchanger could provide triggering for
Ca-induced SR Ca-release (based on lower sensitivity to nifedipine block of twitches), while
Adachi-Akahane et al. (1997) found no evidence for Na/Ca exchange-mediated Ca-induced Ca-
release in the transgenic mice. Clearly more work is needed to clarify some of these points.
In the absence of highly selective blockers ofNa/Ca exchange some groups have used an
antisense mRNA approach to knock-down Na/Ca exchanger expression in myocytes (Lipp et al.,
1995; Slodzinski & Blaustein, 1998a,b). While the technique looks promising, it has not yet
been applied to new mechanistic questions.
In conclusion, it is evident that the Na/Ca exchange system is very important in
myocardial Ca regulation. Key fundamental characteristics of this system and its function are
becoming increasingly clear. Na/Ca exchange is the main means by which Ca is extruded from
the cell, during both relaxation and diastole. By comparison, the sarcolemmal Ca-pump seems
relatively unimportant in cardiac muscle (particularly because net Ca movement in either
direction via Na/Ca exchange would normaJly appear able to overwhelm this pump). Indeed,
Na/Ca exchange can even compete with the powerful SR Ca-pump for cytoplasmic Ca and
thereby contribute to relaxation. The Na/Ca exchange can also mediate Ca influx sufficient to
activate contraction, but this probably does not occur under nonnal physiological conditions
(where the main role of the Na/Ca exchange seems to be to extrude Ca from the cell). In fact,
Na/Ca exchange must extrude as much Ca as enters the cell via Ca current in each cardiac cycle,
in order for a steady state to be achieved. Finally, since the Na/Ca exchange is the main means
by which the cell extrudes Ca, anything which prevents this Ca extrusion will increase cellular
Ca loading and can lead to Ca overload. These issues will be addressed further in Chapter 10.
D.M. Bers. 161
CHAPTER 7
SARCOPLASMIC RETICULUM
Ca UPTAKE, CONTENT AND RELEASE
SRCa-PUMP
Kielley & Meyerhoff (1948) first described a Mg-activated ATPase in a microsomal
fraction from muscle. Ebashi (1961; Ebashi & Lipmann, 1962) and Hasselbach & Makinose
(1961) later identified this as the membrane associated Ca-ATPase or "relaxing factor" in muscle
responsible for lowering cytoplasmic [Ca). This Ca-pump has been the subject of intensive study
since that time (see reviews by Inesi, 1985, 1987; Fleischer & Tonomura, 1985; Entman & Van
Winkle, 1986; Schatzmann, 1989; Mintz & Guillain, 1997; MacLennan et aI., 1997; Anderson &
Vilsen, 1998; Tada, 2001).
Skeletal and cardiac muscle SR vesicles isolated on sucrose density gradients can be
separated into two types (Meissner, 1975; Campbell et al., 1980; Jones & Cala, 1981; Saito et
al., 1984). A heavy SR fraction is obtained at higher density and contains terminal cisternae and
the Ca-release channel (or ryanodine receptor) as well as the Ca-pump. At lower density, a light
SR fraction is obtained in which most of the protein (-90%) is the Ca-pump (Meissner, 1975;
Campbell, 1986; Fleischer & Inui, 1989). The light SR fraction probably originates from the
longitudinal SR. Thus, Ca is pumped into the SR along the longitudinal tubules (and terminal
cisternae) and is released from the terminal cisternae, where the SR "foot" processes span the gap
from the SR to the sarcolemma (see Chapter I). I will focus initially on the SR Ca-pump, while
the release process will be discussed below (pg 186). However, it should be noted that many
characteristics of the Ca-pump have been studied in mixed populations of SR vesicles (heavy
plus light). In heavy SR vesicles Ca uptake can be "short circuited" by open Ca-release channels
(which seems to be the usual state of cardiac SR release channels in isolated vesicles). Ca uptake
in cardiac heavy SR vesicles can be dramatically increased by inclusion of agents which block
the Ca release channel (e.g. ruthenium red or Mg;, Meissner & Henderson, 1987).
The sarco(endo)plasmic reticulum Ca-ATPase (SERCA) is a member of the P-type ion
transporting ATPase family (as are Na/K-ATPase, sarcolemmal Ca-ATPase and H/K-ATPase).
Two striated muscle SERCA proteins have been sequenced and cloned and exhibit 84% amino
acid identity (MacLennan et aI., 1985; Brandl et al., 1986). One of these is from fast twitch
muscle (SERCAI, MW =110,331 Da) and the other from slow twitch skeletal and cardiac muscle
(SERCA2a, which has 4 less amino acids; Brandl et at., 1986, 1987; MacLennan et al., 1987). In
fast twitch skeletal muscle the SERCAla isoform predominates in adult, whereas SERCAlb is
present in fetal and neonatal stages. SERCA2b and a third isoform (SERCA3; Dode et al., 1996)
are ubiquitous in the endoplasmic reticulum of non-muscle cells. While SERCAI and SERCA2a
have very similar properties, SERCA2b has a two-fold higher Ca affinity with slower Vmax and
Model for Calcium Transport by the Ca 2+·ATPase
Figure 81. Schematic of SR Ca-ATPase structure. Most of the protein (70%) is on the cytoplasmic side
of the SR membrane including p-strand, phosphorylation (D351, P), nucleotide binding, stalk domains and
a Hinge. Ten transmembrane spans (M l - M IO) are shown as 1-10. Amino acids on M4-M 6 and M s are
important in Ca-binding and transport (Glu-309 in M4, Glu-77 I in M s, Asn-796, Thr-799 & Asp-SOO in M 6
and Glu-90S in M s). Left shows Ca entry from cytosol and right shows Ca release into the SR lumen (From
MacLennan et at., 1992, with permission).
SERCA3 has a comparable tumover rate, but much lower Ca affinity (Lytton et al., 1992;
Verboomen et aI., 1994; Anderson & Vilsen, 1998). Figure 81 shows an early proposed
secondary structure of SERCA by MacLennan's group, indicating where ATP binds (nucleotide
binding domain), phosphorylation site (Asp-351), a p-strand domain and a hinge region. The
protein has 10 membrane spanning regions (MI-M IO), where M I-M5 each have an additional (J.-
helical "stalk" region on the cytoplasmic side (SI-S5)' Crucial high affinity Ca binding sites
initially proposed to reside in the anionic stalk region (Brandl et al., 1986), have now been
localized in the transmembrane domains (M 4-M 6 and M s, Clarke et al., 1989a,b). Figure 82
shows a newer look, based on crystal structure from X-ray diffraction (Toyoshima et aI., 2000)
greatly extending prior electron microscopic reconstructions (e.g. Stokes & Green, 2000).
The transport reaction starts with two Ca ions and one ATP molecule binding to high
affinity binding sites on the cytoplasmic side of the pump (see Figs 81 & 83). The terminal
phosphate of ATP is then transferred to Asp-351 on the Ca-pump inducing Ca ion "occlusion"
(which means that Ca cannot be readily released to either side). Based on the results of Clarke et
ai. (1 989a,b), Ca binds to sites in transmembrane regions M 4 -M 6 & M s which may form a
channel, and phosphorylation alters the structure such that Ca cannot retum to the cytoplasmic
side from which it came (occlusion). The phosphorylation causes a transition from the EIP to
EzP state, thereby reducing Ca affinity such that Ca can be released to the lumen of the SR via
the channel. This reduction in affinity is important for the rapid release of Ca into the SR lumen
Chapter 7 Sarcoplasmic Reticulum 163
Figure 82. Structure of the skeletal muscle SR Ca-pump. Diagram by MacLennan & Green (2000) based
on the crystal structure by Toyoshima et at. (2000). M j seems to provide a central axis, Ca sites in M4 -M 6
and M s are formed in part by disruption of the M4 and M6 helices. The nucleotide binding domain is above
and must tilt to bring ATP to the phosphorylation site, which lies in a region of 7 stranded parallel ~-sheets
(arrows). The actuator domain (was ~ domain) may rotate 90° toward the phosphorylation domain. The
cytosolic phospholamban interaction site is at the bottom of the nucleotide binding domain.
where ambient [Ca] is higher. The catalytic site for ATP is at least 4 nm away from the pore,
requiring long range functional linkages to couple the ATPase to channel reorientation.
Two Ca ions are transported by the Ca-pump for each ATP molecule consumed in both
skeletal and cardiac SR (Tada et aI., 1982; Reddy et al., 1996). The lower stoichiometries often
reported for the in vitro cardiac Ca-pump probably reflect Ca-pump-independent leak of Ca from
the vesicles or contaminating ATPases. The lipid environment is also important for this enzyme
and removal of the -30 lipid molecules associated with the SR Ca-pump decreases Ca-dependent
ATPase activity (Hidalgo et al., 1976; Hesketh et al., 1976) .
The number of SR Ca-pumps estimated from phosphoenzyme fonnation in guinea-pig
and dog ventricle was 14 and 47 flmol/L cytosol respectively (Levitsky et aI., 1981; Feher &
Briggs, 1982). Hove-Madsen & Bers (1993 b) used thapsigargin titration to estimate the number
of pump sites in penneabilized myocytes from rat vs. rabbit ventricle. We estimated 19 flmol/L
cytosol in rabbit and several fold higher in rat (perhaps as high as 100 flmol/L cytosol). While
these are only estimates and I used 47 flM in Chapter 3, I consider that the likely range is 15-75
flmol/L cytosol and species-dependent (e.g. guinea-pig < rabbit< dog < rat - mouse). The
maximum turnover rate for the cardiac SR Ca-pump has been estimated to be 10-15 Ca ions/
pump/sec in dog and guinea-pig (Shigekawa et al., 1976; Levitsky et al., 1981). This is similar
to the turnover rate of the skeletal muscle SR Ca-pump and Shigekawa et al. (1976) attributed the
t!
ATF>-
....L
Occluded
Figure 83. SR Ca-pump transport scheme. Starting from top left, 2 Ca ions bind with high affinity, then
bound ATP is used to phosphorylate aspartate-35I and modify the conformation of the protein so that the
Ca ions are occluded. The Ca2E-P undergoes a conformational change (to E2) and the Ca ions are released
to the interior of the SR (due to a lower Ca affinity). Protons are carried during the lower part of the cycle
to return from £2 to £, state where Ca can be reloaded.
slower cardiac Ca-pumping rate to ~4-fold lower pump site density and lower Ca affinity in
heart. These values mean that for the cardiac SR to take up 50 /lmol CalL cytosol during
relaxation each SR Ca-pump would only need to cycle only about once!
Regulation ofthe Cardiac SR Ca-pump by Phospholamban

A major difference between the cardiac and skeletal muscle Ca-pumps is that cardiac
muscle contains the regulatory protein phospholamban (PLB; Tada & Katz, 1982; see reviews by
Koss & Kranias, 1996; Simmerman & Jones, 1998; Tada & Toyofuku, 1998; MacLennan et al.,
1997). PLB exists as a homopentamer (apparent total MW -22,000 Da) and the amino acid
sequence has been deduced from eDNA (Fujii et al., 1987). The monomer is 6080 Da (52 amino
acids) and it exhibits one hydrophobic and one hydrophilic domain. Simmerman et al. (1986)
proposed a structural model of the pentamer which could have a hydrophilic pore through the SR
membrane with phosphorylation sites on the cytoplasmic surface (Fig 84A). Provocative initial
observations suggesting that dephosphorylated PLB might form Ca-selective channels in lipid
bilayers (Kovacs et al., 1988) are still not widely accepted. Figure 84 shows the pentameric
structure of PLB in the membrane, and that transmembrane a-helices may form a leucine zipper
to stabilize this complex (Simmerman et al., 1996; Kimura et al., 1996, 1997). That is, a series
of leucines and isoleucines at the a and d positions of the helical wheel plot (Fig 84B) can
interdigitate to stabilize the inuraction throughout the membrane (Fig 84C). Moreover, these
authors showed that mutations along this leucine zipper could prevent pentamerization.
PLB is an endogenous inhibitor of SERCA (Hicks et al., 1979; Inui et al., 1986) and
decreases Ca transport and ATPase activity, especially at low [Ca] because it increases Km(Ca)
without altering Vmax (see Fig 85; WT with PLB vs. PLB knockout or PLB (-1-». PLB inhibits
both SERCAla and SERCA2a, but not SERCA3 (Toyofuku et al., 1993). PLB is present at high
concentration in ventricular myocytes, probably comparable to the concentration of the SR Ca-
B. Helical Wheel c. Pentameric Interaction
M50
C46 L43
L39 ~
~
g~
V49 L42 f b "~,37
138 e L44
LSI
145 C41
L52 148
Trans-membrane domains
Figure 84. Phospholamban structure and pentamerization. A. Domain I of PLB extends above the SR
membrane surface and includes phosphorylation sites Ser-16 and Thr-17 (Domain IA is residues 1-20 and
IE is 21-29). The transmembrane domain II (residues 30-52) interacts with neighboring monomers to
stabilize a pentameric structure (based on Simmerman et at., 1986). B. Helical wheel plot of the
transmembrane residues (i.e. 7 residues /2 full turns) in a monomer where the a and d positions are
occupied by either leucine (L) or isoleucine (I). C. The a domain of one PLB monomer may interdigitate
with the d domain of another forming leucine zippers stabilizing the pentameric array (8 and C are based on
Simmerman et aI., 1996, with images generously provided by L.R. Jones).
ATPase (Tada et al., 1983; Louis et al., 1987). Colyer & Wang (1991) estimated that ventricular
SR has 0.4 mol PLB pentamer per SR Ca-ATPase (or 2 PLB monomers/ per pump molecule).
This would imply that there is ~IOO fLmol PLBIL cytosol (also comparable to [TnC». There is
~ IO times less PLB in atrial muscle and lower concentrations are also present in slow skeletal
and smooth muscle (Briggs et aI., 1992). However, even in ventricle PLB overexpression in
transgenic mice (doubling PLB protein with respect to the SR Ca-ATPase) produces a proportio-
nal shift to lower Ca affinity (Fig 85), suggesting that the Ca-pumps are not nearly saturated with
PLB in situ (Kadambi et aI., 1996; Chu et aI., 1997). In heterologous coexpression studies
Reddy et al. (1995) found maximal inhibition with 3 PLB monomers per SR Ca-ATPase.
Titration of PLB overexpression in transgenic mice indicated that ~40% of the SR Ca-pumps are
normally regulated by PLB, and that maximal pump inhibition took place at 2.6-fold
overexpression of PLB (Brittsan et al., 2000).
Figure 85 shows the [Cal dependence of forward SR Ca-pumping. With endogenous
PLB in ventricular SR (WT) the Km(Ca) is typically ~300 nM. The maximal Ca-pump rate is
shown in the usual cellular units and typical of that in rat ventricular myocytes (Bassani et al.,
1994a; Balke et al., 1994). Recent values for K m in the literature vary considerably (e.g. from
234 nM to > I fLM; Sasaki et aI., 1992; Toyofuku et al., 1993; Hove-Madsen & Bers, 1993b; Kiss
et al., 1994; Bassani et aI., 1994a; Balke et al., 1994; Mattiazzi et al., 1994; Lu & Kirchberger,
1994; Reddy et al., 1996; Luo et al., 1994; Odermatt et aI., 1996). Part of the variation may be
the difficulty in making precisely calibrated Ca-EGTA buffers. Thus a K mvalue of300 nM is an
educated guess.
PLB can be phosphorylated by cAMP-dependent protein kinase (PKA) at serine-16
(Kirchberger et al., 1974; Tada et al., 1974; Simmerman et al., 1986). This largely reverses the
_ 200
(,)
Q)
I/)
:E:::1. 150
-
~
2Q. 100
;j
CO
() 50 PLB 2-fold
~
rn overexpression
o-1--==;;.....~~:;:;::.~or~"s~u~p~e!..;rs~h~if~ti!.;.ng~'_'P~LB~-.-,--,--,,...,..,.,
0.01 0.1 1 10
[Cal (J.lM)
Figure 85. Influence of phospholamban on SR Ca transport. Curves of Ca uptake (200/(1 + {KnI(Ca]) 2»
in ~mol/L cytosol/sec using Km values from various sources (see text). For wild type (WT) SR Ca-pump
Km= 300 nM. Either PLB phosphorylation (PKA-P04 ), gene knockout (-/-) or application of an antibody
that prevents the PLB-SERCA interaction (PLB-Ab) reduce Km to 100 nM. SR with half the normal
amount ofPLB (+/-) has an intermediate K m (178 nM) and two-fold overexpression ofWT PLB or certain
"supershifting" mutant PLBs (which don't form homopentamers) can increase K m to higher values (500
nM). Note the different Ca transport rates expected at 0.1-0.3 ~M [Cali'
PLB-induced Ca affinity shift, increasing the Ca affinity of the SR Ca-pump by 2-3 fold (e.g.
changing K m from 300 to 100 nM in Fig 85). Thus for most relevant [Cali values (0.1 -I flM)
there is a substantial increase in Ca-pump rate. Application of a PLB antibody which interferes
with the interaction between PLB and the SR Ca-ATPase produces similar effects (PLB-Ab in
Fig 85; Sham el al., 1991; Suzuki and Wang, 1986). Furthermore when either the cardiac or
skeletal muscle SR Ca-pump (SERCA2A or SERCAI) is expressed without PLB the Ca pumping
and ATPase activity properties are like the endogenous PKA phosphorylated Ca-pump (e.g.
Toyofuku el aI., 1993; Reddy et al., 1996).
PKA-dependent phosphorylation of PLB at Ser16 has been observed in intact perfused
hearts in response to catecholamines (Le Peuch et al., 1980; Kranias & Solaro, 1982; Lindeman
et al., 1983). This stimulation of Ca uptake rate by catecholamines appears to be the main means
by which ~-adrenergic agonists accelerate relaxation in the heart (vs. TnI phosphorylation; see pg
277-278; McIvor et aI., 1988; Li el al., 2000). Moreover, the activation and washout of the
lusitropic effect (acceleration of relaxation) of catecholamines in heart occurs in parallel to the
phosphorylation and dephosphorylation of PLB at Ser-16, while dephosphorylation of Thr-17
(site of CamKII phosphorylation, see below) or TnI occur more slowly (Wegener et al., 1989;
Talosi et aI., 1993). Catecholamines would also bias the competition between the SR Ca-pump
and the sarcolemmal Na/Ca exchange in favor of the former, increasing SR Ca load and limiting
Ca extrusion from the cell via Na/Ca exchange (see Chapters 3, 6 & 10). In combination with the
potent stimulation of sarcolemmal Ca current by ~-adrenergic agonists (see Chapter 5), this
increase in SR Ca-pumping normally results in a substantial increase in SR Ca content available
for release.
PLB is also phosphorylated by CaMKII at Thr-17 (Le Peuch et aI., 1979; Simmerman et
aI., 1986) which produces a similar lowering of Km(Ca) as PKA phosphorylation of Ser-16
(Kranias, 1985; Sasaki et al., 1992; Odermatt et aI., 1996). While less generally accepted there
are also reports that suggest an increase of V max due to CaMKII phosphorylation of PLB with less
effect on K m (vs. PKA; Mattiazzi et aI., 1994; Antipenko et aI., I997a,b). Bassani et al. (1995c)
showed that CaMKII phosphorylation might be responsible for a frequency-dependent
acceleration of SR Ca uptake and relaxation in intact cells (see pg 270). However, this effect is
still observed in mice in which the PLB gene has been knocked out (Luo et al., 1994; Li et al.,
1998). In intact hearts, ~-adrenergic agonists produce phosphorylation at Serl6, but also more
gradually at Thr-17 (Wegener et al., 1989; Talosi et aI., 1993; Kuschel et al., 1999a). This may
be partly due to adrenergic enhancement of Ca transients. However, it may also be due to
cAMP-dependent inhibition of phosphatase I and also PKA-dependent stimulation of a
phosphatase inhibitor (Ahmad et al., 1989; Neumann et al., 1991). Indeed, elevation of Ca
transients alone (by high [Ca]o or Bay K 8644) does not normally result in Thr-17
phosphorylation (Lindemann & Watanabe, 1985b; Napolitano et al., 1992; Vittone et al., 1993)
except when low concentrations of the phosphatase inhibitor okadaic acid is included or the pH
is acidic (Mundifia-Weilenmann et aI., 1996; Vittone et al., 1998). On the other hand, Hagemann
et al. (2000) recently showed a frequency-dependent increase in PLB Thr-17 phosphorylation in
rat myocytes in the absence of Ser-16 phosphorylation.
PLB can also be phosphorylated by PKC at serine 10 in vitro (Iwasa & Hosey, 1984;
Movesian et al., 1984). While Rogers et al. (1990) found that PKC decreased SR Ca uptake in
permeabilized myocytes, it is not clear whether PKC-dependent PLB phosphorylation actually
occurs in vivo or has any functional impact. PLB can also be phosphorylated at Ser-16 by
cGMP-dependent protein kinase, but whether this occurs physiologically is not clear (Huggins et
al., 1989; Bartel etal., 1995).
Some reports suggest that CaMKII can directly phosphorylate cardiac (but not skeletal)
SR Ca-ATPase increasing the V max for Ca transport (Xu et al., 1993; Toyofuku et al., 1994c).
However, this result is controversial and two major studies have directly contradicted it (Reddy
et aI., 1996; Odermatt et al., 1996). Thus, PLB (and its phosphorylation state) seems to be the
main endogenous regulator of SR Ca-pump function. Of course, the Ca-ATPase is also sensitive
to the concentrations of substrates (Ca and ATP) as well as pH and other small molecules (see pg
169; Tada, 2001).
James et al. (1989) crosslinked Lys-3 on PLB to either Lys-397 or 401 on the Ca-pump,
but this only worked when PLB was dephosphorylated. Toyofuku et al. (1993, 1994a) showed
that residues 397-402 of SERCA2a (KDDKPV) were essential for PLB interaction, by measuring
Ca transport function with SERCA2a/ SERCA3 chimeras. An elegant series of functional muta-
tional analyses of PLB coexpressed with SERCA has resulted in unique insights and the hypo-
thetical model in Fig 86 (Toyofuku et aI., 1994b; Kimura et al., 1996, 1997; Autry & Jones,
1997; Cornea et al., 1997). This intriguing working hypothesis considers PLB monomers to be
the active inhibitors of the pump, whereas the pentamers constitute a PLB reservoir. Some loss
ofPLB function occurs with alanine substitutions for cytosolic residues 2,4,7,9,12 and 14. It was
suggested that the cytoplasmic domain ofPLB is not intrinsically inhibitory, but based on charge
and hydrophobicity this region can interact with SERCA and modulate the inhibitory effects,
OJ
....J
~ Ca
a. ~ ~ Ca
Ph I d PLB
osphory ate /d2 lpLB-SERCA
Kd2
heterodimer
,--...
Ca bound SERCA
f= f= --l
I---- t----
;+ ;+
..
I-'-' '-'-'
•
Kd1
PLB monomers PLB pentamer
Figure 86. Model of phospholamban (PLB)-SERCA2 interaction. PLB can be either mono- or
pentameric with a dissociation constant (KdJ ). PLB mutations which disturb the leucine zipper (Fig 84) shift
KdJ toward more monomers without altering the intrinsic affinity of monomeric PLB for the SR Ca-ATPase
(K,n), which causes greater pump inhibition. Other PLB mutations can increase or decrease K d2 without
altering K dJ . Heterodimeric PLB-SERCA inhibits SR Ca-ATPase (Fig 85), but either phosphorylation of
PLB or Ca binding to the pump can reduce PLB binding and relieve inhibition. Based on diagram in
Kimura et al. (1997), modified and kindly provided by D.H. MacLennan.
which are caused mainly by interactions in the transmembrane regions. Interestingly, PLB and
SERCA2a still co-immuno-precipitate when the inhibitory effect is reversed by either PLB phos-
phorylation or antibody, but they are dissociated at high [Ca] (Asahi et al., 2000). Thus
elevation of rCa]; could reduce the fraction of SR Ca-pumps inhibited by PLB and this might
contribute to frequency-dependent acceleration of relaxation by speeding SR Ca transport (when
time-averaged rCa]; is high).
Mutations in the PLB transmembrane domain which disturb the leucine zipper in Fig 84
(e.g. residues 37,40,44,47) result in de-pentamerization and greater Ca-pump inhibition. These
monomeric mutants or "supershifters" (Fig 85) were suggested to be more effective simply
because they were not in pentameric complexes, and so were free to interact with SERCA2a (i.e.
a leftward shift along the K dJ balance in Fig 86, resulting in more PLB-SERCA heterodimers).
Conversely, mutations on the opposite side of the PLB helix lost inhibitory function (without
pentamer changes). This was attributed to reduced inhibitory interaction ofPLB monomers with
SERCA2a (a downward shift along K d2 in Fig 86). Phosphorylation drives the PLB equilibrium
in favor of pentamers and reduces the inhibitory effect of PLB on the Ca-pump. This might
simply be a downward shift along K d2 in Fig 86, resulting in more pentamers (or phosphorylated
PLB may cause the K dJ to shift in favor of pentamers). A remarkable gain of PLB inhibitory
function was seen when Arg-27 was mutated to alanine (N27A). This resulted in a lO-fold
reduction in Ca affinity, without any loss of pentamers. Moreover, transgenic mice expressing
this pentameric N27A mutant PLB exhibit decrease of SR Ca transport, cellular contraction and
relaxation, and prolongation ofisovolumic relaxation of the ventricle in vivo (Zhai et al., 2000).
There is also evidence to suggest that PLB may decrease the energetic efficiency of the
SR Ca-ATPase (Frank et al., 2000; Shannon et aI., 2001) and the effect can be reversed by PKA
phosphorylation. This would mean that in the presence of unphosphorylated PLB the SR Ca-
ATPase would not be able to establish as large a [Cal gradient (see Thermodynamics, pg 173).
Thus PLB is an extremely important physiological regulator of cardiac contraction. The
remarkable enhancement of cardiac function in the PLB knockout mouse (Luo et aI., 1994)
without any striking down-sides may indicate that selective inhibition of PLB expression or PLB-
SERCA2a interaction could be a powerful cardiac inotropic strategy. Indeed, an advantage of
this explicit PLB target is that one can increase SR Ca transport and Ca transients, without the
physiological concomitant effects of PKA activation. That is, one could avoid PKA-dependent:
a) reduction of myofilament Ca sensitivity (which would limit inotropy), b) increased lea (which
could lead to Ca overload), c) activation of the ryanodine receptor (which could increase
spontaneous SR Ca release) and d) reduced energetic efficiency of contraction and metabolism.
SR Ca-ATPase inhibitors
While there are many modulators of SR Ca-ATPase, there is a group of three key agents
which are widely used and potent inhibitors. These are thapsigargin (TG; Thastrup et al., 1990;
Sagara & Inesi, 1991), cyclopiazonic acid (CPA, Goeger et aI., 1988; Seidler et aI., 1989) and
2,5-di(tert-butyl)-1,4-benzohydroquinone (TBQ, Nakamura et al., 1992). TG is the highest
affinity and most selective of these tools (Kd < 2 pM, Davidson & Varhol, 1995). This extremely
high affinity allows TG to be used very selectively to inhibit SERCA pumps. However, one must
keep in mind that the concentration of SR Ca-ATPase molecules in myocytes is -50 11M, so
higher [TG] than 1-10 nM are often used as a practical matter to obtain rapid and complete block
of SR Ca-ATPase. It is often better to consider [TG] in terms of nmol/mg protein to avoid not
having enough TG molecules to bind to all of the SR Ca-pumps. In permeabilized myocytes in a
cuvette -I nmol/ mg protein is sufficient for complete block, and at 5 nmol/mg it takes less than
5 sec for >95% block (Hove-Madsen & Bers, 1993b). In superfused intact myocytes, exposure to
5 11M TG blocks SR Ca-ATPase by -95% in 60 sec and completely in <90 sec (Bassani et al.,
1993b). Curiously, when TG (or CPA) are superfused around thin multicellular preparations, it
is almost impossible to completely block the SR Ca-ATPase (Baudet et al., 1993). The high
affinity of TG for the SR Ca-ATPase makes TG exposure essentially irreversible. CPA and TBQ
can be used as more reversible SR Ca-pump inhibitors. CPA also has submicromolar affinity and
~10 11M is typically used to inhibit SR Ca-ATPase. TBQ inhibits the SR Ca-ATPase with a
K m-1.5 11M (Nakamura et al., 1992) and 10 11M is typically used experimentally. While these
agents are quite selective for SERCA pumps (as opposed to other P-type pumps) they may not be
without complicating effects (e.g. Dettbarn & Palade, 1998).
Regulation 01 the SR Ca-pump by Ca, pH, ATP and Mg

SERCA has a high affinity ATP site (Kd -111M) which is the substrate site and a second
lower ATP affinity site (Kd -200 11M) which serves a regulatory role (deMeis & Vianna, 1979;
DuPont, 1977; Verjovski-Almeida & Inesi, 1979). Generally [ATP] is in excess for the SR Ca-
ATPase and [Ca]j is the limiting substrate. When cellular ATP levels fall during ischemia, there
may be some decline in SR Ca-pumping and slowing of relaxation due to the allosteric effect, but
[ATP] would have to be extremely low to prevent ATP binding to the substrate site (but see also
Thermodynamics below). The actual substrate for the Ca-pump is probably MgATP, but other
nucleotides can also be used (Tada et ai., 1978). However, the GTPase activity of the cardiac
Ca-pump has different characteristics (e.g. very low Ca sensitivity; Tate et al., 1985, 1989).
The SR Ca-pump is also pH-sensitive. Shigekawa et al. (1976) found a broad pH
optimum around 8, which was more alkaline than that observed for skeletal muscle. Fabiato &
Fabiato (1978a; Fabiato, 1985e) found that the Ca uptake by cardiac SR in mechanically skinned
cell was progressively reduced as pH was decreased from 7.4. Thus, acidosis associated with
ischemia may be expected to depress the rate of SR Ca-pumping and thus slow relaxation as is
observed during acidosis. Of course acidosis can also reduce the Ca sensitivity of the
myofilaments, Ca current, Na/Ca exchange etc. (see Chapter 10).
It should also be noted that there is a broad range of values reported for the optimum pH
and this may depend on the assay conditions (e.g. [Cal, oxalate, [Mg], temperature, [ATP],
phosphorylation state, the type of SR vesicles etc.). Indeed, this consideration is valid for the
[Cal and [ATP] dependence as well and it is a constant challenge to mimic accurately the
intracellular physiological conditions for in vitro experiments aimed at determining the in vivo
Ca-pump dependence on [Cal, [Mg], [ATP], etc. The situation is further complicated by the Ca
release channel which alters net Ca transport by the Ca-pump and the gating of this channel is
also affected by [Cal, ATP, Mg and pH (see below). For example, Fabiato & Fabiato (1978a)
reported that at pH 7.4 the Ca uptake by the SR was optimal at [Ca]=18 nM, since under their
particular conditions higher [Cal induced Ca release. Indeed, Wimsatt et al. (1990) found
maximal SR Ca uptake rate in permeabilized rat myocytes at -500 nM Ca and lower uptake at 1-
100 flM Ca. They attributed this decrease to Ca-induced Ca-release, because addition of SR Ca
release channel blockers (procaine or ruthenium red) converted this to a monotonically
increasing function (with much higher Ca uptake at 10 flM than at 500 nM Ca).
Figure 87 shows an on-line approach we have used to measure SR Ca uptake rate in both
digitonin-permeabilized and intact ventricular myocytes (Hove-Madsen & Bers, 1993b; Bassani
et al., 1994a). The free Ca transient is converted to a total cytoplasmic transient (t.[Ca]tot in Fig
87A) using the Ca buffering characteristics measured in cells (as in Chapter 3) or in
permeabilized cells (after pump blockade). The derivative with respect to time of the t.[Ca].ot
signal is a Ca transport rate and under our conditions this Ca transport was entirely due to the SR
Ca-ATPase. Plotting d[Ca]to/dt as a function of [Ca]j for each individual point during [Cali
decline (Fig 87B) gives the [Cali dependence of SR Ca transport rate (with Km values -200-300
nM and Hill coefficients of 2-4). The maximal SR Ca uptake rate in rat is -2.4 times higher than
in rabbit myocytes, and this is true for both intact and permeabilized cells. The extrapolated Ca
transport rate in permeabilized cells was -50% lower than that obtained in intact myocytes. It is
not clear whether this difference is due to decreased function of some permeabilized cells in the
cuvette (vs. intact cells) or other systematic differences in analysis.
Oxalate is frequently used as a precipitating anion in studies of SR Ca uptake. The
principle is that oxalate is rapidly transported across the SR membrane (by an anion transporter)
600
A ... 0 I>
n ~U200 B
Rat-intact cell
"
100
-u
"- "-~[Caltot ~
~~
<1>
~g
0 ~
:E 400 :;: i: 0 150
.s "-
"- 3
.~ ~
~ "- ...... 30 0 "'>' o ~.

U :5 a~ 100 Rabbit-intact cell
~ 200
....... n :>'"
Cl.
'S. co 0 ()
0
UI
U§. 50 / pe("meabilized cell <1>
iff
-d[Caltot/dt = SR Ca uptake rate
.g, 0::-
VI
0
0 200 400 600 800 0 200 400 600 800 1000 1200
time (ms) [Cali (nM)
Figure 87. SR Ca transport measured in intact and permeabilized ventricular myocytes. A. Ca transient
([Ca];) from a rat ventricular myocyte where the Na/Ca exchange is prevented by complete Na depletion (as
in Bassani et al., 1994a). The [Cali is converted to total cytosolic Ca ([Ca]tot) using buffering data as in Fig
26, pg 43: [Ca]tot = (236 ~M/{I+(498 nM/[Ca];)}) -39 ~M). B. The d[Ca]to.ldt is plotted as a function of
[Cali for each point in time during [Cali decline (a few example points are shown). These curves are then fit
by a Hill equation Y = Ymax/(I+ {Kn/[Ca];}n). The same analysis was used for intact rabbit myocytes and a
similar approach was used for digitonin-permeabilized myocytes. Intact cell data are from Bassani et al.
(I 994a) and permeabilized cell data are from Hove-Madsen & Bers (I 993b).
and as intra-SR free [Ca] ([Ca]sR) increases it binds to oxalate and then precipitates, thereby
limiting further rise in [Ca]SR which would prevent net uptake (see pg 175). This allows SR Ca
uptake to continue almost linearly as a function of time at a given [Ca]. This greatly simplifies
measurement of the [Ca]-dependence of SR Ca transport. Otherwise one would be restricted to
an initial uptake rate at very short times (before [Ca]sR changes appreciably). However, the
oxalate method is not physiological and the precipitated SR Ca is not readily releasable.
Calsequestrin
Physiologically intra-SR [Ca] is buffered in large part by calsequestrin, which was first
described in skeletal muscle SR by MacLennan & Wong (1971). Meissner (1975) suggested that
calsequestrin was primarily localized in the terminal cisternae of the SR. Calsequestrin in
cardiac muscle was first identified and purified by Campbell et al. (1983) and Jorgensen &
Campbell (1984) provided evidence that cardiac calsequestrin is localized to junctional SR (and
corbular SR). The primary structure of cardiac calsequestrin has been deduced by cDNA cloning
(MW = 45,269; Scott et al., 1988) and is only 63% homologous with the fast twitch skeletal
muscle calsequestrin (MW = 42,435; Fliegel et al., 1987). As with the SR Ca-pump and PLB,
"cardiac" calsequestrin is also expressed in slow twitch skeletal muscle (Scott et al., 1988). The
crystal structure of skeletal calsequestrin has been elucidated (Wang et al., 1998b) and it has 3
thioredoxin folds which condense upon binding Ca. Ca binding seems to be by bridging of
negatively charged surfaces between domains, rather than in EF-hand type loops as seen in TnC
and calmodulin.
Cardiac calsequestrin is highly acidic and each molecule binds ~35-40 Ca ions (or ~900
nmol Ca/mg protein, Mitchell et al., 1988) with an apparent Km(Ca) ~ 500 /lM. Assuming the
maximum yield of isolated cardiac calsequestrin reported by Campbell (1986, 40 mg/kg wet wt)
represents a true yield of 25-50% of whole heart calsequestrin, and the extrapolation conventions
from Chapter 3, this corresponds to 175- 350 /lmol of Ca binding sites per liter cytosol (3.2-6.4
Cytosol
SR membrane
Figure 88. Association of calseguestrin (CSQ) with junctional SR proteins. These include the ryanodine
receptor (RyR), triadin and junctin. Ca binds to negative charges (-) on CSQ, bridging the domains and
limiting association with triadin and junctin. Higher [CaJsR may lead to tighter CSQ condensation and
multimerization (Can-CSQ). Based on diagrams by Zhang et al. (1997b) and Wang et al. (l998b).
mM of Ca binding sites in the SR). Thus, these sites could buffer a substantial fraction of the Ca
taken up by the SR with an appropriately low affinity. Shannon & Bers (1997) measured intra-
SR free [Cal and also total SR Ca content in rat ventricular myocytes. From these measurements
we determined that the B lTUlx for intra-SR Ca was ~14 mM with a K d of 0.63 mM. This K d is very
close to the in vitro value above and the B max for Ca is a bit higher than expected. Related recent
results in intact myocytes give a B lTUlx of -3 mM intra-SR Ca sites (Shannon et al., 2000a).
Calsequestrin appears to be attached to the junctional face membrane in the terminal
cisternae of the SR (e.g. Franzini-Armstrong et al., 1987) and when Ca binds to calsequestrin the
shape of the molecule changes (Ikemoto et aI., 1972, 1974; Cozens & Reithmeier, 1984).
Ikemoto et al. (1989, 1991) have even proposed that calsequestrin is involved in the regulation of
the SR Ca release channel, but more work will be needed to clarify this possibility. Triadin and
the structurally related protein junctin (Caswell et al., 1991; Knudson et al., 1993a,b; Jones et
al., 1995; Zhang et al., 1997b) are also co-localized to the junctional region and interact with
each other, calsequestrin and the ryanodine receptor. Thus there may be a physical interaction
among these proteins as illustrated in the schematic in Fig 88. Three cardiac triadins exist (MW
35, 40 & 92 kDa for type I, 2 & 3) with triadin-l being dominant in heart. Skeletal muscle
expresses a 95 kDa triadin, and junctin (26 kDa) is expressed in both cardiac and skeletal muscle.
While functional information is limited, Zhang et al. (1997b) suggested that triadin and junctin
may physically couple calsequestrin to the RyR as a junctional complex (Fig 88). They also
showed that interactions between these proteins and calsequestrin are Ca-sensitive. Thus, as
[Ca]SR falls during SR Ca release, calsequestrin may bind more strongly to triadin andlor junctin.
This cooperative "zipping up" of this association could, in principle, facilitate Ca unloading of
calsequestrin and thereby facilitate SR Ca release. This schematic model is speculative, but
could help to explain the intriguing results of Ikemoto et al. (1989, 1991).
Calsequestrin has been overexpressed >IO-fold in mouse heart (Jones et al., 1998; Sato
et al., 1998b) and the mice develop cardiac hypertrophy. In myocytes there is a massive increase
in SR Ca content (assessed by caffeine application) as expected for a greater amount of intra-SR
Ca buffer. There was also a marked reduction in twitch Ca transients and contractions. One
possible explanation for this somewhat unexpected result is that the free [CalsR may be low due
to exceptionally heavy Ca buffering, and low [Ca]sR can strongly depress fractional SR Ca
release (Bassani et al., 1995b; Shannon et al., 2000b).
Several other Ca binding proteins are in the SR. A 53 kDa SR glycoprotein appears to be
the C-terminal half of sarcalumenin, which is 160 kDa in skeletal muscle and -130 kDa in heart
(Leberer et al., 1989a,b, 1990). Sarcalumenin does not alter Ca-pump function in cotransfection
studies, but the amino-terminal half is highly acidic, and like calsequestrin binds -35 mol Ca/mol
protein. The carboxy end (i.e. the 53 kDa glycoprotein) does not bind Ca appreciably. A
histidine rich Ca-binding protein (-140 kDa) also exists in the SR lumen, but is a minor protein
(Hoffman et al.1989). Another minor luminal Ca binding SR protein calreticulin (MW = 46,567,
Fliegel et al., 1989b) binds 1 Ca with high affinity and -25 Ca with low affinity (MacLennan et
aI., 1972; Ostwald & MacLennan, 1974) and is present in both muscle and non-muscle cells
(Fliegel et al., 1989a,b). Indeed, calreticulin terminates with the ER retention signal peptide
KDEL, whereas the muscle-specific proteins calsequestrin, sarcalumenin and histidine rich Ca-
binding protein lack this sequence (Milner et al., 1992). Calreticulin and the analogous (and
ubiquitous) calnexin may both function primarily as molecular chaperones, assisting in protein
folding and stabilization (John et al., 1998; Danilczyk et al., 2000). Thus, there are other SR Ca
binding proteins, but from a quantitative standpoint calsequestrin is overwhelmingly dominant.
Thermodynamics and Ca-pump backjlux

The SR Ca-pump can work in both directions. That is, it is a reversible enzymatic
reaction and Ca can move out of the SR and even make ATP in doing so (Takenaka et aI., 1982;
Feher, 1984). As such, the net direction of Ca-pump movement is dictated by thermodynamics
(as was the case for Na/Ca exchange, Chapter 6). The free energy from ATP required to build
the [Cal gradient is 6GSR-C'p = 2RT.ln([Ca] sR/[Ca],) and provided that 6GATP + 6G SR.C'p is
negative, net Ca uptake occurs. Shannon & Bers (1997) measured [Ca]SR and thus the [Cal
gradient that could be generated by the cardiac SR Ca-pump. We found that [Ca]sR/[Ca]; was
constant at -7000 over the whole range where measurements could be made (10-300 nM [Cal;).
Thus, for [Cal; = 150 nM, [Ca]SR would be 1 roM. Chen et al. (I996b) also measured [Cab
using a low affinity Ca-binding NMR probe (TF-BAPTA) and found [Ca]SR in the intact beating
heart to be -1.5 roM. Figure 89 shows that the [Cal gradient of 7000 corresponds to 6G SR -C'p of
44 kJ/mol, which is 74% of the energy available from ATP (for 6GATP=59 kJ/mol, Allen et al.,
1985a). This is a fairly high energetic efficiency, but seems to be typical of ion transport pumps
(see Table 13, page 62).
Net reverse Ca-pump flux is not expected physiologically, because the [Cal gradient
would have to exceed that required to make 6GATP (and of course it is 6GATP that builds the [Cal
gradient in the first place). Nevertheless it is important to consider the backwards flux through
- ..
-
50
l: ~
.!!! 0 0
'0
...
ctI
0)_
46
44 kJ/mol
l>
G>
..... 0 »
-"
-i
ctI
..,E 42
...... -
0 I
I
01
I
-g"
.5~ (l)
Il,OOO-fold 5
>. 38
...
0)
Q)
: gradient
I
c..
3
l: 0
w 34 t.G=2RT.ln([Ca]SR/[Ca]i) :
55
1,000 10,000
[CalsR I [Cali
Figure 89. Energy in the trans-SR [Ca] gradient. Energy to transport 2 Ca ions/ATP is a logarithmic
function of [Ca]sR/[Ca]j (inset equation) and measurements indicate the pump can achieve a gradient of
7,000, equivalent to 44 kllmol, requiring 74% of the energy of t.G ATP (Based on Shannon & Bers., 1997).
the SR Ca-pump, as emphasized by Fig 90. Based on the forward Ca-pump rate (V Fo ,) one would
expect Ca influx into the SR at 100 nM [Cali to be 21 Ilmol/L cytosol/sec. If this unidirectional
influx were balanced only by a leak flux, the leak flux rate (VLeak) would also have to be 21
Ilmol/L cytosol/sec for the SR Ca content to be at a steady state. Indeed, this sort of VLeak has
been required to analyze SR Ca fluxes in cardiac myocytes (e.g. Balke et a!., 1994; Bassani et al.,
I 994a). However, this is almost 100 times higher than the measured unidirectional Ca leak rate
with the SR Ca-pump blocked in intact cells (0.3 Ilmol/L cytosol/sec; Bassani & Bers, 1995).
With this V Fo , of 21 and V Leak of 0.3, the reverse Ca-pump rate (VRev) would need to be 20.7
Ilmol/L cytosol/sec for the SR to be in Ca balance (Fig 90B). The implication of this is that the
SR Ca-ATPase may approach thennodynamic equilibrium at physiological [Cali (i.e. with
forward and reverse rates nearly balanced). Is it reasonable that V Rev is this high? This requires
a bit more quantitative consideration. Ca-pump flux can be written as
VmFo, ([Ca]/K mFOr)2 - VmRev ([CaJsRfKmRevi

V net (7. I)
1 + ([Ca]/KmFOr)2 + ([CahfK mRev)2
where VmFor and VmRev are the forward and reverse maximum rates, and K mFor and K mRev are the
forward and reverse dissociation constants. Hill coefficients were assumed to be 2 for flux in
both directions. With [CaJsR = 0, the last tenn in both numerator and denominator drop out and
Eq 7.1 reduces to the standard Hill equation (V=VmaJ(l+{KmI[CaJJ}n)), but as [Ca]SR rises
reverse flux becomes more important and limits net Ca-pump flux. Indeed, at steady state (V ne ! =
0), the numerator of Eq 7.1 is O. If we assume that maximal pump velocity is the same forward
and reverse (Takenaka et a!., 1982), then V mFor = VmRev = Vmax and it follows that KmRcv!KmFor =
[CaJsR f[Cak Thus for a [Cal gradient of 7000 and K mFo , of 300 nM, K mRev would be 2 mM.
Moreover, if[CaJsR is 1 mM the V Rev value in Fig 90B is predicted and this low affinity K mRev is
consistent with kinetic analysis of the SR Ca-pump transport cycle (e.g. see Fig 83).
A. Forward Ca Pump Rate B. Unidirectional SR Ca flux

~ 200 -----------V~~;;2fiT~M7s---------
~
"£ 150
~
o 100 ..---........ .
VRev= 20.71JM/s
"0
E IK m= 300 nM V For = 21 IJM/s
2: 50 inHiI/= 2
(;
u.
21
> O+---==-......-+--L..--_--
10 100 1000
[Cal,(nM)
Figure 90. Unidirectional SR Ca-pump flux. A. Forward pump rate based on simple Hill relations as in
Fig 85 (V For = Vmaxl(1 +{K,J[Ca];} n). B. For [Cali = 100 nM and [Ca]SR =700 ~M, values indicate expected
forward and reverse Ca-pump flux rates and also SR Ca leak rate.
We tested the impact of SR Ca leak on the maximum steady state SR Ca load by

measuring maximal SR Ca load in intact voltage clamped ventricular myocytes (Ginsburg et al.,
1998). If the leak does limit SR Ca load, then accelerating the SR Ca-pump (with isoproterenol)
or slowing the pump (with submaximal thapsigargin exposure) should alter maximal SR Ca load
accordingly. On the other hand, if the leak is inconsequential, then the Ca-pump should
approach the thermodynamically limiting gradient, even when the pump is significantly slowed
(it just takes more time). Essentially, the same maximal SR Ca load was reached when the SR
Ca-pump was activated by isoproterenol, partially blocked by thapsigargin or untreated. Of
course a different number of pulses were needed with isoproterenol or thapsigargin. The impli-
cation is that the normal resting SR Ca leak does not appreciably limit the SR Ca load.
This can also be appreciated on quantitative theoretical grounds. Figure 91 shows that
the SR Ca load is not expected to be greatly decreased by the Ca leak until V Leak approaches the
forward rate of the pump (42 Jlmol/L cytosol/sec at 150 nM rCa];). Furthermore, at physiological
leak rates (0.3 Jlmol/L cytosol/s) slowing or accelerating the Ca-pump does not affect the steady
state SR Ca load, although the time required to attain that load is altered accordingly. For pump
inhibition by thapsigargin V max was decreased by 50% and for Ca-pump stimulation by
isoproterenol, K m was reduced by 50%. Even a V Leak ten times higher than measured would
reduce SR Ca by less than 10%. This is consistent with observed results of Ginsburg et al.
(1998) and re-emphasizes the importance of considering this backward pump flux in realistic
models of SR Ca regulation in intact cells.
Not all results agree with this interpretation. Indeed, blocking leak via the ryanodine
receptor with tetracaine can dramatically increase SR Ca content (Gyorke et al., 1997; Overend
et al., 1998), an effect that would not be expected if leak were very small. More work is needed
to clarify this discrepancy. However, a possible explanation may be that V Leak increases very
steeply as both [Ca]sR and [Cali rise (see Regulation of SR Ca release, pg 192-195 and Chapters
8-9). Thus, at relatively high SR Ca loads (and [Cal;) leak may be much higher and may more
strongly affect SR Ca load.
--
200
0
l::
-
Q)
0
150
l/)
0.3 IJM/s
l::_
0>- Resting leak
U 0 100
nJ~
u"o
~E
C/)::::L 50
[Ca]j=150 nM
0
0.1 1 10 100
Leak rate (IJM/sec)
Figure 91. Effect of SR Ca leak rate on SR Ca content. Curves are based on Eq 7.J in the steady state
(V oet =V For - VRev) and co~sideration that Voet is balanyed by VLeak . Then [<;;~JsR is a function ofV Leak an~
[Cali: [Ca]sR=7000{([Ca]i (VmaxlVLeak - I) - (K OlFor))/ (VmaxNLeak -I- I))} and total SR Ca ([Ca]SRtot) IS
related to [CaJsR: [Ca]sRtot = [Ca]SR -I- (B maxSR/(1+{KIsR/[Ca]SR}), where BOlaxSR is the intra-SR binding
capacity and KdSR is the dissociation constant. Thus [Ca]SR and [Ca]SRtot can be predicted as a function of
[Cali, VOlax , VL and K mFor ' The control values used are Vo"x = 210 funol/L cytosol/s, KOlFor = 300 nM,
BmaxSR = 3.95 mM and KdSR = 600 flM. For [Cali = 150 nM at low leak levels [Ca]sR =1 mM and [Ca]SRtot
of 191 flmollL cytosol corresponds to 3.5 mM intra-SR Ca (see Ginsburg et ai., 1998).
Figure 92 shows incorporation of Eq 7.1 into detailed quantitative analysis of real

cellular Ca transients (Shannon et aI., 2000a). Associated with SR Ca release there is a decline
in SR Ca content (and [Ca]sR) and consequently reverse Ca-pump flux is decreased, while
forward Ca-pump flux is stimulated by high [Cali' Thus there is an increase in net SR Ca uptake
by the pump. As [Cali declines and [Ca]sR rises the forward Ca-pump rate falls and reverse Ca-
pump rate increases, until the net pump flux is almost zero (only balancing the small leak). At
that point the SR Ca content has returned, and in this case the amount of Ca which entered via lea
was also accumulated by the SR because these experiments were done in Na-free conditions to
avoid complicating Na/Ca exchange fluxes.
The functional consequence of this thermodynamic consideration is that Ca uptake by
the SR (especially during late relaxation and diastole) will be sensitive to energetic limitations
that may occur under pathophysiological conditions. For example, if [ATP] declines or [ADP] or
[P04] rise, the t.G ATP available to the Ca-pump will be reduced. While this may not alter Vmax or
initial rates of [Cali decline, it will reduce the [Cal gradient that the SR Ca-pump can generate.
This will have preferential effects on the latter phase of [Cali decline and on diastolic [Cali as the
pump approaches a different thermodynamic equilibrium (at lower [Ca]sR and higher [Cali)' A
lower SR Ca content may also have a disproportionately depressant impact on Ca transients by
reducing both the amount of SR Ca available and also the fraction released (see pg 224-225).
As a protective mechanism with respect to changes in t.G ATP , many glycolytic enzymes
appear to be closely associated with the SR Ca-pump (Xu et al., 1995; Xu & Becker, 1998).
These enzymes can produce ATP locally (and consume local ADP) and this ATP appears to have
preferential access to the SR Ca-pump. This may ensure optimal t.G ATP availability for Ca
~
loa]
.i % /---~,!~~~~(!~q)---
....-....-...._--...- ._._ ...__...__....-.... _....--_ ..---... --_.--.
J ~E SR Ca content
nMT
1
0::
500
~I
<J)<J)
(/J .:!: 50 Reverse Pum
"'0 [Cali 50
~~
Q)
~
G>
ellS
(.)0
o::E ~ Ii) o-j::-----=-"':':::=:::::======
(/JE SR Ca Release =~
"'::1.
U -.50
0::
(/J
·100
Figure 92. SR Ca-pump fluxes during a cellular Ca transient. Left panel shows a Ca transient and SR Ca
release flux in a voltage clamped rabbit ventricular myocyte in Na-free conditions to block Na/Ca exchange
(data from Shannon et al., 2000b). Data were analyzed to provide running information about forward and
reverse SR Ca-pump rate (using Eq 7.1) and SR Ca leak (k([CaJsR -[Cali)' During this twitch SR Ca
content fell by -45% during SR Ca release, but was higher at the end of the trace by the amount of
integrated Ca entry via Ica (i.e. this cell was being progressively Ca loaded).
transport. On the other hand, Chen et al. (1998) showed that [Ca]sR/[Ca]i varies in parallel to
f>G ATP and Tian et al. (1998) found that inhibition of creatine kinase (which phosphorylates ADP
to ATP) limits SR Ca handling and thereby limits contractile reserve in the intact heart.
This raises the issue of energetic requirements for Ca transport (see also pg 62). In
guinea-pig ventricle Schrarrun et al. (1994) determined that 15% of cardiac energy expenditure is
due to the SR Ca-ATPase vs. 76% for myofilament ATPase and 9% for Na/K-ATPase (remember
that ~ 75% of the Na extruded was that used to extrude Ca via Na/Ca exchange). This ~20% of
total energy expenditure for Ca transport is a major energetic investment. Two Ca ions can be
pumped into the SR per molecule of ATP, while only one Ca ion per ATP can be extruded via
Na/Ca exchange (indirectly, since 3 Na are moved per cycle by Na/Ca exchange and Na/K-
ATPase). Thus, any shifts from SR Ca transport to greater transsarcolemrnal fluxes, as may
occur in heart failure, will also carry an extra energetic cost.
It is clear from the foregoing that the rate of Ca transport by the SR Ca-ATPase is
sufficient to drive cardiac relaxation, even if there is also a variable parallel contribution from
the NaiCa exchange (as discussed in Chapter 6). The amount of Ca stored in the SR is obviously
a crucial parameter in understanding SR function and that will be discussed here.
SR Ca CONTENT: ASSESSMENT IN
INTACT CARDIAC MUSCLE AND MYOCYTES
A number of different experimental approaches have been used to measure the SR Ca
content in ventricular muscle. These have included 45Ca fluxes in intact cells and tissue as well
as in homogenates and SR vesicles. Some of the more recent measurements in intact cells have
come from the amplitude of caffeine-induced Ca transients as well as integration of INa/ca during
caffeine-induced contractures. Table 21 shows a compilation of some measurements, with all
converted to the units ~mollL cytosol. The top group are relatively high and are generally at an
ambient [Ca] higher than the diastolic [Ca]i of 100-150 nM. Most of the values in the lower
section of Table 21 are at physiological [Ca] and many are from intact cells. Most of these
values range from 32-58 ~mollL cytosol in guinea-pig ventricular myocytes to 210-260 ~mollL
cytosol in rat or ferret ventricle. This provides a realistic consensus range despite the variety of
experimental approaches, conditions and laboratories. For now, 50-150 ~mol/L cytosol is a
likely range of steady state SR Ca content in intact ventricular myocytes at typical stimulation
frequencies. For an SR volume of 3.5% of the cell this would correspond to 1.5-3 mM total SR
Ca (with about half bound to calsequestrin). Let's consider how SR Ca content is measured.
Electron Probe Microanalysis

Electron probe microanalysis (EPMA) can directly assess Ca in junctional SR (jSR) in
electron microscopic specimens, using emitted X-ray spectra (Wheeler-Clark & Tormey, 1987;
Jorgensen et aI., 1988; Wendt-Gallitelli & Isenberg, 1989; Moravec & Bond, 1990). These
studies have indicated jSR Ca contents (under non-Ca-depleting conditions) of -15 mmollkg dry
wt. Under conditions expected to deplete the SR of Ca (e.g. long rest periods, ryanodine or
freeze clamping during the rising phase of a contraction) values of 3-7 mmol/kg dry wt were
reported. Extrapolation of these values to either mmol CalL jSR (or ~mollL cytosol) must take
technical limitations into account (e.g. dispersion of the X-ray beam into non-jSR regions and
geometry of jSR within the depth of section, -200 nm). At the simplest level, assuming 4 kg wet
jSR/kg dry jSR, a jSR density of 1.1 g/cm3 and 0.3% of cell volume as jSR or terminal cisternae
(see Table 3, page 7), 15 mmol/kg dry jSR would correspond to -4 mM total [Ca] in the jSR or
-20 ~mol/L cytosol. The jSR is only -10% of the SR volume, but may have 5-10 times more
total CalL than network SR, due to the localization of calsequestrin. This may imply that there is
about the same amount of total Ca in network and jSR. This would make total SR Ca load -40
~mol/L cytosol, which is about 50% of the SR Ca content estimated by biochemical or
physiological approaches. It is also similar to the Ca required to activate a normal contraction
(see page 47). Therefore, while electron probe microanalysis is probably the most direct and
absolute method of measurement of SR Ca content, it may slightly underestimate SR Ca and it is
technically difficult, tissue destructive and challenging to extrapolate to the intact cell.
Caffeine-Induced Contractures, iJ.[Caji and fI Nalcn

Two of the most useful means to assess SR Ca "on line," with intact, contracting cardiac
muscle or myocytes are rapid application of caffeine or cold solution (-1°C). Caffeine and
cooling induce SR Ca release at a desired time and the amplitude of the resulting contracture or
Ca transient can be used as an index of the SR that is Ca available for release at that time. With
these indirect approaches the actual amount of Ca released (in ~mol/L cytosol) is inferred, but
this limitation can be modest and may not outweigh the great advantages of these approaches
(on-line, reproducible, sensitive and non-destructive).
Caffeine-induced contractures were first described by Endo (1975b) in mechanically
skinned skeletal muscle fibers and by Fabiato (l985b; Fabiato & Fabiato, 1975a,1978a) in
mechanically skinned cardiac myocytes. Early uses of this approach in intact cardiac muscle and
TABLE 21
SR Ca Content Measurements
Reference Species Prep SRCa Load Conditions
~mol/L cytosol
Solaro & Briggs, 1974 dog V homog 427 I !1M [Cal, 45Ca
Dani et. al., 1979 rabbit perm V myo 171-512 7.5 ~M [Cal
Shannon & Bers, 1997 rat perm V myo 880 -I ~M [Cal; 4 Ca
Kawai & Konishi, 1994 ferret perm papillary 372 I uM [Cal, fl
Solaro & Briggs, 1974 dog V homog 68 100 nM [Cal, 45Ca
Hunter et. aI., 1981 rat perfused heart 119 2.5 mM [Ca]o, rest-dep 45Ca loss
Fabiato, 1983 rat skinned fiber 142 -IOOnM rCa]
Moravec & Bond, 1990 hamster papillary 40 0.2 Hz, 1.9 mM [Ca]o, EPMA
Callewaert et aI., 1989 rat isol V myo 90 66 nM [Ca]" caff, INa/C.
g-p 32 -70 nM [Ca]" caff, 'Na/C,
H-M & Bers, 1993a rabbit perm V myo -100 -100 nM [Ca],
Shannon & Bers 1997 rat perm V myo 415 100 nM [Cal; 45Ca
Varro, et. al., 1993 rat isol V myo 185 I mM [Ca]o, caff, INa/C.
Trafford et al., J997 ferret isol V myo 118 0.33 Hz, 2 mM [Ca]o, caff, , a/C.
Pytkowski 1989 rabbit papillary 408* I Hz, 1.8 mM [Ca]o, 45Ca
Langer & Rich 1993 rat isol V myo 210* I mM [Ca]o;5Ca flux, caff
Kawai & Konishi, 1994 ferret perm papillary 260 100 nM [Ca]" fl
Bassani et. al., 1995a ferret isol V myo 141 0.5 Hz, 2 mM [Ca]o, caff, fl
Bassani & Bers, 1995 rat isol V myo 114 0.5 Hz, I mM [Ca]o, caff, fl
rabbit isol V myo 106 0.5 Hz, 2 mM [Ca]o, caff, fl
Terracciano et aI., 1995 g-p isol V myo 58 0.5 Hz, 2 mM [Ca]o, caff, l Na/Ca
Terr. & MacLeod, 1997 g-p isol V myo 38 0.5 Hz, I mM [Ca]o, caff, INa/C.
rat isol V myo 73 0.5 Hz, I mM [Ca]o, caff, 1Na/c.
Delbridge et. al., 1996 rabbit isol V myo 87 0.5 Hz, 2 mM [Ca]o, caff, fl, INa/C.
Ginsburg et aI., 1998 ferret isol V myo 149-190 0.5 Hz, 2 mM [Ca]o, caff, fl, INa/Ca
The line separates measurements of maximum SR Ca content (above) from those under more normal
cellular conditions. All values are converted to flmol/L cytosol. H-M is Hove-Madsen, Terr. is Terracciano,
g-p is guinea-pig. *represents caffeine and ryanodine sensitive component of kinetically defined 45Ca
washout (-20% of total component). aa= atomic absorption spectroscopy, caff= caffeine-induced release, fl
= Ca-sensitive fluorescence, isol V myo. = isolated ventricular myocytes, 'Na/C. = Ca efflux measured via
Na/Ca exchange current, EPMA=electron probe microanalysis, perm = sarcolemma permeabilized by
digitonin or saponin. Table prepared with help from T.R. Shannon.
myocytes were by Chapman & Leoty (1976), Bers (l987a), Smith et at. (1988) and O'Neill et al.
(1990a). One can use the myofilaments as a Ca bioassay, measuring the caffeine-induced
contracture, and these have been widely useful. Of course whenever tension is being measured
to assess SR Ca load, one must be careful that an intervention under study does not alter
myofilament Ca sensitivity (e.g. as is the case for p-adrenergic agonists). Caffeine itself
increases myofilament Ca sensitivity (Fabiato, 1981b; Wendt & Stephenson, 1983; Eisner &
Valdeolmillos, 1985), which means that force (or cell shortening) in the presence of caffeine
cannot be directly related to the force in its absence (e.g. during a twitch). More robust
quantitative versions of this approach include measurement of [Ca]; and INa/C•.
Figure 93A shows caffeine-induced Ca transients and INa/c., which can both be used
independently to assess SR Ca load. The amplitude of the Ca transient (~[Ca];) can be used as a
A. SR Ca Load B. Ca Buffering
-- -[Cal; --> [Ca]'ot (peak) 120
1000
~ 90
:E
2: 60
o
:? -10°i-"t~·....n77·-z..:;.;H> -_ 600 900 1200
Co
f =106 ~moVL cytosol
..
- ; -200 INalCa [Cali (nM)
u
~-300
FO
..?.rnM.. < _
.. tm~1..
I 1
1400 ms l
Figure 93. Caffeine-induced Ca transient, SR Ca content and cytosolic buffering in a rabbit ventricular
myocyte under voltage clamp. A. Caffeine (10 mM) was rapidly applied and the Ca-independent indo-]
fluorescence signal was used to infer [caffeine]i (bottom; O'Niell el aI., 1990a). Amplitude of the Ca
transient (top) is used to calculate total SR Ca released after conversion to total cytosolic Ca (using
buffering curve #1, pg 43). SR Ca content is also obtained from the integral of I, alC, (x6.44 pF/pL
cytosol, +96,490 C/mol and 0.93 to account for non-INa/ca mediated Ca transport). B. Ca buffering curve is
obtained by backward integration of I ,IC.. conversion to [CallOt and fitting as a function of [Cal; ([Ca].ot
= {Bma/(J+KnI[Cali)}+BnU ", Trafford el aI., 1999). Data here were recorded by K.S. Ginsburg.
crude index of SR Ca content, but can be made more quantitative by using cytosolic Ca buffering
values (see pages 42-46) to translate [Cali to total cytosolic [Cal ([Ca].o.). Then the difference
between resting and peak [Ca].ot provides the SR Ca content (in this case 118 Ilmol/L cytosol).
This method, as shown, can underestimate peak [Cali because Ca extrusion by INalC , begins as
[Cal; rises and hence can curtail the peak [Cali (by up to -25%, Bassani et at., ]994a). This
limitation can be circumvented by applying caffeine in ONa, OCa solution.
Indo-I fluorescence is strongly quenched by caffeine, but in a wavelength-independent
manner. Thus, fluorescence ratios used to infer [Cali are unaffected (if background fluorescences
are appropriately subtracted). O'Neill et ai. (1990a) turned this potential problem into an
advantage by using indo-l quench to indicate intracellular [caffeine]. The two usual indo-I
fluorescence signals can be combined to provide a Ca-independent signal that indicates the time
course of quench. That signal can then be translated to the intracellular [caffeine] as in the lower
panel of Fig 93A. In this case [caffeine]; rose to I mM (sufficient to trigger SR Ca release) in
less than 60 ms. Caffeine must be applied very rapidly. Otherwise contractions can be biphasic,
because myofilament Ca sensitization occurs progressively after SR Ca release. Slow caffeine
application also causes asynchronous cellular SR Ca release, which damps the global Ca
transient (lower & slower) as cellular release and extrusion progress as in propagating Ca waves.
During a caffeine-induced Ca transient the [Cali decline is almost entirely due to Ca
extrusion from the cell via NalCa exchange (93% in rabbit, Table 20). Varro et ai. (1993) first
used integrated INa/C. to measure SR Ca content. The Ca-activated inward current in rabbit cells
(Fig 93A) is entirely INa/ca, because it is abolished in ONa, OCa solution, whereas other candidate
Ca-activated currents (IO(Ca) & Ins(Ca» should still have functioned (Delbridge et aI., 1996). Thus,
caffeine-induced INa/ca can be integrated to measure the number of Ca ions released and extruded
(and divided by 0.68-0.93 to correct for the non-INa/ca Ca removal, depending on species). In this
rabbit myocyte we find 106 flmollL cytosol, in close agreement with that based on 6[Ca];
(especially considering that the two methods have very different assumptions and limitations).
In addition, since we know the rate of total Ca removal (measured by INa/ca) and the [Cali
at each point in time, this data can also be used as an on-line cytosolic Ca buffering titration (Fig
93B). This method, devised by Trafford et al. (1999), integrates INalCa from the end of the
caffeine-induced contracture backward in time for the falling phase of the Ca transient. The
analysis in Fig 93B yielded buffering values almost identical to curve #1 in Fig 26 (which were
used to calculate 6[Ca].01 in Fig 93A), so it would produce the same resultant MCa]IOI'
Caffeine-induced SR Ca release is thus a very powerful and useful approach, especially
in isolated myocytes where diffusional limitations are minimal. Other effects of caffeine such as
myofilament Ca sensitization (above) and phosphodiesterase inhibition (Butcher & Sutherland,
1962) which can increase cAMP and activate protein kinase A, can complicate interpretations
(but are generally not problematic during the few sec of caffeine exposure). The fact that the Ca
released is immediately subject to transport systems such as Na/Ca exchange (above) is also a
limitation. Finally, due to the transient nature of the [Cali rise diffusional barriers in
multicellular preparations mean that the [Cali transient will occur at different times throughout
the preparation. This lack of synchrony can prevent measurable overall muscle contraction or
global 6[Ca]; from being observed. Indeed, we find that caffeine-induced contracture amplitude
is inversely related to the diameter of the multicellular preparation. For multicellular
preparations rapid cooling contractures are far more useful because heat diffuses much faster
than caffeine and the cold also greatly inhibits Ca transport rates.
Rapid Cooling Contractures (RCCs)

Rapidly cooling cardiac muscle (to -1°C) induces contractures attributable to SR Ca
release. Rapid cooling contractures (RCCs) were first described in skeletal muscle fibers (Sakai,
1965), but required pretreatment with 0.3-1 ruM caffeine. This contrasts with mammalian
cardiac muscle, where RCCs are induced normally in the absence of caffeine (Kurihara & Sakai,
1985; Bridge, 1986) and prior caffeine exposure only inhibits RCCs (K m -1 ruM, with abolition
at 5-10 ruM caffeine, Bers et al., 1987; Bers & Bridge, 1988).
Figures 94 and 95A show typical RCCs in rabbit ventricular muscle and guinea-pig
ventricular myocytes after stimulation was terminated. Rapid cooling from 29°C to O-IOC
induces a rapid release of the available SR Ca to the cytoplasm. This Ca then activates a
contracture (slowly at 1°C), the amplitude of which indicates the SR Ca available for release at
the time of cooling. During the time at O-loC, ion transport mechanisms (e.g. Na/Ca exchange
and Ca-pumps) are strongly inhibited so [Cali declines slowly. Upon rewanning, a "rewarming
spike" is observed on the tension record, but not on the [Cali trace. This rewarming spike is due
to the rapid increase in myofilament Ca sensitivity induced by rewarming, before [Cali declines
(as expected from page 29; Harrison & Bers, 1989a). Rewarming also reactivates the ion
RCC
c
.Q
en
c
29°C~
Q)
l-
15 sec
I
Figure 94. Rapid cooling contracture (RCC) in rabbit ventricular muscle. Steady state field stimulation
(0.5 Hz) was terminated and 2 sec later (i) superfusate was switched from 29°C to 1°C. The surface
temperature of the muscle decreased rapidly. After the RCC reached a maximum solution was switched
back to 29°C, inducing a rewarming spike (due to increased myofilament sensitivity) and rapid relaxation.
transport mechanisms which had been inhibited by the cold (e.g. SR Ca-pump and Na/Ca
exchange). Reactivation of these processes causes rapid relaxation, and by changing solution
composition during the cold and rewarming one can evaluate the Ca transport mechanisms
responsible for relaxation (Bers & Bridge, 1989, see pg 52 & 152-4).
The muscle surface in Fig 94 was cooled below 5°C in < 300 msec, reaching a stable
value of ~ 1°C in about 1.5 sec. We estimate the core of a 400 f!m diameter muscle to be cooled
to <5°C in <2 sec (very much faster than caffeine would equilibrate in this muscle). Addition-
ally, because Ca transport systems are largely inhibited at 1°C, the [Cali rise during an RCC is
less transient than with caffeine. This makes RCCs of greater practical utility in multicellular
preparations. While RCCs are not as quantitative (in absolute Ca terms) as caffeine-induced Ca
transients (above), RCCs can be repeated to assess changes in SR Ca load on-line.
[Cali can rise very rapidly during an RCC (see Figs 76 & 95). This Ca comes from the
SR, since RCCs can be abolished by depleting the SR with caffeine, ryanodine and long rest
periods (Bers et at., 1987). RCCs also do not depend on Ca influx since their amplitude is
unaffected by the absence of [Ca]o (Bers et at., 1989; Fig 95A vs. D), even after long times (Fig
9SD-F). Sitsapesan et at. (1991) showed that cooling single cardiac Ca-release channels (in
bilayers) to 5°C dramatically increases the channel open probability (from Po = 0.004 to 0.35 at
100 nM Ca), mainly by tremendous increase in open time. Much evidence also suggests that
RCCs can release all of the available SR Ca (e.g. Bers et at., 1989).
RCCs measured as isometric force are generally smaller than twitch contractions, despite
the high [Cali. This is due to decreased myofilament Ca sensitivity and maximal force at 1°C (pg
29). When RCCs are measured as unloaded shortening in isolated myocytes or muscle they can
greatly exceed twitch amplitude (Fig 95; Hryshko et al., 1989c). The explanation is that
prolonged [Cali elevation in the RCC allows the cell to progressively shorten (since force needs
only overcome passive restoring force). Fig 9SA-C shows that increasing rest duration results in
gradual decreases in RCC amplitude (rest decay, see pg 157 & Chapter 9). When Ca extrusion
via Na/Ca exchange is prevented during the rest (ONa,OCa solution) then this loss of SR Ca is
2 sec rest 3D sec rest 2 min rest
RCC
in NT
D l°C--.-
RCCin
ONa,OCa
[caJI~~~
lD,um[W'(\~
DNa,DCa DNa,DCa DNa,DCa
Figure 95. RCCs and Ca transients in a guinea-pig ventricular myocyte, Shortening (in 11m) and [Cali
during the last stimulated twitches (at 0.5 Hz in A and D) and during RCCs induced 2 sec after the last
twitch (A and D), 30 sec after termination of another train of pulses (B and E) and after a similar 2 min rest
(C and F). In A-C the superfusate during rest and RCCs was NT. In D-F the superfusate during rest and
RCCs was Na-free and Ca-free, with 500 11M EGTA). The horizontal bar indicates the time during which
the superfusate was at 1°C (from Bers et ai" 1989, with permission).
also prevented (Fig 95D-F). [Cal does slowly decline during long RCCs, reflecting incomplete
inhibition of Ca transport systems (and RCCs relax faster at 3-5 QC than at 0-1 QC),
Ryanodine effects on SR Ca were extensively probed using RCCs (Bers et al., 1987,
1989; Bers & Bridge, 1989; Hryshko et aI., 1989c). After equilibration with 100-500 nM
ryanodine, RCCs of similar amplitude could still be measured in rabbit ventricular muscle, but
only immediately after a series of contractions (see Fig 96). With ryanodine, RCC amplitude
declined as a function of rest with a tY2=0.73 sec, which is ~IOO times faster than control (ty,=81
sec). These results indicated that ryanodine did not stop the SR from accumulating Ca when
[Ca]i was high, but that it greatly accelerated the leak of Ca back to the cytoplasm. This agrees
with observations that ryanodine preferentially slows relaxation at low rCa]; and depresses post-
rest contractions at lower [ryanodine] than that required to depress steady state twitches (Bers &
Bridge, 1989; MaU:cot & Katzung, 1987). This relates to the pump-leak considerations above.
That is, when rCa]; is high (and SR [Ca] is low) the Ca-pumping rate exceeds the rate of
ryanodine-induced Ca leak. However as rCa]; declines (and [Ca]SR rises) the leak becomes
greater and the SR loses Ca. These results agree with the action of ryanodine to "lock" the SR
Ca release channel in a submaximal open state (see pg 187,;Rousseau et aI., 1987).
Cumulative Extracellular Ca Depletions

A surprisingly useful tool to study changes in SR Ca is extracellular Ca microelectrodes
used to monitor net Ca gain or loss (as local [Ca]o depletions or accumulations, Bers, 1983, 1985,
1987; Bers & MacLeod, 1986; MacLeod & Bers, 1987). In the steady state Ca entry at one beat
must be extruded prior to the next beat; otherwise progressive Ca loading will occur (note steady
initial [Ca]o traces in Fig 97). However, under non-steady state conditions, such as termination
of stimulation, the cell may lose Ca (rest decay), observable as a rise in [Ca]o. With a long
i=' 70 Cl>
Q)
Z
l: 60 "~ 50
:::l
Ctl
'0.-
;j~ E
:t::u 50 <{
U 25
c.:t::
40
U
[t:: •
~~ Ryan
o ----------------------------
utn 30 o 234 5
UCf) Rest (sec)
0::0 20
-
0~ 10
0
Ryanodine
0 60 120 180 240 300

Rest Duration (sec)
Figure 96_ Ryanodine (100 nM) accelerates rest decay of SR Ca content in rabbit ventricular muscle.
Stimulation at 0.5 Hz was tenninated and the muscle cooled after rest time indicated on the abscissa. Inset
shows an expanded time scale (redrawn from data in Bers et aI., 1987)_
enough rest interstitial [Ca]o gradually returns to steady state. Resumption of stimulation results
in net Ca cellular uptake as the SR refills toward steady state (see [Ca]o depletions in Fig 97).
These cumulative cellular Ca losses and gains are due mainly to changes in SR Ca. They are
inhibited by caffeine and require transsarcolemmal Ca flux, since they can be blocked by Co and
nifedipine (Bers & MacLeod, 1986). Furthermore, larger Cao depletions are observed after long
rest intervals where the SR has lost its Ca (MacLeod & Bers, 1987). Since the SR is then empty,
a larger net Ca uptake is required to return it to steady state (hence the larger Cao depletion).
With ryanodine (Fig 97B) termination of stimulation results in a large and rapid Ca
extrusion from the cell ([Ca]o rise). This is because all of the SR Ca is rapidly extruded from the
cell by Na/Ca exchange (due to the rapid Ca leak from the SR). This fits with the above
mechanism of ryanodine action based on RCCs. That is, during trains of pulses the SR may
accumulate large amounts of Ca, because Cao depletions (& RCCs) in the presence of ryanodine
are as large as control, and are caffeine sensitive (MacLeod & Bers, 1987; Bers & MacLeod,
1986). During SR Ca leak and efflux [Cali remains low, because resting force is not elevated.
Reduction of the transsarcolemmal [Na] gradient inhibits Cao accumulations and
depletions (Fig 165B; Bers & MacLeod, 1986; Bers, 1987b). This is because the lower [Na]
gradient limits Ca extrusion by Na/Ca exchange. If Na/Ca exchange is unable to extrude Ca
from the cell during rest, the SR stays full of Ca (see pp 156-9), there will be no Cao
accumulation during rest and no Cao depletion with resumption of stimulation (i.e. the SR doesn't
lose Ca during rest, so it doesn't need more to be reloaded to reach steady state).
It may seem surprising that measuring [Ca]o is useful as an on-line monitor of changing
SR Ca content, but this emphasizes that Ca can shift from SR to interstitium and back (depending
on SR Ca-pumplNa/Ca exchange competition) without much change in diastolic [Cak One
A. Control
[Ca], [.#Ioj.l----"'''''''''1!II
1 mV 0.2 mM
[Cal,
B. Ryanodine
[ca],[ O.2mM
1 mV ··-·tCal,
Figure 97 Extracellular Ca depletions in rabbit ventricular muscle. Local [Ca]o (upper traces) and
tension before (A, bath [Ca]o =0.2 mM) and after equilibration with I I-tM ryanodine (8). Both panels
begin with steady state stimulation (0.5 Hz), 30 sec rest, I Hz for - 1 min and 0.5 Hz again after a 1 min
rest period. The spikes on the [Ca]o trace are stimulus artifacts. Extracellular [Cal was measured with a
double-barreled Ca-selective microelectrode (from Bers & MacLeod, 1986, with permission).
limitation of this approach is that Ca o depletions are difficult to extrapolate in absolute units of
I-tmol/L cytosoL A lower limit estimate of Ca uptake into the SR from this approach is 64
I-tmollL cytosol (MacLeod & Bers, 1987), but this does not account for interstitial Ca binding
sites. Hilgemann et a!' (1983, Hi1gemann & Langer, 1984) also studied cumulative cardiac Cao
depletions using extracellular Ca indicator dyes. These 2 types of [CaJo measurements also sense
net sarcolemmal Ca fluxes during single contractions (Bers, 1983, 1985, 1987b; Dresdner &
Kline, 1985; Pizarro et a!., 1985; Hilgemann, 1986a,b; Shattock & Bers, 1989).
Direct Chemical and Radiotracer Techniques

Classical chemical measurements (e.g. atomic absorption spectrometry and 45Ca flux)
can provide values that are easily extrapolated in units of I-tmol/L cytosol. However, the precise
intracellular location of the Ca and temporal resolution of these approaches is limited. Long rest
periods (after rest decay) were associated with Ca losses of >400 I-tmol/L cytosol in rabbit and
guinea-pig ventricle (Pytkowski et al., 1983; Lewartowski et al., 1984; Bridge, 1986;
Lewartowski & Pytkowski, 1987; Pierce et al., 1987; Pytkowski, 1989). These values are high
and may include Ca from compartments other than the SR. Pytkowski (1989) also measured the
caffeine-induced decrease in 45Ca content in rabbit heart (408 I-tmol/L cytosol), which was only
44% of the rest dependent loss of 45 Ca. Langer et al. (1990; Langer & Rich, 1993) also estimated
a caffeine-sensitive fraction of cellular Ca content in rat ventricle (210-240 /lmol/L cytosol)
which accounted for 21 % of the Ca in an "intermediate compartment" of 45Ca washout (ty, = 3-
19 sec). Unfortunately, interpretations using this general approach are complicated by contri-
butions of non-SR compartments and the observation that caffeine did not decrease the 45Ca
content in guinea-pig ventricle (Lewartowski & Pytkowski, 1987). On the other hand, radio-
tracer approaches have the unique advantage that one can measure unidirectional (as opposed to
net) fluxes. That is because one can have 45Ca on one side and 40Ca on the other and we assume
that the membrane does not distinguish between these isotopes.
In conclusion, the absolute SR Ca content varies in different species and under different
conditions. On the other hand, we are approaching a consensus where SR Ca content is probably
in the general range of 50-150 ~mol!L cytosol. For an SR Ca content of 100 ~mol!L cytosol we
would require about 50% of this amount to be released in terms of the Ca requirements for
activation of contraction (see Chapter 3). That conveniently matches estimates of fractional SR
Ca release during the twitch (Bassani et at., 1993b, 1995b).
SR Ca RELEASE CHANNEL OR RYANODINE RECEPTOR

Isolation and molecular identification of the SR Ca-release channel was greatly
accelerated by the recognition that the neutral plant alkaloid ryanodine is a selective and specific
ligand for the channel (also called the ryanodine receptor, RyR). Ryanodine produces
irreversible contracture of skeletal muscle, but progressive decline in cardiac muscle twitches
(Jenden & Fairhurst, 1969; Hajdu & Leonard, 1961; Sutko & Willerson, 1980). Ryanodine
opens the SR Ca release channel in both skeletal and cardiac muscle (see below). In cardiac
muscle the Ca lost from the SR is extruded by Na/Ca exchange, thereby unloading the SR and
cell of Ca, and depressing contractions. In skeletal muscle with much weaker Na/Ca exchange
and higher SR Ca content, much of the SR Ca remains cytosolic, activating a contracture which
consumes cellular ATP before the [Cali declines (making contracture irreversible). Some useful
RyR reviews are by Coronado et at. (1994), Meissner (1994), Sutko & Airey (1996), Xu et at.,
(I 998a), Zucchi & Ronca-Testoni (1997), Shoshan-Barrnatz & Ashley (1998) and Sitsapesan &
Williams (1998).
Ryanodine, at low concentrations (1-1000 nM), accelerates Ca loss from heavy SR
vesicles, but at very high concentrations (> 100 ~M) it slows Ca efflux (Fig 98D; Nayler et at.,
1970; Fairhurst & Hasselbach, 1970; Jones et at., 1979; Jones & Cala, 1981; Fabiato, 1985d;
Meissner, 1986a). Smith et af. (l985a, 1986) demonstrated that the Ca release from these heavy
SR vesicles from skeletal muscle could be attributed to a high conductance Ca channel which
they incorporated into lipid bilayers (i.e. the channel was similarly modulated by [Ca], ATP,
ruthenium red and Mg). Rousseau et at. (1986, 1987) demonstrated similar channels in heart and
also showed that ryanodine induced a long lived subconducting state of the SR Ca-release
channel (Fig 98C). That is, ryanodine (at least up to 10 ~M) appears to lock the Ca release
channel into an open, but lower than normal conducting state. Williams and coworkers have
shown that different ryanoids produce a different fraction of maximal conductance and also alter
the dwell time in the open state (Tanna et at., 1998,2000). These studies have provided unique
insights into the kinetics of ryanoid binding to the channel and also into conductance properties
(see also review by Sutko et at., 1997).
The ryanodine receptor (RyR) has a very high single channel conductance, especially
when monovalent cations are the charge carrier (see Table 22). The RyR channel also has a
relatively low Ca selectivity (PC'!P K -6 VS. 3000 for the sarcolemmal Ca channel, Smith et af.,
1988 and see Table 17, pg 109). Indeed, even some relatively large organic monovalent cations
can permeate, but the limiting pore diameter appears to be -3.4 A (Tinker & Williams, 1993).
The length of the RyR pore (i.e. the narrow region where ions must shed their aqueous shell) has
been estimated to be -lOA, by both streaming potential measurements and use of different
length organic divalent cations (Tu et at., 1994; Tinker & Williams, 1995).
A 1.2 f!M Ca 2 +
o 3
~~~
--7Mg+RR
(to block loss
during assay)
00 .601 0.01 0.1 1 1'0 100

[Ryanodine] (f!M)
~3PA
200 msec
Figure 98. Ryanodine effects on cardiac SR Ca release charmel. A-C. Recording from a single cardiac Ca
release charmel incorporated in a lipid bilayer. Currents are shown as upward deflections (from Rousseau et
aI., 1987, with permission). The cis (cytoplasmic) side of the membrane contained 2.5 flM free rCa) and
the trans side contained 50 mM Ba as the charge carrier (pH 7.4, 0 mV holding potential). Ryanodine was
added I min before panel B was recorded. C. shows the transition from normal gating to a stable
subconductance state on an expanded time scale. D. Dependence of 45Ca efflux from skeletal muscle SR
vesicles on [ryanodine). Vesicles were passively loaded with 45Ca (100 flM) and incubated for 45 min with
ryanodine. They were then diluted into a Mg + ruthenium red (RR) medium, which blocks efflux (unless
pretreated with 5 nM - 30 flM ryanodine) or diluted into 5 flM Ca medium which quickly emptied the
vesicles (except at high [ryanodineJ). Ryanodine causes SR Ca release at 5 nM to -30 flM, but blocks
release at 100 flM (from Meissner, 1986a, with permission).
When Ca is used as the charge carrier, the conductance is lower than for monovalents
and :2:50 mM Ca is typically used to obtain clear single channel data. Tinker et al. (1993)
predicted that under more physiological conditions ([Cah=2.4 mM, 120 mM K and 0.5 mM
Mg) the single channel Ca current would be 2 pA. This model prediction required extensive
extrapolation. Mejia-Alvarez et al. (1999) made a concerted effort to approach physiological
conditions with their single channel measurements and a unitary current of ~0.30 pA can be
inferred (at 2 mM [Cah, 150 mM [K], and I mM [Mg]). This single channel RyR current is
only slightly larger than that of the sarcolemmal Ca channel (~0.2 pA), but this is ~ I million
ions/sec or 105 times greater than turnover rate of the SR Ca-pump. The typical open time for a
RyR is ~3 ms (Tinker et al., 1993).
Molecular Identity and Structure ofRyanodine Receptors

Ryanodine was used as a specific ligand in the purification of the RyR from skeletal
muscle (RyRI, Inui et al., 1987a; Campbell et al., 1987; Imagawa et al., 1987b; Lai et aI., 1987,
1988a) and cardiac muscle (RyR2, Inui et al., 1987b; Lai et aI., 1988b). Mammalian RyRI has
been cloned (MW = 565,223 Da; Takeshima et al., 1989; Marks et aI., 1989; Zorzato et aI.,
1990). The cardiac RyR2 has also been cloned (MW=564,71I Da; Otsu et al., 1990; Nakai et
al., 1990) and a third isoform (RyR3) has also been cloned from brain (Hakamata et al., 1992).
These RyRs are products of 3 separate genes, but RyRI & RyR2 are 66% identical and RyR3 is
67-70% identical to RyRI and RyR2. Amphibian and avian skeletal muscle express a, ~ and
cardiac isofofills and a & ~ are similar to RyRI & RyR3 (Sutko & Airey, 1996). Notably, with
respect to E-C coupling issues to be discussed in the next chapter a & ~ isoforms coexist in fast
twitch frog skeletal muscle. Moreover, brain expresses both RyR2 and RyRI in addition to
RyR3 (and the related intracellular Ca release channel, the IP) receptor) and there is a very small
amount of RyR3 in mammalian skeletal muscle. Knocking out the RyR3 gene in mice does not
prevent striated muscle function (Takeshima et al., 1996). However, knocking out RyRI in mice
results in perinatal death (due to skeletal muscle failure, Takeshima et aI., 1994). Knockout of
RyR2 is embryonically lethal, but this may not be due to defective cardiac E-C coupling at this
developmental stage (Takeshima et al., 1998).
Table 22
Cardiac Ryanodine Receptor Permeability and Conductance
P,/P K Conductance (pS)* Radius (A)
K 1.00 723 1.33
Na I.l5 446 0.97
Cs 0.61 460 1.67
Li 0.99 215 0.68
Rb 0.87 621 1.47
Ca 6.5 135 0.99
Ba 5.8 202 1.34
Sr 6.7 166 I.l2
Mg 5.9 89 0.66
ammonium 1.42 594 1.7
methylamine 0.67 286 1.9
ethylamine 0.51 105 2.2
dimeth1alamine 0.09 10 3.1
triethy amine :":0.04 :":10 3.6
Relative permeability is based on reversal potential shifts in bionic conditions.
'Conductances in 210 mM permeant ion. Data are from Williams (1998).
The tetrameric nature of the ryanodine receptor in vivo implies a 2,260,000 Da structure.
The protein appears to exist mainly as a homotetramer, based on its quatrefoil appearance (Saito
et al., 1988; Lai et aI., 1988a; Wagenknecht et al., 1989), gel permeation chromatography (Inui et
al., 1987a) and stoichiometry of high affinity ryanodine binding (Lai et al., 1988a, 1989). The
large size of this homotetramer has helped to identify it ultrastructurally as the junctional foot
process which spans the gap between the SR and sarcolemmal membranes at their junctions.
Thus, it traverses the SR membrane providing a channel for SR Ca release and also extends
toward the sarcolemmal membrane. This proximity is undoubtedly important in the process of
triggering SR Ca release during E-C coupling (see Chapter 8).
Three-dimensional reconstructions of the RyR based on electron microcsopic images
have been continuously refined (Wagenknecht et al., 1989; 1994, 1996; 1997; Sasmo et aI.,
1999; Serysheva et al., 1995, 1999). Figure 99 shows three different views of the RyR. Sites are
also indicated where calmodulin and FK-506 binding proteins (FKBP) interact with the RyR (see
pg 198). The complex is -28 nm along each side and -14 nm high above the SR membrane,
which correspond to the width and length of the junctional "feet" observed ultrastructurally in
electron micrographs of intact muscle (see Chapter I). The RyR reconstructions are intriguing
because some suggest a channel for Ca flux going through the center of the molecule from the
SR lumen and possibly coming out the sides of the RyR into the junctional space (Fig 88 and
A. Top (outside SR) B. Side (along SR) c. Bottom (inside SR)
FKBP CaM D2
Figure 99 Three-dimensional reconstruction of the skeletal muscle ryanodine receptor. Three views A.
from top or T-tubule, B. from along the plane of the SR membrane and C. from within the SR lumen.
Selected cytoplasmic domains are numbered. "clamp" (dashed circle) refers to domains 5-10 that form
each corners of the cytoplasmic region; TM, transmembrane region; IpTxA, Imperatoxin A ; CaM,
calmodulin; D2, divergency region 2 (amino acids 1303-1406); FKBP, FK506-binding protein. Figure
generously provided by T. Wagenknecht.
Serysheva et al., 1999). In Fig 99 the FKBP site is ~9 nm away from the calmodulin site. The
FKBP location may be relevant to functional observations which suggest that FKBP is important
in coupling monomers within the tetrameric array as well as between tetramers (Brilliantes et al.,
1994; Kaftan et al., 1996; Marx et al., 1998a). There is also some initial information about
which RyR I sites might interact with the skeletal L-type Ca channel, (XIS or imperatoxin A
(Nakai et al., 1998a; Sasmo & Wagenknecht, 1998; Grabner et al., 1999).
The high affinity effects of ryanodine on SR vesicles coupled with observations from
more intact preparations (e.g. Sutko et at., 1985) led to the use ofryanodine as a specific ligand
in binding studies with SR vesicles (Pessah et al., 1985; Fleischer et al., 1985; Alderson & Feher,
1987; Imagawa et at., 1987b; Inui et at., 1987a; Lattanzio et al., 1987; Meissner & Henderson,
1987). The affinity of the receptor for ryanodine is dependent on rCa] and the presence of
nucleotides (e.g. ATP), but in the conditions typically used KI values are 4-36 nM.
Figure 100 shows some key RyR domains. The number of suggested transmembrane
spanning domains ranges from 4-12 (Takeshima et at., 1989; Otsu et al., 1990) with at least MI-
M4 consistent with most data (Balshaw et at., 1999). Results from the related IP 3 receptor are
more compelling for 6 transmembrane spans (Michikawa et al., 1994; Mignery et al., 1989;
Galvan et al., 1999) making 4-6 seem plausible for RyR2 until clearer data are available. A
human RyRI mutation (I4898T) produces central core disease (Lynch et al., 1999; see below)
and site directed mutagenesis studies in this M3-M4 region (including GIG, as in Na/Ca
exchanger & GYG in K channels) has identified this as the pore loop in RyR2 and RyRI (Zhao
et al., 1999; Balshaw et al., 1999; Gao et at., 2000). This is analogous to the IP 3 receptor domain
th th
where the channel pore has been shown to reside (the 5 _6 transmembrane spans including an
intervening GGVG sequence, Ramos-Franco et at., 1999). Marx et al. (2000) demonstrated that
the cardiac RyR is really a megacomplex including FKBPI2.6, a PKA anchoring protein
__ ~10!202.0 p~50~0!...-MiHii~n-----NH2
PP1 MH/CCD
02 2000 DHPRlRyR1
PP2A
MH/CCD
Ca-Activation
__ ------:~~~;;:----1~3~00~0~1~;
3500
Calmodulin
mAKAP
RII·PKA
PKAlCamKII
PO. site
Cytosol
SR
Membrane
Lumen
Figure 100 Schematic of domains in cardiac ryanodine receptor sequence. The 4 transmembrane
domains Ml-M4 are according to Takeshima et al.(l989) and there may be 2 more. Approximate locations
along the primary structure of several sites of either interaction (e.g. phosphatases I & 2A, PP I & PP2A;
mAKAP, kinase anchoring protein), a putative pore region (GIG), PKAlCaMKII phosphorylation site (P)
and Ca effector sites. A few sites important in RyR I are also shown, e.g. mutation sites associated with
malignant hyperthermia or central core disease (MH/CCD) and sites where skeletal muscle DHPRs may
interact (1635-2636 & 2659-3720, Nakai et al., 1998a). Figure kindly supplied by A.R. Marks.
(mAKAP) and two phosphatases (PPI & PP2A) in addition to interactions with calmodulin and
junctin/triadin described above. These associated proteins will be discussed further below.
Ca sparks: Fundamental Cellular SR Ca Release Events

SR Ca release in the intact myocyte appears to occur via relatively stereotypical local
events referred to as Ca sparks (Fig 101, sensed by fluorescent Ca indicators). These Ca sparks
(first described by Cheng et al., 1993) occur during rest (at very low frequency) in a stochastic
manner, even in the absence of Ca influx. The normal twitch Ca transient in ventricular
myocytes is also likely composed of a temporal and spatial summation of thousands of Ca spark
events which are synchronized by the AP and lea via Ca-induced Ca-release (Cannell et al.,
1994,1995; Lopez-Lopez et al., 1994, 1995).
Figure lOlA shows two spontaneous Ca sparks in a resting mouse ventricular myocyte
during acquisition of a 2-dimensional image. To enhance temporal resolution it is customary to
use the line scan mode of the confocal microscope. The whole length of this cell was scanned
every 2 ms along a line avoiding nuclei. Figure 10lB shows a line scan image with a single
prominent Ca spark, where distance along the cell length is shown in the vertical dimension and
time along the horizontal dimension. Figure lOlC shows the [Cali in the narrow region of the
cell where the Ca spark occurs. The surface plot (Fig lOID) shows the time and spatial depen-
dence oflocal [Cali during this single Ca spark. Ca sparks originate at the T-tubule (Cheng et al.,
B
10 ~m I .""j
,/
c
300
[Cal, (nM)
I
100
200 ms
300
D
ICa],
(nM)
::---:-"
'2.00 m
S
Figure 101 Ca sparks in isolated mouse ventricular myocyte. A. Two dimensional laser scanning
confocal fluorescence image of myocyte loaded with the Ca-sensitive indicator fluo-3, exhibiting two Ca
sparks (arrows). B. Line scan image along the long axis of the myocyte (only part is shown). Scans were
repeated every 4 ms and stacked from left to right. Distance along the cell is in the vertical direction. C.
Line graph of rCa]; at the spot indicated by the bar in B (-] /lm). D. Surface plot of rCa]; during a Ca
spark, indicating the temporal and spatial spread ofCa (figure kindly supplied by L.A. Blatter).
1996; Parker et aI., 1996) and typically reach a peak lCa]; of 200-300 nM in -10 ms, have a
spatial spread of -2 /lm (full width half-maximum) and lCa]; declines with a time constant of -25
ms. The decline of local lCa]; during the Ca spark is largely due to Ca diffusing away from the
site of release, However, we showed that when the SR Ca-ATPase was blocked in rat (by
thapsigargin) lCa]; decline during a spark was slowed by 26% (and spatial spread also
broadened, Gomez et al., 1996). This represents lCa]; decline attributable to diffusion away from
the source (i.e. both SR Ca-ATPase and Na/Ca exchange were blocked). Conversely, when we
stimulated SR Ca-ATPase by PKA activation, local lCa]; decline was accelerated by 33% (or
50% compared to diffusion alone). Thus Ca transport rate can effect spatial and temporal spread
ofCa sparks and influence their activation of neighboring RyRs via Ca-induced Ca-release.
Quantitative Aspects ofSR Ca release Flux

Cheng et al. (1993) estimated the Ca flux associated with a single Ca spark as -2x10- 19
mol (or 40 fC) and proposed that this might be due to a single RyR channel event (4 pAx 10 ms).
A more realistic single RyR channel flux is $ 2 fC (0.4 pA x 4 ms, see pg 187). This would be
consistent with a cluster of -20 release channels contributing to a single Ca spark. This is in the
range of the clusters of 50-200 feet/RyRs at dyadic junctions in heart (Franzini-Arrnstrong et al.,
1999; see pg 14). Attempts to measure the number of RyRs involved in a Ca spark have been
challenging (e.g. measuring smaller events or titrating some of the RyRs with blockers), but have
given values in the range of 6-20 (Parker et al., 1996; Lipp & Niggli, 1996; Blatter et al., 1997;
Bridge et al., 1999; Lukyanenko et al., 2000). It is clear now that a Ca spark is due to a cluster of
RyRs working as a functional unit (discussed further in Chapter 8).
To explain a resting SR Ca leak rate of 0.3 f.lmol/L cytosol (Bassani and Bers, 1995)
requires about 50 Ca sparks/sec in the cell (or -2 sparks/pLlsec). This is typical of the resting Ca
spark frequency observed in ventricular myocytes (Cheng et al., 1993; Satoh et al., 1997) and is
consistent with virtually all of the resting leak of Ca from the SR being attributed to these
occasional Ca sparks.
How many RyRs are there in a typical ventricular myocyte? Bers & Stiffel (1993)
measured 504, 656, 833 and 1,144 fmol/mg protein RyR in ventricular myocytes from rabbit,
guinea-pig, rat and ferret, respectively. This corresponds to 0.08-0.19 f.lmol/L cytosol RyR or
1.5-3.5 million RyR in a 30 pL myocyte (500 times fewer than SR Ca-pumps). For a resting rate
of 50 Ca sparks/sec, only 1,000 RyR need to open each second (or 0.02% of the cell's RyRs). To
attain a peak SR Ca release flux of 3 mM/s estimated by Wier et al. (1994), would require simul-
taneous activation of about 40,000 RyR (only ~2% of the cell's complement of RyRs). Further-
more, a total SR Ca release flux of 50 f.lmol/L cytosol would also require only ~ 7,500 Ca sparks
(based on 40 fC/spark) or -5% of the cellular RyRs (based on 2 fC/RyR). Thus, normal twitch
activation only requires a small fraction of available RyRs to function at any given twitch.
It is of interest to note here that opening of a similarly modest fraction of L-type Ca
channels (2-3%) is required to produce the measured whole cell lea (pg 114; Lew et aI., 1991).
For example, there may be -250,000 dihydropyridine receptors in a 30 pL rat ventricular
myocyte, but only ~5,000 Ca channels need to open (with a single channel current of 0.2 pA to
produce a peak whole cell current of 1 nA).
Regulation ofSR Ca Release

The most direct and compelling data about SR Ca release channel regulation come from
measurements of single RyR currents in lipid biJayers. Those studies are challenging and still
have the caveats that a) the channels are not in their native physiological environment and b) the
behavior of one channel is assumed to be representative of the population of RyRs. For many
aspects there is also corroborative evidence from measurements of Ca efflux from heavy SR
vesicles, ryanodine binding and cellular experiments. Cellular experiments are necessarily more
complex to interpret, but have the advantage of being in a more physiological context.
Measurement of 3H-ryanodine binding is simple, but useful because ryanodine binds strongly to
the open RyR channel, thus higher ryanodine binding (at sub-B max conditions) can correlate with
greater open probability (Xu et al., 1998a), although the inference is indirect. Table 23 shows
effects of several factors on RyR gating (see also reviews by Palade, 1987a,b,c; Coronado et aI.,
1994; Zuechi & Ronca-Testoni, 1997, Shoshan-Barmatz & Ashley; 1998; Xu et al., 1998a).
Figure 102 shows that Ca activation of the cardiac RyR begins at sub-micromolar rCa],
reaches a broad maximum (at very high Po) near 100 f.lM Ca and decreases at very high rCa] (5-
10 mM, Rousseau & Meissner, 1989; Xu et al., 1998a). The skeletal muscle RyR is less strongly
activated by Ca alone, requiring more Ca for activation, reaching a lower peak Po (near 10 f.lM
Ca) and almost completely inactivating by I mM Ca. ATP (and other adenine nucleotides)
activate cardiac RyR channels, but only if rCa] is high enough to partially activate the channel
2.5 ~M Ca F ,., 0.8 o ~

:= o Q:
:c
~-~
A ([>
"' 0.6
.c o
([>
3;
*
0
c
D: x
60 nM Ca cQ) 0.4
B 30
~~~~~1l;~'''~\;;'''1~:''
Q,
0
,.,
0:: 0.2
20 -=
q
0:: 10 '
en,,-
+ 3 mM Caffeine 0.0
C . ~.. 01 10 100 1000 10000
. .. [Cal (~M)
I 3pA
G 1.0
•
-400ms ~ ~o
+ 6 mM Caffeine :c 08 +2 mMATP
D "'0
.c
D: 0.6
c
[ 0.4
+ 4 mM Mg 0
E 0::
,., 0.2
"-~I;~;I~-.~~.J~,,,:~~,~Iw~~·· 0::
O.O~~-r-r~,..,....,~~,.....~...-~,...,...,
o 10 15 20 25
[Caffeine] (mM)
Figure 102. Caffeine, Mg and [Cal-dependence ofRyR gating (channels incorporated into lipid bilayers).
A-E. Single cardiac Ca release channel records from Rousseau & Meissner (1989) show that lowering cis-
(cytosolic) [Cal reduces channel opening (B, o=open c=c1osed), that caffeine activates the channel at low
[Cal (C & D) and that Mg blocks the channel (E). Current was carried by 50 mM Ca on the trans (luminal)
side. F. Ca dependence of channel open probability (f§') as in A-E (for cardiac and skeletal Ca release
ch3lmel; data from Xu et al., 1998a) or of the rate of Ca efflux from cardiac SR vesicles (Meissner &
Henderson, 1987). G. Effect of caffeine on cardiac and skeletal RyR in bilayers with -60 nM cis Ca and
-50 mM trans Ca as charge carrier. Addition of 2 I-tM ATP caused the channels activated by caffeine to
become almost fully open(P 0 -1; data from Smith et al., 1986; Rousseau et al., 1988).
(Rousseau et al., 1986). In contrast RyRI can be strongly activated by ATP alone, in the absence
of Ca or Mg (Xu et al., 1998a). Mg potently inhibits cardiac RyR opening in the mM range (Fig
102E) and free [Mg]j is normally 0.5-1 mM in cardiac myocytes. With the cardiac RyR activated
by Ca (in the absence of ATP), Mg inhibits Po half-maximally at 2.3 mM (Xu et al., 1996).
However, at physiological [ATP] (5 mM) the inhibitory effect of free [Mg] is modest at 2 mM,
and is still only half-inhibited at 5 mM free [Mg]. The precise in situ [Cali VS. Po relationship is
not known, but ATP shifts activation to the left and Mg shifts it to the right (vs. Fig 102F).
While Ca, Mg and ATP are likely to be central physiological modulators ofRyR gating,
local [ATP] and [Mg] are unlikely to change rapidly during E-C coupling and thus are not likely
to be actively involved in the process per se. Rather, the local concentrations of ATP and Mg are
critical in establishing how the RyR responds to a given physiological Ca signal. For example,
while mM Mg inhibits steady state RyR2 open probability (Po) for any given free [Cal, it also
accelerates the decline in Po induced by a rapid increase in local [Cal (Valdivia et al., 1995).
Free intracellular [Mg] can also increase several-fold during ischemia as [ATP] falls, presumably
because ATP is a major buffer of intracellular Mg (Murphy et al., 1989b). Ischemia is also
accompanied by intracellular acidosis and RyR2 open probability is reduced by >50% when pH
is lowered from 7.3 to 6.5 (Ma et al., 1988; Rousseau & Pinkos, 1990; Xu et al., 1996). Thus,
ischemia may greatly depress the responsiveness of the RyR to a given local activating Ca.
Much of the bilayer work is consistent with results from cardiac SR vesicles, where 45Ca efflux
rate was stimulated by IlM Ca, mM ATP and inhibited by mM Mg (Ky, = 0.3 mM), IlM
ruthenium red, acidosis and calmodulin (Meissner & Henderson, 1987). Furthermore the Ca flux
that they measured is of the order required to activate the myofilaments in the cell.
Caffeine and other methylxanthines (e.g. theophylline, theobromine, pentifylline)
activate both the cardiac and skeletal RyR, with cardiac RyR being more sensitive (Fig 102,
Rousseau & Meissner, 1989; Rousseau et ai., 1988; Liu & Meissner, 1997). Caffeine (1-5 mM)
appears to shift the Ca-dependence of RyR gating to ~1O times lower [Cal. Thus, the RyR in
resting cardiac myocytes is strongly activated by caffeine (even at resting [Cali)' MBED (9-
methyl-7-bromoeudistomin D) appears to have caffeine-like effects on RyRs, but is ~1000 times
more potent (Seino et al., 1991).
Ryanodine, at 1 nM-I0 IlM in bilayer studies, causes the RyR to open permanently to a
subconductance level (~half of the fully open state, Rousseau et aI., 1987). At very high
concentration (0.3-2 mM) ryanodine appears to bind to lower affinity sites and completely block
the RyR (Rousseau et ai., 1987; Lai et ai., 1989). Lai et al. (1989) demonstrated that there is
one high affinity and three low affinity ryanodine binding sites per RyR tetramer. Ryanodine
causes similar functional effects on the RyR in isolated SR vesicles (Meissner, 1986a) and in
intact cardiac muscle and myocytes (Bers et ai., 1987, 1989). The binding of ryanodine to the
RyR is very slow and it is practically irreversible. Consequently, the effects of ryanodine are
slow to develop. In intact cells and tissues the result is that only a small fraction of RyR are
typically activated. This creates a leak which is sufficient to drain the SR of Ca rapidly during
rest, but typically the SR can still transiently accumulate Ca (Bers et al., 1987, 1989). In vesicles
blocking 99% of the RyRs by ryanodine may reduce Ca loss by ~99% (since there may be ~1
RyRlvesicle). However, in the intact cell, where RyRs are in parallel. blocking 99% of RyRs
(with 1% in the open mode) may still result in draining the entire SR. Thus, ryanodine can be
trickier to use in intact cells than thapsigargin or caffeine (which is also rapidly reversible).
Higher [Ca]sR intrinsically increases Ca conductance through the RyR2 channel, but
higher luminal [Cal also increases RyR2 open probability (Sitsapesan & Williams, 1994, 1997;
Lukyanenko et aI., 1996; Tripathy & Meissner, 1996; Xu & Meissner, 1998; Gyorke & Gyorke,
1998). Figure 103A shows that increasing [Ca]sR increases the sensitivity of RyR2 to activation
by cytosolic Ca. One group suggested that this effect was due to more Ca passing through the
channel and acting at the cytoplasmic activating site (Tripathy & Meissner, 1996; Xu &
Meissner, 1998). This may well occur, but cannot explain the results of Gyorke & Gyorke
(1998, Fig 103A), because the [CalsR effect was independent of driving voltage direction. This
luminal Ca allosteric effect seems genuine. It does require the presence of a cytosolic activator
(e.g. ATP or sulmazole) and there may even be more complex Ca allosteric regulation (including
a luminal inhibitory site, Ching et al., 2000).
RyR Adaptation or Inactivation

The Ca-sensitivity of RyR2 activation is higher when the [Cal is raised very rapidly (Fig
103B, Fabiato, 1985b; Gyorke & Fill, 1993). Abrupt elevation of [Cal by flash photolysis of
caged Ca causes opening of RyR2 in 1-2 ms and after this initial peak the Po relaxes back to that
predicted by the steady state [Cal dependence (in ~2 sec, Gyorke & Fill, 1993). This decrease in
A. Lumenal Ca Activation B. Peak vs Adapted

300 [Cal SR = 5 mM 1.0
>.
,
0 :!:
a.. :cnl
-
u
...
"0 200
c:
0
.Q
...0
a..
c:
0.5
Q)
~ 100 C- "Adapted" •
o
0
or 55 V
0... ===....---.----.--....---.
0.01 '0.1 10 100 1000 0.1 10
[Cali (IJM)
Figure 103. Luminal SR [Cal and adaptation shift [Cal dependence of cardiac RyR gating. A. Increasing
[Cal on the luminal (trans) side of the bilayer (i.e. [Ca]SR) shifts the [Cali-dependence of RyR Po. Half
activation is shifted by 3.7-fold and maximal Po is increased 2.7-fold. Data are taken from
Gyorke & Gyorke, (1998). B. Rapid photolytic release of Ca from Ca-DM-nitrophen activates the RyR
in 1-2 ms (Peak data). The channel open probability (Po) gradually decreases (arrows) to a much lower Po,
referred to as the adapted state, which is similar to the steady state (SS) [Cali-dependence. Current was
carried by 250 mM Cs (from cis side). Data from Gyorke & Fill (1993) was regraphed.
release channel opening is essential for limiting the positive feedback inherent in Ca-induced Ca-
release. Gyorke & Fill referred to this process as adaptation, because after Po declined the same
Ca channel could still be reactivated by a larger Ca pulse. That is, it did not appear to reach an
absorbing inactivation state. Very similar results were found by Valdivia et al. (1995).
However, they found that inclusion of relatively physiological [Mg] accelerated the time course
of adaptation so that it occurred in -100 ms. This brings it closer to the time frame where it
could be involved in the turn-off of SR Ca release during a single cardiac contraction.
Several other groups have confirmed a time-dependent decline in RyR2 activation after
local [Cal is raised rapidly (Schiefer et aI., 1995; Sitsapesan et al., 1995; Laver & Curtis, 1996).
However, some of these studies have been more consistent with an absorbing inactivation state
than the adapted state described by Gyorke and Fill (1993). Whether the term adaptation or
inactivation is used to describe this phenomenon, it may serve functionally in the turning off of
SR Ca release (which is otherwise inherently regenerative, see pg 227-228). It is also very
similar to observations of Fabiato (1985b) in mechanically skinned single ventricular myocytes,
where the rate of Ca application was a crucial modulator of SR Ca release produced by a given
""[Ca] trigger, and whee recovery of the E-C coupling process required time (and low [Cali)'
Zahradnikova et al. (1999a) showed that rapid jumps of [Cal produced by flash
photolysis mimic the physiological Ca signal produced by abrupt opening of an L-type Ca
channel (where local [Cal rises to 1-20 11M in tens of llS). This rapid rise of [Cal caused opening
of individual RyR2s with a time constant of 0.15 ms and was consistent with activation by -4 Ca
ions. There is still no consensus on the ideal gating scheme for RyR2 (Stern et al., 1999).
However, rapid activation appears to favor initially a mode where the open time is longer, and
then gating shifts to a mode with shorter openings (possibly accounting for adaptation;
Zahradnikova et al., 1999b). True RyR2 inactivation might require the very high [Ca] or [Mg]
(5-10 mM) at which steady state Po declines (Fig 102F). This issue will be discussed further in
Chapter 8.
Malignant Hyperthermia, cyclic ADP ribose and Toxins

Halothane is used clinically as an inhalation anesthetic and its use can trigger episodes of
malignant hyperthermia (MH) in predisposed individuals. MH (and the related central core
disease, CCD) is attributed to mutations in RyRI which, when certain anesthetics are used (e.g.
halothane or isoflurane), cause inappropriate RyR activation, SR Ca release, skeletal muscle
hypercontracture, massive ATP consumption and consequent potentially fatal elevation in body
temperature (Mickelson & Louis, 1996; Loke & MacLennan, 1998). In MH-susceptible human
and pig skeletal muscle SR Ca release is more halothane- and Ca-sensitive (Endo et aI., 1983;
Nelson, 1983; Kim et al., 1984), has higher Ca release rate and greater RyRI Po (Mickelson et
aI., 1988, 1990; Fill et al., 1990, 1991). Dantrolene is a skeletal muscle relaxant which is used
clinically to curtail the MH-induced contractures above. Dantrolene blocks SR Ca release, but
interestingly has only weak effects on cardiac muscle (Ellis et al., 1976; Van Winkle, 1976;
Danko et aI., 1985). 4-chloro-m-cresol also activates RyRs (Hermann-Frank et al., 1996). It is
used diagnostically to distinguish between normal and MH-susceptible muscles.
Cyclic ADP-ribose (cADPR) is a metabolite of ~-nicotinamide adenine dinucleotide
(NAD), is present in myocytes at 20-200 nM and (along with the related NAADP) releases Ca in
sea urchin eggs (Gallione et al., 1991; Walseth et al., 1991; Lee, 1999,2000). The effect was
thought to be mediated by RyR2, but not RyRI (Meszaros et aI., 1993; Galione et aI., 1993).
Sitsapesan et al. (1994) showed that NAD, cADPR and its metabolite ADP-ribose can all
activate RyR2. However, at physiological [ATP] no activation was seen at all. They concluded
that cADPR, NAD & ADP-ribose compete weakly at the ATP site, but cannot serve as
physiological modulators of SR Ca release. Guo et al. (1996) also found that flash photolysis of
intracellular cADPR caused Ca release in sea urchin eggs, but not in ventricular myocytes.
Toxins isolated from the African scorpion Pandinus imperator can selectively activate
(IpTxA) or inhibit (IpTxl) SR Ca release channels (Valdivia et al., 1992). Tripathy et al. (1998)
found that IpTxA (33 amino acids) alters both RyRI and RyR2 gating by inducing long-lived
subconductance states (28 & 43% of normal conductance), reminiscent of ryanodine-modified or
FKBP-depleted channels (see Fig 98C and below). Based on Em-dependence and enhancement
of ryanodine binding, they inferred that IpTxA binds at a point 23% of the way through the Em
drop into the SR and not at the ryanodine site. Samso et al. (1999) identified the physical
location of IpTxA on the RyR (Fig 99), II nm away from the transmembrane pore and tucked
under the crown, away from the sarcolemma. This is relevant because Gurrola et al. (1999)
found IpTxA to mimic effects of peptides from the II-III loop of the skeletal muscle DHPR on
RyRI (see Chapter 8). While this would be a long physical reach for this II-III loop (Samso et
al., 1999), it raises intriguing possibilities with respect to protein-protein interactions. Bastidin-
10, a macrocyclic compound from the sea sponge Ianthella basta, activates RyRI gating, by
stabilizing the channel open state and makes gating almost independent of physiological Ca and
Table 23
Factors Which Alter Ca Release from the SR
Effective Muscle
Concentration ~ Reference
Enhancers of Ca Release
Ca 0.3-10 flM hrt M & H'87; Rousseau & Meissner,l989
Caffeine 1-10 mM hrt Fabiato, 1983; O'Neill et 01., 1990a;
Rousseau & Meissner, 1989
ATP (or AMP-PCP) 1-5mM hrt Rousseau et 01., 1986; M & H'87
Ryanodine 0.01-30 flM hrtlsk Rousseau et 01., 1987; Meissner, 1986a
Bastadin 10 5flM sk Chen et 01., 1999
Bromo-eudistomin D 10flM sk Nakamura et 01., 1986
MBED 1-10flM hrt/sk Seino et 01., 1991
4-chloro-m-cresol 0.5mM hrtlsk Hennann-Frank et 01.,1996; Xu et 01., 1998a
cyclic ADP-ribose 2-10flM hrt Meszaros et 01., 1993; Sitsapesan et 01., 1994
Doxorubicin 7-25 flM hrtlsk Zorzato et 01.,1985; Nagasaki & Fleischer,
1989; Ondrias et 01., 1990
Halothane -0.5 mM hrtlsk Ohnishi, 1979; Su & Kerrick, 1979
Palade, 1987b; Frazer & Lynch, 1992
Imperatoxin A 15nM hrtlsk Valdivia et 01.,1992; Tripathy et 01.,1998
Polylysine 1-10 flg/ml sk Cifuentes et 01.,1989
Quercetin 10-300 flM sk Kirino & Shimizu, 1982; Palade, 1987b
Sulmazole (AR-L I 15BS) ImM hrt Williams & Holmberg, 1990
Suramin 50flM hrt Sitsapesan & Williams, 1996; Xu et 01., 1998a
Sulfhydryl reagents
AgNO} 0.1-15 flM sk Salama & Abramson, 1984
Ag+ or Hg 2+ 10-25 flM hrt Prabhu & Salama, 1990
Cu 2+ICystei ne 2-10 flM sk Trimm et 01., 1986
Nitrosylation (by NO) hrt Xu et 01., 1998b
Ins(1,4,5)P 3 ]0-30 flM Iut Fabiato, 1990
10-20 flM sk Volpe et 01., 1985
0.5 flM sm Walker et 01., 1987
Inhibitors of Ca Release
Ca 5-10mM hrt Xu et 01., 1998a
Mg 1-3mM Iutlsk M & H'87; Fabiato, 1983
Acidosis pH 7.5~6.5 hrt Xu et 01., 1996
Ryanodine >IOOflM hrt/sk Meissner, 1986a; Jones et 01., 1979;
Lai et 01., 1989
Ruthenium red 10flM hrtlsk M &H'87
Calmodulin IflM hrt Smith et 01., 1989; M & H'87
Dantrolene 2flM sk Danko et 01., 1985
Imperatoxin I InM hrtlsk Valdivia et 01., 1992
Neomycin, gentamycin 60-200 nM sk Palade, 1987c
Spennine, spermidine 20-200 flM sk Palade, 1987c
Tetracaine, procaine 0.1,1 mM hrt/sk Palade, 1987a; Antoniu et 01., 1985;
M & H'87, Xu et 01., 1998a
M & H'87 is Meissner & Henderson, 1987; hrt=heart, sk=skeletal and sm=smooth muscle, MBED = 9-
methyl-7-bromoeudistomin D. This table is based on tables compiled by Fleischer & Inui (l989) and more
extensive tables by Palade (l987b, Palade et 01., 1989) which were focused on skeletal muscle SR vesicles.
This table is intended to focus on cardiac SR Ca release where data are available.
Mg (Chen et al., 1999b). These bastadin-IO effects were abolished ifFKBP was displaced from
the RyR, suggesting interaction with the RyR-FKBP complex.
Regulation by Protein Kinases. Calmodulin and FKBP

Phosphorylation of RyR2 by cAMP-dependent protein kinase (PKA) produces intriguing
effects on channel gating (Valdivia et al., 1995). Basal Po was decreased by PKA (at 100 nM
[Ca]). However, PKA greatly increased peak Po (to nearly 1.0) during a rapid photolytic increase
of local [Cal, while accelerating the subsequent decline in Po. Thus, phosphorylation by PKA
may activate RyR2 gating in the same dynamic way that PKA modifies cardiac contractile force
and cellular Ca transients during a twitch (i.e. increasing both amplitude and rate of decline).
Marx et al. (2000) found that RyR2 phosphorylation by PKA occurs at Ser-2809.
Ca-Calmodulin dependent protein kinase (CaMKII) also phosphorylates the cardiac RyR
at Ser-2809 (Witcher et al., 1991). Notably, Witcher et al. (1991) found that either endogenous
SR CaMKII or exogenous PKA would incorporate 1 P0 4 per RyR2 tetramer, but that exogenous
CaMKII could produce higher phosphorylation (4 P04 per RyR2 tetramer). Bilayer recordings
with skeletal muscle RyR showed that CaMKII either increased or decreased RyR channel
openings (Takasago et aI., 1991; Wang & Best, 1992). In cardiac RyR CaMKII appears to
increase channel Po (Witcher et aI., 1991; Hain et aI., 1995), but Lokuta et al. (1995) reported
that CaMKII decreased RyR2 open probability. This discrepancy may be partly explained by
dynamic changes of RyR gating, as discussed above for PKA (but similar data are not available
for CaMKII). In voltage clamped myocytes, we found that inhibition of CaMKII prevented a
[Ca];-dependent increase in the fraction of SR Ca released for the same lea and SR Ca content (Li
et al., 1997b). Introduction of phosphatases (PPI & PP2A) into myocytes also depresses E-C
coupling gain (duBell el al., 1996). Thus, in the intact cardiac cell repeated Ca transients may
activate CaMKII, phosphorylate RyR2 and enhance the efficacy ofE-C coupling (see pg 268).
A complicating aspect of CaMKII effects on the RyR is that calmodulin (CaM) also has
independent effects on the RyR. At [Cal >100 nM CaM inhibits Ca-induced, caffeine-induced,
and AMP-induced Ca release from cardiac and skeletal SR (lC so -100-200 nM, Meissner, 1986a;
Meissner & Henderson, 1987; Plank et al., 1988; Fuentes et al., 1994; Tripathy el al., 1995).
These effects were ATP-independent, so CaMKII was not involved. Similarly, Tripathy et al.
(1995) found that CaM inhibited RyRI Po at [Ca] ~1 f!M, but stimulated it at [Cal <100 nM.
This dual mode of CaM action was confirmed in skinned skeletal muscle (lkemoto et al., 1995).
CaM binding to the RyR is also Ca-dependent. Tripathy et al. (1995) found that one
CaM binds per RyRI monomer at 100 f!M [Ca], but four calmodulins bind per RyRI monomer at
[Ca]<IOO nM (Kr 10 nM). Fruen et al. (2000) provided valuable comparative data on effects of
calmodulin on RyRI and RyR2. They found that only I CaM binds per RyRI monomer at both
low and high [Cal. The same is true for RyR2 at 200 f!M [Cal, but at 100 nM [Cal RyR2 binds
only I CaM per RyR tetramer (and K d increases from 16 to 84 nM [CaM]). They extended the
RyRI results above, confirming the inhibitory effect of CaM on Ca flux for RyRl and RyR2 at
f!M [Ca]. On the other hand, they found no effect of CaM on RyR2 flux or ryanodine binding at
100 nM [Cal (in the presence or absence of Mg). Thus, it is unclear at this time whether CaM
exerts direct functional effects on cardiac RyR (other than activating CaMKII).
FK-506 and rapamycin are immunosuppressant drugs that bind to immunophillin target
FK-binding proteins (FKBPs). In T-Iymphocytes Ca-CaM normally activates the phosphatase
calcineurin which dephosphorylates the nuclear transcription factor NFAT allowing its entry into
the nucleus where it stimulates interleukin-2 transcription and T-cell proliferation. The FKBP-
FK-506 complex binds to calcineurin preventing its activation and thereby suppressing the
immune response (Schreiber & Crabtree, 1992; Marks, 1996). FKBPs are peptide isomerases
which also bind to and co-purify with the RyR (Jayaraman et al., 1992; Timerman et a!., 1993,
1994,1996), but the isomerase activity is not essential for RyR effects (Marks, 2000). FKBP-12
(MW 12,000) binds tightly to RyRI (Fig 99). Heart expresses both FKBP-12 and -12.6 and
despite a larger amount ofFKBP-12, it is FKBP-12.6 which associates with RyR2 (due to 600x
higher affinity, Timerman et al., 1996).
FK-506 and rapamycin cause dissociation ofFKBP from the RyR and modify RyRI and
RyR2 gating in bilayer studies (Brilliantes et al., 1994; Ahern et al., 1994; Chen et al., 1994;
Kaftan et al., 1996; Barg et a!., 1997), although Barg et al. (1997) found no effect of FK-506 on
RyR2. FKBP removal from RyRI shows clear appearance of subconductance states (with 14, Y2,
& ~ of the normal conductance). Kaftan et al. (1996) found analogous results with RyR2, where
the net effect was an increase in overall Po (despite the 3 lower conductance states). Indeed,
when exogenous recombinant FKBP was added to recombinant RyR in bilayers the normal
channel gating properties with FKBP were restored. FK-506 also inhibits RyR2 adaptation (Xiao
et al., 1997) and this may relate to altered Ca regulation of RyR2 in the absence of FKBP
(below). Complementary measurements in intact cells show that FK-506 increases resting Ca
spark frequency and causes resting SR Ca content to decline (McCall et al., 1996a; Xiao et al.,
1997). This confirms that the overall enhanced Po of RyR2 in bilayers after FKBP removal,
extends functionally to resting Ca leak in intact ventricular myocytes.
A working hypothesis from this work is that FKBP physically stabilizes the coordinated
gating of the 4 RyRs in one homotetramer so that openings go from the fully closed to the fully
open state, but with reduced overall Po for a given [Ca] (e.g. shifting the Po vs. [Ca] relationship
to higher [Ca)). The four conductance levels raise an intriguing question about the RyR tetramer,
especially in light of the apparent pore(s) down its center (see pg 188): Does each RyR monomer
contribute a channel with 14 of the full conductance, or does gating in each subunit contribute to
enhancing the conductance of a single central pore by 25%? The former seems more likely.
Marx et al. (I 998a) demonstrated that FKBP may also be involved in physical coupling between
RyR tetramers. They showed that addition of FKBP induced simultaneous gating of 2 or more
full RyR channels, and this effect could be reversed by removal of FKBP with rapamycin. This
may be a redundant mechanism which, together with Ca-induced Ca-release, allows individual
RyR channels to activate neighboring RyRs and coordinates closure (Bers & Fill, 1998).
FKBP effects may be clinically relevant and FK-506 treatment can be associated with
cardiomyopathy (Atkinson et al., 1995). Marx et al. (2000) showed that PKA-dependent phos-
phorylation of RyR2 causes displacement of FKBP from RyR2 and produces the same sort of
uncoupled gating as does FK-506 or rapamycin. In heart failure (human & canine) they found
that RyR2 is hyperphosphorylated (4 P04 per tetramer) with channels showing the FKBP-
depleted phenotype (multiple conductances, increased overall flux). The higher phosphorylation
could be partly due to less phosphatase (PPI) detected as bound to RyR2 in heart failure (despite
higher global PPI levels). Lower, physiological levels of phosphorylation with PKA (I/tetramer)
may increase Ca sensitivity, enhancing E-C coupling (Valdivia et al., 1995). In contrast, hyper-
phosphorylation (4/tetramer) could cause persistent diastolic SR Ca leak, limiting SR Ca content
and contraction in the failing heart. A puzzling aspect of this work is that CaMKII
phosphorylates the same RyR2 site as PKA, but does not produce the same RyR effects as PKA.
Sorcin is a ubiquitous 22 kDa Ca binding protein (Kn~Ca)=1 IlM) reported to associate
with both cardiac RyR and DHPR (Meyers et 01.,1995,1998). Lokuta et al. (1997) showed that
sorcin reduced RyR2 open probability and ryanodine binding (lC so =480-700 nM), but this
inhibitory effect could be relieved by PKA-dependent phosphorylation of sorcin. While
additional work is required, it is possible that sorcin, like FKBP, serves as a kind of inherent
brake on SR Ca release, relievable by PKA-dependent phosphorylation (Valdivia, 1998).
Inositol 1,4,5 trisphosphate (IP3) Receptor

IP 3 is a well documented activator of Ca release from internal stores in non-muscle cells
(Berridge, 1987; Berridge & Galione, 1988; Berridge & Irvine, 1989), but the role in cardiac E-C
coupling is controversial (see pg 237-243). The IP 3 receptor (lP 3R) is closely related to the RyR,
and these combine to form the superfamily of intracellular Ca release channels. The IP 3R was
initially isolated from neural tissue (Supattapone et al., 1988) and smooth muscle (Chadwick et
01., 1990). The neuronal IP 3R was cloned (MW = 313 kDa, Furuichi et al., 1989; Mignery et al.,
1989) and there are certain sequence homologies with RyRs. Chadwick et al. (1990) showed that
the smooth muscle IP 3R has the same quatrefoil structure (25 x 25 nm) as the skeletal muscle
ryanodine receptor (despite the smaller molecular weight) and they suggested a similar tetrameric
arrangement. There are IP 3R subtypes (type-I, type-2 & type-3). Type-l is the predominant
smooth muscle and neuronal IP 3R, and it is particularly highly expressed in cerebellar Purkinje
neurons. Type-2 and -3 have also been cloned (Siidhof et 01.,1991; Blondel et al., 1993) and are
69 & 64% identical to type-I. IP 3Rs are expressed in a wide variety of tissues, probably
constituting the more general SR/ER Ca release channel than RyRs (which are more specific to
muscle). In whole heart all three types of IP 3Rs are expressed, but in isolated ventricular
myocytes only the type-2 IP 3R is expressed (Perez et al., 1997). The number of IP 3Rs in
ventricular myocytes is probably 2-10% of the number of RyRs (Moschella & Marks, 1993;
Perez et al., 1997), raising the question (see Chapter 8): what is the function of all of these IP 3Rs
in ventricular myocytes? The other IP3R types in whole heart are probably in other cell types
(e.g. vascular smooth muscle & endothelial). However, cardiac Purkinje fibers seem to express
type-I IP 3R and also RyR3 rather than the RyR2 in ventricular myocytes (Gorza et al., 1993).
The IP 3Rs have 3 domains going from amino to carboxy ends: 1) ligand binding, 2)
coupling and 3) channel (Mignery & Siidhof, 1990; Siidhof et al., 1991). Despite their sequence
and domain similarity, the 3 types of IP 3R are differentially regulated by IP 3 and Ca. The type-2
IP 3R has the highest IP 3 affinity (Kd -25 nM) and this is similar to the Ko.s for channel activation
(58 nM IP 3, Perez et al., 1997; Ramos-Franco et al., 1998). Type-l has -4 fold lower affinity
and type-3 has ~IO times lower IP 3 affinity than the others (Hagar & Ehrlich, 2000). Figure
104A shows the [IP 3 ]-dependence of channel activation for the three types. IP 3R activation is
also sensitive to Ca, but the Ca-dependence also differs among the isoforrns (Fig 104B). Type-l
IP 3R shows a striking bell-shaped Ca-dependence (Bezprozvanny et 01., 1991), whereas Type-2
and -3 do not show the prominent decline in Po at high [Cal (Ramos-Franco et aI., 1998; Hagar et
01., 1998). The bell shaped Ca-dependene of type-l IP 3R gating may be functionally important
for the generation of Ca waves or oscillations in some cell types (Thomas et al., 1996). That is,
A. 1P3-Dependence B. [Cal-Dependence
06 0.8 0.08
u:-
>-
:= • ......... _..-
~ 0.6
:0 0.4 ~
co
..c Type-3
0
~
/~~pe-3 :0
co
..c 0.4 Hagar 0.04 ~
I
...I
0 ~
c: ~ c-
Cll
o. 0.2 ,/ c:
0 ../ Cll
0.2 0.02 ~
..•...
0.
0
........ 1IJM 1P3
0.0
250 nM Ca o. 0.00
0.01 0.1 10 0.01 0.1 10 100
[IP 3] (IJM) [Cal (IJM)
Figure 104. IP]- and Ca-dependence of IP] receptor (IP]R) activation (A and B respectively). Single
channel data for the type-I, -2 and -3 IP]R Ca channels was from Ramos-Franco et al. (1998, R-F, solid
curves) and Hagar (dotted curves, Hagar et al., 1998; Hagar & Ehrlich, 2000). Open probability (Po) for
[IP]J-dependence of type-3 is scaled up 5.5x so the indicated ratio of maximum Po for type-3/type-l is as
reported by Ehrlich's group (measured maximum Po was 0.08, Hagar & Ehrlich, 2000).
the strong inhibition at 1-lD flM Ca may allow local [Ca] to shut off local release. This may also
tend to make the IP3-induced Ca transient more restricted in both time and space with the type- 1
IP 3R. Thus, type-I IP]R is truly co-regulated by IP 3 and Ca. The type-2 IP 3R in ventricular
myocytes is both more sensitive to IP3 and almost insensitive to [Ca] over the physiological range
of [Ca]; (0.1-10 flM). Thus, the cardiac IP 3R may function more as a pure IP 3 sensor.
The conductance of the IP 3R channel is about half of that seen for the RyR channel, but
the selectivity among divalent cations and the PB./P K of 6.3 are similar to that for RyR (see Table
22, Bezprozvanny & Ehrlich, 1994, 1995). The IP 3R, like the RyR, is also modulated by
numerous factors, but I will not present as much detail. ATP and non-hydrolyzable ATP
analogues potentiate IP 3R channel opening and Ca release (Ferris et al., 1990; Bezprozvanny &
Ehrlich, 1993). Heparin is the classical inhibitor ofIP]R channels, but it activates RyR channels,
making it a useful diagnostic tool (along with caffeine, which does not activate IP 3R channels).
The type-I IP3R can also be phosphorylated by protein kinases A, C, G, CaMKIl and tyrosine
kinase (Ferris et al., 199Ia,b, 1992; Komolavilis & Lincoln, 1994; Nakade et al., 1994;
Jayaraman et al., 1995). PKA-dependent phosphorylation increases the IPrsensitivity of the
channel (Burgess et aI., 1991), but less is known about regulatory effects of phosphorylation by
the other kinases (or for the type-2 IP 3R in general). FKBP binds to IP 3Rs (in a rapamycin- and
FK-506-sensitive manner), decreases IPrsensitivity and can also form ternary FKBP-IP3 R-
calcineurin complexes (Cameron et al., 1995, 1997). Calmodulin also binds to the type-l IP 3R at
a site which also exists in type-2, but not type-3 IP 3R (Yamada et aI., 1995). Michikawa et al.
(J 999) also showed that calmodulin may be responsible for mediating the Ca-dependent
inhibitory limb of the type-l IP 3R curve in Fig 104B. This is reminiscent of the role of
calmodul in in mediating the Ca-dependent inactivation of L-type Ca channels (see pg 117).
Other SR Channels Related to Ca Release

The penneability of the SR membrane to monovalent ions is very high (Meissner,
1986b). High conductance K- and anion-selective channels exist in the SR membrane (Coronado
& Miller, 1979, 1980; Coronado et al., 1980; Hals et al., 1989). Also, there do not appear to be
any appreciable concentration gradients of monovalent ions between the inside of the SR and the
cytoplasm (Somlyo et aI., 1977a,b; Somlyo & Somlyo, 1986). This has three important
functional consequences. First, it implies that there is no membrane potential between the
cytoplasm and the interior of the SR (which has implications for certain possible models of E-C
coupling). Second, it means that flux through the channel will be carried mainly by Ca, despite
the poor selectivity of the RyR channel. This is because there is a large driving force for Ca, but
not monovalent cations. Third, it allows Ca release to proceed rapidly with monovalent fluxes
compensating quickly for the divalent charges (Ca) leaving the SR. Otherwise, the rate of Ca
release could be limited, in part by the development of a large negative intra-SR potential (which
would oppose further Ca flux from the SR).
In conclusion, it is clear that the SR can accumulate sufficient Ca and release it fast
enough to activate cardiac muscle contraction. Indeed, a great deal is now known about how the
SR Ca-pump and SR Ca release channel work in isolated systems (such as SR vesicles and in
bilayers), and there are often correlates in more intact preparations. The next couple of chapters
will focus more directly on the regulation of SR Ca release in the intact cell (E-C coupling
mechanisms) and also how the SR Ca transport mechanisms interact with the other Ca transport
systems in a dynamic way in the intact cardiac myocyte.
D.M. Bers. 203
CHAPTER 8
EXCITATION-CONTRACTION COUPLING
Since the classic experiments of Ringer (1883) demonstrated that frog heart would not
contract in the absence of extracellular Ca, the crucial nature of Ca in muscle contraction has
become increasingly clear. Figure 105A shows a modem version of Ringer's experiment where
Ca o is removed quickly from the medium around a rat ventricular myocyte causing an immediate
abolition of contraction (in < 1 sec; Rich et al., 1988). This contrasts strikingly with skeletal
muscle (Fig 105B) which can contract for many minutes in the complete absence of extracellular
Ca (Armstrong et al., 1972). Figure 106A shows the Em-dependence of several parameters
during voltage-clamp of isolated guinea-pig ventricular myocytes. The Em-dependence of Ca
transients and contractions is bell-shaped, just like ICa in cardiac preparations (McDonald et al.,
1975; London & Krueger, 1986; Cannell et al., 1987; Beuckelmann & Wier, 1988; Callewaert et
al., 1988; duBell & Houser, 1989). This is also true for an intrinsic birefringence signal in
cardiac muscle which is thought to be associated with SR Ca release (Maylie & Morad, 1984).
However, Em-dependence of intramembrane charge movement (related to Ca channel activation)
in heart is sigmoidal (Field et al., 1988; Bean & Rios, 1989; Hadley & Lederer, 1989, 1991).
Thus, in heart, depolarization causes charge movement, resulting in Ica and Ca transients. At Em
above 10 mY, l ca is smaller (due to lower driving force) and results in both smaller L'l.[Ca]; and
A. Isolated Rat 'I' II

'l'l'l''I'l
Ventricular
Myocyte
Shortening
ICa), (mM) 1 --;LJ LJ 10 sec
B. Single
Frog
Skeletal
Fiber
I I I I I I I
o 5 10 20 25 30 35
Time (min) in OCa+EGTA solution
Figure 105. Cao·free solution abolishes contractions immediately « I sec) in cardiac myocytes (A.), but
not for >25 min in single skeletal muscle fibers (B.). A. [CaJo was changed rapidly between stimuli. The
cell was stimulated continuously (at 0.2 Hz) and arrowheads indicate stimulations in Cao-free solution (from
Rich et aI., 1988, with permission). B. A single frog skeletal fiber stimulated at 0.1 Hz except during the
switch to a Cao-free solution containing I mM EGTA. The eventual decline in force after -26 min was
attributed to gradual membrane depolarization (from Armstrong et al., 1972, with permission).
A. Guinea-Pig Ventricle B. Frog Skeletal Muscle
E Charge
~ 1.0 Movement
.~
-o-I ea
~ ~ ~ Birefri ngence
:E --~[Ca]i
'0 - - Contraction
g 0.5
~
at
O.Oh'::::'iI<'=~~~.j-~~~~~""';'..:;...c~
-60 -30 o 30 60 -60 -30 0 30 60
Em (mV) Em (mV)
Figure 106. Voltage dependence of ICa, Ca transients, contraction and charge movement in isolated
guinea-pig ventricular myocytes (A) and frog skeletal muscle (B). A. Data are from BeuckelmalU1 & Wier,
1988 (lca and MCa]; using fura-2), Hadley & Lederer, 1991 (charge movement) and Bers, unpublished
(shortening) and are nonnalized to their maxima (0.9 nA for ICa, 459 nM for ""[Ca]; and 12 ~m for
shortening). Charge movement was fit with Q=QmaA I+exp[-(E m-V*)lk])where Qmax was 5 nC/~F, V*= 7.5
mV and k=II.5 mV. Holding Em ::; -40 mV and test pulses were 20 msec (for Q) and 200-300 msec for
other curves. B. Skeletal muscle data from Miledi et aI., 1977 (""[Ca]; using arsenazo Ill), Chandler et al.,
1976a (Q), Caputo et al., 1984 (Force), Baylor & Chandler, 1978 (birefringence) and Sanchez & Stefani,
1983 (lca)' Values were normalized to their maxima (-2 ~M for ""[Ca];, 21.5 nC/~F for Q, -3 kg/cm for
tension and 110 ~AJcm2 for ICa)' Qmax was set to I, V*=47.7 mV andk=8 mV. The raw [Cal; data were fit
with the same equation, but with V*=23 mV and k=9 mV. Holding Em was between -100 and -75 mV and
pulses were 100 msec for Q, tension and birefringence, 10 msec for [Cali and 1.8 sec for ICa.
contraction. The parallel nature of lea and Ca transient amplitude in heart sometimes makes it
difficult to distinguish unequivocally between direct effects of Ca entry and the SR Ca release
induced by the Ca entry (see Chapter 9). However, three lines of evidence indicate that SR Ca
release contributes in a major way to contractile activation in cardiac muscle: I) the inhibition of
cardiac contractions by agents which affect SR Ca (caffeine, ryanodine & thapsigargin), 2)
quantitative estimates of Ca entry via ICa and requirements for myofilament activation, and 3)
interpretation of force-frequency relationships. Moreover the bell-shaped Em-dependence of ICa
and il[Ca]; is a hallmark of cardiac E-C coupling and the dependence of SR Ca release on Ca
influx has led to the moniker, Ca-induced Ca-release (CICR), which will be examined below.
In skeletal muscle charge movement is also sigmoidal as a function of Em (Fig 106B,
Schneider & Chandler, 1973), but il[Ca];, force and birefringence signal all follow this E m-
dependence, while ICa remains bell-shaped as in heart. Indeed, since skeletal muscle ICa activates
so slowly (peak ICa at 22°C is ~200 msec VS. -5 msec for cardiac ICa), that little ICa flows during a
normal twitch (Sanchez & Stefani, 1978, 1983; Gonzalez-Serratos et al., 1982). This more
intrinsically Em-dependent E-C coupling mechanism in skeletal muscle is referred to as charge-
coupled SR Ca release or voltage-dependent Ca release (VDCR) and will be discussed below.
Thus, there are striking fundamental differences in E-C coupling between cardiac and
skeletal muscle, despite qualitative similarities discussed in preceding chapters. The following
list (and Fig 107) indicates 7 possible mechanisms that have been suggested by various
investigators to contribute to SR Ca release in cardiac muscle.
Chapter 8 E-C coupling 205
® @ ® ~
Slip I
Mode Na Voltage- lea Tor
depenaent
ea~
lea:TTx ~
Figure 107. Potential E-C coupling mechanisms in cardiac muscle. Diagram

mechanisms that have been proposed to contribute to cardiac E-C coupling refers to 7 potential
(see below).
Possible Activators of Cardiac SR Ca release

Ca-induced Ca-release COCR) variants
I. L-type Ica .
2. T-type or TTX sensitive ICa (ICa,TTX).
3. Ca influx via Na/Ca exchange driven directly by Em-dependence
ofI NaiCa '
4. Ca influx via Na/Ca exchange driven by local high [Na]j, seconda
ry to INa'
5. Altered selectivity ofNa channels (allowing Ca permeation) with
PKA activation.
Ca influx-independent variants
6. Voltage-dependent SR Ca release (VDCR)
7. Inositol (I ,4,5)-trisphosphate (IPJ)-iduced SR Ca release (IPJICR)
The following sections will discuss three major mechanisms, with

focus on the muscle
types in which they are most promine nt (i.e. VDCR in skeletal muscle,
CICR in cardiac muscle
and IPJICR in smooth muscle). However, I will especially focus on
the roles of depolarization-,
Ca- and IPJ-induced Ca release in cardiac muscle.
VOLTAGE-DEPENDENT Ca RELEASE (VDCR)

& SKELETAL MUSC LE E-C COUPLING
Skeletal muscle activation is strongly Em-dependent (Hodgkin &
Horowicz, 1960) and
Schneid er & Chandler (1973) described intramembrane charge moveme
nt that might drive SR Ca
release and hence contraction. This charge movement is recorded
as an outward current upon
depolarization where all known ionic currents are blocked and
linear capacita nce current is
subtracted (see Fig 108B). It is thought to be membra ne delimited
because the same amount of
charge moves back upon repolarization. This charge movement (charge
1) can be broken down
into two components (~, y) by voltage-clamp protocols, kinetics
and pharmacological agents.
One component (o.y), often apparent as a hump, seems closely related
to SR Ca release (Hui,
1983; Rios et ai., 1992). Qy shows the same threshold (near -60 mY)
and steep Em-dependence
as Ca transients (2.5 mV for e-fold change, Hui & Chandler, 1990). Rios & Pizarro (1991)
summarized data that has lead to the working model that Qy is a result of Ca release rather than
causing Ca release. They suggested that locally released Ca (due to RyRI channels activated by
depolarization and Q~) binds to negative charges on the inside face of the voltage sensor (DHPR)
increasing the local Em, which in turn causes more molecules to undergo the charge movement
transition. After several seconds at strongly activating Em the charge movement goes into an
immobilized or inactivated state, where SR Ca release is also prevented. This can cause inequity
of on- and off-charge movement (as in Na channel gating) and requires a more negative Em for
charge to return to the resting state (sometimes called charge 2).
Chandler et al. (l976a,b) followed up the work of Schneider & Chandler (1973) and
proposed a physical "plunger" model by which the charge movement in the T-tubule membrane
might activate Ca release from the SR (Fig 108A). In this model, a charged particle +ZJ
(valence = +3) would move across the T-tubule membrane, pulling a plunger (spanning the T-
tubule-SR gap) out of the SR allowing Ca release to the cytoplasm. The release mechanism
could then move more slowly to a refractory state when -2Z 2 (total valence more negative than
-3) pulls +ZJ back to the position where the channel is plugged. How accurately this conceptual
model reflects the physical interaction is unclear, but it was a remarkably prescient idea.
Eisenberg et al. (1983) showed that skeletal muscle could be paralyzed by the
sarcolemmal Ca channel antagonist, D600, using a specific protocol of cooling and
depolarization. Hui et al. (1984) showed that charge movement was also inhibited under these
conditions. Dihydropyridine Ca channel antagonists (nifedipine and PN200-110) can also inhibit
charge movement as well as contraction, particularly in partially depolarized skeletal muscle
(Lamb, 1986; Lamb & Walsh, 1987; Rios & Brum, 1987). Rios & Brum (1987) suggested that
the dihydropyridine receptors (DHPRs) are the voltage sensors for skeletal muscle E-C coupling
(and thus are also the locus of the intramembrane charge movement). Since intramembrane
charge movement is associated with ion channel gating, the DHPR, which can function as a Ca
channel, was a good candidate.
Baylor et at. (1983) and Melzer et al. (1987) developed a means to estimate the time
course of the Ca release flux from the SR of skeletal muscle, based on the measured Ca transient
and assumptions about Ca buffering and SR transport. Figure 108B shows a Ca transient, the
calculated Ca release flux and the intramembrane charge movement during a voltage-clamp pulse
in a frog skeletal muscle fiber (Rios & Pizarro, 1988). Schneider & Simon (1988) showed that
the rapid decline in Ca release (arrows in Fig 108B) depended on [Cali' It was unclear whether
the transient component of Ca release flux was due to: a) transient CICR activation (and Ca-
dependent inactivation) of RyRs due to Ca released from neighboring VDCR release channels or
b) Ca-dependent inactivation of some Em-dependent release channels (Jacquemond et al., 1991;
Hollingworth et al., 1992). This issue is not entirely resolved, but at least 3 factors favor an extra
CICR component: 1) the transient component can be selectively blocked by low tetracaine
concentrations, known to block Ca-dependent RyR channel gating (Pizarro et al., 1992;
Shirokova & Rios, 1997), 2) the physical arrangement of RyRs, where only alternating ones are
associated with T-tubule particles (Figs 9, 10 & 109; Block et al., 1988) and 3) the known Ca-
dependence of RyRs (Fig 102). These factors make the sum of a steady Em-dependent plus a
Tubular
A. S-R
membrane membrane
B. ~ 10"M]J
Resting
j' ~
;"'~"R", •._]
Activating
Refractory
.... .o,r:n v
J
P
100ms
I ·90 rnV
Figure 108. Intramembrane charge movement and SR Ca release in skeletal muscle. A. Hypothetical
model of how intramembrane charge movement may regulate SR Ca release (from Chandler et af., 1976b,
with permission). The "plunger" blocking the SR Ca release channel is pulled out by the voltage-dependent
movement of three positive charges (+ZI) across the membrane electrical field (Activating). With
maintained depolarization, the slower moving anionic groups (two -Z2, with total charge magnitude> 3)
gradually allow the SR release channel to close in a refractory state. B. Intracellular Ca transient,
calculated Ca release flux and charge movement in response to a depolarizing voltage clamp pulse in frog
semitendinosus fiber (from Rios & Pizarro, 1988, with permission). The charge movement may activate the
SR Ca release channel, which in tum partially inactivates during the long pulse (arrows).
transient CICR an attractive working hypothesis (where CICR at unlinked RyRs is triggered by
neighboring VDCR at RyRs linked to DHPRs).
It is also interesting to note that the sustained Em-dependent component of SR Ca release
does not show Ca-dependent inactivation. Indeed, Ca release flux in skeletal muscle is also
immediately and completely turned off by repolarization. Thus, it may be that the apparent
physical linking of RyRI to overlying DHPRs bestows an Em-dependence to the RyRs, which
also results in lower sensitivity to Ca-dependent inactivation. Interestingly, when skeletal muscle
is treated with a low concentration of caffeine, the shut-off of Ca release is not as tightly coupled
to repolarization (Simon et af., 1989; Klein et af., 1990). What makes this notable is that low
concentrations of caffeine make skeletal SR Ca release more like that in cardiac (in terms of [Ca1
sensitivity and RCCs, Endo, 1975b; Rousseau et af., 1988; Sakai, 1965; Konishi et af., 1985).
Indeed, CICR can be observed in skinned skeletal muscle (Fabiato, 1984). Thus, while Ca
release in skeletal muscle SR appears to be under tight Em control, VDCR probably coexists with
CICR at adjacent RyRs in skeletal muscle.
Shirokova et af. (1996) showed that the ratio of peak to steady release flux was higher in
frog (4-6) than in mammalian skeletal muscle (-2). They correlated this with the higher ratio of
RyR:DHPR measured in frog than mammalian skeletal muscle, suggesting that in frog there may
be a larger fraction of RyRs that are not coupled to DHPRs and thus exhibit CICR rather than
VDCR. Figure 109 shows the model based on the ultrastructural results of Block et af. (1988) in
toadfish swimbladder, where alternating RyRs are associated with a tetrad of 4 DHPRs. Since
each RyR tetramer has only one high affinity ryanodine binding site, the expected ratio of RyR:
DHPR is 2:4 or 0.5. This is just what has been reported for rabbit skeletal muscle (Bers &
Stiffel, 1993; Anderson et a!., 1994b), while in frog skeletal muscle this value is higher (1-2;
Anderson et al., 1994b; Margreth et a!., 1993) implying more unlinked RyRs in frog (;:::75%) than
rabbit (50%). For comparison, this ratio is 4-10 in mammalian ventricle (Bers & Stiffel, 1993),
so in heart ";10-25% ofRyR could possibly link to DHPRs (see Fig 10 & below). In frog skeletal
muscle there is the additional twist that both a and ~ RyRs exist (Sutko & Airey, 1996), and the
possibility exists that the a-RyR participates in VDCR, while the ~-RyR participates in CICR.
While most mammalian skeletal muscle contains only RyRl, some tissues contain low levels of
RyR3 ·(e.g, diaphragm and soleus) especially during earlier developmental stages (Conti et al.,
1996; Taroni et al., 1997). Several lines of evidence suggest that the RyR3 contributes more to
peak SR Ca release and CICR rather than VDCR (Shirokova et al., 1999; Conklin et al., 1999;
Ward et al., 2000). Thus, skeletal muscle gating of the RyRs which are physically coupled to
DHPRs may be mainly Em-dependent, whereas the other RyRs may be activated by CICR.
Local SR Ca release events or Ca sparks (pg 191) are also seen in skeletal muscle and
initiate from T-tubule/SR junctions (Tsugorka et al., 1995; Klein et al., 1996). In skeletal muscle
Ca sparks are more tightly controlled by depolarization (lower stochastic occurrence at resting
[Cali and Em than in heart). Based on prolonged release events induced by ryanodine, IpTxA and
bastadin 10, and their known effects on RyR conductance, Gonzalez et al. (2000) and Shtifman
et al. (2000) inferred that Ca sparks in skeletal muscle (as in cardiac muscle) are due to more
than..one RyR, but these groups estimated different values (;:::6 or 2-4 RyR per spark).
While Em directly controls SR Ca release in skeletal muscle, some reports suggested a
role for Cao (e.g. Frank, 1980), despite the results in Fig 105B. Brum et al. (I 988a,b) studied the
effects of low [Cal o on E-C coupling in frog skeletal muscle in detail. They concluded that the
effects· of low [Cal o could be attributed to effects on the T-tubular voltage sensor (or charge
movement) rather than on Ca fluxes per se. Moreover, in skeletal muscle Ca entry does not have
to occur to activate Ca release. Any of group Ia and IIa elements of the periodic table can
support charge movement and contraction (Ca > Sr > Mg > Ba » Li> Na > K > Rb > Cs;
Pizarro et al., 1989). This relative affinity sequence is strikingly similar to that reported for the
cardiac sarcolemmal L-type Ca channel (see Table 17, page 109), with the notable exception of
Mg. Mg does not seem to permeate the cardiac L-type channel (Hess et al., 1986), but can
permeate the skeletal muscle L-type Ca channel (McCleskey & Almers, 1985). It is possible that
one of these ions must occupy the DHPR/Ca channel structure for the charge movement to occur,
but that ionic current flow is not required in skeletal muscle. Indeed, a pore-mutant skeletal Ca
channel which does not pass Ca ions can still trigger SR Ca release (Dirkson & Beam, 1999). As
we will see below, this is in striking contrast to results in cardiac muscle, where Ca entry appears
to b~an absolute requirement for SR Ca release.
Murine Muscular Dysgenesis: A Model System

Muscular dysgenesis (mdg) is an autosomal recessive genetic mutation in mice that
results in f~ilure ofE-C coupling in skeletal muscle (e.g. Klaus et al., 1983). It has proven to be
a valuable disease model in which to study E-C coupling (Adams & Beam, 1990). Both DHPRs
SR Transmembrane
Domain of T-tubule
Ryanodine Receptor Particle (DHPR)
Figure 109. Spatial relationships between the components at the SR-T-tubule junction in skeletal muscle
of toadfish swimbladder. The "foot" processes of the ryanodine receptor (RyR, shaded) span the gap
between the SR membrane (in which RyRs are imbedded, white) and the T-tubule membrane in which
dihydropyridine receptors (DHPRs) are imbedded (black). Note that DHPR tetrads (made up of 4 <Xl
subunits) overlie the RyR tetramers at alternating "feet" (redrawn after Block et al., 1988).
and the (XI subunit of the Ca channel are lacking in skeletal muscle from mdg mice (Pinyon-
Raymond et al., 1985; Knudson et aI., 1989). The slow Ca current, intramembrane charge
movement and ordered arrays of intramembrane particles (tetrads) are also absent in these
skeletal muscles (Beam et al., 1986; Beam & Adams, 1990; Shimahara et al., 1990; Takekura et
aI., 1994). Normal E-C coupling, charge movement, lea and tetrads were all restored in mdg
myotubes by injection of eDNA encoding the normal skeletal DHPR (XiS (Tanabe et aI., 1988;
Adams et aI., 1990; Takekura et aI., 1994). These results further support the hypothesis that the
DHPR is the voltage sensor that produces the charge movement critical to skeletal muscle E-C
coupling. It also provides evidence that the intramembrane charge movement, tetrad particles
and Ca current are all associated with the same DHPR molecule.
Tanabe et al. (1990a,b) also showed that if eDNA encoding the cardiac Ca channel «(Xie,
rather than the skeletal (XiS) was injected into dysgenic myotubes, lea and E-C coupling were
again restored. However, in this case the lea was more like that observed in cardiac muscle (i.e.
with faster activation and inactivation kinetics) and E-C coupling was also more like cardiac
muscle (e.g. contractions were quickly abolished in the absence of extracellular Ca). Using
chimeric eDNA they also found that replacing a single region of the cardiac DHPR with the
skeletal counterpart (the cytoplasmic loop between domains II & III, see Figs 51 & 110) was
sufficient to cause E-C coupling to be skeletal muscle-type VDCR. Thus, it would seem that the
different DHPRs in cardiac and skeletal muscle are sufficient to explain a major difference in E-
C coupling in these muscle types. That is, in skeletal muscle the DHPR causes release by virtue
of the charge movement (and the mechanical effect which that produces), with the lea being
incidental. In cardiac muscle the Ca entry via the Ca channel (or DHPR) appears to be the
critical event, although charge movement is stiJI important in activation of lea.
Skeletal Muscle DHPR-RyR Interaction

The simplest molecular model for skeletal muscle E-C coupling would be that the DHPR
is the voltage sensor and translates a signal through direct mechanical interaction to open the
RyR (as envisioned by Chandler et al., 1976b). However, there is remarkably little direct
biochemical evidence of DHPR-RyR interaction (e.g. crosslinking studies of Murray &
Ohendeck, 1997). Caswell et al. (1979) showed that SR-T-tubule junctions (triads) disrupted by
French Press treatment could reform in cacodylate buffer. Ikemoto et al. (1984) showed that
such reformed triads also had restored depolarization-induced SR Ca release (presumably due to
T-tubule depolarization, see pg 213-214). Triad reformation was also promoted by GAPD
(glyceraldehyde 3-phosphate dehydrogenase, Corbett et aI., 1985; Caswell & Corbett, 1985).
The glycolytic enzymes, GAPD and aldolase, as well as triadin and sorcin have been reported to
bind to both DHPRs and RyRs (Thieleczek et al., 1989; Brandt et aI., 1990; Kim et al., 1990;
Fan et al., 1995; Meyers et al., 1995, 1998) and could bridge between them. However, it is
unknown whether any of these proteins playa direct role in E-C coupling.
The chimeric DHPR studies of Tanabe et al. (I 990a,b) caused particular interest in the
DHPR II-III loop and how it might affect RyR properties (see Fig 110). Several groups have
shown that peptides from the skeletal II-III loop could alter RyRI gating in bilayers and also Ca
release & ryanodine binding in vesicles (Lu et al., 1994; El-Hayek et al., 1995; Marx et al.,
1998b; Dulhunty et al., 1999; Gurrola et aI., 1999; Zhu et al., 1999). EI-Hayek et al. (1995) split
the II-III loop into 4 peptides (A-D) and found that only peptide A (Thr671_Leu690 or its first 10
residues) altered ryanodine binding to RyRI and SR Ca release (activating both), and that this
effect could be blocked by the C peptide (Glu724_Pr076o, see also Saiki et aI., 1999). In a more
intact system (mechanically skinned skeletal muscle), Lamb et al. (2000) showed that peptide A
could enhance spontaneous and Em-dependent SR Ca release and that the triggered release could
be partially inhibited by peptide C. Nakai et al. (I 998b) showed that part of peptide C (residues
711-765) incorporated into an otherwise cardiac DHPR (XIC was sufficient to support skeletal
type VDCR when expressed in dysgenic myotubes with RyRI, and even just 18 amino acids
from skeletal (XIS (725-742) produced moderate VDCR.
Nakai et al. (1996, 1987, 1998a) also showed that in addition to this orthograde signaling
from skeletal DHPR to RyRl, there is a retrograde signal from the RyRl to DHPR. That is, in
dyspedic mouse myotubes (which lack "feet" or RyRl, Takeshima et al., 1994), but which
express skeletal DHPRs, there was a normal amount of Ca channel gating charge, but very little
Ica and no E-C coupling. Expression of RyRl in these dyspedic myotubes restored both E-C
coupling (orthograde signal) and also Ica function (retrograde signal). Expression of cardiac
RyR2 was not able to restore either E-C coupling or Ica via (XIS. This suggested that RyRl feeds
directly back on (XIS to facilitate the transition from gating charge movement to opening of the Ca
channel. Grabner et al. (1999) showed that the retrograde signal from RyRl could be endowed
on the cardiac DHPR simply by replacing part of the II-III loop with the skeletal DHPR (720-
765). Thus, the DHPR II-III loop appears to be responsible for retrograde as well as orthograde
signaling between (XIS and RyRl.
So, what about partner regions for the DHPR II-III loop on RyRl? Using RyR chimeras,
Nakai et al. (l998a) identified 2 large regions of RyRl which could restore ortho- and retrograde
signaling (1635-2636) or retrograde signaling only (2659-3720). Leong & MacLennan (1998a,b)
Chapter 8 E-C coupling 21 I
Figure 110. Possible interaction sites between skeletal DHPR and RyRl. Numbers refer to amino acid
sequences of (XIS and RyR I. Numbered sections have been shown to interact physically or functionally with
the other protein, but detailed matching between proteins is fanciful at best. Some long stretches (e.g. 1635-
2636 on RyRJ) are unlikely to interact throughout, but may include some key sites. Large arrows show
orthograde signaling from DHPR to RyR (down) or retrograde from RyR to DHPR (up).
found another potentially important RyRl region (922-1112) which bound to the II-III loop of
(XIS (especially 1076-1112 of RyRl) and this region also bound to the III-IV loop of (XIS.
Notably, this RyRI region did not bind to II-III or III-IV loops from cardiac (XIC. Yamamoto et
al. (1997) showed that a region of major divergence between RyRl and RyR2 (known as D2,
1303-1406 in RyRl) was also important in skeletal-type E-C coupling. This elegant ongoing
body of work has established that the skeletal muscle DHPR-RyR connection has unique
interaction sites and that the highly homologous cardiac (X,C or RyR2 cannot functionally
substitute with the skeletal counterparts.
While the II-III loop of (XIS (especially 681-690) has received a lot of attention as a
mediator of skeletal E-C coupling, Proenza et al. (2000) found that scrambling this 10 amino acid
sequence in (XIS expressed in dysgenic myotubes made no difference in restoring E-C coupling
(or retrograde lea signaling). There is also a 20 amino acid stretch of the DHPR in the proximal
carboxy tail (1487-1506 in (X,s) that is identical in (Xle, and this peptide inhibits ryanodine
binding to both RyRI and RyR2 and reduces RyRl open probability in bilayers (Slavik et al.,
1997). Notably, this region is in the carboxy tail, near sites implicated in Ca- and calmodulin-
dependent inactivation and facilitation of cardiac lea (see pg 117-120). Mice lacking the nonnal
Ca channel ~ subunit gene (~Ia) also lack gating charge movement, Ica and effective E-C
coupling, despite RyRI expression and replete SR Ca stores (Beurg et al., 1997, I999a,b).
Expression of the cardiac ~2a in these myotubes only partially restored ICa and Ca transients, but
~Ia (especially the carboxy half in chimeras) could completely restore E-C coupling. Thus, there
are multiple potential regions of the DHPR complex that might interact functionally with RyR
(even ifnot physically), particularly in skeletal muscle.
Cardiac Muscle DHPR-RyR Interaction?

The situation in cardiac muscle is less clear. While data below support some interaction
in heart, the emerging picture is of a much less robust DHPR-RyR interaction than in skeletal
muscle. This is entirely consistent in heart with the apparent lack of VDCR (below), the less
ordered physical array ofDHPR over RyR injunctions (Fig 10) and the 4-1O-fold excess ofRyR
over DHPR (Bers & Stiffel, 1993). This excess of RyR implies that at most 10-25% of RyR
could possibly interact with a DHPR. Nevertheless, cardiac DHPRs do appear to be concentrated
at sarcolemmal junctions with the SR, albeit not as tetrads (Franzini-Armstrong & Protasi, 1997).
El-Hayek & Ikemoto (1998) found that the carboxy half of the II-III loop cardiac peptide A (Ac-
10C, KERKKLARTA) could activate RyRl and Lamb et al. (2000) also found that Ac-lOC
enhanced SR Ca release in skinned skeletal muscle. There is limited data for Ac-l OC effects on
RyR2, and the effects are in the opposite direction. We found that Ac-lOC can inhibit RyR2
open probability in bilayers (IC so ~ I flM, preliminary data only), and when included in the
dialyzing patch pipette this peptide depresses Ca spark frequency by 63% in voltage clamped
ventricular myocytes, for the same SR Ca load and diastolic [Cali (Li et aI., 1999). As mentioned
above the carboxy peptide that is common to (XIC and (XIS inhibits ryanodine binding to RyR2
(Slavik et aI., 1997). Thus, it is possible that the analogous cardiac (XIC and RyR2 domains
interact, but more work is needed to clarify this.
Bay K 8644, the dihydropyridine L-type Ca-channel agonist, has provided evidence in
favor of intermolecular communication between cardiac (XIC and RyR2 in intact ferret ventricular
myocytes. Bay K 8644 (100 nM) accelerates resting loss of SR Ca in ventricular myocytes in a
manner that is completely independent of Ca influx and which is competitively inhibited by DHP
antagonists (Hryshko et al., 1989a,b; McCall et al., 1996b; Satoh et al., 1998; Katoh et al.,2000).
This is apparent as a 400% increase in resting Ca spark frequency (e.g. Fig lilA) in the
complete absence of extracellular Ca. This effect is maximal within 10 sec of exposure of cells
to Bay K 8644, is unaltered by stimulation of APs (in Ca-free solution) and is completely block-
ed by nifedipine. Bay K 8644, even at 100 times higher concentration, had no direct effect on
cardiac RyR channel gating in bilayer experiments. Another Ca channel agonist which does not
bind to the same DHPR site (FPL-64176) had no effect on Ca sparks, but similar effects
enhancing Ica . Bay K 8644 also increased ryanodine binding in intact cells, but not after mecha-
nical disruption. Our working hypothesis (Fig 1lOB) is that Bay K 8644 binds to the DHPR and
transmits a Ca-independent signal to the RyR, altering its resting open probability. While this
effect appears to be mediated by Bay K 8644 binding to the DHPR, it differs from effects on Ica
which occur both more slowly and in a highly depolarization-dependent manner (Katoh et aI.,
2000). We concluded that after binding to the DHPR the pathways diverge for the Ica gating
effect and the intramolecular effect on the RyR, manifest as increased resting Ca sparks.
A. 80 --0- control
DHPR site
(.) ---'-+Bay K Bay K 8644

:ll 60 at time =0 J
::J
.Eo
~ 40
...<1l
Q.
If) 20
<1l
U
0+r~~~""T""T~~~~"""-~~~"'"
o 10 20 30
Rest duration in OCaONa/EGTA (sec)
Figure 111. Bay K 8644 alters cardiac RyR2 function in Ca-independent manner. A. Ferret ventricular
myocytes were stimulated to steady state (I Hz) in normal Tyrode's and then stimulation was stopped and
superfusion switched (in <I sec) to a Ca-free, Na-free solution with I mM EGTA (±Bay K 8644, 500 nM).
Ca Spark frequency was monitored for 30 sec. During the middle 10 sec the cells were stimulated at I Hz
to see if depolarization altered Ca sparks. B. Schematic of Bay K 8644 binding to the DHPR and producing
divergent effects on ICa gating and on RyR gating (independent of Ca flux). There mayor may not be an
intermediate protein @ (modified versions of figures in Katoh et al., 2000).
In Fig I I IA it is notable that there were Ca sparks in the complete absence of

extracellular Ca, emphasizing that Ca sparks are due to SR Ca release which occurs at a very
low, but detectable frequency even at diastolic [Cali' At this microscopic level it is clear (as in
Fig I05A) that action potentials in the absence of [Ca]o produce no SR Ca release or change in
either resting Ca sparks or [Cali (±Bay K 8644). Thus, while these Bay K 8644 studies imply
some weak intramolecular link between DHPR-RyR in heart (changing Po from ~0.0001 to
0.0005), they do not provide any support for VDCR in cardiac muscle. With respect to E-C
coupling, it is notable that Bay K 8644 depresses E-C coupling (lower Ca release for a given lea
and SR Ca load; McCall & Bers, 1996; Adachi-Akahani et aI., 1999). While one could propose
altered RyR Ca sensitivity, this effect is readily explained by the prolonged open times character-
istic of Bay K 8644 modified L-type Ca channels (see Fig 61). That is, a comparable whole cell
lea in the presence of Bay K 8644 will include fewer total channel openings (because some will
be open for very long times). Since only the first ms (or so) of opening is needed to trigger SR
Ca release, much of the Ca influx will be wasted with respect to triggering SR Ca release (i.e.
lower Ca release for a given lea). So, there may only be a weak DHPR-RyR link in heart.
Direcl Depolarization ofthe SR?

Peachey & Porter (1959) raised the possibility that skeletal muscle T-tubular depolar-
ization could depolarize the SR membrane, causing Ca release. This hypothesis was tested in
mechanically skinned skeletal muscle fibers using ionic substitution (Costantin & Podolsky,
1967; Nakajima & Endo, 1973). In this approach, a relatively imperrneant anion (e.g. propionate,
gluconate or methanesulfonate) is replaced by a perrneant one (e.g. CI), or a perrneant cation (e.g.
K) is replaced by a relatively imperrneant one (e.g. choline, Tris, Li or Na). This could set up a
diffusion potential which changes upon the solution switch. Although this can induce Ca release
(see Endo, 1985), two major factors make this mechanism seem unlikely physiologically.
First, in these skinned fibers, T-tubules seal off, and with ATP present (required for
skinned fiber solutions) they can re-establish the normal transsarcolemmal ion gradients (high
[Na] and more positive potential in the sealed off T-tubules). Then ionic substitution can
depolarize T-tubules, so that Ca release still depends on charge movement and VDCR as above.
A compelling argument for this explanation is that such depolarization-induced Ca release in
skinned fibers can be prevented by blocking Na/K-ATPase (preventing T-tubule polarization,
Donaldson, 1985; Stephenson, 1985; Volpe & Stephenson, 1986). This argument also holds for
heavy SR preparations which may include intact T-tubule-SR junctions. Second, as described at
the end of Chapter 7, the permeability of the SR to physiological monovalent ions is very high
and there are no appreciable monovalent ion gradients between SR and cytoplasm (Somlyo et aI.,
1977a,b; Meissner, 1986b; Somlyo & Somlyo, 1986). This makes it likely that Em = 0 across the
SR membrane. Fabiato (1985f) also found no evidence for direct SR depolarization-induced SR
Ca release in skinned pigeon cardiac myocytes (which lack T-tubules, but exhibit CICR). Thus,
a direct SR depolarization-induced Ca release seems untenable.
Mg as a Possible Mediator ojVDCR in Skeletal Muscle

Lamb & Stephenson (1990, 1994) and others have extensively used the mechanically
skinned skeletal muscle preparations just described above (with sealed off T-tubules) to directly
study VDCR in skeletal muscle where the cellular ionic conditions can be readily manipulated.
Lamb (2000) made the intriguing proposal that that VDCR works by removing a tonic Mg-
dependent inhibition of RyRI (such that RyRl gating in Fig 102 looks more like that for
cardiac). Note that 1 mM [Mg]j (or [Cali) strongly inactivates skeletal, but not cardiac RyR. Mg
also shifts RyR activation curve to higher [Cal. Thus, depolarization would allow ambient [Cali
to activate SR Ca release and repolarization would cause rapid Mg-dependent RyR inhibition.
In conclusion, in skeletal muscle there is clear and compelling evidence for VDCR. The
key molecules involved have been identified (DHPR and RyR), these proteins are physically
adjoining in the junctional space and active efforts are underway to understand this interaction
better in terms of molecular interactions as well as biophysical and theoretical aspects. CICR
also occurs in skeletal muscle and may be triggered by the Ca released by VDCR. Thus, while
VDCR seems essential to initiate SR Ca release, CICR may boost the initial rate of release.
In cardiac muscle several lines of evidence suggest that VDCR is not functional. 1) The
Em-dependence of L'l[Ca]i and contraction follow lea and not charge movement (Fig 106). 2) Ca
entry seems to be an absolute requirement in cardiac muscle (Figs 105A & see below), 3)
Depolarization does not induce SR Ca release by itself and does not appear to modify Ca-induced
Ca-release from the SR (Figs 114, 115 & 117), 4) Ca release from cardiac corbular and extended
junctional SR is physically too distant for VDCR (if, indeed Ca release from corbular SR occurs
physiologically), 5) The exquisite structural DHPR-RyR organization and interaction apparent in
skeletal muscle (Fig 109-110) does not seem to exist in heart. Nevertheless, several groups have
argued for the presence of VDCR in cardiac muscle, and this possibility will be addressed (pg
235-236) after considering CICR and the evidence for its role in cardiac E-C coupling.
CICR is the more primitive form of E-C coupling from a phylogenetic standpoint. Most
invertebrate skeletal muscle (e.g. crayfish) is functionally very similar to mammalian cardiac
muscle in this regard (Gyorke & Palade, 1993, 1994). My perspective is that CICR is very old
phylogenetically and has, in general, served well as an adaptive mechanism in heart (where speed
of contraction is not of paramount importance). Thus, there may be weak physical interactions
(direct or indirect) between cardiac DHPR and RyR, but these function mainly to keep sarco-
lemmal Ca channels in the neighborhood of clusters of RyRs. In fast-twitch skeletal muscle,
there is considerable adaptive advantage (survival) to high speeds of contraction. The vertebrate
skeletal DHPR and RyRl may have evolved additional points of interaction which are both more
robust and also able to transmit the crucial E-C coupling signal very rapidly using the voltage
sensor signal, without the need for Ca influx. In this context lea in vertebrate skeletal muscle is
essentially vestigial (for E-C coupling) and consequently the Ca channel function deteriorated
with further evolution. This is consistent with the very slow activation of lea (vs. charge
movement or cardiac lea) in vertebrate skeletal muscle. These are just musings, of course.
Ca-INDUCED Ca-RELEASE (CICR)

Ca-Induced Ca-Release in Skeletal Muscle
CICR was first described in skinned skeletal muscle fibers (Endo et al., 1970; Ford &
Podolsky, 1970). While CICR exists in both cardiac and skeletal muscle (Chapter 7), the major
question is whether it occurs physiologically and how it interacts with other possible
mechanisms. Endo (1975a, 1977) argued that CICR was only demonstrable in skeletal muscle
fibers at unphysiologically low [Mg], required heavy SR Ca loading and very high trigger [Ca]
(e.g. 100 /lM Ca with 0.9 roM Mg, or 10 /lM Ca with 50 /lM Mg). Fabiato (1984, 1985g),
however, demonstrated CICR in skeletal muscle with the SR loaded at 100 nM Ca and -3 roM
free [Mg], triggered by a rapid [Ca] increase to 200-600 nM. The preceding section described
how CICR and VDCR may coexist and function together in vertebrate skeletal muscle.
Ca-Induced Ca-Release in Mechanically Skinned Cardiac Muscle.

Fabiato & Fabiato extensively characterized CICR in an elegant and formidable series of
studies in mechanically skinned single cardiac myocytes (Fabiato & Fabiato, 1973, 1975a,b,
1978a,b, 1979; Fabiato 1981 a, 1983, 1985a-c). In a culminating experimental series, Fabiato
(1985a-c) used mechanically skinned canine Purkinje fibers because they lack T-tubules (which
could reseal and complicate interpretations). The solutions included 5 J.lM calmodulin (which
slightly increased Ca release) and -3 roM free Mg. The largest CICR was seen in 1-3 roM Mg,
although the threshold [Ca] for CICR is higher than at lower [Mg] (Fabiato, 1983). Ca entry via
lea was simulated by very rapid Ca application, which induced SR Ca release. Solutions of
various [Ca] at various rates could be applied as fast as -I msec to these skinned cells and SR Ca
release was measured using aequorin luminescence and force. Since CICR implies positive
feedback, one might expect that Ca release would proceed to completion (as released Ca would
cause more and more Ca release). However, a remarkable feature of CICR is that the amount of
Ca released is graded with the amount of trigger Ca (Fabiato, 1983, 1985b). Indeed, at higher
[Ca] CICR could be inhibited or inactivated (see below).
Figure 112A illustrates Fabiato's approach and shows inactivation of CICR at high [Ca).
In control contractions (C) the 100 nM Ca solution used to load the SR is withdrawn and 250 nM
Ca solution applied briefly to activate SR Ca release (note that this [Ca] was insufficient to
directly activate contraction; Fig 2IA). In the third contraction (test) a higher [Ca] (10 J.lM) is
A. Tension A t~
25
1--1
~ Apply
Apply
015 10 ~M
Solution (test only)
~ 100 nM
Switch ----,,-,',,, ,--- -1'-_
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100nM Remove 10 ~M
[Cal 250 nM (test only)
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6.8 6.5 6.0 5.5 5.0

Bulk Solution Ca Trigger (pCa)
Figure 112. Fabiato's CICR in mechanically skinned canine cardiac Purkinje fiber. A. Tension recorded
in response to rapid application and removal of experimental solutions of indicated [Cal (lower trace is
expanded time scale, as indicated by bar). In 100 nM [Cal buffer the SR accumulates Ca. Removal of that
solution and application of 250 nM Ca solution for 30 msec induces SR Ca release, peak [Cal of 1.7 11M
and contraction (control traces, C). For the test contraction only, [Cal was raised to 10 11M for -150 msec
immediately after initiation of Ca release by 250 nM Ca. This extra elevation of [Cal led to a smaller
contraction and peak [Cal (1.2 11M), indicating Ca-dependent inactivation of SR Ca release. B.
Relationship between trigger [Cal for SR Ca release (as pCa= -log [Ca]) and the contraction amplitude
resulting from CICR. Experiments were done like the controls in A, except trigger Ca was varied and the
time taken to reach the trigger [Cal was varied from I to 50 msec. Ca release depended on both trigger [Cal
and the rate of[Ca] change around the SR (modified from Fabiato, 1985b, with permission).
applied (for -ISO ms) right after the 30 ms pulse of 250 nM Ca. This higher [Cal results in a
smaller contraction and Ll[Ca);. This indicates that the higher [Cal inactivates the usual CICR.
Figure ll2B shows the trigger [Cal dependence of the tension transient produced by
CICR (Fabiato, 1985b). Data are shown for several different durations taken to reach the
indicated trigger [Cal. It can be appreciated that the Ca released depends on both the trigger [Cal
and the time taken to reach that [Cal. At high (or supra-optimal) trigger [Cal, the SR Ca release
Sarcolemma
Ca
Binding -_~_
Sites
SR
Resting Activated Inactivated

Figure 113. Diagram ofCICR based on Fabiato's work. From the resting state (channel closed), Ca may
bind rapidly to a relatively low affinity site (I), thereby activating the RyR. Ca may then bind more slowly
to a second higher affinity site (2) moving the release channel to an inactivated state. As cytoplasmic [Cal
decreases, Ca would be expected to dissociate from the lower affinity activating site first and then more
slowly from the inactivating site to return the channel to the resting state.
was inhibited. CICR also exhibited a refractory period where a second Ca release could not be
induced. This was not the case for caffeine-induced Ca-release. Fabiato (1985b) likened this to
the inactivation (and recovery from inactivation) described for sarcolemmal Ca channels. He
also showed that some steady state inactivation existed at 63 nM Ca, but that this inactivation
could be removed by a few seconds at [Ca]= 12 nM (analogous to lea inactivation and recovery).
On the basis of these findings Fabiato (1985b) proposed a model (Fig 113) where Ca
binds to an activating site with a high on rate (but modest affinity) and also binds to a second
inactivating site which has a higher affinity, but a slower association constant (-0.7 sec at [Ca]=
63 nM). Thus, when [Cali increases rapidly the activation site is occupied and SR Ca release
occurs. The inactivation site binds Ca more slowly to turn off Ca release. Rapid application of
very high [Cal can still produce inactivation more directly since binding to the inactivation site is
expected to be proportional to the product of on rate and [Cal. Thus, the very high [Cal can
partially overcome the limitation of the slow on rate. This model provides a useful framework
for understanding global behavior ofE-C coupling at the cellular level in cardiac myocytes.
Ca-Induced Ca-Release: Supportfrom Intact Cardiac Myocytes.

Figure 113 puts the sarcolemmal Ca channel close to the SR Ca release channel (RyR).
Thus, Ca entry via lea may have ready access to the activation (and inactivation sites) of the SR
Ca release channel. The similar Em-dependence of Ie., contraction and Cai transient in cardiac
myocytes (Fig 106A) are classic findings which are consistent with CICR in heart (London &
Krueger, 1986; Cannell et al., 1987; Beuckelmann & Wier, 1988; Callewaert et al., 1988; duBell
& Houser, 1989). Further support comes from the observation of lea "tail transients" (Fig 114,
Cannell et al., 1987; Beuckelmann & Wier, 1988). These occur when a cell is first voltage
clamped at positive Em (e.g. +100 mY), where Ca channels are open, but no inward lea is seen,
Current '[
(nA)
o
Deactivating tail ICa
[Cal,
signal "'[
1.0 +100mV
l--...J "tail transient"
100 msec
Figure 114. Activation of SR Ca release by tail Ie, during repolarization in a rat ventricular myocyte.
Current (top) and [Cali (lower Fura-2 fluorescence traces) in response to depolarization to either + 10 mY or
+100 mY. Depolarization to +10 mY produces the usual lea and ~[Ca]i' Depolarization to +100 mY
produces no inward lea or Ca transient, but upon repolarization to -50 mY, a briefI ea tail current activates a
robust Ca transient due to CICR (modified from Cannell eta!., 1987, with permission).
and no Ca transient occurs. Then when Em is suddenly clamped back to negative Em (e.g. -50
mY) Ca channels deactivate, but as the Ca channels are closing, a large, short-lived inward lea
flows through open Ca channels (tail current) and induces a Ca transient and contraction. This is
likely to reflect CICR (vs. YDCR), although a complex Em-dependence could be contrived to
explain this (Cannell el aI., 1987). These "tail Caj transients" are not observed in skeletal
muscle, where voltage appears to control SR Ca release more directly.
iibauer el al. (1989) provided compelling evidence for CICR (vs. VDCR) in rat
ventricular myocytes (Fig 115). The first pulse shows a control depolarization with a large lea
and Ca transient. For the second pulse, 4 mM EGTA was added to decrease [Ca]o to
submicromolar levels. Under these conditions the Ca channel carries a large Na current (see
Chapter 5) which inactivates very slowly compared to lea. There is no Ca transient associated
with this pulse despite the facts that J) ionic current flowed through the Ca channel, 2) there was
presumably charge movement associated with Ca channel activation and 3) there was plenty of
SR Ca (as shown by the large caffeine-induced Ca transient). They also showed that Ba current
could not induce SR Ca release. Thus, E-C coupling cannot be due to Ca channel charge
movement or current per se. Ca entry appears to be an absolute requirement for induction of Ca
release in cardiac muscle.
Two photolabile Ca chelators Nitr-5 (Adams el al., 1988) and DM-nitrophen (Kaplan &
Ellis-Davies, 1988) in which Ca affinity is decreased upon illumination make it possible to
produce rapid increases in [Cali in intact cells. Kentish el al. (1990) used Nitr-5 to demonstrate
CICR in saponin skinned cardiac muscle for comparison with IP3-induced Ca-release (see pg
241). Yaldeolmillos el al. (1989) demonstrated that photolysis of Ca-Nitr-5 induced ryanodine-
sensitive contractions in rat ventricular myocytes that persisted in the presence of 10 mM Ni
Vm
Jl A.
'--___ JlL.-
B. C. lomv
-BOmV
3 Ca I 4 EGTA
Current
6Ft?
Increasing
[Cali
J.
i1iD'mS
Figure 115. Ca entry is required for induction of SR Ca release in rat ventricular myocytes. A. depol-
arization activates ICa and a large Ca transient (fura-2 fluorescence). B. After replacing extracellular Ca
with 4 mM EGTA, depolarization activates Na current through Ca channels (INS, with characteristically
slow inactivation). Na current through the Ca channel does not induce SR Ca release. C. Application of 5
mM caffeine induces a large Ca transient (indicating SR Ca available for release) in B, and activates an
inward current, likely to be INa/C. (from Niibauer et al., 1989, with permission).
(which would block both Ic• and Na/Ca exchange). These contractions probably reflect CICR,
but Em was not measured. Niibauer & Morad (1990) and Niggli & Lederer (1990) performed this
type of photolysis experiment in isolated myocytes under voltage clamp. The contractions were
largely suppressed by caffeine or ryanodine and were observed at constant Em (excluding any
possibility that the Ca release was depolarization-dependent). Niggli & Lederer (1990) also
showed that the CICR was the same whether Em was -100, 0 or + I00 mY, suggesting that CICR
in cardiac muscle is not modified by Em (see also below). Niibauer & Morad (1990) also tried to
reproduce Fabiato's experiment (shown in Fig 112A) by increasing [Cali with a flash during the
rising phase of the contraction. They did not see any evidence of inactivation of SR Ca release
with elevated [Cali' They saw only enhancement of contraction. While the [Cali reached by
DM-nitrophen photolysis may not have been high enough (or for long enough) to produce
inactivation, no intact cellular data have confirmed the result in Fig 112A of CICR inactivation
with supra-optimal Ca trigger. CICR inactivation and termination of SR Ca release will be
discussed in more detail below (pg 227-230).
Repolarization Induced Turn-offofCa-Release in Cardiac Muscle?

Cannell et al. (1987) showed that the rise in [Cali can be curtailed by repolarization, even
after the peak of Ie. has occurred (see Fig 116). They suggested that this might reflect an
intrinsic Em-dependence of the CICR process or that repolarization turned off SR Ca release (as
in skeletal muscle). Another possibility is that repolarization hastened Ca extrusion via Na/Ca
exchange, thereby limiting the rise of [Cali' However, this possibility seems unlikely since Bers
400
5 msec 10 msec 20 msec 40 msec 80 msec
!'1'~~
:E
.s
';'
~
100
1 sec
100
x
E
Ii 50
U
~
50 msec
20 40 60 80
-2.0
Depolarization time (ms)
Figure 116. Duration dependence of Ca transients in a rat ventricular myocyte (using indo-! as Ca
indicator). Test pulses to 0 mY (from -50 mY) for the indicated times were given after 5 conditioning
pulses to 0 mY to ensure that SR Ca load was the same at each test pulse. Superimposed lea traces (leak
subtracted) recorded simultaneously are shown. The I'.[Ca], increases despite a constant peak lea. Graph
shows duration dependence of I'.[Ca], for different test Em from a different cell in which Na/Ca exchange
was prevented by no Na in the pipette or bath solution (from Bers et al., 1990, with permission).
et al. (1990) demonstrated that duration-dependence of Li[Ca]j was still apparent when Na/Ca
exchange had been eliminated (in Na-free solutions, Fig 116).
An intrinsic Em-dependence of CICR is unlikely, based on three types of experiments.
First, Niggli & Lederer (1990) found the same contraction at -100, 0 and +100 mV when
contraction was initiated by photolysis of Ca-Nitr-5 (see above). Second, in experiments like
that in Fig 116, Cleeman & Morad (1991) showed that Ca transients were equally well curtailed
when the Ca entry via lea was stopped by repolarization to -40 mV (deactivating Iea) or by
further depolarization to + 100 mV <preventing Ca influx electrochemically). Third, the Em-
dependence of lea is similar to Ca transients and contractions (Fig 106), although substantial Ca
transients can be seen with very small lea (Cannell et al., 1987). Figure 117 shows another way
to look at this type of result. If SR Ca release depended on Em (in addition to Ca), then one
would expect higher Li[Ca]i for a given lea at more positive Em. The opposite is observed in Fig
117 (Beuckelmann & Wier, 1988; Wier et aI., 1994; but see pg 233). Less Li[Ca]i is seen at more
positive Em for the same value of lea. This is explained by the size of the single channel lea (i ea )
at different Em. That is, at more negative Em the iea amplitude is larger simply because of a
greater electrochemical driving force (Em -Eea, and see Fig 117B). Thus, a Ca channel opening
at more negative Em yields a larger iea and is consequently more likely to trigger SR Ca release.
This single channel perspective also explains the duration-dependence of E-C coupling shown in
Fig I 16. The duration-dependence is more prominent at more negative Em (Fig I 16), consistent
with longer latencies for single L-type Ca channel opening, especially apparent at Em just
A. ~[Ca]i vs lea B. Em-dependence of Gain

+10
1.0
OmV
~ 2.5
+20 ~ .03
'6" -10
"
.!:! 7; 2.0
~"TJ
§ +30 !2.
E. S .02
I
...
0.5 -20 c
u
+40 'm
(.? rr
<r (.)
(.)
.01
W 0.5
Psparks 'lea
0.0 0.0 .00
0.0 0.2 0.4 0.6 0.8 1.0 -40 ·20 20 40
Peak Ie. (normalized) Em (mV)
Figure 117. Dependence of ~[Ca]; on Ic. and E-C coupling gain in guinea-pig ventricular myocytes. A.
Data from Beuckelmann & Wier (1988) from Fig I06A. Note that for the same Ic. amplitude the induced
MCa]; is higher at more negative Em. B. The ratio of ~[Ca]; to the Ic. needed to induce it is a convenient
crude index of E-C coupling gain. The Em-dependence of Ca spark probability (normalized to Ie.) is also
shown (Data from Santana et aI., 1996). The expected Em-dependence of single channel Ic. (ic.), based on
the Nemst-Plank equation is similar to both sets of data.
positive to the activation threshold (Rose et al., 1992). Thus, duration-dependence at -20 mV
may reflect time-dependent recruitment of SR Ca release units, based on ie• latency. Together
these results strongly suggest that Ca-induced Ca-release is not directly modulated by Em. Higher
~[Ca]; for a given Ic• at positive Em has been reported (e.g. when [Na]; is elevated, Isenberg et aI.,
1988), consistent with Ca entry via INa/C. or VDCR (see discussion on pg 232-236).
LOCAL CONTROL AND CICR

A more quantitative consideration of CICR requires much more detailed information
about the local physical environment in the junction and diffusional limitations in this region. It
is useful to consider the [Cali which might be expected in the vicinity of the inner mouth of a
sarcolemmal Ca channel. This can be easily done using the solution to the diffusion equation for
radial dependence of [Ca] from a point source in a hemisphere (Crank, 1975):
q r
[Ca],.• = [CaIto + - - x erfc(-- ,-) (8.1 )
2nDr 2 '1Dr
where [Ca].o and [CaIn are the initial [Cal; and the [Cali at a radial distance r, from the channel
mouth at time t after a current q is activated, and D is the diffusion coefficient.
Figure 118A shows [Ca]i as a function of radial distance from the inner mouth of a sarco-
lemmal Ca channel (for two different D values). Inclusion of Ca binding to the inner sarcolem-
mal surface delays the steady state, but does not change the final value (Bers & Peskoff, 1991).
The [Cal rises to very high values near the channel (r ~0.2 nm) and is 10-100 I-tM at the SR side
of the junctional cleft (r -15 nm) where RyR Ca binding sites may be. For r <20 nm local [Ca]i
is at its steady state value in <I msec (the last factor in eq. 8.1 is -I). This means that Ca diffu-
sion away from this region is "keeping up" with Ca entry via the channel. It is thus implicit that
[Cal; in this region will decrease rapidly when Ie. stops. This may partly explain rapid
deactivation of SR Ca release when lea is stopped by early repolarization.
A. Diffusion near Ca Channel B. Channel Mouth 15 nm

1000 0=60 ~m2/s -15 bound Ca ions
100 [Ca]= 10 ~M
!E
2:
10
'ii
~
C. Junctional Cleft
0.1
15
10 100 1000 1 freeCa ion nm
Distance from Ca Channel (nm) -100 bound Ca ions
D. Local [Ca] in Cleft 2.5 om from

Ca channel
_----------- -- ,-ForChannelclosingatO.3ms
100
i;;;~e- _-----------'-=-;.-~--I;r--I'100 .2.5 nm
+~~--~:-
+ u ,'"
+
!i;,Q,J'io-l2y.ff.~!ll~r-=-L
45 nm 10 -.:
....,...., / ,.:~,
0.2 pA
0.1 +--.-......"""';;y:.--.-~~"'~~~"'-I- 0.1-1-~~~~~-;=== ..... -';;:""_+
0.001 0.01 0.1 0.3 1
lime (ms) after opening time (ms) after closing
Figure 118. Predicted [Ca]i near the inner mouth of a Ca channel and junctional cleft. A. Solid curves are
from Eq. 8.1 (q =0.1 pA, [Ca]IO =100 nM, t= 1 ms). Thin broken curves are at 100 lls and the thick broken
curve includes linear Ca binding to the inner sarcolemmal surface (as in Bers & Peskoff, 1991). B. Within
a 15 nm radius of a Ca channel mouth for free [Ca]=10 llM there is Jess than one free Ca ion and only - 15
bound to the membrane. C. For the whole junctional cleft (at 15 llM free [Ca)) there is only I Ca ion free
and - 100 bound. D. [Ca] predicted in the junctional cleft by Soeller & Cannell (1997) for a Ca channel
opening at the cleft center (0.2 pA), including local membrane Ca buffering and eleclrodiffusion (ED).
Right panel assumes that the channel closes at 0.3 ms (data for 0 kindly supplied by M.B. Cannell).
It is worth considering the actual numbers of Ca ions in the hemisphere around a Ca

channel or in the dyadic cleft space. Figure 118B shows that at 10 J.l.M [Cal there is less than one
free Ca ion (0.04) within this small hemisphere and -15 Ca ions bound to the membrane. For the
whole dyadic cleft (Fig 118C) at 15 J.l.M [Cal there is still only I free Ca ion total (and -100 Ca
ions surface bound). Thus, one should think about [Cal in this environment as a stochastic
probability, rather than as an extensive quantity.
Langer & Peskoff (1996) and Soeller & Cannell (1997) extended our work on diffusion
near Ca channels, by including important geometric constraints, local buffering and electro-
diffusion. Figure 118D shows that inclusion of electrodiffusion (+ED) limits steady state [Cal
(due to charge repulsion by higher local [Ca]). Without buffers, local [Cal rises throughout the
cleft to the steady state value in <10 J.l.s. Even with buffers [Cal throughout the cleft is >10 J.l.M
in < I ms (Soeller & Cannell, 1997). When the Ca channel closes (typically after 0.3 ms, Rose et
aI., 1992) the local [Cal falls more slowly with Ca buffers, but still within -5 ms. Cannell &
Soeller (1997) extended this simulation to activation of RyRs and illustrated how the junctional
geometry contributes to the optimal tuning of the E-C coupling process.
The notion oflocal control ofRyR by Ca in cardiac E-C coupling was a logical extension
from Fabiato's 1985 work, where the rate of [Ca] change in the RyR environment could activate
or inactivate SR Ca release. This, combined with the apparent junctional colocalization of
DHPR with RyRs and simple diffusional modeling, made local control appealing. Leblanc &
Hume (1990) brought the issue into sharp focus by results suggesting that Na entry via INa could
raise local submembrane [Na]; to cause local elevation of rCa]; (via Na/Ca exchange) and
consequent triggering of SR Ca release. Lederer et al. (1990) coined the term "fuzzy space" for
the junctional cleft, emphasizing the importance of local spatial concentration gradients in this
restricted junctional space and their consequences. This captured the spirit which was apparent
in many papers and labs at about that time; clearly the field had collectively embraced the
importance oflocal concentration gradients in E-C coupling. Stern (1992) crystallized some key
aspects in a series of mathematical models which indicated that common pool models (where
trigger [Ca]=global [Ca)) could not explain the graded nature of CICR. To obtain a reasonable
amplification factor required the common pool system to teeter on the brink of spontaneous
release. He also considered two models of local control: I) a "Ca-synapse" where I DHPR
triggers only I RyR, and 2) a "cluster bomb" where I DHPR triggers a cluster of RyRs. Either
model could explain both graded release and high gain, but the synapse model required an
unrealistically large single RyR Ca flux.
The initial observation of localized SR Ca release events (Ca sparks) by Cheng et al.
(1993) was timely and gave immediate physical evidence for elementary Ca release events that
were restricted in space (see pg 190-192). Ca sparks initiate along z-lines, raise local [Ca] by
~200 nM (rise in 10 ms, fall with 't~ 25 ms) and have a spatial spread of ~2 /-lm (Cheng et aI.,
1993, 1995, 1996; Lopez-Lopez et aI., 1994, 1995; Cannell et aI., 1994, 1995; Shacklock et aI.,
1995; Gomez et aI., 1996; Satoh et aI., 1997). It should be noted that the scale of these optical
signals (~1 /-lm) is 100 times larger than the depth of the junctional cleft (and 10,000 times larger
volume). So even Ca spark measurements, while quite valuable, still don't directly reflect local
[Ca]i in the cleft. Ca sparks occur randomly at very low frequency in the resting cell (~100/s,
independent of Ca entry), but several thousand Ca sparks can be synchronized by lea (and ClCR)
during depolarization, and the released [Ca] summates in time and space to produce the whole
cell Ca transient. To visualize discrete Ca sparks during a twitch requires the almost complete
blockade of lea (by nifedipine, D600 or Cd) so that there are fewer local signals to overlap in
time and space (which normally obscures their resolution). Cheng et aI. (1993) initially proposed
that Ca sparks might represent single RyR events, but it now seems likely that a cluster of RyRs
(6-20) function in concert to produce a Ca spark (see pg 191-2). However, the precise number of
RyR is not known and how they are synchronized is still not completely clear. Of course local
CICR within a cluster of RyRs almost surely occurs, but RyRs within a cluster may also be
physically linked and undergo simultaneous coupled gating, providing redundancy of control
(Marx et aI., 1998a; Bers & Fill, 1998). Thus, the data seem to suggest that the cluster bomb
model is most appropriate.
In the local control theory of Stern (1992) it is acceptable that within a cluster of RyRs
(perhaps a junctional region or couplon) Ca release can be effectively all-or-none. That is, the
release within a cluster can be regenerative. However, the physical separation between clusters
and the high local [Ca] required to ignite a cluster prevents one cluster from activating another.
This is really the heart of local control theory. Indeed, Ca sparks normally do not trigger Ca
sparks in neighboring region (except during Ca waves). Parker et al. (1996) showed that
occasionally a Ca spark can activate a lateral neighbor along a z-line (-0.76 ftm away), but never
longitudinally from one z-line to the next (-1.8 ftm), despite the observed slower Ca diffusion in
the lateral vs. longitudinal direction. Thus, CICR gradation comes largely from the recruitment
of different numbers of RyR clusters, rather than varying the flux at each release cluster.
SR Ca release can also be induced by Ca entry via Na/Ca exchange and by flash
photolysis of caged Ca (Lopez-Lopez et al., 1995; Lipp & Niggli, 1996, 1998). These Ca
triggers are not necessarily as localized to the junctional region, nor are they likely to produce as
large a rise in local [Cali- Interestingly these approaches can produce SR Ca release which also
appears more spatially uniform (vs. Ca sparks), leading Lipp & Niggli (1996) to propose
unresolvable events or "Ca quarks," several times smaller than Ca sparks. It is not clear why
RyRs activated this way do not produce a locally regenerative Ca spark (cluster bomb). Niggli
(1999) speculated that there may be 2 classes of RyRs in the cleft: 1) those immediately apposed
to DHPRs which may have low Ca-sensitivity, but large release flux and 2) the others with
higher Ca-sensitivity, but lower Ca release flux (e.g. very brief openings). When lea is activated
the first class of RyR is readily activated and the large release flux recruits the neighbors in the
cluster (a Ca spark). With a weaker global elevation of [Ca]j a few of the second class of RyRs
might fire, but not produce a strong enough trigger to activate the first class or a Ca spark (hence
producing only a Ca quark). Its not clear if this conceptual model would work, but it is an
interesting notion that is yet to be tested.
Gain and Fractional SR Ca release

So what is the real amplification factor of CICR? This is often referred to as gain, where
a measure of SR Ca release is divided by the Ca trigger signal that produces it. In the most literal
sense it should be the amount of total SR Ca released (or rate of release) divided by the
integrated triggering lea (or influx rate). Moreover, one should correct for the contribution ofl ea
to the Ca transient, so gain = (Li[Ca]IOI - fleadt)/ fleadt. Wier et al. (1994) reported a gain of -16
at Em =0 mV in rat ventricle (using peak flux rates), which varies with Em as in Fig 117B (higher
at more negative Em, due to higher unitary iea). We find a gain of 3-8 in rabbit using total Ca
fluxes at E m= 0 mY, which varies with SR Ca load (Shannon et a!., 2000b). These values are
generally consistent with the overall Ca fluxes in these species (Chapters 3 & 9). Many
investigators use variations on this gain factor which are also quite useful, but require less
detailed quantitative analysis of Ca fluxes (e.g. Li[Ca]/peak lea or d[Ca]/dt/peak lea). This is a
reasonable simplification to allow comparison of gains under different conditions (especially
when varying Ca trigger at constant SR Ca load).
Since the amount of SR Ca release also depends on the amount of available SR Ca, gain
would be expected to vary as a function of SR Ca load, even if the intrinsic E-C coupling process
is not changed. Thus, we also use fractional release as an index of the efficacy of E-C coupling,
especially when SR Ca load is being varied (Bassani et al., 1993b, 1995b; Shannon et al.,
2000b). Notably, knowledge of SR Ca load is required for fractional release measurements.
Figure 119 shows both gain and fractional release determined from the same data. We showed
that during a normal twitch the SR releases 43, 35 & 55% of its Ca in intact rabbit, ferret and rat
A. Gain of E-C Coupling B. Fractional SR Ca Release

0.4 0 on
~
De iil
n
VI
'i? 0.3 g.
::l
~~
!!!.
(Jl
u
=. 0.2 Constant Fractional ;:0
-0
---Reiease------ 0
III
.;. ;:0
Constant Fractional c.>

0.1
'"
10
III
______~':I~-~§ll.------------ ~ 10
lR
o+",;==:r~--,-B-"-''-;::~~......----r 0.0
o 20 40 60 80 100 120 0 20 40 60 80 100 120 ~
SR Ca load (/-lmol/l cytosol)

Figure 119. Gain ofE-C coupling and fractional SR Ca release in rabbit ventricular myocytes. Data from
voltage clamp studies measuring Ie" [Ca); (indo-I) and SR Ca load (as in Fig 93). Na-free solutions were
used to prevent Na/Ca exchange from complicating Ie, measurements. A. Gain was measured in individual
cells as total amount ofSR Ca released / total fIe, (typical maximal gain was 6-10). To illustrate the SR Ca
load-dependence of gain for all pulses in all cells the definition was relaxed as shown. B. Fractional SR
release (total Ca release flux! SR Ca load) was sometimes> 100% at very highest loads (i.e. some released
Ca was taken up and re-released during a pulse). The maximal % SR Ca depletion during a twitch was
rarely >50% at any time. Left axis chosen (as for A) to define the shape of the curve, right axis is
approximate for true fractional release. Dotted lines indicate expectations if fractional release was
unchanged by SR Ca load (data are taken from Shannon et at., 2000b).
ventricular myocytes, for SR Ca contents of ~100 )..lmol/L cytosol (Bassani et al., 1993b, 1995b;
Delbridge et al., 1997). The fractional release changes in parallel to the lea amplitude (for a
given SR Ca load), as expected for the graded nature of CICR (Bassani et al., 1995b). When SR
Ca load is varied a striking finding is that fractional release (& gain) become almost zero at an
SR Ca content which is still ~50% of normal (see Fig 119). That is, Ie, cannot release this Ca,
but caffeine can. We suspect that this is an effect ofIow luminal [Ca]sR to reduce the sensitivity
of the RyR to trigger lea. This would serve to prevent further SR Ca depletion and also
encourage refilling, but it is remarkable that CICR seems to shut off at moderate [Ca]sR. This
modulation of Ca release flux by [Ca]sR may even be involved in the termination of normal SR
Ca release (see below). At high SR Ca load both gain and fractional release increase steeply, and
this may be due to high [Cah causing sensitization of RyR gating to cytosolic [Ca] (see Fig
103A). Thus, there may be a continuous modulation of SR Ca release by luminal Ca. This
would also explain the apparent "spontaneous" release ofCa associated with SR Ca overload. At
very high [Ca]sR, the RyR may be sufficiently sensitized so that even diastolic rCa]; is sufficient
to activate SR Ca release, which may propagate as a wave (because neighboring RyR clusters are
also sensitized). In this sense spontaneous SR Ca release may really be triggered by high [Ca]sR.
Under these conditions diastolic rCa]; is often elevated as well and could conspire with [Ca]sR to
cause SR Ca release (Edgell et al., 2000). This increased fractional release with SR Ca load
(Bassani et al., 1995b; Shannon et al., 2000b) is consistent with the effects of [Ca]sR on single
RyR gating (pg 194) and work in intact myocytes (Han et al., 1994b; Spencer & Berlin, 1995).
The frequency of Ca sparks certainly increases with increasing SR Ca load and at a
certain point macrosparks (more than one locus firing together) and Ca waves are seen (Cheng et
al., 1996; Satoh et al., 1997). It also seems that the Ca spark frequency declines at lower SR Ca
load at unaltered rCa]; (Satoh et al., 1997) and this is consistent with a continuous modulation of
RyR gating by [Ca]sR. However, at low [Ca]sR this point is complicated because Ca spark
amplitude decreases, making them more difficult to detect (Song et al., 1997). Thus, high [Ca]sR
may increase a) individual RyR open probability in bilayer recording, b) Ca spark frequency
during rest in intact cells and c) fractional SR Ca release due to CICR during E-C coupling.
Activation of SR Ca Release and Time Course of Ca Release Flux

The Em-dependence and latency of Ca spark activation match that of Ic• activation near
the threshold for activation (Lopez-Lopez et aI., 1995; Cannell et aI., 1995; Santana et aI., 1996;
Collier et al., 1999). These studies uniformly demonstrated that the opening of a single L-type
Ca channel is sufficient to activate a Ca spark. It is more controversial how many Ca ions must
bind to activate SR Ca release. Lopez-Lopez et al. (1995) showed that the probability of Ca
sparks (P sP 3<ks) as a function of Em was roughly that expected for single channel Ca current (ica)'
Since local [Ca] in the cleft is expected to be proportional to iCa (Soeller & Cannell, 1997), this
would be consistent with a direct dependence of Psp.rks on local [Ca] and thus activation of a
spark by one Ca ion. Whole cell rCa]; data of Fan & Palade (1999) also seemed to support this.
Santana et al. (1996) followed up this issue with additional Ca spark data and divided Psparks by
Ie" which can intrinsically correct for the Em-dependence of Ica gating. Then the Em-dependence
of Psparks/Ica should be linear if one Ca ion is required, but their data showed Em-dependence like
ic" indicating that two Ca ions are needed for activation (see iCa in Fig 117B). The single RyR2
in bilayers seems to require -4 Ca ions to be activated (Zahradnikova, 1999a). Thus, the opening
one Ca channel can trigger a Ca spark and I - 4 Ca ions are probably required to bind to an RyR
to start a Ca spark, in which a cluster of RyRs are recruited by CICR (or coupled gating).
During a strong activation of Ica , as during the AP, there is rapid activation of SR Ca
release (as Ca sparks) and there appears to be very little delay (perhaps < I ms) between peak Ica
and the peak of SR Ca release flux (pg 122). Figure 120 shows the time course of SR Ca release
measured by several different approaches. The slowest SR Ca release flux signal is based on
deconvolution of the whole cell Ca transient (Sipido & Wier, 1991; Wier et aI., 1994; Shannon et
al.,2000b). This is not surprising because it uses a global rCa]; signal (even though we correct
the signal for dye kinetics). This global signal may be what the myofilaments sense and is thus
functionally important, but it represents a damped and distorted version of the release flux
produced by the RyRs. Measuring the timing of Ca spark occurrence during a Ca transient
provides a closer optical signal and one can differentiate that to get a rather rapid release flux
time course (Lopez-Lopez et al.,1995; Cannell et al., 1995; Blatter et al., 1997; Collier et aI.,
1999; Bridge et al., 1999). However, the Ca spark is still distributed over a relatively large area
of the sarcomere, so this signal is still too slow. Song et al. (1998) devised a "Ca spike"
approach to measure SR Ca release flux, using a combination of a low affinity fast Ca indicator
(Oregon Green 488 BAPTA-5N or OG-5N; at I mM) plus 4 mM EGTA (a slow high affinity Ca
buffer) in the patch pipette. As Ca is released it binds rapidly to OG-5N increasing fluorescence,
but as it diffuses away from the source Ca binds to EGTA, limiting spatial spread of the
fluorescence signal (see also Cleeman et al., 1998). Song et al. (1998) showed that the time
course of these Ca spikes was similar to d[Ca]/dt during a Ca spark.
SR Ca Release Flux by Several Strategies

,----------
-
100
Sparks
~ Ca Spikes (Song et al)

ii:
III
II)
Deconvolved
'"
III 50
Cell Ca flux
~ (Shannon et al)
'"
(,)
0::
l/)
o ---
o 50 100 150 o 5 10 15 20 25 30
time (ms) time (ms)
Figure 120. SR Ca release flux measured by different approaches in ventricular myocytes. The slowest
curve is based on quantitative deconvolution of total Ca fluxes and global [Cal; measures in rabbit with
kinetic corrections for Ca binding to indicators and buffers (Shannon et af., 2000b). Ca sparks curve is
based on the occurrence of Ca sparks in guinea-pig ventricular myocytes reported by Lopez-Lopez et af.
(1995). The time course of Ca spikes in rat is taken from the work of Song et af. (1998). Time course of
SR Ca release-dependent inactivation of lea is also shown for rabbit ventricular myocytes during square
voltage clamp pulses at 25°C (Shannon et al., 2000b) and AP-clamp pulses at 35°C (Puglisi et al., 1999;
see Fig 60 for methods). Right panel is on an expanded time base.
In addition to these optical signals, SR Ca release causes lea inactivation and this can be
used as an indicator ofCa release rate (see Figs 60 & 120, Puglisi et aI., 1999; Shannon et al.,
2000b). This electrophysiological signal is not subject to indicator kinetics and uses a sensor
(the L-type Ca channel) which is perfectly positioned to detect local [Cal changes in the cleft.
With an AP at 35°C the peak SR Ca release flux may occur as early as 2.5 ms and coincide with
the peak lea (i.e. with no detectable delay). The disadvantage of this lea signal is that it may be
nonlinear and cannot be calibrated. With the simplifying assumption that all of the integrated Ca
release flux from the global Ca measurements is compressed into the more rapid kinetics of the
local lea signals, the peak release flux would be 3-10 times higher than the value of -3 mmol/L
cytosol/s for the deconvolved global Ca (Wier et al., 1994; Shannon et al., 2000b). Thus, the
apparent Ca release flux sensed by global Ca indicators (and the myofilaments) is slower and
lower than that sensed locally near the RyRs. This would also increase the required fraction of
the cell's RyRs needed to explain the measured Ca release flux, from -2% (pg 192) to -6-20%.
Termination ofSR Ca Release

Since CICR has intrinsic positive feedback, what turns off SR Ca release? In principle
there are 3 possibilities: I) local depletion of SR Ca, 2) inactivation (or adaptation) and 3)
stochastic attrition (e.g. Stem, 1992; Sham et al., 1998 Lukyanenko et al., 1998). Stochastic
attrition means that if the L-type Ca channel and all relevant RyRs in a junction happen to be
closed at the same moment (as channels gate stochastically), the local [Cal; would fall very
rapidly (Fig 118D) which could interrupt the otherwise regenerative release. This could work
well if only one DHPR and one RyR are involved, but the larger the number of RyRs responsible
for a Ca spark, the less likely it will be that they all close at once. SR Ca depletion is not the key
factor because when cells are treated with ryanodine or caffeine, very long lasting local [Cal;
elevations (>200 ms) are seen and these do not decline with time (Cheng et aI., 1993; Satoh el
aI., 1997). If local SR Ca could be depleted on the time scale of a Ca spark one would expect
rCa]; to sag during long events. Thus, diffusion from other regions of the SR may prevent local
SR Ca depletion. However, during a global Ca transient the entire [Ca]sR declines. Given the
evidence supporting a modulatory role of [Ca]sR on RyR gating, [Ca]sR depletion might play
some role in shutting off global SR Ca release during a twitch. This may be why we do not find
SR depletion to much greater than -50% during large Ca releases (Shannon et al., 2000b), which
coincides with the SR Ca load where gain and fractional release are zero (Fig 119, and see
Bassani et al., 1995b). However, as stated above, this can't explain why Ca sparks turn off, so
depletion is not the main factor controlling termination of SR Ca release.
Sham et al. (1998) addressed termination of SR Ca release in an elegant Ca spike study.
They confirmed that SR Ca depletion was not the cause. Early L-type channel openings
produced Ca spikes, but reopenings (or prolonged opening) of L-type Ca channels did not
reactivate SR Ca release. Even large tail lea (due to the Ca channel agonist FPL-64 176) did not
seem to induce local Ca release, unless the region hadn't already fired. These results argue
strongly against stochastic attrition and suggest that some sort of inactivation process must occur.
The inability to re-activate with large tail lea would also favor a more absorbing inactivation,
rather than adaptation as seen in isolated RyRs (Gyiirke & Fill, 1993; pg 194-5). Functionally, it
may not matter in the intact cell whether inactivation is absorbing or not, especially if the local
rCa]; cannot be physiologically driven high enough to reactivate the RyR (as in bilayers).
Ca spikes are a clever strategy to monitor Ca release flux more directly. However,
trapping the Ca which leaves the SR has several major consequences which may alter the time
course of SR Ca release. First, it prevents released Ca from potentially activating neighboring
RyRs, tending to reduce release time and spatial spread. Second, it lowers local rCa]; around the
RyR, which may either reduce local activation (reducing release) or limit inactivation (enhancing
Ca release). Third, it greatly delays reuptake of Ca by the SR which, may make local [Ca]sR
depletion more severe (reducing total Ca release). This could be due to the electrochemical
effect of lower [Ca]sR on release flux, but also the possible regulatory effect of [Ca]sR.
While it is clear that RyR Ca flux inactivates, the mechanism is unresolved. While there
is evidence for Ca-dependent inactivation (and adaptation; Chapter 7), models which include a
fateful inactivation linked to activation (independent of Ca) can also explain cellular experiments
(Stern et aI., 1999). There may even be two Ca-dependent steps 1) Ca-dependent adaptation,
causing reduced mean open time (Zahradnikova et al., 1999b), and 2) a separate absorbing Ca-
dependent inactivation caused only by very high [Ca] (1-10 mM, see Figs 102-3) on the
downward side of the RyR2 Po dependence on [Ca). However, since local [Ca] would rarely
reach this very high level, this a lsorbing state might not be readily achieved.
Recoveryfrom Inactivation/Adaptation
Whether RyRs tum off by inactivation or adaptation, there must be some time required
for these channels to return to their initial Ca sensitivity. This is analogous to recovery from
inactivation for lea or I a. Figure 121 shows an AP and Ca transient along with the recovery of
Ica , SR Ca content, RyR availability and tension. Full recovery of contractile force in
mammalian ventricle generally requires -1-2 sec depending on temperature, species and other
A. AP and Ca transient B. Recovery of E-C Coupling

100 SR Ca
[CaJ; ~ Content
40 r, > \
/
/ \
\ o
Q) ' \
\
<.> \
.s> 0
\Contraction
\
\
~
1: 50
\
E \ "'~ Q)
W \ ~o
~
I~ Qi
-40 \
" '---
Q) Contraction
a.
-80 ~
200 400 600 800 5' - 0 -200 200 400 600 800 1000
time (ms) time (ms)
Figure 121. Recovery of E-C coupling in heart. A. AP, Ca transient and contraction recorded in rabbit
ventricular myocyte at 37°C (by K. Schlotthauer). B. The AP is shown with time=O reset to the end of
repolarization. The SR Ca content is plotted as the reciprocal of the Ca transient assuming -50% SR Ca
release (recovery 't -200 ms). An adjacent dashed curve (barely discernible) shows a 5 ms lag between SR
Ca uptake and availability for release. Ie" RyR and tension recovery are described by 1=100 ms (Fig 57)
1=650 ms and 1= J. 7 s, respectively, starting after AP repolarization.
conditions (Gibbons & Fozzard, 1975; Edman & J6hannsson, 1976; Wolthart, 1979; Lipsius et
al., 1982; Yue et al., 1985; Wier & Yue, 1986).
lea recovers from inactivation with a time constant (1) of -100 msec at -80 mY, but
recovery is slower at more positive Em (Fig 57, e.g. Kass & Sanguinetti, 1984; Josephson et aI.,
1984; Lee et al., 1985; Fischmeister & Hartzell, 1986; Hadley & Hume, 1987; Fedida et al.,
1987a; Tseng, 1988; Argibay et aI., 1988). lea recovery can also overshoot and this may be
related to Ca-dependent lea facilitation (see pg 119). Early AP restitution may also influence lea
due to its Em-dependence (e.g. see Boyett & Jewell, 1978, 1980).
The SR Ca content also recovers rapidly. Figure 121B shows that SR Ca content falls
and recovers during the Ca transient (and SR Ca uptake is largely reflected in [Cali decline). In
some early models of the force-frequency relationship it was supposed that time was required for
Ca to move from an uptake to a release compartment (e.g. from longitudinal to junctional SR,
Morad & Goldman, 1973; Edman & J6hannsson, 1976; Wolthart, 1979; Yue et al., 1985). This
hypothetical construct was useful in explaining the observed 1-2 sec delay between [Cali decline
and ability to release Ca during an AP. However, Ca diffusion from longitudinal SR to
junctional SR « 1 /-lm) without membrane barriers should take« 5 msec (shown in Fig 121B as
a broken curve, lagging the SR Ca content curve by 5 ms)_ Indeed, we now know that the Ca in
the SR can be released in < 200 ms by application of caffeine or cold solution, despite a much
longer recovery time for responsiveness to lea or rapid Ca application (Fabiato, 1985b; Bers et
al., 1987; Sham et al., 1998). Thus, the SR Ca release channel is refractory to activation via
CICR, but not to activation by caffeine or cooling.
Fabiato (1985b) showed that recovery time of RyRs was accelerated by low [Ca]i in
much the way lea recovery is hastened by more negative Em (Fig 57). Cheng et al. (1996) showed
this recovery process (Fig 122) by triggering a depolarization-induced SR Ca release in the wake
of a Ca wave passing through the cell. Figure 122 shows that for cell regions where the Ca wave
passed before the image started (top) the local Ca transient has fully recovered and is uniform
along the cell length. However, at the bottom of the cell, where the Ca wave has recently passed,
the local RyRs are refractory to activation. The 1: of recovery of local E-C coupling (Fig 122C)
was ~650 ms. DelPrincipe et at. (1999) found that cellular CICR triggered by flash photolysis of
caged Ca recovered with a 1: of 320 ms, but after a localized SR Ca release they could not detect
local refractoriness after 250 ms. They concluded that global SR depletion and rebinding of Ca
to key intra-SR sites might be involved in the restitution process. The local Ca transient ends in
<100 ms compared to > 1 s for the global Ca transient. I suspect that Ca-dependent recovery (as
proposed by Fabiato) is still important in restitution of E-C coupling, but intra-SR Ca may also
be an important modulator. There may also be a slower phase of recovery of some RyRs from
inactivation both macroscopically and microscopically, which takes several seconds (Satoh et al.,
1997). That will be discussed with respect to rest potentiation in Chapter 9 (Fig 138).
Thus, it appears that locally the gating of the RyR in the intact ventricular myocyte is
governed by three key intrinsic factors: 1) local [Ca]j via CICR, inactivation and recovery, 2)
[Ca]sR, perhaps by modulating the [Cakdependence of channel gating and 3) recent history,
reflecting inactivation and recovery from inactivation (which are both time- and Ca-dependent).
Spontaneous SR Ca Release, Cyclic Contractions and Ca Waves

In general, the inactivation of SR Ca release and time required for recovery from
inactivation limit positive feedback of CICR, allowing cardiac relaxation and stabilizing resting
[Ca]j. Indeed, immediately after a twitch there is a reduction in Ca spark frequency which slowly
recovers during rest without changes in [Ca]j or SR Ca load (Satoh et al., 1997). However, when
SR Ca load is increased above a certain level (e.g. by raising [Ca]o) Ca spark frequency is greatly
increased and many Ca sparks appear immediately after the twitch. These can trigger Ca waves
which propagate through the cell via CICR (Cheng et al., 1996; Satoh et at., 1997). Clearly
elevation of SR Ca content can partially defeat the intrinsic E-C coupling safeguards against the
positive feedback inherent in CICR. High [Ca]SR may alter RyR gating in two ways: 1) it may
increase the sensitivity to activation by [Ca]j (Fig 103A), and 2) it may greatly hasten the
recovery of RyRs from inactivation (allowing Ca waves). These effects could be interdependent
and should be incorporated into future refinements ofRyR gating schemes.
Cheng et al. (1996) detected discrete Ca sparks which initiate Ca waves, and for slowly
propagating waves, Ca sparks could be detected along the wavefront. Thus, Ca waves, like
normal global Ca transients, are still caused by the temporal and spatial summation of Ca sparks
(although they are not temporally synchronized during waves). The combination of reduced
refractoriness and increased Ca-sensitivity of the RyRs allow longitudinal Ca wave propagation
by CICR, which normally does not occur (Parker et at., 1996). The shortening of refractory
period cannot be complete, because when two Ca waves approach each other from opposite
directions they almost invariably annihilate each other on contact (i.e. they don't pass through
each other; Lipp & Niggli, 1993). While these Ca waves can be arrhythmogenic and contribute
to mechanical dysfunction (see Chapter 10), they are also beneficial in limiting Ca overload.
This is because spontaneous SR Ca release at resting Em gives the Na/Ca exchanger a
thermodynamic advantage in extruding Ca from the cell (Chapter 6), and 15-20% of the SR Ca
content is extruded during a wave (Diaz et al., 1997a,b). This means that if the cell can extrude
A. Cell B. Line Scan C. Recovery
o 5
~
ex:
(j)
u
c
(j)
U
l/I
~
o
::>
u::
I ! t I I )
0.2 04 06 08 1.0 1.2

time (s)
Figure 122. Microscopic recovery of E-C coupling in a rat ventricular myocyte. A. schematic cell
showing a longitudinal scan line. B. Ca wave (measured with fluo-3) is propagating from the top of the cell
toward the bottom at constant velocity (angle implies -120 flmJS). At the point indicated an AP is
stimulated causing a spatially uniform L'.[Ca]; in the top half of cell, but the region near the bottom is
partially refractory due to the recent passage of the wave there. C. [Cal; measured at the points indicated in
B, time aligned to the wave front. Note the uniform and large local [Cal during the wave at each point and
the progressive increase in L'.[Ca]; as the local RyRs recovery from prior activation/inactivation during the
wave. Data from Cheng et al. (1996); image kindly supplied by W.J. Lederer.
Ca, these waves will tend to progressively decline. However, when Na/Ca exchange is inhibited
(e.g. by Na-pump blockade or [Na]a removal) waves can continue repeatedly as oscillations.
Fabiato & Fabiato (1972) showed that elevated [Ca] in skinned cardiac myocytes
induced cyclic contractions. These are also observed in intact myocytes and muscles, where they
are also manifest as contractile or Ca waves (propagating at 100->2000 flmlS) or as a series of
aftercontractions (Kass et al., 1978; Orchard et al., 1983; Stern et al., 1983; Wier et al., 1983;
Allen et al., 1984b; Kort & Lakatta, 1984; Capogrossi et al., 1986a,b; Mulder et aI., 1989; Backx
et aI., 1989; Takamatsu & Wier, 1990). In general these phenomena are observed where Ca
overload is expected (e.g. high [Ca]a, low [Na]a, high stimulation frequency, Na-pump inhibition,
long depolarizations and reduced sarcolemmal permeability barrier). Fabiato (1985b) argued that
cyclical Ca release is mechanistically different from CICR, partly because it is seen at [Ca]
where CICR can be inactivated (e.g. 30 flM Ca). He also argued that they represent a purely
pathological state of Ca overload. Indeed, the lack of spontaneous contractions is often used as a
criterion for healthy isolated cardiac myocytes. O'Neill et al. (1990b) showed that the normal
systolic L'l[Ca]; cannot propagate in rat ventricular myocytes without Ca overload. While Ca
waves may be pathophysiological, I prefer to think of the behavior of CICR in a continuum,
where very high [CaJsR modulates intrinsic RyR properties (increasing [Ca];-sensitivity and
decreasing refractoriness), allowing local release to propagate.
Ca waves could propagate by saltatory conduction of CICR via Ca sparks, as above. An
alternative would be that a heavily Ca loaded SR region reaches its limit first (dumping its Ca to
the cytoplasm), then extra Ca uptake in neighboring SR could contribute to activation of release
in the next region by raising [CaJsR (Takamatsu & Wier, 1990). By blocking the SR Ca-ATPase
Lukyanenko et al. (1999) showed that this sort of Ca uptake by the SR ahead of the wave front is
not important in wave propagation. Moreover, they showed that sensitizing the RyR to [Cali by
low [caffeine], allowed propagating Ca waves to be observed at much lower SR Ca loads. Thus,
sensitization of CICR to [Cali, abbreviated refractoriness and the inherent larger rate of Ca
release at higher [Ca]sR must be the predominant factors in allowing propagating Ca waves in
cardiac myocytes.
In conclusion, CICR triggered by ICa.L is probably the central means by which SR Ca
release is controlled in cardiac muscle. However, some other sources of trigger Ca, VDCR and
IP 3 ICR have also been proposed to play roles. These are considered below.
OTHER E-C COUPLING MECHANISMS IN HEART

Alternative Ca Triggers in Cardiac CICR
There can be little doubt that ICa .L is a robust and sufficient Ca trigger in cardiac E-C
coupling via CICR. There are several other ways that Ca can enter the cell during depolarization
that might also contribute: I) ICa,T, 2) Na/Ca exchange (either driven by Em or by local [Na];
secondary to INa) and 3) ICa,TTx or Ca entry via Na channels.
T-type Ica (lCa,T) is undetectable in most ventricular myocyte types, but can be significant
in Purkinje and some atrial cells (see pg 102). It is also unknown whether T-type Ca channels
are located at the sarcolemmal-SR junctions. Thus, ICa.T cannot be a major player in CICR in
most ventricular myocytes. Even in guinea-pig ventricular myocytes Ica,T is small compared to
ICa,L. In principle, ICa,T could work like Ica,L if the T-type Ca channels were preferentially
localized at junctions, and the effectiveness of a given ICa.T trigger would be expected to be
comparable to that of ICa,L. This was directly tested by Sipido et al. (1998a) in guinea-pig
ventricular myocytes and Zhou & January (1998) in canine Purkinje fibers. They measured SR
Ca release induced by comparable (and measured) Ica,T and ICa,L. SR Ca release triggered by ICa,T
was greatly delayed in onset and also slower than that triggered by a comparable Ica,L trigger.
Indeed, the ~[Ca]; induced by a larger Ica,T (at -40 mY) was ~6 times smaller than for a smaller
ICa,L (at -30 mY, Sipido et al., 1998a). These results suggest that T-type Ca channels are not
preferentially located in junctional regions in these cells. Thus, ICa,T can trigger SR Ca release,
but obviously not in ventricular myocytes which lack Ica.T. Even in cells which exhibit Ica.T, it is
probably only a very minor contributor to cardiac E-C coupling, overshadowed by ICa,L.
. Ca influx via Na/Ca exchange might also trigger SR Ca release, as suggested by our
earlier work with elevated [Na]; (Bers et al., 1988). Leblanc & Hume (1990) showed a
tetrodotoxin-sensitive component of contraction. Their data gave credence to the hypothesis that
Na influx via Na channels raised local [Na]; and that this caused Ca entry via Na/Ca exchange to
trigger SR Ca release (see Fig 74). Subsequent studies have supported these results (Levesque et
al., 1994; Lipp & Niggli, 1994; Vites & Wasserstrom, 1996). However, other results suggest that
this mechanism does not contribute appreciably to the physiological activation of SR Ca release
(Sham et al., 1992; Bouchard et al., 1993; Sipido et aI., I995b). They suggested that the above
observations were due to either Em-escape during voltage clamp or gradual changes in SR Ca
load. Before dismissal of this hypothesis, it would be valuable to know how large and how long-
lasting local elevation of [Na]; produced by INa might be, and whether it occurs in the junctional
cleft (see Fig 74).
A. [Na] alters Em-dependence B. Contraction-dependence of lea

[Nalp;p +60
15
'01.5
(l)
.!:l
~ 1.0 10 ~'- 1.0

o
o ~
~ <:
g 0.5
o ~
~
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U .,..0/
10& 0 g ._:~B···/ [Nalp;p=O mM
j} 0.0.1...qJ_f:'-¥-~--,-~---,~---,,---"---::'r15 U o.o..,=.-----,~_,--._-..,..-
-40 -20 o 20 40 60 0.0 0.2 0.4 0.6 0.8 1.0
Em (mV) lea (normalized)
Figure 123. [Na]; and Em-dependence of contraction. A. Em-dependence of contraction and ICa in voltage
clamped rabbit ventricular myocyte (Em steps from -50 mV to the indicated Em) with different [Na] in the
patch pipette ([Na]p;p), normalized to the value at + 10mV. B. Ica-dependence of contraction for data in A.
Note that for the same ICa amplitude the contraction is higher at more positive Em (mean data from Litwin et
aI., 1998 are replotted).
Ca entry via Na/Ca exchange can also be activated directly by depolarization (Chapter
6). The reversal potential for this Na/Ca exchange current at rest is typically -30 to -80 mY.
Thus, during the rapid upstroke of the action potential to +50 mV Ca influx via outward INalCa is
favored thermodynamically. Several labs have provided evidence that this Ca entry via Na/Ca
exchange can trigger SR Ca release and contraction, especially at large positive Em and in the
absence of Ica,L (Levi et al., 1994; Baartscheer et al., 1996; Wasserstrom & Vites, 1996; Litwin et
al., 1998). Figure 123 shows that with higher [Na] in the dialyzing patch pipette the E m-
dependence of contraction becomes less bell-shaped (vs. Fig 106A and 117). Thus, there is
greater contraction and presumed SR Ca release for a given Ica at more positive Em (Fig 123B),
especially with high pipette [Na]. This is what would be expected for Ca entry via INa/ca (Chapter
6). Surprisingly this was even true with zero Na in the pipette.
Sipido et al. (1997) found the usual (pg 203, Fig 106) bell-shaped Em-dependence of both
peak Ica and L\[Ca]; during the first 20 ms, even with 20 ruM [Na] in the patch pipette. ICa
triggered a rapid [Cal; rise (Fig 124B) and an additional very slow [Cali rise at positive Em (likely
due to Ca entry via 1Na/ca)' The eventual peak [Cali (at ~250 ms with 20 ruM [Na];) showed an
Em-dependence like that in Fig 123A for 10-15 ruM [Na]p;p. These two kinetic components of
[Cali rise are difficult to resolve in contraction records. With Ica blocked, Sipido et al. (1997)
showed that outward 1Na/ca could trigger SR Ca release at positive Em. At Em = +70 mY, INa/ca-
induced Ca release rate was almost as high as that induced by Ica at + 10 mV (Fig 124A-B).
However, the INa/ca-induced Ca release was delayed, requiring 60-120 ms (vs. 10 ms for Ica ), and
the efficacy of a given Ca influx in triggering SR Ca release was -4-fold lower for INalCa vs. Ica
(Fig 124C-D). The apparent delay is important, because it means that even at +60 mY, Ica
activates CICR long before the INa/ca-mediated Ca influx is sufficient to activate release. Thus,
when both Ica and INa/ca triggers are functional CICR is controlled almost entirely by Ica . It may
be noted that this is even the case for this unphysiologically high [Na];, using 20 ruM [Na] in the
dialyzing pipette. Normal [Na]; is less than half of that in rabbit ventricular myocytes.
A. Ca entry B. Peak d[Ca]/dt

_100
)(
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~
80
~ 150
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60
~ 100
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"
VI
co
.!: .91
U
co 50 c::" 40
co
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20
20 40 60 80 0 20 40 60 80
Em (mV) Em (mV)
C. Delay for Release 20 D. Efficacy of E-C Coupling

150
"'.§.. 120
"~
VI
90
~
B
.
60
.§" 30
lea
...... • ~ I !
20 40 60 80 2 4 6
Em (mV) ea influx (~moll L cytosol)
Figure 124. Comparison of ICa and INa/ca in triggering SR Ca release. Data are from voltage clamped
guinea-pig ventricular myocytes (225 ms depolarizations from Em = --45 mY) with K-aspartate and 20 mM
Na in the pipette. A. Em-dependence of Ca entry via ICa or INa/Ca' B. Maximal rate of rise of [Cali (from
fluo-3 signal), normalized to the maximum in the same cell. C. Time from depolarization to maximum rate
of [Cali rise. D. E-C coupling efficacy of Ca influx via ICa vs. INa/Ca. Fluxes were converted assuming
6.44 pF/pL (data from Sipido et aI., 1997, kindly supplied by K.R. Sipido).
Indeed, when an L-type Ca channel opens (early in the AP) high local [Cali can prevent
further Ca influx via Na/Ca exchange (see Fig 74). Thus, Ca entry via INa/ca may provide a back-
up or redundant system for activation of SR Ca release, or one that allows gradual rise of local
[Cali that works synergistically with the L-type Ca channel opening. Lopez-Lopez et al., (1995)
found that Ca entry via Na/Ca exchange produced slow unifonn rise in [Cali throughout the cell
(rather than Ca sparks). Thus, Na/Ca exchange may help set local junctional [Cali for local lea
activation, or may bring some Ca in during the latent time before a particular Ca channel opens.
Initial studies reported that Na/Ca exchangers reside mainly in T-tubules (Frank et aI.,
1992) or uniformly in the sarcolemma (Kieval et al., 1992). Scriven et aI., (2000) found that the
Na/Ca exchanger was not co-localized with either Na channels or RyRs, whereas DHPRs were
highly colocalized with RyRs (Table 4). This suggests that the Na/Ca exchanger is not in the
ideal place for either responding to INa or directly triggering SR Ca release. In addition, the
unitary flux though the Na/Ca exchanger is perhaps 1000 times lower than ICa,L. Thus, a Ca
influx trigger comparable to a single L-type Ca channel would require -1000 Na/Ca exchanger
molecules. This would place physical constraints on the ability ofNa/Ca exchange to produce a
4
comparable localized Ca trigger signal for E-C coupling (requiring> 10 exchangers/11m2 in the
junction; see pg 146). Thus, Ca influx via Na/Ca exchange can trigger SR Ca release, but the
physiological role is unclear. On the other hand, Na/Ca exchange may become more important in
E-C coupling when Ica , is reduced, [Na]; is increased or Na/Ca exchange expression is
upregulated as in heart failure (Chapter 10).
Ca entry via tetrodotoxin-sensitive Ca current (Ica,nx) was reported in the absence of
[Na]o and attributed to a distinct subpopulation ofNa channels (LeMaire et al.,1995; Aggarwal et
al., 1997). While this ICa,nx could mediate CICR, it is unclear that any appreciable Ca entry
occurs in the presence of physiological [Na]o' Santana et al. (1998; Cruz et aI., 1999) showed
provocative data to suggest that cardiac Na channel selectivity could be altered dramatically by
either ~-adrenergic agonists, ouabain or digoxin. Based on shifts of the INa reversal potential
they inferred that Na channel PC.!P Na increased from essentially zero to > 1, making the Na
channel prefer Ca over Na (termed "slip-mode conductance"). This TTX-sensitive Ca entry
appeared able to trigger SR Ca release. The authors proposed this as a novel mechanism
explaining the inotropic effects of ~-adrenergic agonists and cardiac glycosides (effects generally
attributed to increased Ica & SR Ca-pump activity for ~-adrenergic agonists, or to Na/K-ATPase
inhibition and Na/Ca exchange for glycosides), Nuss & Marb{lll (1999) found no evidence at all
for altered PCa/PNa in cells with heterologous expression of cardiac a and ~ I Na channel subunits,
although Cruz et al. (1999) in similar heterologous expression experiments found effects which
were quite like their results in cardiac myocytes (Santana et al., 1998). The provocative finding
of a huge change in Na channel selectivity induced by cAMP or ouabain has eluded detection by
other groups and is not widely accepted. For example, DelPrincipe et al. (2000) could not detect
any Ca influx via INa with ~-adrenergic agonists (using optical measurement of Ca influx),
indicating a maximal PCalPNa of 0.04 for I 'a' Chandra et al. (1999) found PC.lP Na = 0.017 for
cardiac INa, which was unchanged by adrenergic stimulation. We also find no effects at all of
ouabain or digoxin in myocytes which have been depleted of intra- and extracellular Na, to
prevent Na/Ca exchange (Altamirano et al., 1999). It is also unclear whether ICa,TTx and slip-
mode conductance are related (Wier & Balke, 2000). Further clarification would be useful.
In conclusion, the central physiological Ca trigger in CICR appears to be IcaL. T-type
channels are either not present at all or not localized at the junction, making them weak
substitutes. Ca entry via Na/Ca exchange can induce SR Ca release, but in normal physiological
conditions it brings in too little Ca, too late, and again, not as well focused at the RyR, when
compared to ICa,L' However, Na/Ca exchange may modulate E-C coupling and under certain
conditions its role in CICR may be enhanced. TTX-sensitive Ca entry will require further study.
For now I consider its role in E-C coupling as speculative.
Voltage-Dependent Ca Release (VDCR) in Heart

Previous sections provide compelling evidence for VDCR in skeletal muscle and over-
whelming experimental evidence against VDCR in cardiac muscle (even though some RyRs
might interact with DHPRs, see pg 212-213), Despite this evidence a series of studies have
suggested a VDCR mechanism in cardiac myocytes, similar to that in skeletal muscle, with an
Em-dependence negative to the normal Ica,L activation and this VDCR is apparently not blocked
by nifedipine (Ferrier & Howlett, 1995; Hobai et al., 1997b; Howlett et al., 1998; Ferrier et al.,
1998, 2000; Mason & Ferrier, 1999). Depolarization appears to require intracellular cAMP, but
not Ca influx to trigger SR Ca release. On the other hand, this VDCR (unlike that in skeletal
muscle) requires extracellular Ca and this Ca-dependence makes it somewhat less compelling as
a Ca-influx independent E-C coupling mechanism. Most of these studies have focused on a two-
step Em protocol to separately activate VDCR and CICR. The first step to --40 mV activates
VDCR and the second step from --40 to 0 mV activates CICR. They reported that the VDCR
component was more sensitive to 30 nM ryanodine and 200 flM tetracaine (opposite to skeletal
muscle) and that CICR was more sensitive to block by Cd and nifedipine. There are several
issues that have limited the widespread acceptance of VDCR in heart, despite these intriguing
observations.
First, extracellular Ca is required and other divalent or trivalent cations do not substitute.
This certainly differs from VDCR in skeletal muscle where many mono- and divalent cations
readily support VDCR (pg 208). Being able to demonstrate VDCR without Ca would allow
clearer distinction from CICR. Moreover, since CICR is very selective for Ca over all other ions,
the similar high Ca-selectivity of putative VDCR is a concern. Second, cardiac VDCR requires
strong stimulation of cAMP or PKA. The powerful stimulation of lea by cAMP makes it very
difficult to completely block lea and the shift ofI ea activation to more negative Em (Fig 64) means
that lea will indeed be activated during the VDCR step to --40 mY. It might also explain why
they find apparent VDCR activation and availability at Em negative to that for basal lea
activation. These studies use K-containing solutions, making it particularly difficult to verify
complete block of lea (an essential point to rule out CICR). Indeed, cAMP broadens the normal
bell-shaped ~[Ca];-dependence (Hussain & Orchard, 1997; Piacentino et aI., 2000) such that a
very small fraction of maximal lea can trigger a large SR Ca release (at more positive and
negative Em). This relates to the next point. Third, the SR Ca load in the myocytes is very high
where VDCR is reported (due to PKA phosphorylation of phospholamban and enhanced SR Ca
uptake). The high SR Ca content strongly sensitizes SR Ca release to trigger Ca (seen in Fig
119, even without PKA). Thus, even a tiny fraction of unblocked lea can lead to substantial SR
Ca release and the efficacy of a single channel opening at --40 mV to trigger Ca release is very
high (Fig 117B). Since cAMP is such a powerful promoter of CICR, this makes it harder to
isolate any VDCR. Fourth, if VDCR is important in cardiac E-C coupling it should be very
robust, and not require special conditions to be observed (e.g. high cAMP). Many investigators
over the years have used very similar (if not identical) conditions and found only evidence
supporting CICR and refuting VDCR in heart (see CICR section). We have never seen results
consistent with VDCR in heart in my lab. Piacentino et al. (2000) made a concerted effort to
mimic the conditions used in Ferrier & Howlett's work and saw no evidence for VDCR. All of
their data were fully explained by CICR. In conclusion, some of the data suggesting VDCR in
heart are interesting, but I am not convinced that VDCR is functional in cardiac myocytes.
Other E-C Coupling Proposals

Alkalosis was proposed as an SR Ca release activator (Nakamura & Schwartz, 1972) and
can increase RyR open probability (Ma et al., 1988), but also increases SR Ca accumulation
(Fabiato, 1985e). It seems unlikely that alkalosis is a physiological activator of SR Ca release,
although the pH sensitivity of SR Ca uptake and release may modify SR Ca content, Ca release
and contraction (Orchard & Kentish, 1990). Early observations ofNa effects on Ca uptake in SR
vesicles raised the possibility that local elevation of [Na]j during the cardiac action potential
might directly induce Ca release from the SR (Palmer & Posey, 1967; Vassort, 1973; Caille et
al., 1979). However, cardiac SR vesicle and skinned fiber studies showed this to be unlikely
(Jones et al., 1977; Fabiato, 1986c). These phenomena may be related to displacement of Ca
bound at the inner sarcolemmal surface by roM Na (see pg 51), or local [Na]; elevation near the
sarcolemma due to INa (Akera et aI., 1976). However, these are still variants of CICR (see pg
232-235) and contractions are abolished in the absence of Ca o, despite maintained Na entry (Figs
105A & 115). Heavy metals (Ag+, Hg 2+, Cu 2+, Zn 2+, Cd2+) and sulfhydryl oxidation can induce
rapid Ca release in isolated SR vesicles (Abramson et aI., 1983, 1987; Salama & Abramson,
1984; Trimm et al., 1986; Prabhu & Salama, 1990). These may certainly modulate RyR gating
(see pg 197), but surely are not the normal driving factors in cardiac E-C coupling.
IP3-INDUCED Ca RELEASE (IP3ICR)

Inositol(I, 4, 5)-trisphosphate Induced Ca Release in Smooth Muscle
IP) induces endoplasmic reticulum (ER) Ca release in many cell types (Berridge, 1987,
1993, 1995; Berridge & Galione, 1988; Berridge & Irvine, 1989). In smooth muscle IP) induces
SR Ca release in both skinned fibers and SR vesicles (Suematsu et aI., 1984; Carsten & Miller,
1985; Smith et aI., 1985b; Somlyo et al., 1985; Yamamato & van Breemen, 1985; Watras &
Benevolensky, 1987). IP)-activated Ca channels have also been described in SR vesicles from
brain, smooth muscle and cardiac muscle (see pg 200-201; Ehrlich & Watras, 1988). The IP)
activated Ca release in smooth muscle appears to be large enough and fast enough to explain
adrenergic agonist induced E-C coupling (Somlyo et aI., 1985, 1988; Walker et aI., 1987).
A challenge in studying IP)ICR in isolated muscles is overcoming diffusional limitations
such that IP3 concentration rises very quickly at the SR surface (and is not degraded on its way to
the SR). The experiments in Fig 125 overcome these limitations in permeabilized smooth muscle
by laser photolysis of caged IP 3 and caged phenylephrine (an <XI-adrenergic agonist; Somlyo et
al., 1988). The release of -1 flM IP 3 activated contraction with a 0.4 sec lag. This latency is less
100 ~~~.~-~
..
Ins (1,4,5) P3 ~
~. J ~~"'""~....
".-r
/
~
~
/ /
/·I
Laser ,/ I
pulse
/PhenYlePhrine
! IJ
I
I '
o ~"
6
time (sec)
Figure 125. Activation of contraction in guinea-pig smooth muscle strips by photolysis of caged IP3 or
caged phenylephrine (from Somlyo et aI., 1988, with permission). Portal vein strips were depolarized (143
mM KCl) for the phenylephrine experiment or permeabilized (50 mg/ml saponin) for the IP 3 experiment
(-10% of the 50 flM phenylephrine & 10 flM IP 3 were released by a 50 nsec laser pulse).
than when the muscle was activated by release of -5 liM phenylephrine (1.8 sec). These results
are consistent with the relatively slow contractile activation of smooth muscle and the additional
steps involved with phenylephrine activation (e.g. receptor activation of phospholipase C which
forms IP 3 and diacylglycerol). Smooth muscle also has a well developed metabolic pathway for
production ofIP 3 (coupled to adrenergic receptors and G-proteins) and also for degradation ofIP 3
(Sasaguri et aI., 1985; Saida & Van Breeman, 1987; Walker et at., 1987; Somlyo et aI., 1988).
Heparin, which blocks the IP 3 receptor (Worley et at., 1987), can also inhibit phenylephrine and
IP 3-induced contractions in smooth muscle (Kobayashi et aI., 1988, 1989). Thus, IP31CR is
clearly physiologically important in smooth muscle (especially in the case of pharmaco-
mechanical coupling).
CICR is also apparent in smooth muscle (Saida, 1982) and there are ryanodine-sensitive
Ca pools in vascular smooth muscle (Ito et at., 1986; Hwang & van Breemen, 1987; Ashida et
aI., 1988; Kanmura et at., 1988). lino et at. (1988) showed that guinea-pig smooth muscle has
two intracellular Ca pools: one which is both IP 3 and caffeine/ryanodine sensitive and a second
which is only IP 3 sensitive. Thus, IP 3ICR and CICR mechanisms coexist in SR regions in smooth
muscle. The importance of both IP31CR and CICR in smooth muscle E-C coupling is compelling
(Somlyo et aI., 1988; van Breemen & Saida, 1989; Somlyo & Himpens, 1989; Somlyo &
Somlyo, 1990, 1994; Himpens et aI., 1995; Van Breeman et at., 1995; Bolton et at., 1999; Austin
& Wray, 2000; Jaggar et at., 2000). However, it is not as clear exactly how these two
mechanisms interact. Indeed, the relative predominance of IP 31CR and CICR varies in different
smooth muscle cell types. IP 3R and RyR are each located in both superficial and deep SR in
smooth muscle myocytes (Nixon et at., 1994; Lesh et aI., 1998) and in intestinal smooth muscle
there are -10 times as many IP3Rs as RyRs (Wibo & Godfraind, 1994).
Smooth muscle is activated by receptor agonists (pharmacomechanical coupling, inde-
pendent of Em) as well as by action potentials and tonic depolarizations. This additional
heterogeneity makes it particularly difficult to generalize, and I will not go into detail here with
respect to smooth muscle (for more detail see the reviews cited above). Some smooth muscles,
like urinary bladder, exhibit phasic contractions triggered by APs (Heppner et at., 1997). In
these muscles, there appears to be prominent CICR via RyR as in cardiac muscle (contributing
-70% of the activating Ca). In tonic smooth muscle (e.g. arteries) the majority of activating Ca
appears to come from Ca influx via L-type Ca channels, probably as a window current at
depolarized Em (Knot & Nelson, 1998; Knot et aI., 1998). There are also receptor-operated
channels (typically non-selective cation channels) which can initiate depolarization (pharmaco-
electrical coupling). This can result in either an AP in phasic smooth muscle (with Ie, causing
the AP upstroke), or bring Em into the range where a significant Ca window current occurs (--40
to -20 !'IV, Fig 57) allowing some steady Ca entry.
Ca activated K currents carried by big conductance K channels (BKe,) are involved in
negative feedback by causing hyper- or repolarization, thereby turning off further Ca entry. BKe,
channels are JrJlown to be responsible for spontaneous transient outward current spikes (STOCs)
observed in smooth muscle cells (Benham & Bolton, 1986). These STOCs and the high [Ca]
required to activate BKe, channels (2-10 f!M), led to the suggestion that local submembrane rCa];
may transiently be much higher than bulk [Ca]i (see review by Jaggar et at., 2000). This would
Ca Ca
~ Ca4Ca~ MLCK iI5\
I
d!!!k
CaM a J-'
MLCK; .diliilib
Figure 126. E-C coupling in smooth muscle (lP 3ICR & CICR). Receptor agonist (e.g. a)-adrenergic)
activate the receptor and a GTP-binding protein coupled to that receptor activates phospholipase C (PLC),
which produces IP 3 and diacylglycerol (DAG). IP 3 stimulates SR Ca release and DAG activates protein
kinase C (PKC) which can modify contractile proteins, ion channels and Na/H exchange. Depolarization
can be initiated by receptor activated channels (ROC) which can promote L-type Ca channel opening.
CICR also occurs via RyRs in smooth muscle and can contribute to propagating Ca waves. SR Ca release
via RyRs as Ca sparks also opens Ca-activated K channels (BKca ), which produce spontaneous transient
outward currents (STOCs), which can hyperpolarize Em and thereby deactivate lea. Depletion of SR Ca
stores can also activate capacitative Ca entry (CCE) via a mechanical signal from the IP3R to the TRP
(transient receptor potential) channel. Ca activates smooth muscle contraction by binding to calmodulin
(CaM), which activates myosin light chain kinase (MLCK) to phosphorylate myosin light chain (MLC) such
that actin and myosin to interact. This contrasts with striated muscle where Ca activates myofilaments by
direct binding to troponin C (TnC).
also be consistent with a diffusion restricted space between the SR and sarcolemma, similar to
that proposed in the superficial buffer barrier hypothesis (van Breeman et at., 1995).
Figure 126 shows E-C coupling in smooth muscle (Kamm & Stull, 1989; Somlyo &
Himpens, 1989; Somlyo & Somlyo, 1990, 1994; van Breemen et al., 1995; Bolton et at., 1999;
Jaggar et at., 2000). Pharmacomechanical coupling is started by a receptor agonist, rather than
an action potential as in striated muscle. Agonist occupied receptor activates a GTP binding
protein which stimulates phospholipase C to cleave phosphatidylinositol-(4,5)-bisphosphate
(PIP 2) into IP 3 and 1,2-diacylglycerol (DAG). DAG activates PKC which stimulates Na/H
exchange causing alkalosis and increased myofilament Ca sensitivity. PKC also phosphorylates
myofilament proteins and membrane channels directly to modulate their function. Depolari-
zation can be induced by receptor activated channels (or depolarization in a neighboring
electrically coupled cell). This can activate L-type Ca channels, and in these cells with large
surface: volume ratio a given Ca flux (pA/pF) can have a strong impact on [Cali' Ca entry can
trigger CICR involving RyRs, and RyRs and IP 3Rs can interact in producing both Ca waves and
Ca oscillations in smooth muscle. Smooth muscle myofilaments are activated by Ca differently
than striated muscle. Ca binds to calmodulin to activate a myosin light chain kinase (MLCK).
Phosphorylation of the regulatory myosin light chains then allows actin-activation of myosin
ATPase activity. Crossbridge cycling is also much slower in smooth muscle, which allows tonic
force (attached crossbridges) at relatively low energy cost. Several chemical reactions must
occur along the E-C coupling pathway, accounting for the slow onset of force development in
smooth muscle compared to striated muscle.
Ca sparks in Smooth Muscle: SR Ca Release can Contribute to Relaxation

Nelson et al. (1995) demonstrated Ca sparks in vascular smooth muscle cells. These Ca
sparks are longer in duration than those in cardiac myocytes (20 ms rise time and 50-60 ms half-
decay time). It is now clear that Ca sparks are seen in most smooth muscle and are undoubtedly
the direct cause of the STOCs which are mediated by BKca channels (Jaggar et al., 2000). A
particularly intriguing aspect of this work is that local SR Ca release as Ca sparks can serve to
relax smooth muscle by local activation of BKca channels and STOCs which hyperpolarize the
whole cell (thereby shutting off Ca entry via Ica). Thus, a local [Cali rise which does not
appreciably alter global [Cali or contractile activation, can induce global [Cali decline and
relaxation. That is, local CICR can lead to global relaxation (rather than contraction as in cardiac
muscle). BKca channels have a very large unitary conductance (120 pS) and one channel may
contribute a ~ 10 pA current in the cell. Thus a cluster of 10-100 BKca channels can produce 100-
1000 pA of outward current. With the high input impedance of smooth muscle cells, this can
strongly hyperpolarize the cell.
There are also small conductance Ca activated K channels (SKca ) which may participate
in this negative feedback loop and Ca-activated CI channels which can contribute to
depolarization (but of course can only raise Em as far as E CI), and activation of these CI channels
can lead to spontaneous transient inward currents (STICs). Although STOCs and STICs can
even coexist in the same cells (ZhuGe et al., 1998), STOCs decays much faster than STICs. This
is presumably due to the lower [Cali required to activate ICl(Ca) in smooth muscle (365 nM,
Pacaud et aI., 1992; note that this differs markedly from cardiac IC1(Ca), pg 85). BKca channels
would only be briefly activated while local [Cali is> 111M. Blocking RyR (or BK ca channels)
can lead to ~ 10mV depolarization and an increase in tonic force in cerebral arteries (Knot et al.,
1998), consistent with a tonic basal contribution of Ca spark- and BKca-dependent
hyperpolarization in this tissue. Thus, there is a complex interplay of factors in smooth muscle,
but clearly CICR from RyRs can contribute either in a positive way to E-C coupling (especially
in phasic muscles) or in negative feedback producing relaxation. There do not appear to be Ca-
activated K channels in cardiac myocytes.
Capacitative Ca entry in Smooth Muscle

Depletion of intracellular Ca stores in a wide variety of cell types results in the activation
ofCa influx, known as capacitative Ca entry (CCE, Putney, 1986, 1997; Putney & Ribeiro, 2000;
Berridge, 1995). Hoth & Penner (1992, 1993) showed that the Ca entry pathway was a Ca-
selective current, which they called Ierae (Ca-release activated current). CCE does not occur in
cardiac muscle, but has been seen in several vascular smooth muscle preparations (Missiaen et
al., 1990; Baro & Eisner, 1992; Blatter, 1995). There was substantial controversy about whether
the signal from the SRiER was mediated by a diffusible messenger molecule or by direct
conformational coupling between an ERiSR Ca store sensor and the Ierae channel (see Putney &
Ribeiro, 2000, for review). Hardie & Minke (1993) suggested that the Drosophila transient
receptor potential (TRP) channel protein might be invlved in CCE. Kiselyov et al. (1998, 1999)
showed that IP 3R and TRP3 directly interact, that the IP 3R (even added exogenously) could
activate 66 pS TRP3 channels and that the N-tenninal IP 3 binding domain was sufficient to
activate TRP3. Additionally, an endogenous Ca channel was similarly modified by IP 3 and IP 3R
(Zubov et al., 1999). The N-terminal of the IP 3R may activate TRP channels, but when the
ERJSR is Ca-Ioaded the activation is repressed. Thus, the IP 3R might serve as a sensor of the
ERJSR [Cal and transmit this signal to the cytoplasmic side of the IP3R where it transmits
regulatory infonnation to the TRP channel Fig 126). This may relate to the apparent luminal
[Ca]sR-dependence ofRyR2 gating (Pg 194-195,225) and to the retrograde molecular feedback
from RyRl to the skeletal muscle DHPR (pg 211). Indeed, like cardiac cells and isolated RyR2,
the SR Ca release via RyR in smooth muscle and via IP 3R seems to be sensitive to luminal [Cal
(ZhuGe et al., 1999; Missiaen et al., 1994; Berridge, 1997). The precise role of CCE in smooth
muscle and its interaction with CICR, IP3ICR and other Ca channels will require further study.
IP3 Induced Ca Release in Skeletal Muscle

The possibility that IP 3 is involved in skeletal muscle E-C coupling was raised by
observations that application of IP 3 could induce SR Ca release in skinned skeletal muscle fibers
and SR vesicles (Vergara et a!., 1985; Volpe et a!., 1985; Donaldson et al., 1987). IP 3
production was increased during tetanic contractions (Vergara et a!., 1985) and the enzymes
required for IP 3 synthesis and degradation are present (Vergara et al., 1985, 1987; Hidalgo et al.,
1986; Hidalgo & Jaimovich, 1989; Varsanyi et al., 1989; Lagos & Vergara, 1990). It has also
been suggested that the effect of IP3 on skeletal muscle is [Cal and Em-dependent (Volpe et al.,
1986; Donaldson et al., 1988).
On the other hand, numerous reports show that IP 3 does not induce SR Ca release in
skeletal muscle (Scherer & Ferguson, 1985; Lea et al., 1986; Mikos & Snow, 1987; Palade,
1987c). Using photolysis of caged IP3 (as in Fig 125), Walker et al. (1987) showed that IP 3-
induced contractions were too small and much too slow to be physiologically relevant. Indeed,
the rapid turn-off of SR Ca release with repolarization (Simon et al., 1989) is several orders of
magnitude faster than degradation of the IP3 (Somlyo et al., 1988). The enzymes and substrates
involved in rapid IP 3 turnover are present, but not at levels which would make this feasible
(Walker et a!., 1987; Somlyo et a!., 1988; Hidalgo & Jaimovich, 1989; Varsanyi et al., 1989).
Blinks et al. (1987) found apparent IP 3ICR in detubulated skeletal muscle, proposing that IP3
could induce depolarization of sealed off T-tubules, but not in intact fibers. Finally, blocking the
IP3 receptor with intracellular heparin produced no effect on skeletal muscle E-C coupling (Pape
et al., 1988), in contrast to the profound depression seen in smooth muscle (Kobayashi et a!.,
1988). In conclusion, IP 3ICR is unlikely to play any substantial role in skeletal muscle E-C
coupling. However, there are IP 3R in skeletal muscle, and more recent work seems to point to a
perinuclear localization and a possible role in nuclear Ca signaling (Jaimovich et al., 2000).
IP3 Induced Ca Release in Cardiac Muscle

Hirata et al. (1984) first showed IP3ICR from cardiac SR vesicles. However, Movsesian
et al. (1985) found no effect of IP3 in isolated cardiac SR or in myocytes permeabilized by
saponin. Nosek et a!. (1986) found that IP3 potentiated spontaneous and caffeine-induced Ca-
release in cardiac muscle. Vites & Pappano (1990) showed IP 3 induced contractions in small
LJ
1P B. CICR
A. I~
3
-5x,~werL
Tension ~
~ "'--
!n':'
(I)
~
Inj.lAsp.
Signals
I .......
Inj. pCa 7.75 Inj. pCa 6.25 Inj. pCa 6.25 25
5 I'M IPg 10l'M IPg
Inj.lAsp. I 1
Signals -J-- _ .1
-J--- >-i
0.15
T T 1
Asp. pCa 7.75 Asp. pCa 7.75 Asp. pCa 7.75
Figure 127. IP) and Ca-induced Ca-release in a mechanically skinned rat ventricular myocyte (from
Fabiato, 1986b, with pennission). Rapid injection (lnj.) of 5 f.lM IP 3 induced a small and slow contraction
(A), compared to that induced by a rapid increase in [Cal from 20 nM to 560 nM (i.e. via CICR in B). Note
different tension scales. Simultaneous application of these two stimuli produced a contraction in which the
major part was not much different than with CICR (C). Aspiration (Asp.) of20 nM precedes injections.
skinned multicellular preparations from chick atria which were caffeine- and ryanodine-sensitive.
Fabiato (1986a,b, 1990) showed that rapid application of IP) to skinned rat myocytes could
induce SR Ca release, but that it was much smaller and slower than that induced by CICR (Fig
127). Kentish et al. (1990) confirmed this in skinned rat ventricular muscle, using flash photo-
lysis of caged Ca and caged IP 3 • Very high [IP)] could induce SR Ca release, but the rate and
extent of Ca release was much lower than for CICR.
These studies made it clear that IP)ICR is not centrally important in cardiac E-C
coupling. However, IP) could still serve as a modulator of CICR (Nosek et al., 1986; Suarez-Isla
el aI., 1988). Activation of cardiac lXI-adrenergic and muscarinic receptors increases IP)
production as well as contractile force (Gilmour & Zipes, 1985; Brown & Jones, 1986; Poggioli
el aI., 1986; Jones el aI., 1988; Otani et al., 1988; Scholz el al., 1988). On the other hand, the
increase in Ca transients and contractions in response to a-adrenergic activation in mammalian
ventricle appear to be mediated mainly by PKC, rather than IP) (Endoh, 1996; Mattiazzi, 1997;
Gambassi et al., 1998). Thus, while it is clear that several cardiac membrane receptors stimulate
phospholipase C to produce both IP) and diacylglycerols (DAG), the DAG stimulation of PKC
appears to predominate in alteration of acute contractile function.
Kijima et al. (1993) found 1P) binding sites in ventricular homogenates. In membrane
fractionation studies, RyR was enriched in SRisarcolemmal fractions, while IP)R was especially
enriched in a different fraction that included intercalated disks. This agreed with their immuno-
localization, suggesting IP)R preferentially at intercalated disks rather than in SR. Perez et al.
(1997) found that the IP)R expressed in ventricular myocyte is the type-2 isoform (while other
cells in ventricular homogenate express type-l IP)R). The number ofIP)R is as much as 10% of
the number of RyR. Lipp et al. (2000) confirmed that cardiac myocytes express type-2 IP)R and
found six times higher IP)R expression in atrial than ventricular myocytes. They found atrial
myocyte IP)Rs to be sub-sarcolemmal and apparently co-localized with surface RyRs. They
found that IP) could produce SR Ca release in skinned atrial cells and that a membrane permeant
A. Skeletal - VDCR+ B. Cardiac - CICR

Ca Ca
Figure 128. E-C coupling in skeletal and cardiac muscle (VDCR & CICR). A. In skeletal muscle the
physical link between the sarco.lemmal Ca channel (or DHPR) is critical for VDCR. Ca released by VDCR
can then activate uncoupled RyRs via ClCR. Ca influx is not required in skeletal muscle E-C coupling and
Ca cycles mainly berween the SR and the cytoplasm. B. In cardiac muscle Ca entry via lea activates RyR
via CICR. Ca from lea or one RyR can activate a neighboring RyR via CICR. Physical links between the
sarcolemmal and SR Ca channels in cardiac muscle probably do not transmit a signal for VDCR.
IP 3 analogue enhanced Ca spark frequency and twitch Li[Ca]i in intact atrial myocytes. They
suggested that IP 3 might interact with RyR-mediated CICR in atrial myocytes. However, a
specific role for IP 31CR in ventricular myocytes is still not clear.
Preliminary immunolocalization studies with type-2-specific IP 3R antibodies show a
perinuclear localization in ventricular myocytes (with G.A. Mignery, unpublished). We
speculate that such perinuclear IP 3R may be activated by neurohumoral agents known to cause
increased [IP 3] (e.g. a-adrenergic agents or endothelin). The IP 3 might activate local Ca-
dependent processes at or in the nucleus (e.g. CaMKlI) due to high local [Cal near the IP 3R.
This could contribute to transcriptional regulation that is also seen with these agents. This
possibility seems attractive, because it would allow cardiac myocytes to distinguish between the
regular global dynamic [Cali signals and those neurohumoral signals that might regulate cardiac
transcription in a Ca-dependent manner (Ramirez et al., 1997). There is evidence for ion
channels in nuclei of cardiac myocytes (Rousseau et aI., 1996) and other cell types (Stehno-Bitel
et aI., 1995; Humbert et al., 1996) and there is considerable evidence for Ca-dependent
transcriptional regulation (Karin & Hunter, 1995; Cahill et aI., 1996; Malviya & Rogue, 1998).
While an appealing possibility, I should emphasize that this is mainly speculation at present. On
balance, IP3ICR might playa minor modulatory role in cardiac E-C coupling (especially in atrial
cells), but CICR is clearly the primary mechanism of cardiac E-C coupling.
SUMMARY
In a simplified manner the 3 muscle types can serve as models of the 3 main mechanisms
ofSR Ca release (i.e. VDCR in skeletal, CICR in heart and IP 3ICR in smooth muscle, Figs 128 &
126). It should be emphasized that this is an oversimplification since there is some evidence in
support of every permutation of mechanism and muscle type. For example, VDCR appears to be
the crucial initiating process in skeletal muscle. However, there are at least twice as many RyRs
as T-tubule/DHPR tetrads in skeletal muscle and CICR may be crucial in recruiting the RyRs
which are not physically coupled to T-tubule tetrads. IP 3 can also induce Ca release in skeletal
muscle under some circumstances, although the physiological relevance of this pathway is not
yet clear. In cardiac muscle CICR is the main E-C coupling mechanism. However, IP3 may
modulate cardiac Ca release, and there is some evidence for a functional direct link between the
sarcolemma and the SR (and possibly VDCR). Whether this DHPR-RyR link is important, aside
from just bringing the RyR and DHPR close together, is not known. In smooth muscle there is
compelling evidence for both IP3ICR and CICR. There is even evidence that the IP3R interacts
with a different plasma membrane Ca channel (TRP) involved in CCE where the signal is
retrograde from IP 3R to TRP.
D.M. Bers. 245
CHAPTER 9
CONTROL OF CARDIAC CONTRACTION BY

SR AND SARCOLEMMAL Ca FLUXES
Both Ca influx and SR Ca release are important elements in E-C coupling and Ca from
both sources can contribute to the activation of contraction. In this chapter I will try to clarify
the dynamic interplay of transsarcolemmal and trans-SR Ca fluxes in different cardiac muscle
preparations and under different experimental situations.
While Ca entry via ICa triggers SR Ca release in cardiac E-C coupling, Ca entry may also
contribute to rCa]; elevation during contraction. Since SR Ca release depends on Ie" it can be
difficult to distinguish unequivocally between the direct effects of Ca entry vs. effects of Ca
entry on SR Ca release. For example, interventions which increases lea typically increase Lo.[CaJi
and contraction. How can we distinguish whether this is due to: a) the direct effect of Ca entry,
b) an increased fractional SR Ca release (due to higher lea) or c) an increase in SR Ca load? One
cannot simply block Ca entry to study the contribution of SR Ca to contraction, because CICR
will also be blocked. On the other hand, one can inhibit the SR Ca function (with caffeine,
ryanodine or thapsigargin) to study the activation of contraction by lea in the absence of a
functional SR contribution. This approach is especially valuable because while SR Ca release is
clearly capable of activating the myofilaments (pg 186), this is less clear for lea (pg 120).
SPECIES, REGIONAL AND DEVELOPMENTAL DIFFERENCES

Figure 129 shows the effects of caffeine and ryanodine on steady state twitch
contractions in several cardiac preparations. Variation is apparent among different species (frog
vs. rabbit vs. rat), at different stages of development, (neonatal vs. adult) and regionally in the
heart (rabbit ventricle vs. atrium). While caffeine can prevent net SR Ca uptake by making the
SR extremely leaky to Ca (Weber & Herz, 1968), it has side effects at the mM concentrations
that are required for SR effects_ For example, caffeine increases myofilament Ca sensitivity
(Wendt & Stephenson, 1983, see Chapter 2), can increase Ca influx (Bers, 1983; Tseng, 1988)
and inhibits phosphodiesterases (Butcher & Sutherland, 1962), thereby elevating cyclic AMP.
These other caffeine effects tend to increase force and may be partly why the values for caffeine
are typically higher than for ryanodine. Ryanodine is much more specific in its interaction with
the SR (the Kd is nM), but its action is more complex (Sutko et at., 1985; Bers et at., 1987; see
pg 183- I 85). The concentration dependence of these agents does not vary in different tissues,
despite the difference in maximal effect (Sutko & Willerson, 1980; Shattock & Bers, 1987).
Thus, the variation in tension depression among cardiac tissues in Fig 129 by caffeine and
ryanodine indicates the relative requirement for SR Ca release for myofilament activation.
Effects of Caffeine and Ryanodine

on Steady State Twitch Force
...
'0
'Eo 100
()
'0
~
l: 50
o
'iii
l:
Ql
l-
0...1.-.1..-----'-----
Frog Neonate Adult Rabbit Neonate Adult
Vent Rabbit V Rabbit V Atrium Rat Vent Rat Vent
Figure 129. The effect of caffeine (10 roM) or ryanodine (100 nM) pretreatment on steady state twitch
contractions (0.5 Hz at 30°C or 23°C for frog) in various cardiac muscle preparations (Data are from Bers,
1989; Haddock et al., J999, and unpublished observations).
However, the 20-30% reduction of twitch contractions by ryanodine in rabbit ventricular

muscle cannot be taken to mean that under normal conditions the SR contributes only 20-30% of
the Ca required for myofilament activation. It simply means that in the absence of a functional
SR, Ca influx can supply sufficient Ca to activate a nearly normal amplitude contraction. Under
normal conditions, SR Ca release raises [Ca]j, which decreases slightly the gradient for Ca influx
(a minor effect), but more importantly inactivates lea and shortens the action potential. Figure 60
(pg 122) showed that preventing SR Ca release doubles the amount ofCa influx via lea during the
same AP (e.g. from 6 to 12 flmol/L cytosol). Preventing SR Ca release also prolongs AP
duration (e.g. in rabbit ventricle from 222 to 305 ms at 37°C, Shattock & Bers, 1987), which
could also increase Ca influx by another 10% or so (based on Fig 59). In addition, the lower Ca
transient in the absence of SR Ca release will allow more Ca influx to occur via Na/Ca exchange
(Fig 74). Thus, preventing SR Ca release indirectly increases Ca influx, and so would
underestimate the normal contribution of SR Ca release to the twitch.
On the other hand, ryanodine can diminish the ability of a given Ca influx to activate
contraction. This is because some of the Ca entering the cell may also be transiently accumu-
lated by the SR (see pg 183-185). Indeed, in the presence of ryanodine the SR can accumulate a
similar amount of Ca as under control conditions, albeit only very transiently (Bers et aI., 1987;
MacLeod & Bers, 1987). This transient Ca uptake by ryanodine-treated SR is especially notable
at post-rest contractions. In this case the SR is Ca-depleted, such that the net SR Ca accumula-
tion may more closely keep pace with Ca influx via lea (which is also reduced during post-rest
depolarizations, Fig 58, pg 119). Indeed, ryanodine, even at very low concentrations, prefer-
entially depresses post-rest contractions (Fig 130; Hajdu & Leonard, 1961; Bers, 1985; Malecot
& Katzung, 1987). So, ryanodine can allow the SR to still function as a transient Ca buffer.
Caffeine can prevent SR Ca accumulation altogether (since RCCs are abolished even at
very short rest intervals, Bers et aI., 1987). Thus, with caffeine the SR may be more effectively
short-circuited such that Ca entry has more direct access to the myofilaments. At high ryanodine
Chapter 9 SR Ca Release & Sarcolemmal Ca Fluxes 247
150
Frog Rabbit Rabbit
ec Ventricle Ventricle Atrium
o 100
~
U
~---"~
~
c
,~ 50
C
Q)
I-
10 55 1
Post-Rest Contraction Number

Figure 130. Recovery of twitch force after 30 sec rest in frog, rabbit and rat ventricle and rabbit atrium in
the absence (top) and presence (bottom) of 100 nM ryanodine. Steady state twitch tension is shown by the
end points of each curve (SS). Shading indicates ryanodine-sensitive force (redrawn from Bers, 1985).
concentrations (> 10 flM) or after long exposure times, there is often some recovery of twitch
contraction despite a Ca-depleted SR (Sutko & Willerson, 1980; Bers, 1985; Lewartowski et al.,
1990). This probably reflects a higher percentage of ryanodine-modified (leaky) SR release
channels, such that the effect is more like caffeine (i.e. the transient buffering ability is lost).
Thapsigargin, which blocks the SR Ca-ATPase, also progressively depresses twitch
contractions as SR Ca is gradually depleted. However, acutely blocking the SR Ca-ATPase can
increase twitch contraction and Ca transients. This is true both when the SR Ca content is low
(so SR Ca release does not occur, Bassani et al., 1993b; Janczewski & Lakatta, 1993) or when
SR Ca content and release are normal (Fig 133C; i.e. if SR Ca loss is prevented during pump-
blockade, Bassani et aI., 1994a). The explanation is that the SR Ca-ATPase actively transports
Ca as soon as rCa]; rises, and is rapid enough to limit the peak rCa]; attained during the twitch.
In conclusion, complete SR Ca-ATPase blockade by thapsigargin can prevent both SR Ca uptake
and release, and may most directly indicate the ability of Ca influx to activate contraction (but
only after the SR is Ca-depleted). However, Ca influx will still be higher than during a normal
twitch with SR Ca release (as above), and it can be difficult to block the pump completely with
thapsigargin in multicellular preparations (Baudet et al., 1993; see pg 169).
There are ultrastructural conelates to these pharmacological dissections. For example,
the SR network is most highly-developed in adult rat ventricle, somewhat less in rabbit atrium
and ventricle, and is the most sparse and poorly developed in frog ventricle (Chapter 1).
Furthermore, the small diameter of frog ventricular myocytes (and hence high surface:volume
ratio) makes Ca entry a more plausible mechanism for activation in frog. Therefore, the virtual
insensitivity of frog ventricle to ryanodine is not especially surprising and in frog ventricular
muscle it is possible that the myofilaments can be activated entirely by Ca entering from outside
the cell at each contraction (Morad & Cleeman, 1987; Chapman, 1983).
Fabiato & Fabiato (1978a; Fabiato, 1982) also found variation in CICR among different
preparations, consistent with the above interpretation. For example, CICR could not be observed
in frog or prenatal rat ventricle, but was most prominent in adult rat ventricular myocytes. This
developmental transition to relative SR dependence is also apparent in other mammalian cardiac
preparations (Penefsky, 1974; Maylie, 1982; Seguchi et al., 1986; Haddock et aI., 1999). This
A. Adult Cat Atrium B. Adult Cat Ventricle

- ~~~~t~;";..'
~/;
.fiJi
'1ft,,' •
I
cc ~I t~
:'~~:
.~~~/~ .
. ' .. ," •
I
I
cc
I
1000
~
..s
til
~
100
200 ms
Figure 131. Spatial [Cali gradients in atrial myocytes. A. Transverse line scan in a cat atrial myocyte,
showing that [Cali rises earlier in the subsarcolemmal (ss) region than the cell center (cc). B. Similar
transverse line scan and Ca transients in a cat ventricular myocyte, showing simultaneous [Cali rise across
the cell. Lower traces show superimposed ss and cc traces (figure kindly supplied by L.A. Blatter).
agrees with ultrastructural results which indicate that the T-tubule/SR system is gradually
developing from the prenatal period through the first few weeks of life, albeit at different rates in
different species (Scheibler & Wolff, 1962; Legato, 1979; Olivetti et al., 1980; Hoerter et aI.,
1981; Page & Beucker, 1981; Penefsky, 1983; Goldstein & Traeger, 1985; Artman, 1994).
Furthermore, SR Ca uptake in vesicles increases over this developmental period in rat, rabbit and
sheep ventricle (Nayler & Fassold, 1977; Nakanishi & Jarmakani, 1984; Mahony & Jones, 1986;
Pegg & Michalak, 1987). An interesting twist on this variation is that in chipmunks that
hibernate, the ventricle appears to function like rat ventricle during hibernation, but more like
rabbit ventricle when not hibernating (Kondo & Shibata, 1984; Kondo, 1986, 1988).
Spatial [Cali gradients are generally not observed during twitches in adult ventricular
myocytes (Figs 131, 132). This is because T-tubules conduct depolarization axially to the center
of the cell and local CICR throughout the cell is synchronized by lea activation. This synchrony
also occurs along the myocyte length (Fig 122). Both adult atrial and neonatal ventricular
myocytes lack appreciable T-tubules, and the [Cali rises first at the cell periphery and then
spreads to the center of the cell (Figs 131, 132; Berlin, 1995; Huser et al., 1996; Haddock et al.,
1999). In both atrial and neonatal ventricular myocytes there are RyRs transversely arranged at
the level of the Z-line, despite a lack ofT-tubules (Sedarat et al., 2000). In atrial muscle, SR Ca
release appears to propagate transversely via CICR, because the peak [Cali at the center can be as
high as the subsarcolemmal [Cali, and ryanodine depresses both central and subsarcolemmal Ca
transients. Furthermore, local [Cali at the center can exceed subsarcolemmal [Cali, which is not
expected for simple Ca diffusion from the subsarcolemmal region. The ability of CICR to propa-
gate axially also depends on local SR Ca content, and propagation fails when central SR Ca load
is low (Huser et aI., 1996). This reflects the SR Ca-load-dependence of SR Ca release (pg 225).
In neonatal rabbit ventricular myocytes the Ca transient (subsarcolemmal or central) does
not seem to involve appreciable SR Ca release, but appears to reflect mainly diffusion from the
A. Adult Rabbit Ventricle B. Neonatal Rabbit Ventricle

t _
¥
E
-cc=~ ~
55 ••••••
f1••••••3 t
'0
500
15
Figure 132. Spatial [Cal; gradients in neonatal rabbit ventricular myocytes. A (top). Transverse line scan
in a rabbit ventricular myocyte (25 f.!m wide) showing uniform Ca transients in the subsarcolemmal (ss)
region and the cell center (cc). At bottom are superimposed ss (grey) and cc (black) Ca transients from A.
B. Similar transverse line scan and Ca transients in a 9 f.!m wide neonatal rabbit ventricular myocyte, with.
superimposed ss and cc traces. Data are from study of Haddock et al. (1999).
subsarcolemmal region to the center (Fig 132; Haddock et aI., 1999). In adult myocytes
thapsigargin decreases the rate of rise of [Ca]; for both subsarcolemmal and central location by
80%. In neonatal myocytes the rate of subsarcolemmal and central [Ca]; rise is 44% and 12%,
respectively of that in adult myocytes, but thapsigargin has no effect at all on the neonatal
myocyte Ca transient (peak, rate of rise or spatial gradient). There is, however, Ca stored in the
neonatal myocyte SR, because caffeine-induced Ca transients and contractures are robust, and
are spatially homogeneous (Balaguru et al., 1997; Miller et al., 1997; Haddock et aI., 1999).
However, this SR Ca does not seem to participate in E-C coupling. Ca sparks are seen in the
neonatal myocytes, but preferentially in the subsarcolemmal region. We speculated that RyRs in
neonates may not be clustered or as sensitive to Ca (especially non-junctional RyRs), such that
they are not normally activated during E-C coupling (Haddock et al., 1999).
If neonatal rabbit ventricular myocytes don't have SR Ca release to boost the Ca influx
signal, how is there enough Ca to activate the myofilaments? The density of Ica (in A1F) is
actually lower by ~50% in neonatal ventricular myocytes (vs. adult; Osaka & Joyner, 1991;
Wetzel et al., 1991; Akita et al., 1994). The higher surface:volume ratio in neonatal cells (which
have smaller diameters and lack T-tubules), would increase the impact that a given Ica (in pAlpF)
has on [Ca]; by a factor of -2-3 (Haddock et aI., 1997). Neonatal myocytes also have a longer
AP than adult (especially in rat). Thus, Ic• may still bring in 5-10 /!mol/L cytosol in neonatal
myocytes. Na/Ca exchanger expression and current are -5-fold higher (in pAlpF) in neonatal
than adult rabbit ventricular myocytes (Boerth et aI., 1994; Artman et aI., 1995; Haddock et aI.,
1997). Haddock et al. (1997) showed that 9-14 /!mol/L cytosol enters the neonatal myocyte via
INa/C. during a 300 ms pulse at +60 mY (vs. 1-2 /!mol/L cytosol for adult) and this produced 10%
cell shortening (vs. «I % in adult). The lack of SR Ca release allows greater Ca entry via INa/C.
in the neonate. Thus, Ca influx might be 15-20 Ilmol/L cytosol, with 67-75% due to INa/C.
(consistent with modest twitch inhibition by blockade of Ic., Wetzel et aI., 1995). Intracellular
Ca buffering power is also 2-3 times lower in neonatal ventricular myocytes (Bassani et al.,
1998), consistent with the lower amount of myofilament and SR Ca-ATPase at this stage of
development. This means that neonatal myocytes need only 33-50% as much activator Ca to
reach the same state of activation as adult cells. Thus, Ca influx of 17-25 Ilmol/L cytosol in
neonatal cells may activate as effectively as a combined Ca influx & SR Ca release of 50 Ilmol/L
cytosol in adult cells. In conclusion, neonatal rabbit ventricular myocytes appear to depend on
Ca influx by INa/C. + Ica . This Ca influx by itself may be sufficient to support contraction because
of high Na/Ca exchange expression, high surface:volume ratio and lower cytosolic Ca buffering.
Based on results from several sources a rough sequencing of cardiac muscle preparations
from most to least SR reliant is (V, ventricle; A, atrium): calf Purkinje fiber> adult mouse V ~
adult rat V > dog V - ferret V > cat V > neonate rat V - rabbit A > human V > rabbit V ~ failing
human & rabbit V > guinea-pig V > neonate rabbit V > fetal V (human, cat & rabbit) > trout V >
frog V -toad V (Penefsky, 1974; Sutko & Willerson, 1980; Sutko & Kenyon, 1983; Bers, 1985;
Malecot et aI., 1986; Seguchi et al., 1986; EI-Sayed & Gesser, 1989; Schlotthauer et al., 1998;
Pieske et al., 1999a; Haddock et al., 1999). In a given species, atrial muscle is typically more SR
Ca-dependent (with more active CICR, Fabiato, 1982), and has faster rCa]; decline, due in part to
lower phospholamban content. Shorter atrial AP duration may also limit Ca entry via Ica or
Na/Ca exchange. Of course this sequence is only approximate, and the relative importance of the
SR Ca release also varies under different conditions (e.g. changes in frequency, drugs, etc.).
Ca Removal Fluxes
The amount of Ca which enters the myocyte at each steady state twitch must also be
extruded from the cell; otherwise the cell would be gaining or losing Ca (i.e. not in steady state,
see also pg 52-55 & 152-156). Although there are 4 Ca removal systems (SR Ca-ATPase, Na/Ca
exchange, sarcolemmal Ca-ATPase and mitochondrial uniporter), the analysis of Ca removal
fluxes can be more direct than that for activator Ca. Bassani et al. (1994a) developed a
quantitative method which determines the specific contributions of these Ca transport systems to
relaxation and rCa]; decline in rabbit and rat ventricular myocytes (below & Figs 133-5).
First, Ca transients are recorded with selective inhibition of Ca removal systems (Fig
133). In Fig 133A only the slow systems (mitochondrial Ca uptake and sarcolemmal Ca-
ATPase) can remove [Cali, because Na was not present anywhere and SR Ca uptake was blocked
by either caffeine or thapsigargin. Both caffeine-induced Ca transients and those evoked by an
action potential (with SR Ca-pump completely blocked by thapsigargin) declined with a 1: -12 s.
This was true in both rat and rabbit ventricular myocytes indicating that the combined action of
the mitochondrial Ca uptake and sarcolemmal Ca-pump were comparable in these species.
Moreover, the AP-induced twitch (with thapsigargin) only released about half of the SR Ca load
(which could all be released by caffeine). This is consistent with the findings that -50% of the
SR Ca content is released during a normal twitch. These results also emphasize that the
mitochondria and sarcolemmal Ca-ATPase require >20 s to bring [Cali down to diastolic levels.
Figure 133B shows that the rCa]; decline during the normal rabbit ventricular twitch is
slowed by 30% when Na/Ca exchange is blocked. In this case [Cali decline depends on the
A. Slow Removal B. Slow + SRCa-ATPase C. Slow + NalCaX

1.4 (Mito + SL Ca-ATPase) 0.7
:E 1.0
-=~
(J
~ 0.6
Figure 133. Ca transients in rabbit ventricular myocytes with selective Ca transporter block. A. Twitch
and caffeine-induced Ca transient (Caft) where both SR Ca uptake and Na/Ca exchange were prevented.
Caff was applied in ONa,OCa solution. Twitch in thapsigargin (TG-Tw) was evoked after pre-depletion of
Na; (and in Na-free solution). The SR Ca-ATPase was completely inhibited by thapsigargin, but SR Ca
content nonnal (i.e. prior to gradual SR Ca depletion). B. Twitches evoked in either control or Na-free
solution (in both cases Na; was predepleted to prevent Ca entry via Na/Ca exchange). C. Twitches evoked
in either control or with the SR Ca-ATPase completely blocked by thapsigargin (in both cases cells were
incubated in ONa, OCa solution ±TG to maintain SR Ca while SR Ca-pump blockade was instituted. Data
are regraphed from Bassani et al. (1994a) and time constants 1: are shown for mean data.
combined action of the slow systems plus the SR Ca-ATPase. When the SR Ca-ATPase is
selectively blocked by thapsigargin (Fig 133C), [Cal; decline is slowed by 50%, indicating that
the combined slow systems plus Na/Ca exchange can restore diastolic [Cal; in -2 s. Notably
peak twitch [Cal; is higher than control when either the SR Ca-ATPase or the Na/Ca exchanger is
blocked. This indicates that in rabbit ventricle Ca removal by the SR Ca-ATPase and Na/Ca
exchange can actually curtail the peak of the Ca transient, because removal begins as soon as
[Cal; rises. This was also true in rat ventricular myocytes with respect to SR Ca-pump blockade,
but not Na/Ca exchange inhibition.
To analyze Ca fluxes, the free [Cal; during [Cal; decline must be converted to total
cytosolic [Cal ([Cahot), as in Fig 29 or 87. Then the rate of total Ca removal from the cytosol
(d[Caho,ldt) can be graphed as a function of [Cal; for each point in time (see Fig 87 or 134A).
This transport rate must be the sum of the individual transport rates given by
d[Ca],ldt = J SR + JNalCaX + J S10w - Leak (9.1)
where the three J terms refer to flux through the SR Ca-ATPase, Na/Ca exchange and the
combined slow Ca transport by mitochondria and sarcolenunal Ca-ATPase. Leak is considered
to be small compared to other fluxes during [Cal; decline. For simplicity JSR , JNa/CaX and JS10w can
be empirically described as simple [Cal dependent fluxes via a Hill equation:
V max
(9.2)
J S10w is first fit by using the decline of [Cal; when other systems are blocked (Fig 133A), where
J SR and J NaCaX are zero in Eq 9.1. We then assume that this [Ca];-dependence of J s10w (with a V ma"
Km and n) is valid for other Ca transients in that species. Similarly, parameters for JNaCaX can be
measured during a twitch in thapsigargin (or a caffeine-induced Ca transient) where d[Ca],ldt =
JNa/CaX + J S10w (Fig 133C-thapsigargin). Likewise parameters for J SR are measured using the
A. Analysis of Cell Ca Fluxes B. Relative Ca Fluxes
200
_ _..:.Ra.:;;.;.;tS;;:.R"'--_ _ 207
Rat V!
Mouse V ~;;;;;;;;;I~
Dog V
.2!u
I:=iiiiiiii~~~~
III Q)
0::: ~ 150 Rabbit
Cat VV
'0.5 0~ Ferret V
G-PV
:!! ~ 100 Human V
~::;; HF Rabbit V
HF Human V
!!!!!!!!!!!!=~~~~~
I- 0
III E HF Dog V
U.2: 50 ~~~~!~~~!9.!!.>s---.46
Neo Rat V iiiii~~~~~~t
------- Rat Na/CaX 27
N Rabb;t V 1=========;=;:~~7=l
O-J.-,o~~""'''''9=;=:=Mi=it0i=&#S=.;,L",c,,;a-::::p;:=um;:!::;=; 4 Frog V 1==::;=====;=~I~&~N~a~C~aX~
o 200 400 600 800 1000 o 50 100
Relative SR vs SL Ca Transport (%)
[Ca]; (nM)
Figure 134. [Cali-dependence of Ca transport in ventricular myocytes. A. Ca transport functions derived
from Ca transients in intact rabbit and rat ventricular myocytes. Ca transport rates as functions of [Cal; for
the SR Ca-ATPase, Na/Ca exchange (NaCaX) and combined slow systems (Mito & SL Ca-pump) were
determined as described in the text and with respect to equations 9.1 & 9.2. Independent values obtained
for hR' JNalCaX and J Slow respectively were: Vmax (given at the end of each curve in Ilmol/L cytosol/sec); K m
(in nM) = 264,316, and 362 in rabbit and 184, 257 and 268 in rat; n = 3.7,3.7,3.2 in rabbit and 3.9, 3.4 an
3.5 in rat. (based on data from Bassani et aI., 1994a). B. Relative Ca fluxes in different ventricular (V)
preparations based on analyses like Fig 134A and 135 (see also Table 20, pg 153). Black bars are %
activation and extrusion by SR, white bars are % supply by ICa and removal by NaCaX.
twitch where Na/Ca exchange is blocked (Fig 133B-ONa). This analysis can be checked to see if
total Ca removal during the normal twitch Ca transient is well described by the sum in Eq 9.1
(and this is the case). Figure 134A shows the resulting Ca flux functions estimated in this way
for rabbit and rat. Vmax for the SR Ca-ATPase in rat is larger than in rabbit, but the situation is
reversed for Na/Ca exchange. lSlow is quite small compared even to the lNacax. It should also be
noted that the Hill function (Eq 9.2) used to describe lx does not necessarily have to be a good
mechanistic descriptor of the flux (e.g. it is not for Na/Ca exchange). What is important is that it
provides a reasonably good empirical fit to the dependence of d[Ca].ldt on [Cali over the [Cali
range of interest.
The final piece of this analysis (Fig 135) is to calculate how these systems compete
dynamically and simultaneously during a normal twitch. Here we use the free [Cali during that
twitch to calculate the instantaneous individual fluxes through each system (Eqs 9.1 & 9.2).
Ideally, this analysis would include the backfluxes for each of these transporters as described for
the SR Ca-pump and Na/Ca exchange (pg 174 and 149), but that was not done by Bassani et aZ.
(1994a). This would allow net flux via these systems to more closely approach zero as free [Cali
returns to the resting level. Accounting for local spatial distributions of both Ca transporters and
buffering sites could also make this sort of analysis more comprehensive (e.g. Fig 74).
Figure 135 shows that during a normal twitch the fractions of Ca transported by the SR,
Na/Ca exchange and slow systems are 70, 28 and 2% respectively in rabbit and 92, 7 and I % in
rat myocytes. This 28% estimate of Ca flux by Na/Ca exchange in rabbit agrees with the 25-33%
estimated based only on the ratio of time constants of relaxation or [Cali decline (pg 52-54). The
7% value for rat also agrees with very similar experimental results in rat by Negretti et aZ.
75
A. Rabbit Total 100% 75
B. Rat 75
C. Rabbit HF
100%
XC 92%
::> 0
<;: C/l
Total 100%
.. ~50 70% 50 50
U o
"0 ..
16::
CI> '"
49%
... ==
"'0
25 NCX 28%
25 25
.fl E
.E.=: Slow
(SL Ca·ATPase & MilO 2%
NCX 7%
a a 1%
0.0 0.5 1.0 15 2.0 0.0 0.2 0.4 0.6 0.6 1.0 0.5 1.0 1.5
lime(s) lime(s) lime(s)
Figure 135. Integrated Ca fluxes during twitch relaxation in rabbit and rat ventricular myocytes. Free
[Cali during twitch relaxation was used as a driving function to calculate Ca flux via each system, using the
functions in Fig 134A. Percents indicate the fraction of the total cytosolic Ca removal attributable to each
system when they dynamically interact in the ceIL Data in A and B are from Bassani et al. (I 994a). For C
rabbit heart failure (HF) the Vmax for NCX was increased 116% and SR Ca-ATPase was reduced by 24% as
indicated by Pogwizd et al. (1999).
(1993). Thus, there are clear species differences in this competition between the SR Ca-ATPase
and the Na/Ca exchange. In heart failure the expression of SR Ca-ATPase can be down-
regulated and the Na/Ca exchange up-regulated (see Chapter 10). This shifts the balance of
fluxes during [Cali decline. In a rabbit heart failure model, we found that Na/Ca exchange was
increased ~2-fold at the mRNA, protein and functional level, while SR Ca-ATPase function was
reduced by up to 24% (Pogwizd et al., 1999). This resulted in an almost unchanged rate of [Ca]i
decline and twitch relaxation. We interpreted this as a large increase in Na/Ca exchange
offsetting a small decrease in SR Ca-ATPase function. However, as in Fig 135C, this creates a
situation where SR Ca-ATPase and Na/Ca exchange contribute almost equally to [Cali decline in
heart failure. We also showed that this shift may occur in human heart failure (Schlotthauer et
al., 1998; Pieske et al., 1999a). Table 20 (pg 153) and Fig 134B shows data for several species.
Ca influ'-'; must match Ca efflux

If 28% of Ca removal during twitch relaxation is due to Na/Ca exchange in rabbit (Fig
135A) we would expect Ca influx and SR Ca release in rabbit to supply roughly 28 & 70% of the
activating Ca respectively (and 7 and 92% in rat). This would be required to maintain cellular
and SR Ca balance and prevent net gain or loss of Ca. Delbridge et al. (1996, 1997) assessed Ca
influx and SR Ca release quantitatively in rabbit and rat (see Fig 31, pg 55). We measured Ca
influx via Ie.. the SR Ca content by caffeine-induced INa/ca integral and used measurements of
fractional SR Ca release (Bassani et al., 1993b, Delbridge et al., 1997). Table 24 summarizes
quantitative analysis of data from rabbit and rat ventricular myocytes. After making corrections
for surface:volume ratios, the integrated ICa in rabbit myocytes is sufficient to increase [Cahot by
9.7 f!M and the SR Ca content is 87 f!mol/L cytosol. Using 43% of SR Ca released during a
twitch we have 9.7 plus 43% of the SR Ca content (43 x 87) giving 47 f!mollL cytosol activating
the twitch, with 23% coming from Ica and 77% from SR Ca release. These numbers are in good
agreement with the experiments in Fig 135, where 28% of Ca extrusion was due to Na/Ca
exchange and 70% due to SR Ca uptake. Similar analysis for the rat twitch in Table 24 also
agrees with ~7-8% of activating Ca coming across the sarcolemma with 92% from the SR.
Table 24
Fraction of Activator Ca from lea and SR Ca release
Rabbit Rat
ICa SR (lNa/Ca) ICa INa/cax (SR)
fIdt (fC/pF) 221 ± 14 860± 118 185 ± 12 851±70
t.[Cahot (J.!M) 9.7 ± 0.5 77 ± II 6.5 ± 0.3 120 ± 8
SR Ca (J.!moIlL cytosol) 87 ± 13 t 138 ± 9 t
Twitch t.[Ca]j (J.!moIlL cytosol) 9.7 87x0.43 6.5 138xO.55
Total (J.!moI/L cytosol) = 47 = 82
Activator Ca (%) 23 ± 2% 77 ± 2% 7.9% 92%
Data are from Delbridge et al. (1996) for rabbit and Delbridge et al. (1997) and Yuan et al. (1996)
for rat ventricular myocytes. Columns labeled SR (lNa/C,) show integrated INa/ca to derive SR Ca
content (first 3 Jines) and the amount of SR Ca release contributing to the twitch (vs. Ca influx via
Ic, (last 3 Jines; see Table 2 & 9). tIncrease from 77 to 87 (& 120 to 138) reflects correction for non
Na/Ca exchanger-mediated Ca removal during caffeine-induced contracture (Table 20).
Terracciano & MacLeod (1997) used a slightly different approach to measure Ca entry
during AP-c1amp. In rat ventricular myocytes they found that Ca entry via Ica was 3.5% of the
SR Ca content. This is equivalent to the above 7% results in rat (assuming a 50% fractional SR
Ca release). They also studied guinea-pig ventricular myocytes at both 0.5 and 0.2 Hz and found
that the Ca influx via Ica was a much larger fraction of the SR Ca content at lower frequency
(~30 and 50% respectively). This is consistent with the higher transsarcolemmal Ca flux
dependence in guinea-pig in Fig 134B. At the lower frequency, the SR Ca content was lower
while Ica was slightly higher (secondary to less Ca-dependent inactivation of Ica ). Consequently
the ratio of Ica to SR Ca load was higher at lower frequency. Given the different methodologies
and limitations ([Ca]j decline vs. current integration), this agreement seems remarkably good.
What about Ca entry via Na/Ca exchange during the cardiac action potential? I have
focused here on Ic., but early in the AP some Ca entry via Na/Ca exchange may occur (see Fig
74, pg 151). However, the amount of Ca entry during a nonnal Ca transient is likely to be <I
J.!mollL cytosol (pg 149-151), much less than 10% of that entering via Ica . Thus, under normal
conditions it is expected that most of Ca influx is via Ica and only a very small fraction « I0%) is
via Na/Ca exchange (see also Grantham & Cannell, 1996). On the other hand, when [Na]; is
high, as with digitalis-induced inhibition of the Na/K-A TPase, this situation can change and
substantial Ca can enter during the AP and activate contraction (see Fig 72 and Chapter 10).
BIPHASIC CONTRACTIONS
Under certain conditions two phases of contraction can be seen, where the first
component is often attributed to SR Ca release and latter to Ca influx. Indeed, biphasic contrac-
tions have been reported under a variety of conditions, especially when cAMP is increased or
when the SR is relatively Ca-depleted (Braveny & Sumbera, 1970; Coraboeuf, 1974; Allen et al.,
1976; Beresewicz & Reuter, 1977; Seibel et al., 1978; Bogdanov et al., 1979; Endoh et al., 1982;
King & Bose, 1983; Reiter et al., 1984; Honore et al., 1986, 1987; Malecot et aI., 1986).
A. Milrinone Addition B. Steady State Milrinone

l'
"'~\
+ Caffeine lOmM
,,5' •
'" "mg I 1.~'0'

~~1~~
[400mg
}c~ 1'30 2'
lOOms
lOOms
I
l'
',c
~
. 200m g
+ Ryanodine lOOnM
:\ 20'
20'
t~~.....
.
:~.7i7_~
~o' ~
1100mg
Figure 136. Biphasic contractions in ferret ventricular muscle induced by 240 f!M milrinone (at 28°C and
0.5 Hz). A. Addition of milrinone to two different muscles, C is control and numbers refer to time after
milrinone addition (in min). B. Steady state biphasic contractions in milrinone (M) and changes in
contractions after addition of 10 mM caffeine (top) or 100 nM ryanodine (bottom) for the times indicated.
Both of these agents depress the first phase of contraction and caffeine also increases the second
component. (from Malecot et al., 1986, with permission of the American Heart Association).
Figure 136 shows biphasic contractions induced by the cardiotonic drug milrinone.
Milrinone increases lea (due to phosphodiesterase inhibition) and exerts a mild caffeine-like
action on the SR (Malecot et aI., 1986; Rapundalo et aI., 1986) Initially, contractions are
increased in amplitude and shortened in duration, probably due to cAMP-induced enhancement
of lea. Over the next few minutes the fast peak decreases and a second component becomes
increasingly apparent. We attributed this to a milrinone-induced SR Ca leak (like low
[caffeine», such that the larger Ca entry via lea can activate contraction directly (but more
slowly). Figure 136B shows that the early component is suppressed by either caffeine or
ryanodine. The second component is relatively unaffected by ryanodine, but is enhanced by
caffeine. Since caffeine increases myofilament Ca sensitivity, these results are consistent with
the first component of biphasic contractions being due to SR Ca release and the second
component being due to Ca influx.
Slow or tonic contractions can be readily seen when depolarization is prolonged by
voltage clamp pulses in frog and mammalian heart, even when SR Ca transport is blocked (see
Figs 71, 75 & 78, Morad & Trautwein, 1968; Braveny & Sumbera, 1970; Goto et al., 1971;
Leoty & Raymond, 1972; Coraboeuf, 1974; Horackova & Vassort, 1976, 1979; Chapman &
Tunstall, 1981; Chapman, 1983; Eisner et al., 1983; Isenberg & Wendt-Gallitelli, 1989). These
tonic contractions can be due to either sustained Ca entry via Na/Ca exchange or a Ca window
current. Those due to Ca entry via Na/Ca exchange are sensitive to the [Na] gradient and are
more prominent at positive potentials (Fig 71). Those due to window lea are more commonly
observed at Em = -30 to 0 mV and still occur in the absence ofNa (Fig78).
REST DECAY AND REST POTENTIATION

Here I will discuss dynamic changes in SR Ca content Ca influx and SR Ca release in
non-steady state conditions (e.g. during rest and upon resumption of stimulation after rest).
Early Electrical and Mechanical Restitution

Immediately after a contraction, some time is required before another contraction of the
same amplitude can be activated. This early phase of recovery of contraction (-1 s) is often
termed mechanical restitution and is essentially a relative refractory period. Several systems
contribute to this finite restitution period, but the predominant factor after the first 200 ms is
recovery of the RyR from an inactivated or adapted state (see Chapter 8, pg 228-230).
This explanation for early mechanical restitution also explains the well known functional
response in cardiac muscle known as post-extrasystolic potentiation (PESP, Hoffman et al.,
1956). This is where activation occurs prior to full mechanical restitution, such that only a weak
contraction occurs (extrasystole), but the subsequent contraction is potentiated (PESP). If lea
recovers (even partially) before the extrasystole, some Ca will enter the cell at the extrasystole.
If SR Ca release is refractory, normal Ca release will not occur, resulting in a weak contraction,
and the lower [Cali will allow more Ca influx via lea (due to less Ca-induced inactivation) and
less Ca efflux via Na/Ca exchange. The net gain in Ca during the extrasystole will enhance SR
Ca content. Then, at the next beat, when the SR Ca release channel has recovered, there is a
greater SR Ca release and contraction (PESP). In the intact heart a premature ventricular
contraction (PVC) would cause this same chain of events. However, the reduced ventricular
filling time will further reduce the contraction at the extrasystole (and may not raise ventricular
pressure sufficiently to open the aortic valve). In contrast, at the post-extrasystolic beat the
ventricular preload is higher than normal (especially after a compensatory pause for a missed
sinus beat). The increased preload enhances myofilament Ca sensitivity and contractile force
(Chapter 2) and works synergistically with the enhanced Ca transient to produce a very strong
PESP. This creates the sensation of the heart "skipping a beat."
Mechanical alternans, where even at a constant frequency, contraction amplitude
alternates from one beat to the next (Wolfhart, 1982) may also be explained this way. If the nor-
mal refractory period for the SR Ca release channel is prolonged (e.g. due to elevated [Cali or
low temperature) then Ca release and !l[Ca]j would be small (but Ca influx would still occur).
By the next pulse the SR may no longer be refractory (and at higher Ca load), so a larger than
normal Ca release may occur. The larger Ca transient inactivates lea faster (limiting Ca influx),
and enhances Ca extrusion via Na/Ca exchange. This results in a lower Ca content for the next
contraction. Thus, the smaller contraction might be smaller for two reasons: 1) because the SR
has less Ca and 2) due to refractoriness of the SR release mechanism (see Fig 143).
Slow Ryanodine Receptor Recovery and Rest Potentiation

In addition to the rapid restitution above (and pg 228-230), which occurs in the first
second after a twitch, there is a slower phase which occurs over many seconds. This phase may
be largely responsible for rest potentiation, which refers to the increase in contraction amplitude
observed after a rest period of 10-120 sec (e.g. in rat ventricle, see top trace in Fig 137B). Figure
137B shows that rest potentiation develops in rat to a maximum of -130% of control with a't of
-8 sec and stays elevated for 5 min (Twitch NT). The enhanced contraction (and Ca transient)
occurs despite no change in SR Ca content (assessed by caffeine application, CaffNT).
Rabbit (and guinea-pig) ventricle typically exhibit rest decay rather than rest potentiation
(see Fig 137A, Twitch NT), but as discussed below this is primarily due to a decrease in SR Ca
A. Rabbit Ventricle B. Rat Ventricle

150 150
l:cn
~cn Twitch ONa,OCa
() ... 125 125
nl 0
.b't)
l: ell
o ... 100 100
() <11
CiiN
eIl_ 75 75
c:: 0
--J;~
o <11 50 50
Q. nl
25 25
0 60 120 180 240 300 0 60 120 180 240 300
Rest Interval (s) Rest Interval (s)
C. Ferret Ventricle D. Human Ventricle

150 250
l:cn a ,-,N=on,-,f..=;a",iling-Twitch
~cn
()
nl 0
... 125 200
•
';t) Nonfailing-RCC
l5
()
~
<11
100
150
CiiN 75
eIl_
c:: 0 100
f;;~
o <11 50
Q. nl 50
25
0 60 120 180 240 300 0 60 120
Rest Interval (s) Rest Interval (s)
Figure 137. Rest decay of twitch contraction, 5R Ca content and peak lea. Post-rest twitches, caffeine-
induced contractures (Catl) or RCCs were measured in normal Tyrode's solution (NT) or when Na/Ca
exchange was blocked by ONa,OCa during only the rest (in A), or after pre-depleting [Na]; and rest in Ca-
»
free NT (to stimulate Ca efflux via Na/Ca exchange, OCao in B). 5R Ca load data (Caff & RCC) were fit to
[Ca]SRC = a·exp(-t1'tRD)(I-exp(-th:RF +b, where t is time, 'tRD & 'tRF are time constants of rest decay of5R
Ca content & rest-dependent filling (used to explain 5R Ca increase or delays in SR Ca decline), and a and
b are constants for scaling and baseline respectively. Twitch data were fit with the expression c[CalsRe(l-
exp(-t1'tEec» + d, where 'tEee is the time constant for recovery of E-C coupling (20, 8, 7, & 7 s for rabbit,
rat, ferret and human) and c and d are scaling and baseline constants. A small second 't was included to
describe the slow decline in twitch in rabbit ventricle after long rests in ONa,OCa. Data are at 23°C except
for human (37°C) and pre-rest steady state (55) stimulation was 0.5 Hz except for human (I Hz). Data are
from Hryshko & Bers (1990), Bers et at. (1993), Bassani & Bers (1994) and Pieske et at. (1999a).
content (Caff NT). If the loss of SR Ca is prevented by exposure to ONa,OCa solution during the
rest (CaffONa,OCa), rabbit ventricular myocytes exhibit the same rest potentiation as observed in
rat ventricular myocytes (Twiteh-ONa,OCa, Bassani & Bers, 1994). The amplitude of the Ica
trigger at the post-rest twitch is typically smaller by 5-20% due to the loss of Ca-dependent Ica
facilitation (Iea trace; see also pg 119). Thus, this slow phase of rest potentiation develops with a
1: of 5-10 sec, can occur without any increase in SR Ca load or lea and indicates a time-dependent
increase in fractional SR Ca release.
This slow recovery of SR Ca release can also be seen at the microscopic level using Ca
sparks (Satoh et aI., 1997). Immediately after a twitch in rat ventricular myocytes, there is a
>-
500 250 0
"
gQi" .~,
!!l ~ 400 (1)
!!l.
0"> 200
Ql U t>
at QlI/I
300
'0
~N ~
nl_
c.o 150 ~
-
ennl~
~ 200 0
0
t> ;:,
.,
100-'=:~~~~~~~~~~~~~~,..J- 100 g
o 10 20 30
Rest Duration (sec)
Figure 138. Recovery of Ca spark frequency and rest potentiation in rat. After stimulation at 0.5 Hz, rat
ventricular myocy1es were rested for various periods and Ca spark frequency was observed using confocal
microscopy and post-rest potentiation of Ca transients was also recorded by field stimulation. There was no
change in SR Ca content during this period (assessed by caffeine-induced Ca transients) Time constants fit
to the data were 5 & 7 sec for twitch and Ca spark frequency. Data are from Satoh et at. (1997).
period where spontaneous resting Ca sparks are nearly abolished, but then Ca spark frequency
gradually recovers back to a steady state level. Figure 138 shows that recovery of Ca sparks
parallels that of rest potentiation (Satoh et al., 1997). Therefore, this slow recovery ,of SR Ca
release appears to be an intrinsic property of individual RyRs (or at least of RyR clusters).
At high SR Ca content this quiet period with respect to Ca spark frequency can be
overcome (Satoh et al., 1997). This may be due to the effects of luminal SR [Cal on the gating
properties of the cardiac RyR (pg 194-195,225). This may encroach on a restitution-dependent,
intrinsic safety factor against spontaneous SR Ca release between beats and may contribute to
delayed afterdepolarizations and triggered arrhy1hmias at high SR Ca loads.
Since this slow recovery phase takes several seconds to develop, it may seem irrelevant
to the normal physiological situation with heart rates of ~I Hz. Nevertheless, the experimental
study of post-rest contractions has increased our overall understanding of how E-C coupling
functions in the intact cell and this will be more apparent in subsequent sections in this Chapter.
It should also be appreciated that if some fraction of RyRs are unavailable for SR Ca release for
several seconds after activation, then at steady state heart rates of ~ I Hz, there will be a certain
fraction of RyRs which are in a refractory state and unable to participate in E-C coupling.
Moreover, this fraction will be expected to change with heart rate or stimulation frequency (see
Force-Frequency Relationship below, pg 267).
Rest Decay and SR Ca Depletion

Rest decay refers to the rest-dependent decline in the amplitude of the first post-rest
twitch with increasing rest duration, and is apparent in many mammalian cardiac preparations
(Fig 137A, Twitch NT; Allen et al., 1976). During rest, there is a finite leak of Ca from the SR
(~O.3 Ilmol/L cy1osol/sec in both rabbit and rat ventricular myocy1es; Bassani & Bers, 1995) and
this may occur primarily as Ca sparks, due to occasional stochastic RyR openings (see pg 192).
Once Ca leaks into the Cy1osol, it is subject to the same competition among Ca transport systems
as discussed above for twitch relaxation and [Cali decline (i.e. mainly the SR Ca-ATPase and
Na/Ca exchange). If Ca extrusion is prevented by ONa,OCa solution, there is no serious

competitor with the SR Ca-pump, and consequently the Ca which leaked from the SR is mainly
taken back up by the SR, preventing net loss of SR Ca (see Fig 137A, Caff ONa,OCa). However,
in rabbit ventricular myocytes, there is normally a substantial thermodynamic gradient favoring
Ca extrusion via Na/Ca exchange (see Fig 72). Thus, some Ca leaked from the SR during rest is
extruded from the cell, while the remainder is taken back up by the SR. The Ca which leaves the
cell during rest typically exceeds the transsarcolemrnal Ca leak and results in a net loss of
cellular and SR Ca. Quantitatively, if 33-50% of the leaked SR Ca (0.3 J.tmol/L cytosollsec) is
extruded by Na/Ca exchange the cell would lose 6-10 J.tmollL cytosol/min, such that an SR Ca
load of 100 J.tmollL cytosol could be half-depleted in ~5 min (as in Fig 137A). This decline in
SR Ca load is the main explanation for rest decay as seen prominently in rabbit and guinea-pig
ventricular muscle and myocytes (Fig 137A, NT traces). The amplitude of RCCs also declines
as a function of rest duration in a manner parallel to that of stimulated twitches (Bridge, 1986;
Bers, 1989). Guinea-pig (Fig 79) behaves similar to rabbit ventricle (with somewhat faster rest
decay) and mouse behaves very similarly to rat ventricle.
In addition to the progressive loss of SR Ca available for release during rest decay, a
smaller Ica may also make a minor contribution during the first 10 sec of rest. The ability of a
given Ica trigger to cause SR Ca release may also affect rest decay in two opposing ways. On one
hand, we expect fractional SR Ca release to increase during rest as described above for rest
potentiation. On the other hand, as SR Ca content declines, the lower [Ca]sR decreases fractional
SR Ca release, and this can be profoundly inhibitory at low SR Ca content (see Fig 119). Several
factors may be involved at early times, but the decreased SR Ca content and fractional SR Ca
release are increasingly dominant factors at later times. At some point as SR Ca falls (even
before the SR becomes fully depleted), the SR may stop participating in E-C coupling. This may
occur at an SR Ca load of 30-40 J.tmollL cytosol (as in Fig 119). The so called "rested-state
contraction" (where further rest makes no difference) reflects a state in rabbit and guinea-pig
where the contraction is largely due to Ca influx, rather than SR Ca release (not the case for rat).
It is important to appreciate that the decline in SR Ca content need not be complete after
long rest intervals. [Ca]sR may simply establish a new steady state, where at low SR Ca content,
there is both a reduction in SR Ca leak and enhanced ability of the SR Ca-pump to compete with
Na/Ca exchange. The reduced leak may be due to low [Ca]sR, which decreases Ca spark
frequency and also lowers Ca flux through open Ca release channels. The enhanced SR Ca-
pump competition with Na/Ca exchange may be due to higher net SR Ca-pump rate at low [Ca)SR
(less backflux, pg 173-177) and also to de-activation of the NaiCa exchanger at low [Ca)i (pg
139-140). Indeed, Terracciano & MacLeod (1996) found that after caffeine-induced SR Ca
depletion in guinea-pi,o, ventricular myocytes (which behave much like rabbit myocytes) there
was spontaneous SR refilling during rest, to ~50% of the steady state seen at 0.2 Hz stimulation.
An implication from this framework is that anything which inhibits SR Ca-pumping (e.g.
thapsigargin) or increases SR Ca leak (e.g. ryanodine), would shift the competition toward
extrusion by Na/Ca exchange and be expected to accelerate rest decay. This is seen with both
thapsigargin and ryanodine (Figs 79 & 96; Bassani & Bers, 1995; Sutko et aI., 1986; Bers et aI.,
1987,1989). Furthermore, anything which inhibits Ca extrusion via Na/Ca exchange would bias
the competition toward the SR Ca-pump and slow rest decay. This effect is clearly seen for
elevated [Na]i (with Na/K-ATPase inhibition), reduced [Na]o (Sutko et ai., 1986) and the extreme
case where Na/Ca exchange is completely blocked by ONa, OCa (Figs 137A, 79-80 & 95).
These implications may also explain why rat (& mouse) ventricle typically do not exhibit
rest decay of twitches or SR Ca content (Fig 137B, NT traces). That is, in rat vs. rabbit the SR
Ca-Pump activity is higher and Na/Ca exchange is less able to extrude Ca (see Fig 134A).
Indeed, for the rat, this inability of the Na/Ca exchanger to extrude Ca may be a consequence of a
relatively high resting [Na]i in rat and mouse myocytes (Shattock & Bers, 1989; Yao et ai.,
1998), which means that there is little or no driving force for Ca extrusion at diastolic Em and
[Cali (see Fig 140). However, we can deplete rat cells of [Na]j by incubation in ONa,OCa
solution, and then returning to Ca-free normal Tyrode's for the rest interval (OCa o in Fig 137B).
This shifts the driving force on the Na/Ca exchange, such that it can compete better with the SR
Ca-pump. Fig 137B shows that in rat ventricular myocytes this results in clear rest decay of both
SR Ca (Caff OCa o) and twitches evoked immediately after restoring Cao (Twitch OCao)' Thus,
there is a critical and dynamic balance between SR and sarcolemmal Ca transporters which is
crucial in determining SR Ca available for release.
In the simplest quantitative terms, the amplitude of the post-rest contraction depends
mainly on two factors: 1) the amount of SR Ca available for release ([CalsRC in Fig 137 legend),
multiplied by 2) fractional SR Ca release (which depends on time and [Ca]sRc)' In many cardiac
preparations [Ca]sRC declines during rest, due to the balance of SR Ca transport and Na/Ca
exchange as described above. While [Ca]sRC is declining, the slow phase of post-rest recovery of
E-C coupling is increasing ('tECC ~ 7 sec in Fig 137 legend). This can lead to transient rest
potentiation which is commonly seen in ferret ventricle (Fig 137C), and many other cardiac
preparations (including failing human ventricle, Fig 137D). At longer times the rest potentiation
('tECc) factor may be maximal, but as [CalsRC continues to decline with time, the product of
[CalsRC x fractional release will progressively decline. The fractional release factor (assuming
the Ca trigger is unchanged) will increase at short times, but if SR Ca stays high (as in rat), then
fractional release may not change further. If [Ca]SRC declines, it will also reduce fractional
release and can exacerbate the rest decay of twitch contraction (i.e. both factors decline).
So in ferret ventricle (Fig 137C) there is typically rest potentiation which lasts for up to 2
min, but then rest decay becomes more dominant as [CalsRC declines monotonically. In non-
failing human ventricle, [CalsRC appears to increase during rest (Fig 137D, Pieske et aI., 1999a).
In this case post-rest twitches are greatly enhanced, because both [CalsRC and fractional release
factors are increasing. This has also been reported in some cases for rat ventricle (Bers, 1989;
Bers & Shattock 1989; Lewartowski & Zdanowski, 1990; Banijamali et ai., 1991; Maier et ai.,
2000). We interpret this in the following manner for the rat (and possibly human). If [Na];
increases during pacing (from ~s already high value in rat), the reversal potential for INa/C. may
fall below the diastolic Em, such that net Ca entry occurs during diastole. This implies that net
Ca extrusion occurs during systole in rat. As we will see, this actually occurs (Fig 139). Thus,
rat [CalsRC may increase, remain unchanged or even decrease during rest. Indeed, when [Na]i is
low in individual rat myocytes rest decay can occur (Fig 137B; Frampton et ai., 1991). This
emphasizes the importance of specific ionic conditions in driving the delicate balance of Ca
fluxes across the sarcolemmal and SR membrane. In ferret ventricle, an unusually strong
sarcolemmal Ca-ATPase can partially substitute for Na/Ca exchange in mediating cellular Ca
extrusion during rest decay (Bassani et al., 1994b, 1995a). In this specific case ONa, OCa
solution during rest does not abolish rest decay of SR Ca content (as in Fig 137A), but only
slows this rest decay partially (see Fig 80).
In human heart failure, there is decreased SR Ca-ATPase expression and increased
Na/Ca exchanger expression (e.g. Hasenfuss, 1998a). This shifts the competition between the
SR Ca-ATPase and Na/Ca exchange more in favor of the Na/Ca exchanger during rest.
Consequently, failing human ventricle exhibits rest decay of [CalsRC, and only a transient rest
potentiation is seen. As in ferret, this occurs during the time when the positive effect of RyR
recovery is sufficient to more than offset the initial small loss in [CalsRC'
Clearly this is a simplified consideration of diastolic Ca fluxes since it neglects other
possible Ca fluxes (e.g. mitochondrial Ca fluxes and sarcolemmal Ca leak and Ca-pump).
However, the mechanisms considered above appear to be the most important ones and suffice to
explain the main features of diastolic changes in SR Ca content. Thus, the process of rest decay
in cardiac muscle appears to depend upon Ca extrusion via Na/Ca exchange and the balance of
SR Ca-pump and leak.
Ca Influx and Effiux in Rabbit and Rat Ventricle

The above results suggest major fundamental differences in transsarcolemmal Ca fluxes
in rat vs. rabbit ventricle during both rest and contraction. Shattock & Bers (1989) compared Ca
fluxes in rat vs. rabbit ventricle during the cardiac cycle using extracellular Ca microelectrodes.
We demonstrated that during the twitch in rabbit ventricle net Ca influx occurs, but that during
the twitch in rat ventricle net Ca efflux occurs (see Fig 139). Since Na/Ca exchange is the main
mechanism by which Ca is extruded from cardiac cells, we suggested that the large Ca efflux
seen in rat was due to Ca released by the SR and extruded by Na/Ca exchange. Indeed, Ca efflux
recorded in rat ventricle was caffeine sensitive and larger at the first large post-rest contraction.
We also compared resting intracellular Na activity (aNa;) and found that aNa; was significantly
higher in rat (12.7 ruM or [Na]; =16 ruM) than rabbit ventricle (7.2 ruM or [Na]; =9 ruM).
In rabbit ventricle Ca extrusion via Na/Ca exchange is thermodynamically favored at rest
because the predicted reversal potential for Na/Ca exchange (ENa/ca) is positive to Em (see Fig
140). During the action potential Em exceeds ENa/ca (even though [Cali is elevated) such that
there may be a modest driving force favoring Ca entry during the AP (see also Figs 72 & 74).
This, together with Ic" explains the transient Cao depletions seen during contractions in rabbit
ventricle (Fig 139A) and Ca loss during diastole (as in rest decay). The rate of diastolic Ca
extrusion is small, despite the large thermodynamic driving force. This is because inward INa/ca is
limited by the low resting [Ca];, both kinetically and due to deavtivation (Weber et al., 2001).
The resting [Na]i in rat ventricle is high enough that the ENa/ca is near the resting Em (Fig
140). In particular, after a train of stimuli aNa; would be higher still, such that ENa/ca would be
negative to Em and net Ca uptake would be favored. This can explain the above described rest-
dependent increase in SR Ca sometimes seen in rat ventricle and could contribute to rest
potentiation. While Bassani et al. (I 994a) found no difference in diastolic [Cali in rat vs. rabbit,
DuBell & Houser (1987) reported higher resting [Cali in rat than in cat ventricular myocytes. In
rat ventricle the AP duration is also very short (compared to rabbit ventricle), and normally lacks
A. Rabbit Ventricle B. Rat Ventricle

",Control 520
:iE +Citrate 550
:1 I 1 ""'----.::-
~--r:::::::::;:=:::::::=;-.--- J500 500
480 J490
~ Control
Control
~
01
,200 ms,
J~3
PO
I 100 ms I
Figure 139. Changes in [Ca]o measured with double barreled Ca-selective microelectrodes during
individual contractions in rabbit (A) and rat (B) ventricular muscle (0.5 Hz, 30°C). The traces show [Ca]o
(top) and tension (bottom) in the absence and presence of 10 mM citrate (which limits Ca o depletion by
buffering [Ca]o (but it can also inhibit Ic" Bers et aI., 1991). The bath [Ca]o = 0.5 mM and is indicated by
the dotted line. (A is redrawn from Shattock & Bers, 1989, with permission.).
an appreciable plateau phase (see Fig 140), Thus, during the contraction, when [Cali is high,
ENalCa is positive to Em' so that there is a large driving force favoring Ca extrusion via Na/Ca
exchange. In this way the competition between the SR Ca-pump and the Na/Ca exchanger is
biased toward the latter, and net Ca efflux occurs during the contraction (compare Fig 139B and
140B). During a potentiated post-rest contraction a larger Ca efflux will occur and the SR Ca
content will be lower at the next contraction until a new steady state is achieved (where Ca influx
and efflux must again be equal over a complete cardiac cycle). This may contribute to the
decrease in contraction amplitude during post-rest recovery and also to the well known negative
"staircase" or force-frequency relationship in rat ventricle (see below).
If the depolarization in a rat ventricular myocyte is prolonged near 0 mV by a voltage
clamp pulse (to resemble that in rabbit ventricle), the negative "staircase" can be converted to a
positive "staircase" (Spurgeon et al., 1988), as would be expected from Fig 140. Figure 141
shows a similar result in guinea-pig ventricle which behaves much like rabbit ventricle.
Increasing pulse duration leads to a positive staircase, while reduction of pulse duration to 100
msec leads to a negative staircase (Isenberg & Wendt-Gallitelli, 1989). Thus, prolonging the
duration of depolarization can increase intracellular (and SR) Ca loading by two means: I)
limiting the extrusion of Ca via Na/Ca exchange and 2) allowing continued Ca entry via
sarcolemmal Ca channels (and possibly via Na/Ca exchange, depending on conditions).
Ryanodine greatly accelerates rest decay of RCCs in rabbit ventricle (ty, -I sec, Fig 96),
but does not completely abolish RCCs in rat ventricle (Bers & Christensen, 1990). This is
consistent with the foregoing discussion, since the low transsarcolemmal [Na] gradient may limit
Ca extrusion via Na/Ca exchange (so that even a "leaky" SR may be able to retain some Ca).
Furthermore, by manipulating [Na] and [Cal gradients, rabbit ventricle can be made to behave
more like rat ventricle, and vice versa (Bers & Christensen, 1990; Bassani & Bers, I 994). For
example, in Fig 137A-B, limiting Ca extrusion by ONa,OCa in rabbit ventricle causes rest
30 A. Rabbit Ventricle B. Rat Ventricle

>
S. 0 aNa; = 7.2 mM aNa; = 12.7 mM
.
!,1
z -30 E Na/ Ca
W
(;
E -60
W Em
-90
.. >
g>~E 30 /Caefflux
co ~~
J: 0 E
uu.w
~ r:n I
c ..
o:;E ~ -30
iVO~
Z ~
-60
250 500 0 250 500

time (ms) time (ms)
Figure 140. Schematic diagram of the estimated changes in the reversal potential of the Na/Ca exchange
(ENa/ca) that accompany the action potential and Caj transient in rabbit and rat ventricle (top). The
estimated changes in the net electrochemical driving force for Na/Ca exchange (ENa/ca-Em) are shown in the
bottom panels. We assumed a stoichiometry of3Na:ICa for the Na/Ca exchanger, aNa; values as actually
measured in these preparations (Shattock & Bers, J989) and, for simplicity, the Ca transi~nt accompanying
the contraction has been assumed to be the same for both species. Resting [Ca]j was assumed to be 150 nM,
rising to a peak of 111M, 40 msec after the AP upstroke. The shape of the Ca transient was calculated as
described by Bers (l987b). Note the similarity between the lower panels and the [Ca]o traces in Fig 139.
(Top panels were redrawn after Shattock & Bers, 1989, with permission).
potentiation as seen in rat ventricle. Conversely, boosting Ca extrusion via Na/Ca exchange in
rat causes rest decay which resembles that in rabbit.
While depolarization alters transsarcolemmal Ca fluxes, Ca extrusion via Na/Ca
exchange can also modify the AP. Schouten & ter Keurs (1985) demonstrated that removal of
[Na]o suppressed a slow component of the rat ventricular AP which was most prominent at large
contractions where [Ca]; is high (see Fig 142). They attributed the late low plateau AP phase to
inward INa/C. (i.e. 3 Na influx for I Ca extruded). Hilgemann & Noble (1987) simulated this
- - 0.1s - ......~I--- Pulse Duration 0.5s ----l.~~_-- PulseDur.0.1s - - -
g>~ %
·Ed
~ a::: 3 °1
~ ~ 5
(/J~
Figure 141. Duration of depolarization determines the direction of the contraction staircase in guinea-pig
ventricular myocyte. Em and shortening as a percent of resting cell length (RCL) under voltage-clamp
(3rC, [Ca]0=1.8 mM). (From Isenberg & Wendt-Gallitelli, 1989, with permission).
Rat Ventricle
> Experimental Theoretical

E Hilgemann
o Schouten &
o ter Keurs, 1985 & Noble, 1987
100 msec
Figure 142. Action potentials recorded from rat ventricular muscle (left) stimulated at low frequency
where contractions are large in control superfusate and after reduction of [Na]o from 150 to 30 ruM. This
behavior could be simulated in an AP model simply by reducing the effective [Na] for NaJCa exchange
(right). (from Schouten & ter Keurs, 1985 and Hilgemann & Noble, 1987, with permission).
effect in their model of the rat ventricular action potential (Fig 142).
APs are also influenced by Ca fluxes during paired-pulse stimulation and mechanical
altemans (Wolfhart, 1982; Hilgemann, 1986a,b). Figure 143 shows extracellular Ca depletion,
Em and force in rabbit atrium during paired-pulse stimulation (Hilgemann, 1986b). As the large
contraction develops, [Ca]o reaches a nadir (maximal influx), but net Ca efflux is evident by the
end of the contraction. This is analogous to the result with rat ventricle in Fig 139B where high
[Cali drives Ca efflux via NaiCa exchange. The small paired-pulse beat produces progressive net
Cao depletion, a more prominent AP plateau and only a tiny contraction. As for the extrasystole
above, SR Ca release may be refractory at the small beat, such that no SR Ca is released. Thus
[Cali remains low during the AP, which allows more Ca influx (explaining the larger Cao
depletion and the higher AP plateau). The low [Cali limits Ie. inactivation and Ca extrusion via
Na/Ca exchange during the small beat. The net result is that the SR is more Ca loaded (and non-
refractory) at the time of the next pulse. In this case inward INa/C. may limit rapid repolarization
at the large beat. However, the persistent inward Ie. at the small beat may more than offset the
[Cal o _ V·--\t.: :=- I ~M nf

1 Ca
Tension 10.1 mN
200 ms
Figure 143. Paired-pulse stimulation of rabbit atrium in the presence of 2 ruM 4-aminopyridine (to
suppress transient outward K currents). The [Ca]o is assessed by the absorbance of the extracellular Ca
indicator, tetramethylmurexide. Free [Ca]o is 150 11M, the basic frequency is 0.5 Hz and the smaller paired-
pulse beat is evoked 200 msec after the main pulse (from Hilgemann, 1986b, with permission).
Refilling of Ca Depleted SR
O-l<-~_r-~-,-_~--,-:...;R""ab",b",i:,.t V.:...e",n.;otr:..:;icle"T
o 2 4 6 8 10
Beat Number
Figure 144. Refilling of the SR with Ca after depletion in rabbit ventricle.. The SR Ca content was
depleted by either a 5 min rest period in ventricular muscle (at 30°C) or by a lOs exposure to 10 mM
caffeine in myocy1es (at 23°C). Recovery of SR Ca content upon resumption of stimulation at 0.5 Hz was
assessed by a second caffeine-induced contracture in myocy1es or by an RCC in muscle (data are taken from
Bel'S, 1989 and Bassani et al., I 993b).
lower inward INa/ea at the small beat (with respect to Em)' Since there is little Ca efflux during the
small paired-pulse (where SR Ca release is refractory) one could consider the integrated lea to be
"injected" into the SR. Once the Ca which enters has been pumped into the SR, the SR then has
an extra aliquot of Ca available for the next contraction (as for the PESP on pg 256).
SR Ca Refilling and Post-Rest Recovery

After long rests in rabbit ventricular muscle or after sustained caffeine application, the
SR is depleted of Ca. Thus, when contractions are resumed the SR gradually refills to the steady
state level. Figure 144 shows the time course of SR Ca refilling in rabbit ventricle (using
caffeine or RCCs to assess SR Ca). In general, the recovery of twitches and SR Ca are similar.
However, SR Ca can recover back to steady state faster than stimulated twitches (especially after
long rest intervals). Thus, most, but not all of the twitch recovery is due to refilling of the SR.
Indeed, if 10-15 ~mol CalL cytosol enters via lea, it would take 5-10 beats to restore SR Ca
content to 60-100 ~mol/L cytosol (see Fig 145).
The half-time for refilling the SR is 2-4 beats and it reaches steady state in 10-15 beats.
This agrees with results from extracellular Cao depletion studies in rabbit ventricle (Bel'S &
MacLeod, 1986; MacLeod & Bel'S, 1987; Bel'S, 1987b). These Cao depletions, which reflect net
Ca uptake by the cells, reach a maximum in 6-12 contractions upon resumption of stimulation
after a rest with the half-maximum typically occurring at beat 4-5 (see Figs 97 & 165). lea
amplitude also increases toward steady state during the first 5-10 post-rest pulses (see Fig 58).
However, this lea staircase is small and its contribution to the post-rest recovery is not clear.
Figure 145 shows an elegant quantitative procedure to assess Ca fluxes during refilling
of the SR, after Ca depletion induced by caffeine application in a voltage clamped ferret
ventricular myocyte (Trafford et aI., 1997). They use the reasonable simplification that during a
pulse from - 40 to 0 mV, inward current indicates Ca influx via Ie" and that upon repolarization
A.o.9 B.O.9
~ 06
20
"i?
~ 0.3
0.0 f--------i 0.0

10 s
c. oojl ~Y1 D.
;::j ,~vr,~·--- ~1"

#1
500 ms
~
E. 15
F.16
roE'
~
P
41 ...J 10
#1 12
8~
~
"'-
E
.. ::I. "84
-"
'" "
...J
z'" g
""0
500ms
2:: 12
G. 80
"
:::l
~ 8 Net Ca influx
III
1: u
.!i 60
:5u ~" 40
o _.._....
~~:::1
0:: 20
<Jj-
I-----i
10 s
Figure 145. Ca fluxes and SR Ca content during refilling after depletion. Induced by caffeine. A. Ca
transients during the first 25 voltage clamp pulses from -40 to 0 mV for 100 ms (27°C). B. Expanded time
scale of selected Ca transients. C. Currents for the ISland 21 st pulse. D. Enlarged INa/c, traces from C. E.
Evolution of net integrated transsarcolemmal Ca flux for pulse 1 & 21 (corrected for surface:volume ratio).
F. Unidirectional Ca fluxes and net Ca influx (influx - efflux), based on integrals of data in panels C-E. G.
Cumulative cellular Ca gain, which is assumed to be nearly all taken up by the SR. Data from Trafford et
al. (1997, kindly supplied by the authors) were regraphed.
the slow inward tail current indicates Ca extrusion via INa/c, (Fig 145C). Panel A shows the
recovery of Ca transients during 100 ms pulses at 0.5 Hz. Panels B & C show details of [Ca]i and
st st
current for the 1 and 21 pulses. As the SR reloads the Ca transient is larger and Ic, inactivates
more rapidly, gradually decreasing the integrated Ie, influx to a steady state value (Panels C & F;
see also Fig 60). As the Ca transients increase, the Ca extrusion by INa/c, also increases (enlarged
in panel D) to a steady state where Ca efflux via INa/c, is equal to Ca influx via Ic, (8 /!mol/L cel!
= 12 /!mol/L cytosol, panel F). Thus, at pulse #21 Ca influx = Ca efflux, and no net gain or loss
of cell Ca occurs (panels E & F). In contrast, at pulse #1 Ca influx far exceeds Ca efflux and the
cellular Ca content rises by ~12 /!mol/L cell (panel E and F). Since diastolic [Ca]i doesn't
change, it is reasonable to assume that the cellular Ca gain reflects a gain in SR Ca (panel G).
Thus, over these 21 beats the SR Ca content rises from 0 to 80 /!mol/L cell (120 /!mol/L cytosol).
A. D. ~o )( O.2[
t; fEW 0.1
E
u.. 0.0
Figure 146. Increased SR Ca release enhances Ca transients only transiently. Adding 0.5 mM caffeine
enhances CICR and abruptly increases twitch ~[Ca]i & ~[Cahot (A,B), but this increases Ca extrusion via
INaiCa (C,-D) and reduces SR Ca content (E). Despite a maintained elevation offractional SR Ca release (F)
twitch ~[Ca]i returns to control (where Ca influx and efflux are again equal, C). At this point Ica is
unchanged, and since MCa]i is the same, so is INa/c, ([Ca]o=0.5 mM, pulses are from -40 to 0 mY for 100
ms at 0.5 Hz). Data from Eisner et al. (2000) & Trafford et al. (2000) was kindly supplied by the authors.
SR Ca refilling occurs with a time course similar to that in Fig 144, but the data in Fig 145
provide valuable detailed information about the underlying transsarcolemmal Ca fluxes.
Eisner et al. (1998,2000) also argue that enhancing (or partially inhibiting) RyR release
can only produce transient changes in contractility, but not steady state changes. This is based
on results as in Fig 146 with low caffeine concentrations (~0.5 mM, which enhances CICR,
O'Neill et al., 1990a; Trafford et al., 2000), with tetracaine (which depresses CICR, Overend et
al., 1997, 1998), and the following rationale. If SR Ca release (or CICR) is abruptly enhanced,
the SR will release more Ca at the first twitch or two, but the larger L'l.[Ca]i increases Ca extrusion
via INa/c, (and will limit Ca entry via Ica ), resulting in a reduction of SR Ca content at the next
twitch. The lower SR Ca content causes L'l.[Ca]i and fractional SR Ca release to decline at
subsequent twitches. These twitches could still have a higher Ca transient than control, but this
would cause SR Ca load to decline further. In the steady state in Fig 146, twitch L'l.[Ca]i comes
back essentially to the control level, but this occurs with a lower SR Ca content and a higher
fractional SR Ca release. This is because sarcolemmal Ca efflux and influx must come back into
balance at steady state. Thus, if Ca influx is not changed, Ca efflux must be unchanged and the
same L'l.[Ca]i would be required to drive the same Ca extrusion via INa/Ca. Exactly the converse
was found with inhibition of CICR by tetracaine (or caffeine withdrawal, later in the Fig 146
traces). This sort of autoregulation of Ca transients emphasizes the integral role of SR Ca
content in the regulation of SR Ca release. It also indicates that alteration of RyR gating, by
itself is not a robust way to modulate contractility for more than a few beats. However, if
combined with stimulation of the SR Ca-pump (as occurs with ~-adrenergic activation), this can
create a much faster and stable inotropic change than can be accomplished by SR Ca-pump
activation alone (which would otherwise require 5-20 twitches to reach a new higher SR Ca
content and inotropic state, Eisner et at., 1998).
This paradigm is conceptually instructive and appropriate over a limited linear range, but
must not be extrapolated too broadly. Clearly, if CICR is completely blocked, no SR Ca release
occurs and smaller steady state contractions would result. Likewise, if CICR were hyper-
sensitized (as for caffeine concentrations, >5 mM), the SR is becomes completely Ca-depleted,
and again there is no SR Ca release, and depressed steady state twitches (e.g. see Figs 129-130).
Of course, the lack of SR Ca release will allow Ca influx to rise, but this may not compensate for
the lack of Ca from the SR. Changes in the time course of the AP or Ca transient, and continued
alteration of SR Ca release during diastole can also complicate this analysis.
FORCE-FREQUENCY RELATIONSHIPS
The relationship between stimulation pattern and contractile force has attracted study
since the early work of Bowditch (1871) and Woodworth (1902). This force-frequency
relationship has been reviewed (e.g. Kruta, 1937; Braveny & Kruta, 1958; Blinks & Koch-
Weser, 1961; Koch-Weser & Blinks, 1963; Wood et al., 1969; Allen et aI., 1976; Edman &
J6hannsson, 1976; Johnson, 1979; Wolfhart & Noble, 1982; Lewartowski & Pytkowski, 1987;
Schouten et al., 1987). However, we can now better address the cellular mechanisms involved
than was possible in some of these classic reviews. Most of the key fundamental mechanisms
have been addressed above with respect to mechanical restitution, rest decay (and potentiation),
post-rest recovery, paired-pulses, alternans and post-extrasystolic potentiation. We can draw on
those mechanisms to explain most features of the force-frequency relationship.
Figure 147 shows a classical response to a transient increase in stimulation frequency in
rabbit ventricular muscle. The first pulse at 1.5 Hz is smaller, probably reflecting insufficient
time for the SR Ca release channel recovery from inactivation. Continued pacing at 1.5 Hz leads
to a progressive positive staircase (which overcomes the continued infringement on restitution).
At least three factors could contribute to this increase: I) increased Ie" 2) higher diastolic [Ca]i
(due to greater Ca influx/sec and also less time between contractions for Ca extrusion), and 3)
increased SR Ca load available for release (as a consequence of the above, and the higher
average [Ca];). The higher average [Ca]i may also stimulate CaMKII, which can increase
fractional SR Ca release (Li et al., 1997b). Additionally, higher frequency raises [Na]; (Cohen et
al., 1982; January & Fozzard, 1984; Ellis, 1985; Boyett et al., 1987). This shifts the NaJCa
exchange balance further toward less Ca extrusion and more Ca influx. The result is that there is
more Ca in the cell and in the SR. This is confirmed by extracellular Ca depletions and larger
RCCs when stimulation frequency is increased in rabbit ventricle (Bel's & MacLeod, 1986; Bel's,
1989; Maier et al., 2000). Figure 148A shows that SR Ca rises with increasing frequency in
rabbit and guinea-pig ventricle (based, on RCCs), in parallel with changes in twitch force.
llllldllllliUlillI'1
0.5 Hz 1.5 Hz 0.5 Hz
Figure 147. Frequency-dependent changes in twitch force in rabbit ventricular muscle (30°C).
A. Rat, Rabbit & Guinea-Pig 200 B. Human 500

Q) 200
Twitch
::s
~ 175 00 ;ll
~N 150 (")
(")
:r l'J 150 300 ."
.==:::::::~=:::4'>:=~l.o-_ &
M
Q
'0 100
-'= 125 200 (')
CI>
!:!
~ 100 100 .!:
75
2 3 1 2
Frequency (Hz) Frequency (Hz)
Figure 148. Force-frequency relationship in rabbit, rat, guinea-pig and human ventricular muscle (37°C).
Effect of frequency on twitch force (filled symbols) and SR Ca content (open symbols, assessed by RCCs
initiated within 5 sec of the last steady state stimulated contraction). Data for rabbit and rat are from Maier
et at. (2000), guinea-pig from Kurihara & Sakai (1985) and human from Pieske et al. (1999a).
In Figure 147, switching back to 0.5 Hz results in a large first contraction. This probably
reflects a combination of the relatively high SR Ca content (described above) and also a greater
fraction of SR Ca released (due to the greater time available for recovery from inactivation of the
SR release channel). This large SR Ca release and longer diastolic interval stimulates substantial
Ca extrusion via Na/Ca exchange, and limits Ca entry via Ica . Consequently there is a progress-
sive decline in the amount of Ca in the SR until the initial steady state is re-attained (e.g. where
Ca efflux via Na/Ca exchange over one cycle matches Ca influx via Ica ). Stepwise declines from
potentiated contractions have also been used as an index of the fraction of released Ca that is
resequestered (or recirculated) into the SR (Wolfhart & Noble, 1982; Schouten et al., 1987).
That is, if the second contraction is 70% of the potentiated one, then one might say that ~ 70% of
the Ca released was recycled to the SR. Using "recirculation fraction" this way tacitly assumes
that Ca influx is negligible, trigger Ca is unchanged and that the relation between SR Ca released
and peak force is linear. This limits the true quantitative value of these recirculation fraction
values, but they may be helpful empirically.
Figure 148 shows force-frequency relationships in several types of ventricular muscle,
along with measures of SR Ca content (measured on-line in the same muscles using RCCs).
Rabbit, guinea-pig and nonfailing human ventricle all show classic positive force-frequency
relationships, accompanied by parallel increases in SR Ca content. Thus, despite encroachment
on restitution time in these preparations as frequency increases, the increase in SR Ca appears to
more than compensate, such that the product [CalsRC x fractional release is increased.
In rat and mouse ventricle, SR Ca content is often relatively high, even at very low
stimulation frequencies (as in post-rest contractions, Fig 137B). This may be due in part to
relatively high [Na]; which limits Ca extrusion via Na/Ca exchange (Shattock & Bers, 1989; Yao
et al., 1998). Thus, increasing frequency in rat (Fig 148A or mouse) usually causes little or no
further increase in, or even a slight decrease in SR Ca content (Bers, 1989; Shattock & Bers,
1989; Bouchard & Bose, 1989; Banijamali et a!., 1991; Maier et al., 2000). This makes the
dominant frequency-dependent effect in rat (& mouse) the encroachment into full recovery time
of E-C coupling. This is why rat and mouse myocytes often show negative force-frequency
relationships. On the other hand, rat ventricle can also exhibit a positive force-frequency
relationship (Schouten & ter Keurs, 1986; Layland & Kentish, 1999; Kassiri et al., 2000),
presumably when cells start with low SR Ca content at low frequency (such that [Na]; and SR Ca
can increase with frequency, Frampton et al., 1991; Layland & Kentish, 1999).
In the failing human heart (Fig 148B), SR Ca content increases only slightly with
increasing frequency (mostly between 0.2 and I Hz), and this is associated with some increase in
twitch force. However, SR Ca load does not increase further at higher frequency, and so the
relationship from I to 3 Hz is dominated by the intrinsic depressant effect of higher frequency on
fractional SR Ca release. This results in a flat or negative force-frequency relationship. This
would, of course, limit the functional reserve of the failing human heart and could be a direct
consequence of reduced levels of SR Ca-ATPase and increased levels of Na/Ca exchange
expression in the failing heart (see Chapter 10; Pieske et al., 1999a). Another limiting factor in
the intact heart (normal or failing) is reduced diastolic filling time at higher heart rates.
Thus, a largely positive force-frequency relationship is expected for normal mammalian
cardiac muscle, except for those tissues which have either high SR Ca load at low frequency or
possibly those with short AP duration (such as rat & mouse ventricle and some atrial
preparations). In this case large Ca efflux may occur during the twitch as in rat ventricle (Figs
139-140). Even for these exceptions one can find conditions where a positive staircase is
demonstrable (e.g. rat ventricle at low [Ca]o, low [Na]; or high frequency, Forester & Mainwood,
1974; Henry, 1975; Frampton et al., 1991; Schouten & ter Keurs, 1986; Layland & Kentish,
1999; Kassiri et al., 2000). The potential physiological importance of a positive force-frequency
relationship in maximizing cardiac output at high heart rates, makes the rat and mouse ventricle
results of some concern. Indeed, some investigators (who typically record positive force-
frequency relationships in rat) have suggested that this is the true physiological condition, and
that the negative force-frequency relationship often observed in rat and mouse ventricle result
from effects of tissue or cell isolation (which cause increased SR Ca load at low frequency).
The competition between the SR Ca-ATPase and Na/Ca exchange may also change as a
function of frequency. We used paired RCCs (as described in Fig 76) to determine the ratio of
RCC2/RCCI at 37°C (Pieske et aI., 1999a; Maier et al., 2000). If all Ca released at RCCI is re-
sequestered by the SR during relaxation of RCCI this ratio is 100%. In rabbit and nonfailing
human heart RCC2/RCCI increases monotonically as twitch frequency increases from 0.25 Hz to
2 Hz (28 to 65% in rabbit and 37 to 74% in human). A simple explanation for this effect could
be that as frequency increases, the gradual increase in [Na]; (Cohen et aI., 1982; Maier et aI.,
1997a) limits the ability of the Na/Ca exchange to compete with the SR Ca-ATPase. In addition,
increasing frequency also accelerates [Cali decline due to an increased rate of SR Ca transport,
possibly due to CaMKII (see next section). Thus, the SR Ca-ATPase becomes increasingly
dominant over the Na/Ca exchanger in transporting Ca from the cytosol at higher frequencies. In
both rat and failing human ventricle the RCC2/RCCI ratio was high even at low frequency, and
did not change appreciably with frequency. We interpreted this as a reflection of the strong
dominance of the SR Ca-ATPase in rat at all frequencies, and to a limitation in SR Ca-ATPase in
the failing human heart.
Frequency-dependent acceleration ofrelaxation (FDAR)

Mammalian ventricular muscle shows a marked frequency-dependent acceleration of
relaxation (FDAR, Fig 149; Schouten, 1990; Pieske et aI., 1995; Hussain et al., 1997). This
..s 175
150
A. Human Ventricle
. . 150
E
:; 125
B. Rat Ventricle
1 ~
.;t
.... .
l: 125 100
0 Force
:;:l ~
~
)( 100 .c: 75
.c:
& 75 2
.;t
50
I- 25
0
Frequency (Hz) Frequency (Hz)
Figure 149. Frequency-dependent acceleration of relaxation. With increasing frequency the half-time (or
half-width) of relaxation and [Cali decline get shorter. Data at 37°C are taken from Pieske et al. (1995) for
human (A) and from Layland & Kentish (1999) for rat (B).
FDAR is also readily apparent when comparing twitch relaxation at a steady state (SS) frequency
(e.g. I Hz) vs. a post-rest twitch, and is independent of p-adrenergic activation (Fig ISO; Bassani
et al., 1995c). From a physiological standpoint this effect may be very important in speeding
relaxation at high heart rates, allowing the heart to refill more rapidly between beats. Schouten
(1990) hypothesized that faster relaxation of the SS vs. post-rest beat was due to CaMKIl-
dependent phosphorylation of phospholamban (PLB). That is, with stimulation at SS (or higher
frequency), the average [Cali is higher and that could activate CaMKIl to phosphorylate
phospholamban and thus accelerate SR Ca-ATPase, [Cali decline and relaxation. With a long
rest or lower frequency, PLB may be dephosphorylated, causing slower [Cali decline. We tested
this in rat, ferret and mouse myocytes and found that the CaMKIl inhibitors (KN-62 & KN-93),
could abolish the acceleration of relaxation and [Cali decline during SS vs. post-rest twitches
(Bassani et al., I 995c; Li et al., 1997b, 1998). It takes several beats at I Hz for the acceleration
in [Cali decline to develop (Fig 150A; 't ~5 s) and FDAR was lost during rest with a
biexponential time course ('t = 3.4 and 69 s), consistent with known kinetics of CaMKIl
activation, deactivation and slow deactivation of autophosphorylated CaMKIl (Braun &
Schulman, 1995; De Konnick & Schulman, 1998). Furthermore, phosphatase inhibitors
prevented (or delayed) the post-rest slowing of twitch [Cali decline (Fig ISOC). The accelerated
twitch [Cali decline was SR-dependent, because the effect was abolished or reversed if SR Ca
uptake was prevented with thapsigargin or caffeine (such that relaxation was via Na/Ca
exchange, Fig I SOB). However, PLB phosphorylation is not essential for FDAR, because it still
occurs in the PLB knockout mouse, and is still abolished by KN-93 (Li et al., 1998; DeSantiago
& Bers, 2000). Thus, it appears that accelerated relaxation at high heart rates is due to CaMKIl-
dependent stimulation of SR Ca transport, but does not require phospholamban.
This issue is not yet mechanistically resolved. Some groups found that KN-62 or KN-93
do not abolish FDAR (Hussain et aI., 1997; Layland & Kentish, 1999; Kassiri et aI., 2000).
Bluhm et al. (2000) found that FDAR of isometric force was lower in PLB-KO mice. Hagemann
et al. (2000) also showed frequency-dependent phospholamban phosphorylation (at '6Thr) which
correlated with FDAR. It is possible that direct CaMKIl-dependent phosphorylation of the
cardiac SR Ca-ATPase (Xu et al., 1993) occurs, but this has been challenged (Odermatt et
al.,1996; Reddy et al.. 1996). So, even the appropriate CaMKIl target involved in accelerating
SR Ca transport during FDAR in ventricular myocytes is not yet identified unequivocally.
A. Twitch-Dependent Acceleration of Relaxation
gr 200 PR Twitch-dependent
acceleration
1:= -5 s
'"
.5
U /' \
"'"
Rest-dependent slowi ng
. . 100
1: =
3.4 and 69 sec
£
'0
p
50'-,0!:-'-~-'-:5-!:0~~1~O-='O~'-715:-:0;-'--'--';:2:::-0~O
~~2:-:5:::0~'-';:-30:':-:O;-'--'-':3:-:5-;'O~~4':":OO
time (sec)
B. FDAR depends on SR C. FDAR depends on CaMK

ii) 400
.s _ 400
u
,g
300 !
Q) 300
Jiu
~ 200
~ 200
~ ~
i'
.
100 ~ 100
.
~ '0
'0 .l.....1-. . .-'------..Lo~
Control TG
Figure 150. Frequency-dependent acceleration of relaxation (FDAR) at steady state (SS) vs. post-rest
(PR) contractions. A. Build up of FDAR at 1 Hz, and dissipation during rest. Time constant ('I:) of [Cali
decline is shown for the PR contraction and ensuing 100 twitches at I Hz. Stimulation was then stopped at
100 sec and a single twitch was evoked after different periods of rest (2-300 s). After resumption of SS,
another rest period was tested. B. FDAR was observed for SS vs. PR in control twitches, but not when the
SR Ca-ATPase was blocked by thapsigargin (TG). In this case, relaxation depends on Na/Ca exchange
(note la-fold slower 1: values in TG) C. Inhibition of phosphatase activity by okadaic acid (10 flm)
prevented slowing of [Cal; decline at the PR twitch. Inhibition of CaMKll alone (by KN-62) or together
with PKA inhibition (by H-89, as shown) prevented the acceleration of [Cali decline from PR to the SS.
Data are in rat ventricular myocytes and are from Bassani et al. (I 995c).
In conclusion, there is great variation in details of [Cal; regulation in different cardiac

muscle preparations and conditions. However, this apparent complexity can be largely
understood by considering a small number of common systems which interact and a few key
functional properties that differ among cardiac preparations. Ca influx can activate substantial
contractions in some mammalian as well as amphibian hearts, but under normal conditions the
SR is the major source of Ca for activation of adult mammalian cardiac muscle. Ca influx can
serve to trigger SR Ca release and also contribute to the loading of the SR for the next
contraction. Ca released from the SR can either be re-accumulated by the SR or extruded from
the cell via Na/Ca exchange. In the steady state, however, the Ca extruded by Na/Ca exchange
during one cardiac cycle is balanced with the Ca influx via lea (and possibly Na/Ca exchange).
The Ca content of the SR can be gradually depleted by the sarcolemmal Na/Ca exchange during
rest, and can also be quickly refilled during post-rest recovery by the Ca entering via lea in 5-10
contractions. Depending on the transsarcolemmal [Na] gradient, rest can either deplete the SR or
fill the SR with Ca. Clearly a dynamic yet delicate balance exists in the control of intracellular
Ca in the heart and changes in these -systems can lead to inotropic and lusitropic changes.
D.M. Bers. 273
2nd Ed., Kluwer Academic Publishers, Dordrechl, 2001
CHAPTER 10
CARDIAC INOTROPY AND Ca MISMANAGEMENT
In this chapter I will discuss some general mechanisms involved in cardiac inotropy and
their relationship to cellular Ca overload and mismanagement. It is not intended as a compre-
hensive review of either inotropic agents or cardiac pathophysiology. I will start by considering
five different means of cardiac inotropy: I) hypothermia, 2) ~-adrenergic activation, 3) 0.-
adrenergic activation, 4) CaMKII activation and 5) cardioactive steroids (digitalis glycosides).
Then I will discuss the ways in which cellular Ca regulation can go awry, with particular
emphasis on Ca overload and heart failure. Finally, I will address strategic sites for induction of
cardiac inotropy. This discussion may help to bring some of the characteristics of specific
cellular systems discussed in preceding chapters into a more integrative picture of cellular Ca
regulation.
CARDIAC INOTROPY
Hypothermic Inotropy
Reduction of temperature from 37°C in mammalian cardiac muscle results in an increase
in developed force (Kruta, 1938; Sumbera et aI., 1966; Blinks & Koch-Weser, 1963; Langer &
Brady, 1968). This hypothermic inotropy results in a remarkable 400-500% increase in force at
25°C (see Fig 151). Much of this large inotropic effect occurs immediately, when temperature is
abruptly changed between twitch contractions (see Fig 152). This rapid change suggests that
alteration in SR Ca content or relatively slow biochemical changes are unlikely to be critical for
most of this inotropic effect. Additionally, hypothermic inotropy of similar amplitude is still
observed when normal SR function is depressed by ryanodine or caffeine (Fig 151 B, Shattock &
Bers, 1987). Myofilament Ca sensitivity and maximal force are both decreased by cooling, albeit
only moderately at 29 VS. 36°C (see Fig 20, pg 29; Harrison & Bers, 1989a). This suggests that
[Cali must either increase dramatically or come to more complete equilibration with the
myofilaments at cooler temperature. Figure 60 (pg 122) shows that cooling from 35 to 25°C
reduces peak lea (QIO~3, Cavalie et at., 1985) and increases action potential duration (e.g. from
-220 to -370 msec in rabbit ventricle; Shattock & Bers, 1987; Puglisi et at., 1999). Inter-
estingly, the lower peak lea is compensated by slower Ca channel inactivation at 35°C, such that
total Ca entry via lea is essentially the same at 25 VS. 35°C (Fig 60; Puglisi et at., 1999).
If peak lea is reduced, we would also expect less Ca-induced SR Ca-release (particularly
as SR Ca load is unlikely to change between two consecutive twitches in Fig 152). On the other
hand, since cooling increases the open probability of the cardiac SR Ca release channel (by
increasing the duration of openings, Sitsapesan et at., 1991), a greater fraction of the SR Ca
content might be released for a given lea trigger. Whether this effect would be sufficient to
compensate for the lower peak lea is not completely clear. However, peak [Cali is much larger
Hypothermic Inotropy in Rabbit Ventricle

700
l~l
600
~ 500
cf2. Control ~I
~ 400
I'~odine
o
'iii
c:
QJ
300
I-
200 (1IJM)
Force 100
3mN
o
, i I •
37 33 29 25
200msec Temperature (OC)
Figure 151. Hypothermic inotropy in rabbit ventricular muscle (0.5 Hz). Steady state contractions are
shown at the indicated temperatures (left). Pre-equilibration with I l-lM ryanodine does not prevent this
hypothermic inotropy in rabbit or rat (right panel is from Shattock & Bers, 1987, with permission).
during steady state twitches at 25 vs. 35°C (Puglisi et al., 1996). The maximum rate of rise of
force (+dF/dt) is also not decreased at the first contraction at 25°C in Fig 152C, indicating that
SR Ca release is at least as large as at 35°C. Otherwise the slower development of contractile
force expected from the myofilaments at I+ow temperature (for any given [Ca)) should lead to a
smaller +dF/dt. So peak [Ca]i is increased at lower temperature. While this might reflect higher
fractional SR Ca release, an alternative explanation should be considered.
Relaxation is greatly delayed at 25 vs. 35°C (Fig 152) and the Ca transport systems
which drive relaxation are slowed by cooling. Indeed, the halftimes for twitch relaxation and
[Ca]i decline in rabbit ventricular myocytes are more than doubled (from 50 to 130 ms and 117 to
252 ms respectively) upon cooling from 35 to 25°C (Puglisi et al., 1996). This creates a greatly
prolonged time to peak tension and this allows the myofilaments to more closely approach a
steady state level of activation by [Ca]i. It also allows peak rCa]; to reach a higher level, simply
because Ca transport out of the cytosol is slowed, analogous to the increased peak twitch [Ca]j
when either the SR Ca-pump or Na/Ca exchange are blocked (Fig 133). Thus there is a
prolonged "active state" where the myofilaments are being activated. Moreover, at 25°C there is
a more distinct separation between the activation and relaxation phase. Note that the delay
between maximum +dF/dt and -dF/dt in Fig 152C is -200 ms at 35°C and -950 ms at 25°C.
We also analyzed Ca fluxes via SR Ca-ATPase and Na/Ca exchange during relaxation
and [Ca]i decline at 25 vs. 35°C, as described in Figs 133-135 (Puglisi et aI., 1996). The SR Ca-
ATPase, Na/Ca exchange and slow systems (sarcolemmal Ca-ATPase plus mitochondrial Ca
uniporter) are all slowed by 2-3-fold upon cooling from 35 to 25°C, so each system has a
functional QIO of 2-3. This is consistent with the 2-3 times slower relaxation half-time at 25°C.
However, the relative competition between these Ca transport systems is not appreciably
changed, such that in rabbit the SR Ca-ATPase is still responsible for 70-75% of rCa]; decline,
and the Na/Ca exchanger 20-25%. This parallel slowing of all Ca transporters was also seen in
Chapter 10 Cardiac Inotropy & Ca Mismanagement 275
A.
~ ~ ~
pJ~~~~~
~ ~ I Force
lmN
--.J'------I/ h 35°C
'-----25°C
B.
__-"",-I~
2OOmseo
Figure 152. Hypothennic inotropy in rabbit ventricular muscle is rapidly induced. A quick change in
temperature shows that most of the hypothennic inotropy take place rapidly. The muscle was stimulated at
0.5 Hz with temperature switch >90% complete in -300 ms (using the same set-up as for RCCs). In A, the
break in the force record is -2 min. Force (8) and dF/dt (C) are shown for the first contraction after
cooling to 25°C and the last contraction at 35°C from panel A.
ferret and cat ventricular myocytes. With the prolonged AP duration at 25°C, this could
especially delay the functional contribution of Na/Ca exchange to [Cali decline, creating a slight
additional bias toward the SR vs. extrusion from the cell.
In the steady state at lower temperature the Na-pump is inhibited (QIO ~3, Eisner &
Lederer, 1980) and [Na]; rises (Shattock, 1984; Chapman, 1986). The rise of [Na]; will also shift
Ca fluxes via Na/Ca exchange and tend to increase cellular and SR Ca content and Ca influx.
Indeed, in the steady state SR Ca content is increased by cooling to 23-25°C, measured by
caffeine-induced Ca transients and RCCs (Shattock & Bel'S, 1987; Puglisi et af., 1996). The Ca
loading effect may be responsible for much of the slower phase of hypothermic inotropy which
develops over a minute or two (in contrast to the more immediate changes discussed above).
f3-Adrenergic Agents and Cardiac 1notropy

~-adrenergic receptor (~-AR) activation is particularly important to consider, because it
is the physiological means by which the inotropic, lusitropic and chronotropic states of the heart
are controlled via sympathetic stimulation. Indeed, sympathetic nerve endings and ~-ARs are
broadly distributed throughout the heart. Isoproterenol, a ~-adrenergic agonist, can produce
large increases in cardiac contraction, Ca transient amplitude and the rates of relaxation and [Cali
decline (see Fig 153). Four effects of ~-adrenergic agents have already been discussed: 1)
decreased myofilament Ca sensitivity due to troponin I phosphorylation (Chapter 2),2) increased
Ie. (Chapter 5), 3) enhanced SR Ca-ATPase rate (Chapter 7) and 4) altered RyR gating (Chapter
7). Activation of ~1-ARs can cause all of the major cardiac adrenergic effects (inotropy,
1.6
'0;:
~=L ....
C1I.~
.~~
N VI
=c:
,..:;6 Ez
"'0 0""":;
u" z!1
-:'99 ~OPROTERENOL
129. "
.s:: '0"
g,-
.,E .Ez..
.,0
N;
:.: u
:;-
- ::L
U o~
Zu
"0
SOD ms
107.
Figure 153. Effect of the ~-adrenergic agonist, isoproterenol (0.5 mM) on Cai transients (top) and
shortening of rat ventricular myocytes at 23°C. At left [Ca]o=1 mM in the presence and absence of
isoproterenol. Center panel traces are a different cell with [Ca]o = 3 mM Ca o or I mM Ca o plus 0.5 IlM
isoproterenol, so that similar peak [Cali was reached in both cases (note smaller contraction with
isoproterenol). Right panel shows traces normalized traces from control and isoproterenol (from middle
panel). Note the faster time course with isoproterenol (from Spurgeon et aI., 1990 with permission).
lusitropy and chronotropy) and I will focus on that pathway. ~2- and ~3-ARs will be discussed
on pg 280. ~-adrenergic agonists stimulate adenylyl cyclase (see Fig 65, pg 129), elevating
cyclic AMP, which activates cyclic AMP-dependent protein kinase (PKA) and which
phosphorylates several key proteins, including: I) troponin I (reducing Ca affinity of TnC), 2)
sarcolemmal Ca channels (increasing Ie.), 3) phospholamban (increasing SR Ca pump rate) and
4) SR Ca release channels (modifying RyR gating). The stimulation of Ie. and SR Ca uptake
causes Ca transients to be larger and faster after ~-adrenergic agonists (Figs 153 & 154).
In the presence of a ~-adrenergic agonist, more Ca enters the cell at each excitation (due
to increased Ie.) and the SR accumulates a larger fraction of the cytosolic Ca pool during
relaxation (due to SR Ca-pump stimulation). Thus, a larger SR Ca load is expected. The
increased Ie. (and SR Ca) may also increase the fraction of SR Ca which is released. The result
is a much larger peak [Cali during contraction with isoproterenol (see Figs 153 & 156). Of
course, the SR Ca-pump stimulation also accelerates relaxation and leads to a shorter time to
peak tension an [Cali, and more rapid relaxation and [Cali decline. If Na/Ca exchange is
unaltered, the SR Ca-pump stimulation will also bias the competition between these two
mechanisms in favor of the SR Ca-pump (even though the higher peak rCa]; will also stimulate
Ca extrusion via Na/Ca exchange, see Fig 145). This bias toward the SR Ca-pump (vs. Na/Ca
exchange) can also explain why isoproterenol can abolish the negative post-rest staircase in rat
ventricular myocytes (Fig 154A, Raffaeli et al., 1987). That is, if less Ca is not extruded via
Na/Ca exchange at the large contractions (because the SR Ca-pump competes more effectively),
the SR Ca load will not decrease progressively from beat to beat as discussed on page 267. It is
also possible that isoproterenol shortens the time for the SR Ca release process to recover from
inactivation. This could also contribute to the loss of the negative staircase.
A. Iso Limits Negative B. Most Iso inotropy

Post-rest staircase is due to Phospholamban
~. 100 I~:"""''8=--O--Q==:SJ
::: +Iso *
't;;
&. Control
'0
C MCaJ;
..
50
Control
'tl
::l Shortening
I'" 2 3 4
WT PLB-KO
Post-rest Pulse #
C. Lusitropic effect of Iso is mainly due to Phospholamban

Muscle Force Myocyte Length
PLB-KO
Figure 154. Isoproterenol (Iso) effects on inotropy and lusitropy at n°e. A. Iso abolishes the negative
staircase observed upon resumption of 0.2 Hz stimulation after rest in rat ventricular myocytes at n° (Data
taken from Raffaeli et at., 1987). B. Iso nearly doubles contractile force in wild type (WT) mouse ventricle,
but only increases force by 30% in the phospholamban knockout (PLB-KO) mouse. C. In WT mouse, Iso
reduces relaxation time constant (1) by nearly 50%, whereas in PLB-KO myocyte relaxation was unaffected
by Iso. In PLB-KO muscles developing force, there was a small lusitropic effect of Iso. [Ca]o or sarcomere
length was adjusted to match twitch amplitude (±Iso) in e. Data in B & C are from Li et aI., 2000).
~-adrenergic activation also decreases myofilament Ca sensitivity (due to phosphoryla-

tion of Tnl, see Chapter 2), but the dramatic increase in Ca transients more than compensates for
this, so contractions are increased substantially (but less so than Ca transients, Fig 156). It had
been thought that the lower myofilament Ca sensitivity (and Ca-affinity) might contribute to the
more rapid relaxation (or lusitropic effect) observed with ~-adrenergic agonists (due to faster Ca
dissociation from troponin C). However, data of McIvor et at. (1988) and Endoh & Blinks
(1988) suggested that the lusitropic effect of isoproterenol was attributable mainly to SR Ca-
pump stimulation, rather than decreased myofilament Ca sensitivity. Mechanical factors, such as
the rate of cross-bridge detachment and geometric considerations might also be involved in the
~-adrenergic induced acceleration of relaxation (Hoh et at., 1988). We quantitatively compared
the contributions of Tnl and phospholamban phosphorylation in the lusitropic effect of
isoproterenol (Fig 154C, Li et at., 2000). In mice lacking phospholamban (PLB-KO),
isoproterenol had no effect at all on myocyte or muscle twitch relaxation, despite a substantial
increase in Tnl phosphorylation. However, this was only the case when relengthening was
measured in the absence of an external mechanical load (at right). When muscles from these
PLB-KO mice developed isometric force, the amount of lusitropic effect observed with
isoproterenol was directly related to the amount of force development (left). This emphasizes the
dynamic interplay between force, myofilament properties and Ca transients. Nevertheless, the
lusitropic effect of isoproterenol in the PLB-KO mice was still very small, compared to wild type
mice. We estimated that at maximal force, -85% of the lusitropic effect is due to
phospholamban phosphorylation and acceleration of SR Ca-pumping, while only 15% could be
due to TnI phosphorylation.
PKA-dependent phosphorylation of the cardiac RyR can also increase RyR sensitivity to
activation by a Ca trigger in lipid bilayers (Valdivia et at., 1995; Marx et at., 2000). Thus
isoproterenol might be expected to directly increase SR Ca release during the twitch by an effect
on the RyR. On the other hand, preliminary data in intact myocytes indicate that when we
control for changes in SR Ca load and lea, isoproterenol does not increase SR Ca release
(Ginsburg & Bers, 2001). Thus it is not yet entirely clear how important the PKA-dependent
phosphorylation of the cardiac RyR is in the inotropic response to catecholamines.
Interestingly, there is still a significant inotropic effect of isoproterenol in the PLB-KO
mouse (though much less than in wild type mice or other ventricular myocytes (Wolska et at.,
1996b; Li et at., 2000). This may be mainly due to the large increase of Ie" which progressively
increases SR Ca content in the absence of phospholamban (by simple mass action). That is, there
is more Ca influx, higher average rCa]; and consequently higher SR Ca-pump rate. The higher
lea may also increase contractility by increasing fractional SR Ca release (in WT & PLB-KO).
The enhanced lea with ~-adrenergic agonists tends to elevate the plateau phase of the
cardiac action potential, but ~-adrenergic agonists also increase cardiac K conductance (e.g. I Ks ,
Gadsby, 1983; Walsh & Kass, 1988, 1991) which shortens the AP and tends to hyperpolarize the
diastolic Em. lea also inactivates faster because of the high D.[Cak ~-adrenergic agonists also
activate the CFTR Cl current in cardiac myocytes (pg 84; Harvey & Hume, 1989). The net result
of effects on Ca, K and Cl currents is that AP duration can be increased, decreased or unchanged
depending on species and experimental conditions (Tsien, 1977; Tsien et at., 1986). However,
under physiological conditions AP shortening is typical, and is consistent with the positive
chronotropic effect of ~-adrenergic agonists. That is, shorter AP duration allows channels to
recover from refractoriness, thereby allowing higher heart rates. Earlier repolarization will also
indirectly stimulate Ca extrusion via Na/Ca exchange (Fig 140).
~-adrenergic agonists also influence the inward pacemaker current If (pg 86-87), which is
activated by hyperpolarization. ~-adrenergic agonists shift the Em-dependence of If activation to
more depolarized potentials (see Fig 42), resulting in a more rapid diastolic depolarization
(DiFrancesco, 1986; DiFrancesco et at., 1986). ~-adrenergic agonists also accelerate the decline
in potassium conductance (gK' Bennett et at., 1986; Connors & Terrar, 1990) which may
contribute to pacemaker activity (especially in cells with more positive diastolic potentials, such
as sino-atrial nodal cells (Noble, 1985; Noma, 1996). Stimulation of SR Ca uptake may also
increase spontaneous SR Ca release in pacemaker cells, activating inward (depolarizing) current
(pg 95). Thus, several effects contribute to the positive chronotropic effect of ~-AR stimulation.
This increase in heart rate can increase cardiac output independent of ~-adrenergic
agonists. That is, increased frequency by itself would increase contractility (and speed relaxa-
tion), based purely on the force-frequency relationship and frequency-dependent acceleration of
relaxation discussed in Chapter 9 (i.e. independent of the p-adrenergic activation). Thus, the
increase in contractility in vivo with p-adrenergic agonists is a combination of the intrinsic
inotropic & lusitropic effect of increased frequency and the PKA-mediated effects, which are
directly attributable to p-adrenergic activation (e.g. increased lea and SR Ca pumping). Thus,
cardiac output can be greatly enhanced by p-adrenergic agonists.
Sarcolemmal Na/K-ATPase is also affected by p-adrenergic agonists (see pg 90). While
some results indicated that p-adrenergic agonists can stimulate Na/K-ATPase and reduce [Na];
(Wasserstrom et al., 1982; Lee & Vassalle, 1983; Desilets & Baumgarten, 1986; Kockskiimper et
al., 2000), Gao et al. (1996, 1997c, 1998a) provided compelling evidence for Na/K-ATPase
inhibition by p-adrenergic agonists and PKA. Na/K-ATPase inhibition would cause [Na]; to rise,
further limiting the ability of Na/Ca exchange to compete with the SR Ca-ATPase. This could
also contribute to greater cellular and SR Ca load. It could also exacerbate the rise in [Na];
caused by the accompanying physiological increase in heart rate. Thus, PKA-dependent
regulation of the Na/K-ATPase may contribute indirectly to the enhanced Ca transients and
inotropy induced by activation of PKA, but the net effect is somewhat controversial.
Activation of P-ARs also increases metabolism and glycogenolysis (via PKA, that
activates phosphorylase b kinase, which in tum activates phosphorylase b, resulting in glycogen
breakdown, Hayes & Mayer, 1981). The increased metabolic energy demand, in combination
with the large demands due to the increased inotropic state and frequency, can increase O2
consumption dramatically (Rolett, 1974). This is a shortcoming of p-adrenergic agonists as
inotropic agents. That is, in energetic terms (and in terms of O2 requirement) increasing
contractility and heart rate via p-adrenergic agonists is expensive. P-ARs are also down-
regulated or removed from the membrane during chronic activation and in the failing human
heart (Watanabe et aI., 1982; Stiles et al., 1984; Bristow et al., 1982, 1986). This makes p-
adrenergic agonists more useful for acute rather than chronic inotropic therapy.
P-ARs are members of the 7-transmembrane domain receptor family that couple to
heterotrimeric GTP-binding proteins (with IX & Pr subunits); in the case of PI-AR it is Gs (Figs
65, 155). The third intracellular loop of the receptor interacts with the carboxy-terminal of the
G,,-subunil. When the P-AR is activated by a ligand, bound GDP is exchanged for GTP, and the
Gs" subunit is activated (until GTP is hydrolyzed to GDP). Activated Gs" dissociates from the
ternary complex (agonist-receptor-G-protein) and can activate adenylyl cyclase to produce
cAMP. The amount of adenylyl cyclase may be a rate-limiting step in the cascade (Gao et aI.,
1998b), and the main isoforms in heart are types V & VI (Ishikawa et al., 1994). P-ARs can
become desensitized (or uncoupled) in seconds to minutes, and this process involves
phosphorylation of a serine on its carboxy-terminal tail, by a G-protein coupled receptor kinase
(GRK) or PKA (Post et al., 1999). Two GRKs appear to be involved in heart, GRK2 (also called
P-AR kinase-I, or PARK-I) and GRK5. After P-AR phosphorylation, another protein (P-
arrestin) can bind to the P-AR, and prevents further interaction with Gs (Lefkowitz, 1993). P-
arrestin can also interact with clathrin and this results in the internalization of P-ARs (Goodman
et al., 1996; Ferguson et al., 1996). Once internalized the receptors can either be recycled to the
membrane in a re-sensitized form (after dephosphorylation, Krueger et al., 1997) or degraded as
a final step in receptor down-regulation. Interestingly, this down-regulation and loss of cell
surface ~-ARs, which occurs importantly in heart failure (Bristow et al., 1982), is accompanied
by reduction in ~-AR mRNAs due to decreased message stability (Hadcock et aI., 1989).
The stimulatory effect of ~-adrenergic agonists on adenylyl cyclase and cAMP
production can be inhibited by another G-protein (G;) which is activated by muscarinic M 2
receptors (and some other receptors). Thus, parasympathetic release of acetylcholine can
diminish the effect of sympathetic stimulation of ~-ARs. While this clearly occurs in the sino-
atrial node, and is important in the control of heart rate, there are fewer parasympathetic nerve
endings in ventricular muscle (LOffelholz & Pappano, 1985). Thus, parasympathetic activation
may have less anti-adrenergic effect on contractility than on heart rate. Nevertheless, there are
muscarinic (and other) receptors in ventricular myocytes which can mediate Gj-dependent
limitation of adenylyl cyclase activation. Histamine and serotonin receptors can also couple to
Gs and enhance contractility via stimulation of adenylyl cyclase (Brodde et al., 1998)
~r and ~3-ARs are also present in mammalian ventricle (see Table 25). ~I-AR are more
numerous than ~rAR in ventricle (-75% of total ~-ARs) and ~J-AR also are more sensitive to
the physiological agonist norepinephrine than are ~r or ~rARs (Brodde,1993; Lafontan, 1994;
Post et al., 1999). Thus, the majority of ~-AR response is likely to be mediated by ~J-AR.
Consistent with this idea, knockout of the ~I-AR gene in mice, results in prevention of both
inotropic and chronotropic response to isoproterenol (although most of these knockout mice die
in utero; Rohrer et al., 1996). On the other hand, there is compelling data that ~r and ~3-AR can
alter ventricular myocyte function. There is even evidence that ~rAR are more effectively
coupled to adenylyl cyclase than ~J-ARs (Bristow et aI., 1989; Kaumann et al., 1989).
~rAR activation can produce the same effects as discussed above for ~,-ARs. However,
there is evidence to suggest that ~2-ARs can couple to both Gs and Gj in rat and dog ventricular
myocytes (Xiao et al., 1995, 1999; Kuschel et aI., 1999b; Chen-lzu et aI., 2000). They found
that ~2-AR stimulation induced a PKA-dependent inotropy and increase of Ie" but did not raise
global cAMP levels or phosphorylate phospholamban, Tnl or protein C (all of which were seen
with ~J-AR activation). Moreover, Ca channels in a cell attached patch could be activated by ~J
AR agonist outside the patch (diffusive signaling), but the ~rAR agonist zinterol only activated
lea if it was included in the patch pipette (local signaling only). However, pertussis toxin
treatment (disrupting G; regulation) allowed ~rAR agonist to also activate lea via diffusive
signaling (zinterol outside the pipette). Thus the apparent G,IG; coupling of ~rARs may create
very localized PKA activation which can phosphorylate nearby Ca channels, but not distant
targets like phospholamban and Tn!. Perhaps co-activation of G j limits the amount of cAMP
produced, creating a very local stochastic PKA activation. This intriguing pathway is still
controversial. For example, La Flarnme & Becker (1998) found no evidence that ~rAR activa-
tion modulates Ca handling in rat ventricular myocytes, even with pertussis toxin.
~2-AR overexpression in transgenic mice (by> 100-fold) appears to fully activate the
PKA-mediated inotropic state, even in the absence of ~-adrenergic agonists (Milano et al., 1994;
Bond et al., 1995). This has been taken as evidence that the ~2-AR can shift back and forth
between a resting state and a stochastically rare active state (stimulating Gs). Thus even though a
very small fraction of unoccupied ~2-ARs are in this active state, the massive overexpression
allows strong basal activation and cAMP production. This work also illustrated nicely the
Activation-Desensitization Down-regulation
~-AR types
Figure 155. ~-adrenergic receptor signaling in ventricular myocytes. Top shows the process of activation
by norepinephrine (NE), Gsa-activation of adenyJyl cyclase (Ae) and production of cAMP (which activates
PKA). PKA phosphorylates key functional targets (see Fig 65), and along with GRK2 & GRK5
phosphorylates (P) the ~-AR to cause desensitization. Then ~-arrestin binds to the phosphorylated ~-AR,
causing internalization. Bottom shows differences in coupling of ~I-, ~r & ~3AR to G-proteins (G s and/or
Gi) and different consequences. Note that the ~3AR lacks the GRK phosphorylation sites that lead to
desensitization and down-regulation (see also Fig 65, pg 129).
principle of inverse agonism. That is, drugs that can shift this equilibrium toward the resting
state (e.g. the ~2-AR inverse agonists ICI 118,551) can prevent this basal agonist-independent
activation in the ~rAR transgenic mouse. This differs from classical receptor antagonists, which
block binding and activation by an agonist.
~3-AR activation in ventricular myocytes appears to produce negative inotropic effects
(Gauthier et al., 1996, 1998, 1999,2000; Varghese et al., 2000). These negative inotropic effects
are sensitive to pertussis toxin (implicating G/G o rather than Gs) and appear to be mediated by
nitric oxide (NO) produced by endothelial NO synthase (NOS3, which is constitutively
expressed in ventricular myocytes). NO production in ventricular myocytes stimulates guanylyl
cyclase, cGMP production, and protein kinase G activation. This cascade produces negative
inotropic effects in heart, but the molecular mechanisms are not as clearly identified. Nitric
oxide can decrease myofilament Ca sensitivity via PKG-dependent phosphorylation of TnI, just
as seen for PKA (Pfitzer et aI., 1982; Lincoln & Corbin, 1978; Shah et al., 1994, Kaye et al.,
1999). Nitric oxide can also decrease lea (Mery et aI., 1993; Sumii & Sperelakis, 1995; Wahler
& Dollinger, 1995). ~3-ARs lack the carboxy-tail serines that are the target for phosphorylation
by ~ARK-I (or GRK5 or PKA) and thus do not appear to exhibit desensitization and down-
regulation as seen for ~I- and ~rAR. This may be particularly important in heart failure, where
the nonnally dominant ~-AR (~I-AR) is down-regulated. In this case, the negative inotropic
effect of ~3-AR may become unmasked (i.e. it is nonnally masked by the positive effect of ~I- &
~z-AR, which are more numerous and have higher affinity for noreipnephrine). Nevertheless,
most (if not all) of the ~-AR response of ventricular muscle to the physiological agonist
norepinephrine, appears to be mediated by ~,-AR.
a-Adrenergic Agents and Cardiac Inotropy

Inotropy can also be mediated by a-adrenergic receptors (a-ARs) in the heart, and in
some hearts a-AR are comparable in number to ~-ARs (Bode & Brunton, 1989; Benfey, 1990;
Brodde & Michel, 1999). These a-ARs are 7 transmembrane domain G-protein-coupled
receptors, and cardiac myocytes appear to express only the a,-AR subtype (a2-ARs are primarily
involved in presynaptic inhibition of norepinephrine release, Brodde & Michel, 1999). Table 25
(pg 287) shows densities of receptors and ion transporters in whole tissue (fmol/mg homogenate
protein) and also the estimated surface density (/llm 2) on the sarcolemma (or on the SR).
Schumann et al. (1974, 1975) demonstrated inotropic effects attributable to a-AR activation, and
this inotropic pathway has gradually become better appreciated (reviewed by Endoh, 1996). In
addition to the inotropic effects, al-AR activation is involved in ventricular hypertrophic
signaling (Simpson, 1985; see pg 312). The inotropic and hypertrophic effects are largely
mediated via the G-protein Gq. There are strong parallels between the downstream effects of al-
AR activation and some other Gq-coupled receptors, such as endothelin (ET-l) and angiotensin II
receptors (ETA & AT], Endoh, 1996), but I will restrict the discussion here mainly to a,-AR.
Three subtypes of a]-ARs are expressed in heart (alA, alB & aID)' The pharmacolo-
gically defined aIA-AR subtype turned out to be the clone originally known as aleAR, but to
avoid confusion these are now both referred to as a'A-AR (Zhong & Minneman, 1999). The alA-
AR is selectively activated by the agonist A61603, and blocked by the selective antagonists WB-
4101, (+)-niguldipine, 5-methylurapidil and KMD-3123 (Brodde & Michel, 1999), although WB-
4101 can also block alD-AR with high affinity. The a'B-AR is preferentially alkylated by
chloroethylclonidine (CEC), which creates an irreversible block. The ulD-AR can be selectively
blocked by BMY-7378, but this agent is also a partial agonist at serotonin (5-HT IA ) receptors
(Goetz et aI., 1995). The ulD-AR is also intermediate between UIA- and u'B-AR in sensitivity to
CEC and 5-methyurapidil. Phenylephrine, methoxamine and cirazoline are non-selective u,-AR
agonists, and phenylephrine is the most widely used. The hypertrophic response appears to be
mediated via the UIA receptor subtype, while the inotropic (action is mediated mainly by the UIB-
AR subtype, with some contribution by UIA-AR (Minneman et aI., 1988; Minneman, 1988;
Simpson et al., 1990; Michel et al., 1990; Knowlton et aI., 1993; Endoh, 1996; Gambassi et aI.,
1998; Brodde & Michel, 1999; Rohde et al., 2000). In rabbit ventricle the UINB-AR pool is 40%
u'A-ARs and 60% UIB-AR, while in rat it is 20:80% (Gross et al., 1988; Takanashi et al., 1991;
Salles et aI., 1994). The ulD-AR numbers and functional impact are small in comparison to UIA-
and UIB-AR (Deng et al., 1996; Yang & Endoh, 1997; Wolff et al., 1998).
The magnitude of the inotropic effect of u-AR activation varies in different ventricular
preparations, with rabbit >rat >ferret > guinea-pig> human » dog (Hartmann et al., 1988;
Hescheler et al., 1988; Hiramoto et aI., 1988; Jakob et aI., 1988; Endoh, 1996) and a similar
sequence is seen for ET-I and angiotensin-II (Endoh, 1996). Figure 156 shows Ca transients and
force during u- and ~-AR activation in rabbit ventricle where the maximal u-AR-induced
inotropy is ~50-60% of the maximal ~-adrenergic response (the same is true for ET-l &
Isoproterenol
a- vs. ~-adrenergic inotropy (~), 30 11 M
[Cal,
Phenylephrine «x)
Control 1 ~M 10 ~M
Force
[~ ]~
200ms
Figure 156. The influence of a-adrenergic activation and ~-adrenergic activation on contractions and Cai
transients (the earlier peaks, measured with aequorin) in rabbit ventricular muscle at 37.5°C, stimulated at I
Hz and equilibrated continuously with I ~M bupranolol (a ~-adrenergic blocker). Several [phenylephrine]
were applied and then washed out. Then isoproterenol was added at a high enough concentration to
overcome most of the ~-adrenergic blockade by bupranolol. Note that the Cal transient in the presence of
isoproterenol was recorded at reduced gain (modified from Endoh & Blinks, 19158, with permission).
angiotensin-II in rabbit ventricle, Endoh & Blinks, 1988; Endoh, 1996). With a-AR activation
the Ca transient amplitude is increased (but less than for ~-AR), and there is no acceleration of
[Ca]j decline (as for ~-AR). Despite the smaller increase in Li[Ca]j with phenylephrine vs.
isoproterenol, the contraction amplitude is comparable. This is consistent with the reduced
myofilament Ca sensitivity classically observed with ~-AR/PKA activation (see Fig 157A).
However, a,-AR does just the opposite (i.e. they increase myofilament Ca sensitivity; see Fig
157B; Endoh & Blinks, 1988). This causes a modest negative lusitropy (slowed relaxation),
again contrasting to the prominent positive lusitropy with ~-AR activation.
Enhanced myofilament Ca sensitivity explains part of the a,-AR inotropic effect, but
higher myofilament Ca affinity would tend to decrease the Ca transient amplitude (since more Ca
would bind). Thus, the increase in Li[Ca]j implies that some other changes must also occur (e.g.
more Ca entry or SR release). Despite early suggestive results (Bruckner & Scholz, 1984) a-AR
activation does not appear to increase lea (Hescheler et al., 1988; Hartmann et aI., 1988; see pg
130). In contrast, a-AR agonists can even stimulate cAMP phosphodiesterase activity, reducing
cAMP (Buxton & Brunton, 1985). Thus, increased lea cannot explain the enhanced Li[Ca]j.
AP duration is typically increased by a,-AR activation in parallel to the positive
inotropic effect (Endoh et al., 1991). This may be due primarily to decreased K-currents. For
example, 1'0 is decreased by a,-AR activation in rat and rabbit (Fedida et al., 1990; Apkon &
Nerbonne, 1988; Ravens et aI., 1989; Williamson et aI., 1997; Homma et al., 2000). This can
prolong the AP, especially in rat myocytes where 1'0 is a critical determinant of AP duration. a,-
AR stimulation also decreases IK , in rabbit ventricle (Fedida et aI., 1991). This would both
prolong AP duration and destabilize diastolic Em, which could increase the likelihood of
triggered arrhythmias. In guinea-pig ventricle which lacks 1,0, al-AR shortens AP duration and
produces a PKC-dependent increase in delayed rectifier K current (Dirksen & Sheu, 1990)
A. ~-AR Reduces MF Ca sensitivity B. <X1-AR Increases MF Ca sensitivity

~1D ~1D
E E
~ 2-
Cl Cl
.5 .5
c: c:
.2l
(; o
...
.2l
.l: .l:
l/) l/)
0.5 0.7 0.4 0.5
[Ca); (F 400/F500)
Figure 157. ~-ARs decrease, while (X,-ARs increase myofilament Ca sensitivity. A. Rabbit ventricular
myocyte contraction was plotted as a function of [Cali for each time point during a twitch (23°C, 0.5 Hz)
Arrows show the direction of time. [Cali rises ahead of shortening, but during the relaxation phase (bold)
the relationship between shortening and [Cali provides a crude dynamic index of myofilament Ca sensitivity
(Spurgeon et al., 1992). Note the right shift induced by isoproterenol (Iso), indicating decrease d
myofilament Ca sensitivity. In control [Ca]o was increased to 8 mM to match peak shortening seen with Iso
(2 mM [Ca]o). B. Phenylephrine (Phe) produces the opposite shift in myofilament Ca sensitivity, and this
effect was blocked by the PKC inhibitor calphostin C ([Ca]o was 4 mM in control, to match contraction with
that in Phe). Data were provided by 1. DeSantiago (DeSantiago et al., 1998).
consistent with PKC-mediated stimulation of l Ks (pg 81; Walsh & Kass, 1991). Thus, <x,-AR
effects may be species-dependent, but the AP prolongation seen in most myocytes would tend to
increase Ca influx (by lea and possibly INa/C.) and also decrease Ca efflux via Na/Ca exchange
(allowing greater SR Ca uptake). This could contribute to the enhanced Ca transients seen with
<Xj-AR, but let's look at the signaling cascade initiated by <Xj-AR activation (Fig 158).
<x-AR activation causes G q to activate phospholipase C (PLC), which splits phospha-
tidylinositol 4,5-bisphosphate (PIP 2) into IP 3 and diacylglycerol (DAG) (Fig 158, Brown &
Jones, 1986; Poggioli et aI., 1986; Jones et aI., 1988; Otani et al., 1988; Scholz et aI., 1988).
DAG activates protein kinase C (PKC), while IP 3 can activate or modulate Ca release. There is
little evidence for IP3 causing enhanced Ca transients in ventricular myocyte (see Chapters 7 &
8). Indeed, most groups find that the <XJ-AR-induced increase in ~[Ca]; and inotropy are
abolished by inhibition of PKC, even though IP 3 production remains (Endoh, 1996; Gambassi et
aI., 1998; DeSantiago et aI., 1998). My current speculation (pg 243 & Fig 158) is that IP 3
activates SR/ER Ca release near (or in) the nucleus (without impact on global rCa];) and that this
may activate nearby CaMKII selectively. This may explain the apparent link between CaMKII
activation and transcriptional regulation in the hypertrophic phenotype pathway (Ramirez et al.,
1997). Thus, IP 3 may not be involved in the inotropic effects of <XJ -AR activation.
PKC activation in response to <Xj-AR activation stimulates Na/H exchange to extrude
protons (Wallert & Fr6lich, 1992). This Na/H exchange causes both intracellular pH and [Na]; to
rise (Gambassi et al., 1990, 1998). The alkalinization is mainly responsible for the increased
myofilament Ca sensitivity (see Chapter 2, Fig 157), and the rise in [Na]; contributes to the
increase in Ca transients via Na/Ca exchange (see next section). Thus, <XJ-AR activation
a-Adrenergic Regulation
(fill Na Ca
J.,I & iAPD---7iCell Ca
Na Ca K
ipH & MF
Ca sensitivity
::::::: :::::::
Increased MCa);
& MF sensitivity
Figure 158. (XI-AR transduction pathway in ventricular myocytes. The (Xj-AR activates the G-protein Gq,
which activates phospholipase C (PLC) and D. PLC produces IP 3 and diacylglycerol (DAG) and these
products have divergent effects leading to positive inotropy and hypertrophy (see text).
increases both L'>[Ca]; (less than ~-AR) and myofilament Ca sensitivity, without accelerating Ca
transport. The result is a positive inotropy, with modest negative lusitropy.
(Xl-AR agonists, endothelin and angiotensin-II can also exert negative inotropic effects,
resulting in sometimes bi- or triphasic changes in force upon application. These effects may be
mediated by stimulation of Na/K-ATPase (pg 90), IKs (shortening AP duration, above), IK(Ach)
(Kurachi et al., 1989). In rat ventricular myocytes phenylephrine caused acidification when (Xl A-
ARs were blocked (rather than alkalinization seen with phenylephrine alone or when (X1B-AR are
blocked, Gambassi et al., 1998). Thus (XIB-AR may cause a decrease in pH; (still PKC-
dependent) that limits the increase in pH; induced by activation of the (XIA-AR. PKC can also
phosphorylate the PKA sites on TnI (Ser23 & Ser24 , which reduce myofilament Ca sensitivity)
and also Ser42 & Ser44, which reduce myofilament ATPase rate and unloaded shortening in
response to (XI-AR activation (Noland et al., 1995, 1996; Strang & Moss, 1995). PKC has
multiple isoforms and other targets in heart (including other myofilament proteins, channels and
transporters). These effects help explain multiphasic responses sometimes seen with (X]-AR, ET-
I and angiotensin-II. These agents also stimulate a hypertrophic signaling cascade (see pg 314).
Physiological norepinephrine (and epinephrine) release activates both (X- and ~-ARs, but
the inotropic effect is mediated mainly by ~-adrenergic activation. However, in species with a
prominent (X-adrenergic response (e.g. rabbit), up to one-third of the inotropic response to
norepinephrine can be attributed to (Xj-AR activation (Aass et aI., 1983; Nawrath, 1989; Endoh,
1996; Brodde & Michel, 1999).
Ca-Calmodulin dependent protein kinase (CaMKJI)

The previous sections have described how PKA and PKC modulate contractility.
CaMKII is modulated by changes in [Cali (associated with altered frequency or inotropic states).
Little is known quantitatively about dynamic changes in activation state of CaMKII during the
cardiac cycle. Indeed, one may expect Ca-CaM to change during the Ca transient (Fig 29), which
may activate CaMKII in a cyclical manner. However, activated CaMKII also becomes auto-
phosphorylated, and thus stays active in an autonomous manner, independent of [Cali (Braun &
Schulman, 1995; De Koninck & Schulman, 1998). This gives the CaMKII system memory and
the ability to integrate [Cali signals (e.g. increasing activation state with increasing frequency,
see pg 271).
Local [Cali near the mouth of the L-type Ca channel causes inactivation by binding to
calmodulin (pg 117), and the frequency-dependent facilitation of lea appears to require CaMKII
(pg 119). Thus, CaMKII activation can increase lea. Phospholamban is also phosphorylated by
CaMKII and this can stimulate the SR Ca-ATPase (pg 167) and CaMKII can thus increase SR Ca
content. The ryanodine receptor (RyR) is also phosphorylated by CaMKII, and both calmodulin
and CaMKII alter RyR gating in a complex manner, even in isolation in bilayer studies (Pg 198).
To assess whether CaMKII has any intrinsic effect on E-C coupling, we used voltage clamped
ventricular myocytes and controlled SR Ca load (keeping it constant) and measured SR Ca
release as a function of lea (Li et al., 1997b). We found that the CaMKII inhibitor KN-93
prevented a Ca-dependent increase in SR Ca release for the same SR Ca load and lea trigger.
That is, if the conditioning pulses did not elevate [Cali very much, there was no effect of KN-93.
Thus, endogenous CaMKII may have stimulatory effects on lea, SR Ca uptake and E-C coupling,
and could contribute to dynamic regulation during changes in heart rate and inotropic state.
Cardioactive Steroids: Glycoside lnotropy
Digitalis is the oldest cardiac inotropic agent. Withering (1785) described its use in
heart failure, known then as dropsy, and the related cardioactive steroids are still among the most
efficacious inotropic agents. Since ouabain and digitalis glycosides were recognized as specific
Table 25 compiled with help of M.T. Ziolo, includes the following abbreviations: V=ventricle; A=atrium;
L=left; R=right; HF= heart failure, DCM=dilated cardiomyopathy, ICM= ischemic cardiomyopathy,
G-Pig= guinea-pig CGP=CGP- I2177; ICYP=iodocyanopindolol, isradipine=PN200-110, QNB,
quinuclidinyl benzilate; SL, sarcolemma; E-P04 , Phosphoenzyme.
* SL Bmax values for saxitoxin (678 fmol/mg) and ouabain (365 pmol/mg) were divided by the purification
factor (35x & -60x respectively). Colvin et al., (1985) & Doyle et al. (1985) estimated SL ouabain
and saxitoxin site density at330 and 3.6-7.6/llm2 respectively, using different assumptions.
t Assuming 120 mg homogenate proteinlg wet weight.
t Bmax are from microsomes. Extrapolation to SL surface density uses 2-fold enrichment, 120 mg pnl g wet
wt, 2.43 g wet wt/L cytosol, 7 pF/pL and I Cm2/IlF (or fmol/mg x 0.125, see pg 6 & 42). for DHPR
2
and RyR 109 mg cell Pnl JIll cell volume wa~ used. For the SR Ca-pump 1.96 11m SRlllm was used,
and for the RyR 0.18 11m junctional ~Rlllm cell was used (Table 1). Maximal possible density for
27 nm square RyRs would be 1400/llm and for a 12 nm wide SR Ca-ATPase -6000/llm2
a Endohetal., 1991 j Brodde & Michel, 1999 r Nozawa et aI., 1994
b Anthonio et al., 2000 k Steinfath et aI., 1992 s Regitz-Zagrosek et aI., 1995
c Maurice et al., 1999 1 Salles et al., 1994; t Doyle et al., 1985
d Tse et al., 2000 Gross et al., 1988 u Colvin et al., 1985
e B6hm et al., 1990 m Takanashi et al., 199 I v Cheon & Reeves, 1988
f Brodde, 1996 n Kobayashi et aI., 1999 w Bers& Stiffel, 1993
g Rohrer et al., 1996 o Pieske et al., 1999b x Levitsky et al., 1981
h Musser et al., 1993 p Vatner et aI., 1988
i Kompaetal., 1995 q Ishihata & Endo, 1995
Chapter 10 Cardiac 1notropy & Ca Mismanagement 287
Table 25
Hormone receptors and Ion Transporters in Cardiac Muscle
Kd B max Density
Ligand (nM) (fmol/mg) (/~mz)
B-adrenergic receptors a P,/(P, + pz)
Rat LV CGP 0.83 60 8 60%b
Rabbit LV 1.1 133 a 17 77%~
OogLV 2.5 125 a 16 72%f
Human V 0.52 100e 13 73%f
Human V (OCM) 0.57 31 e 3.2 57%f
Human V (lCM) 0.58 41 e 3.8 75%
g 72%g
Mouse V lCYP 008 50 h 6.5
G-PigY 0.01 59 8 93%1
a-adrenergic receptors a, B/(a'Ma'B) a-ARIp-AR
Rat LV prazosin 0.14 112 a 14 80%1 1.86 a
Rabbit LV " 0.25 19 a 2.4 60%m 0.15 a
OogLV 0.26 26 a 3.2 0.21 a
Human Y lOJ 1.3 0.1
Human Y (HF) 0.063 II k 1.4 0.29
Mouse Y 0.05 15 k 1.9 0.3
G-Pig Heart 0.05 16 k 2 0.27
Endothelin-l ET A/(ET A+ET B)
RatV ET-I 0.030 155 " 19 91%"
Rat V (HF) 0.029 243 0" 30 86%"
Human 0.013 63 0 8 63% 0
Human (HF) 0.028 122 15 73%0
Muscarinic CM zl
Oog Q~B 0.12 157 P 20
Oog (HF) 0.13 121 P 15
Human 0.58 275 e 34
Human (OCM) 0.5 201 e 25
e
Human (lCM) 0.47 24g 31
Angiotensin II ATzI( AT,+ AT z)
Rat, ferret A~FIl 1.5 21,37 q 3-5
Rabbit, dog 1-2 37,76 q 5-9
Human V 1.1 6' 0.7 67%'
Human RA 0.5 II 5 1.4 69%5
5 64%5
Human RA (HF) 0.7 4 0.5
Adenosine (A'L NY
Rat Y DPCPX 1.5 12 h 1.4 1.5~
h
Rabbit V 1.9 9 1.1 1.7 h
G-Pig 3 34 h 4.3 2.2
Na Channel t
Sheep V Saxitoxin* 0.22 20 3
Na/K-ATPase CSL)
u
Bovine Y Ouabain* 33 6000 1032
Na/Ca exchanger
OogV Rate 1087 v 187
OHPR (SL Ca Channel)
w
Rabbit Y my isra~~pine I 137 16
Rat Y my I 114 w 14
G-PY my I 151 w 18
Ferret Y my 2 112 w 13
RyR CSR Ca release channel) RyR/OHPR
Rabbit V my Ryan,?dine 3 504 w 184 4
Rat Y my 4 833 w 304 7
G-P Y my 2 656 w 240 5
Ferret Y my 4 J 144 w 418 10
SR Ca-ATPase
G-P V E-PO.1 49,000x 2600
10
8
•
6
e •
5 •
0 OJ
OJ
E- o
o· E-
c
0
4
oil • c
0 1.0
'iii
'iii 3 0.8
•
0 C
C o. 2 0.'
2 o. s:;
s:; 2 o· .8
0.4
.8 e .§
§ I- 0.2
l- I
0 0.1
8 9 10 11 2. 345678')10 20 3040~
a~a (mM) a~a (mM)

Figure 159. Twitch tension in dog Purkinje fiber depends on aNai (measured with Na-selective micro-
electrodes, 36°C, 1 Hz). At left, measurements were made during the onset (.) and washout (0) ofinotropy
induced by I J.lM strophanthidin in one fiber. The right panel shows data from the same fiber (x) and from
3 other fibers on a log-log plot (from Lee & Dagostino, 1982 and Lee, 1985, with permission).
inhibitors of the Na/K-ATPase (Glynn, 1964; Skou, 1965), it has become increasingly clear that
this action is primarily responsible for the positive inotropic effects (as well as negative inotropic
and arrhythmogenic effects which limit glycoside utility, see pg 294-300). There were
indications that very low glycoside concentrations could stimulate Na/K-ATPase, and reduce
[Na]j, while still producing inotropy (Blood, 1975; Cohen el ai., 1976; Godfraind & Ghysel-
Burton, 1977; Noble, 1980). However, this may be secondary to autonomic catecholamine
secretion and stimulation of ~-ARs (Hougen el ai., 1981). The powerful cardiovascular effect of
Na-pump inhibitors and other results fueled the search for, and discovery of endogenous ouabain
(a natriuretic factor, which may contribute to hypertension; Hamlyn el al., 1991, 1996). Here, I
will focus here on the mechanisms by which glycosides lead to cardiac inotropy and Ca overload.
Inhibition of the Na-pump by strophanthidin or acetylstrophanthidin (ACS, rapid acting
& reversible) increases aNa; and contractility (Figs 159 & 161). Indeed, the aNal-dependence of
twitch force can be very steep, particularly in cardiac Purkinje fibers where force can double
with ~ I roM rise of aNa; (with Hill coefficients of 3-6, Lee & Dagostino, 1982; Wasserstrom el
al., 1983; 1m & Lee, 1984; Eisner el al., 1984). In ventricular muscle the relationship is less
steep, probably because of a ceiling effect. That is, in rabbit ventricle at ~29°C and 0.5 Hz,
control twitches are ~40% of the maximum myofilament force (Harrison & Bers, 1989a).
Glynn (1964), Repke (1964) and Langer (1965) first suggested that reciprocal Na and Ca
movements might be involved in this inotropic effect. This was before Na/Ca exchange was
demonstrated, after which this hypothesis was more clearly stated (Reuter & Seitz, 1968; Baker
et al., 1969; Langer & Serena, 1970). Now it is quite clear that relatively small increases in [Na];
can have a large impact on the balance of Ca fluxes mediated by Na/Ca exchange. For example,
an increase of [Na]; from 9 roM to 12 roM would shift the reversal potential for Na/Ca exchange
(ENalca> by -30 mY; see Fig 72). This will tend to increase Ca influx via Na/Ca exchange during
the AP and also limit Ca extrusion via Na/Ca exchange during relaxation and diastole. The result
Diastolic [Cali rise is Intrinsically Inotropic

E
:::l
80
E
'xIII 60
:=
i----------------
I
I ~144% inotropy
0~
- 40 I
c: I
~65% inotropy
0
'iii I
c:
Q)
20
I
I
I
I- I
I
A B I
0
0 20 40 60 80 100
Activating A[Caltotal (IJmol! I cytosol)
Figure 160. Increased diastolic [Cali increases developed tension, for the same amount of activating Ca.
Tension-dependence on total cytosolic Ca ([Caho,), where Force=100/(1+(600/[Ca]l) and [Cahot=
(244/(1+673/[Ca];»-44.5. A, Band C ([Caho'=O, 10 & 20 flmol/L cytosol) correspond to diastolic [Cali
levels (150, 193 & 241 nM) and A', B' and C' correspond to tension and [Caho! reached when 50 flmol/L
cytosol is added to A, Band C respectively ([Ca]; = 425, 504 & 595 nM).
is that resting [Cali can increase (Lee et al., 1980; Marban et aI., 1980; Bers & Ellis, 1982; Sheu
& Fozzard, 1982; Allen et al., 1984a; Weingart & Hess, 1984; Wier & Hess, 1984). Let's
consider the impact of elevated diastolic [Cali on contraction, if all other things are equal.
Figure 160 shows how force depends on total cytoplasmic Ca (see also Fig 28). In this
case, three diastolic rCa]; values are indicated: control (A=150 nM) and values expected during
inotropic effects of Na-pump inhibition (B=193 nM & C=241 nM), which are at or below the
threshold for myofilament activation. If we assume that activation adds a "bolus" of 50 Ilmol
CalL cytosol, then A goes to A', B to B' and C to C' (peak rCa]; ~600 nM). Thus, increasing
resting [Cali from A to either B or C would increase developed force (by 65 or 144%), despite an
exactly constant 6[Ca]'ot and a 6[Ca]; increase of only 13 and 28%. Therefore increased diastolic
rCa]; can contribute significantly to the inotropic effect of cardiac glycosides.
The rise in [Na]; and shift in ENaiCa will limit the ability of the Na/Ca exchange to
compete with the SR Ca-pump during relaxation, thereby increasing SR Ca uptake. Along with
the higher diastolic and mean rCa]; under these conditions, higher SR Ca load is expected.
Indeed, SR Ca content assessed by RCCs is increased with Na-pump inhibition (Fig 161B). Of
interest, the RCC amplitude remains high at high ACS concentration, despite a progressive
decline in the amplitude of twitch contractions. This negative inotropic effect of high concen-
trations of cardioactive steroids will be addressed below (pg 294-300). The increase in SR Ca
content with glycosides results in greater SR Ca release (Wier & Hess, 1984; Allen et aI.,
1985b), and contributes to the glycoside inotropy. Indeed, some investigators attribute the entire
glycoside inotropy to an increased SR Ca release (Morgan, 1985; Akera, 1990). While SR Ca
loading is likely to be a dominant contributor to the inotropic effect, it may not be that simple.
20 A. Na-pump block raises aNa; B. Na-pump block increases SR Ca

e;: 160
iO o
::ii 15 u 140
c'" C
~ l:3 120
~ 10 c::
::; 100
.J::.
U
:t::
5-'---.-~---,~---,~~r-~,-K,-,;m_-_6r~~M ~ 80+-~---,r-~--,-~....,---'-,-~--.
o 2 4 6 8 10 o 2 4 6 8 10
[Acetylstrophanthidin] (~M) [Acetylstrophanthidin] (~M)
Figure 161. Effect ofNa-pump inhibition by acetylstrophanthidin (ACS) on aNa;, twitch tension and SR
Ca content in rabbit ventricular muscle (30°C, 0.5 Hz). A. Resting aNa; measured with Na-selective
microelectrodes (data from Shattock & Bers, 1989). B. Steady state twitch tension and RCCs induced
immediately after a twitch (to assess SR Ca content; data from Bers & Bridge, 1988).
For example, if the SR is inhibited by caffeine or ryanodine, the inotropic effect of ACS
can be just as large as under control conditions (Fig 162). While increased SR Ca load is surely
important in glycoside inotropy, this result demonstrates that the inotropy can still occur without
a normally functioning SR. Thus, changes in diastolic [Cali and transsarcolemmal Ca fluxes may
also contribute to the glycoside inotropy.
Na-pump inhibition and elevation of [Na]; are expected to favor Ca influx via Na/Ca
exchange and make Ca extrusion less favorable. Under normal conditions the amount of Ca
influx via Na/Ca exchange is insufficient to activate appreciable contraction (pg 150, Fig 74).
This can also be appreciated in Fig 163, where the Ca channel antagonist nifedipine virtually
abolished twitch force under control conditions (and in the presence of caffeine or ryanodine).
However, when [Na]; is increased by ACS (with the SR suppressed by caffeine or ryanodine)
contraction is much less sensitive to nifedipine, despite a large decrease in AP duration (Fig
163). Increasing AP duration back to control under these conditions also returned twitches to
c--,,-- 5min ~
J~
C;; 2 0 0 ' - - - - - - - - - - - - - - ,
V
e
:l
...o Co
150
(1-,,-UUU J~
Caffeine ,---,
C
g 50
'iii
c
CI>
Acetylstrophanthidin (4 ~M) I- ACS Catt Catt ACS Ryan Ryan
+ACS +ACS
Figure 162. Acetylstrophanthidin (ACS) increases twitch force, even with caffeine or ryanodine. Rabbit
ventricular muscle (at 30 D C, 0.5 Hz, 4 flM ACS). After washout, and equilibration with 10 mM caffeine,
ACS was applied again. The ACS-induced increase in force was similar in control and after treatment with
10 mM caffeine or 500 nM ryanodine (right, adapted from Bers, 1987b, with pennission).
Chapter 10 Cardiac 1notropy & Ca Mismanagement 291
~[
~$~'\
~ ,
Caff +ACS
+ACS
+Nif
250ms
Figure 163. The Ca channel blocker nifedipine nearly abolishes control twitch tension, either by itself
(not shown) or in the presence of 5 mM caffeine (top). However, after Na-pump inhibition (by ACS),
nifedipine does not abolish tension, despite reduction in AP duration (right). Experiments were with rabbit
ventricular muscle at 30°C, stimulated at 0.5 Hz (adapted from Bers et aI., 1988, with permission).
control levels (Bers et al., 1988). This indicates that when [Na]; is elevated, enough Ca can enter
the cell via Na/Ca exchange to activate contraction directly. It was also suggested that Ca influx
via Na/Ca exchange can trigger SR Ca release (Bers et aI., 1988; Leblanc & Hume, 1990).
While this triggering action is still controversial (see Fig 71,74 & pg 232-4), it is clear that with
high [Na]; large amounts of Ca can enter the cell via Na/Ca exchange. But does Ca influx via
Na/Ca exchange increase measurably with Na-pump inhibition?
I studied the influence of ACS on sarcolemmal Ca fluxes in rabbit ventricle using extra-
cellular Ca microelectrodes (Bers, 1987b). ACS sometimes increased Cao depletion (or net Ca
uptake) in a simple manner (Fig I64A). At other times ACS increased the initial Cao depletion
rate, but then gave way to net Ca efflux during the contraction (Fig 1MB, trace 2). This may
have the same cellular basis as the Ca efflux observed during twitches in rat ventricle (Fig 139),
or upon abrupt application of low [caffeine] (Fig 146). That is, the large SR Ca release
(contraction was 225% of control in Fig 1MB) and the shorter AP duration in ACS may
stimulate Ca extrusion via Na/Ca exchange, analogous to rat ventricle in Fig 140 (see Fig 164C).
The fact that Ca efflux occurs during contraction and probably overlaps temporally with
Ca influx, makes it ha:'d to assess changes in unidirectional Ca influx. To limit this problem, I
studied the effect of ACS on Ca influx under conditions where Ca efflux is initially minimized
(Fig 165). This occurs at the first few post-rest contractions in the presence of ryanodine (as
discussed on pg 183-5) or after SR Ca depletion (Fig 145). Ryanodine causes rapid SR Ca
depletion at rest, which is reflected by the rapid rise in [Ca]o when stimulation is stopped in Fig
165C vs. A. Thus, during the first post-rest contraction, the SR begins empty and may
accumulate much of the Ca which enters the cell. This post-rest contraction is small and the low
rCa]; will not stimulate much Ca efflux via Na/Ca exchange. Therefore only Ca influx is
B. \
A. 0.4 mV [ Control ~\:;;.:::::
to' 'V
. ./
ACS (2 min)
'F\:
ACS
(6mln)"
ACS (5 min) \'''~... 5.6
Recovery l...a...i!tl',~~',',,'''--_",=
(6 min) t'l"'''~
ACS (10 min) ""t'~, 7.1
,~",."
Recovery ,.....,"""',';c_---- Recovery

(20 min)
t~i\,' " , . '~'(BI
(40 min) " ..............,.•..-.... 4.3 ~
L-......J
l.---J
200ms
200 ms
Figure 164. Extracellular Ca depletions in rabbit ventricle measured with Ca-selective microelectrodes
(30°C, 0.5Hz, [Ca]o=0.3 mM). A. Na-pump inhibition (by ACS) increased the L'>Cao (see numbers at right),
reflecting more Ca influx. B. ACS increased the initial rate of Cao depletion, but then Ca started to come
out of the cells, indicated by the increase in [Ca]o (from Bers, 1987b, with permission). C. Conceptualized
explanation of differences in A and B, based on driving forces as in Figs 72 & 140.
appreciable during the first two post-rest contractions and Cao depletion occurs without recovery
between beats (Fig 165C or E). During the first few pulses, the amount of Cao depletion
increases at each beat, consistent with the lea staircase (Fig 58) and possibly more Ca entry via
Na/Ca exchange.
Addition of ACS in control (Fig 165B) prevents SR Ca loss during rest (i.e. slows rest
decay) and no slow rise in [Cal o is seen. Since the SR and cell did not lose Ca during rest, the
post-rest contraction is large, and no cumulative Cao depletion occurs (i.e, the SR is already Ca
loaded). With ACS in the presence of ryanodine (Fig 165D), the SR is emptied rapidly during
rest, although more slowly than with ryanodine alone (Fig 165C). ACS slows the ryanodine-
induced SR Ca loss in rabbit ventricle (assessed by RCCs; Bers & Christensen, 1990).
With the background above we can now compare the effect of ACS on Ca influx in Fig
Control Ryanodine (100nM), Steady State

A
Pre- 'mv(~~ ~r-----'
ACS,mN[~ ~I.W.
~---
ACS
(5 ~M)
LmJillJJJ 8 sec
2mtn 10Sec
Figure 165. Acetylstrophanthidin (ACS) increases twitch force, even with caffeine or ryanodine. Rabbit
ventricular muscle (at 30°C, 0.5 Hz, 4 ~M ACS). After washout and equilibration with 10 roM caffeine,
ACS was applied again. The ACS-induced increase in force was similar in control (A vs. B) and after
treatment with 500 nM ryanodine (C-F; adapted from Bers, 1987b, with permission).
Chapter 10 Cardiac lnotropy & Ca Mismanagement 293
165E vs. F, where Ca efflux has been minimized. At the first post-rest contraction, ACS greatly
increases the initial rate and extent of Cao depletion (-6- and 2-fold, respectively). As steady
state is approached (where Ca influx = Ca efflux over each cycle), both Ca influx and Ca efflux
appear to be increased by ACS. The greater Ca influx is via Na/Ca exchange favored by the
elevated [Na]i' The greater efflux results from the increased peak [Cali during the twitch which
drives Ca efflux via Na/Ca exchange (and overcomes the shift in ENaiCa favoring Ca influx).
Ryanodine helps to temporally separate Ca influx and efflux phases since peak [Cali occurs later.
This allows us to conclude that Na-pump inhibition can lead to increased Ca influx and efflux
during the AP (although Pizarro et al., 1985 did not detect strophanthidin effects on Ca depletion
in frog ventricle). Le Grand et al. (1990) showed that ouabain increases both L- and T-type Ca
currents. It is possible that the higher average [Cali in the presence of Na-pump inhibition
contributes to this increase of lea as suggested by Marban & Tsien (1982). This lea facilitation
may be related to the lea staircase phenomenon shown in Fig 58.
Two additional Na/Ca exchange-independent modes of glycoside inotropy have been
proposed. First, ouabain has been shown to increase the open probability of single RyR channels
in lipid bilayers (McGarry & Williams, 1993; Rardon & Wasserstrom, 1990). However, this
might be expected to only produce a transient elevation of SR Ca release as seen for low caffeine
concentrations (Fig 146). Second, it has been suggested that ouabain or digoxin changes the
selectivity of cardiac Na channels, such that they become equally permeant to Ca ions (the so-
called slip-mode conductance, Santana et aI., 1998; see pg 235). This would increase the Ca
influx signal in CICR and enhance SR Ca release. Although these two results are intriguing, I
am skeptical that either of these pathways contribute significantly to glycoside inotropy, for the
following simple reason. If cells are depleted of [Na]i and studied in completely Na-free
solutions, we find that glycosides have no effect at all (transient or otherwise) on contractions
(under conditions where inotropy is seen in normal Na-containing solutions, Altamirano et al.,
1999). Thus, glycoside inotropy appears to depend on the presence ofNa, and is presumably due
to Na/K-ATPase inhibition and consequent alterations of [Na]i and [Cak
There are intriguing data indicating that Na/K-ATPase isofonns are differentially
distributed on the sarcolemma of cardiac and smooth muscle cells (McDonough et al., 1996;
Juhaszova & Blaustein, 1997). In rat ventricular myocytes, the <x,-isoform is preferentially
localized to T-tubules, while the <Xrisofonn is ubiquitous. In smooth muscle the low ouabain-
affinity <x,-isoform is ubiquitously distributed, while the higher ouabain-affinity <Xr and <X)-
isoforms are preferentially localized in regions overlying the SR. Ouabain augmented Ca
transients without raising global [Na]i in smooth muscle (Arnon et aI., 2000). This work
indicates that local junctional microdomain [Na]i and [Cali may be controlled more by local
Na/K-ATPase and Na/Ca exchange molecules. Su et al. (2001) also showed that abrupt Na/K-
ATPase inhibition in guinea-pig ventricular myocytes increases the efficacy of a given lea to
trigger SR Ca release. Thus, a nonnally functioning Na/K-ATPase may maintain [Na]i and [Cali
in the junctional cleft at lower values than bulk cytosolic, and this may limit CICR. Goldhaber et
al. (1999) found that abrupt [Na]o removal increased resting Ca spark frequency, attributing this
block of tonic Ca removal from the junctional cleft by Na/Ca exchange (although Ca entry via
Na/Ca exchange may have contributed to this effect).
Glycosides limit Ca efflux via Na/Ca exchange and also favor Ca influx via Na/Ca
exchange. It is hard to distinguish which mode of transport is causing inotropy. Satoh et al.
(2000) used KB-R7943 to selectively block outward INa/c. (Ca entry). KB-R7943 did not prevent
the inotropic effect of strophanthadin, but prevented the spontaneous activity characteristic of
glycoside toxicity and cellular Ca overload. We suggested that slowing Ca extrusion via Na/Ca
exchange is sufficient for the inotropic effect, but that Ca overload and toxicity occur when [Na]j
rises to the level where net Ca influx occurs via outward INa/c. (blocked by KB-R7943).
In conclusion, glycoside inotropy can be attributed primarily to Na-pump inhibition and
consequent shifts in Na/Ca exchange, making Ca influx more favorable and Ca efflux less
favorable. During individual contractions this can occur by increasing diastolic [Ca]j, increasing
SR Ca content (and release) and increasing Ca influx early in the contraction. These are, of
course, all interrelated and it is difficult to determine unequivocally the fractional contribution of
each effect. When the cell gains too much Ca due to the shift in Na/Ca exchange, negative
inotropic and arrhythmogenic effects occur. These will be discussed in the next section.
Ca MISMANAGEMENT AND NEGATIVE INOTROPY

Ca Overload and Spontaneous SR Ca Release
At high glycoside concentrations the positive inotropic action gives way to a negative
inotropic effect (Figs 161 Band 166). Other toxic effects of glycosides also become apparent: I)
elevated resting force, 2) oscillatory after-contractions and 3) oscillatory or delayed
afterdepolarizations (DADs, Fig 166). It now seems clear that all of these effects are secondary
to cellular Ca overload and spontaneous Ca release from the SR during diastole.
Fabiato & Fabiato (1972) observed spontaneous cyclical contractions due to SR Ca
release in skinned rat ventricular myocytes when bathing [Ca] was> 100 nM. In intact rat
ventricle Lakatta & Lappe (1981; Lappe & Lakatta, 1980) reported scattered light intensity
fluctuations (SLIF) attributed to spontaneous SR Ca release-induced local contractions, and these
were enhanced by high [Ca]o or ouabain. Resting rat ventricle often shows spontaneous SR Ca
release, SLIF and a high Ca spark frequency at rest, whereas rabbit ventricle only shows them at
high [Ca]o or with Na-pump inhibition (Kort & Lakatta, 1984, 1988a,b; Capogrossi & Lakatta,
1985; Capogrossi et aI., 1986a; Diaz et al., 1996, 1997a; Satoh et al., 1997). This is consistent
with rat ventricle having high resting SR Ca load, because resting [Na]j is high (Chapter 9).
Furthermore, Ca sparks, spontaneous oscillations and SLIF in rat ventricle are suppressed for
several seconds after a stimulated synchronous contraction.
In intact single cells spontaneous SR Ca release events can propagate as Ca waves,
especially when SR Ca load and [Ca]j are very high (see pg 230-2). Local [Ca]j during a wave is
comparable in amplitude and kinetics to that during a twitch (Fig 122). However, when whole
cell [Ca]j is measured, the tl[Ca]j amplitude appears to be smaller and slower, because of the lack
of spatial uniformity. While the negative consequences of these Ca waves will be discussed
below, there is also a beneficial effect. The high [Ca]j and negative Em drives Ca extrusion from
the cell via Na/Ca exchange, and this unloads the cell and SR of Ca. Diaz et al. (1997a) found
that Ca waves occur at a threshold SR Ca load of ~ 100 I!mollL cytosol, and that a typical Ca
wave in rat ventricular myocytes causes extrusion of ~ 15 I!mol/L cytosol (and we find similar
Control
~,~uabain (100 nM~
Em (mv~1
-sJ
~
[Caj1ar
L1Lm~xoL nA
10[
O ,k.....'. "'" '. *~
3 mgt J""-. _
100ms
Figure 166. Ouabain-induced delayed afterdepolarizations (DADs) and aftercontractions in a canine

cardiac Purkinje fiber at 35°C, I Hz. Addition of the Na-pump inhibitor decreased AP duration, increased
the Ca transients (assessed by aequorin luminescence) and increased contractile force. Diastolic force
eventually rises and developed force declines as DADs and aftercontractions become more apparent
(modified from Wier & Hess, 1984, with permission).
values in rabbit). This allows SR Ca load to fall below threshold. Indeed, if Ca extrusion via
Na/Ca exchange is prevented the cell will continue to oscillate (as observed in skinned cells
where the Ca has nowhere to go). Once the cell has extruded this -15 flmol/L cytosol, it may
then take several seconds for cellular Ca to increase back to the point where spontaneous Ca
release occurs again. With higher Ca overload this delay can be abolished and SLIF, Ca waves
and Ca sparks can be elevated immediately after the twitch (Kort & Lakatta, 1988a; Diaz et aI.,
1996; Satoh et al., 1997). Caffeine and ryanodine abolish both the [CaJj and tension fluctuations
in all of these cases, confirming that the SR is the source.
The functional consequence of spontaneous SR Ca releases are serious at both the
cellular and tissue level (and both mechanically and electrophysiologically). When a stimulated
twitch occurs soon after a spontaneous release, the stimulated Ca transient and contraction are
depressed (Allen et al., 1985b; Capogrossi et al., 1986b; Capogrossi & Lakatta, 1985). The
weaker Ca release can result from a combination of incomplete mechanical restitution (e.g.
refractoriness of the SR Ca release channel, Fig 122, Ishide et al., 1990) and a net loss of Ca
from the cell (as discussed above). In multicellular preparations such spontaneous Ca release can
occur in random cells, but as the number of Ca overloaded cells increases a progressive decline
in contractile force is expected. The negative inotropic effect in multicellular preparations is
much more severe than this simple additive expectation. This is because the cells are not
independent, but linked mechanically in series. A weakly activated cell will have high
compliance and be stretched by more fully activated cells. Such a fully activated cell will also
produce less force and shortening velocity as a consequence of its shorter sarcomere length.
Additionally, the shortening that was required to stretch the more compliant cell will not
contribute to external work. Thus, the negative consequences of these inhomogeneous
spontaneous Ca releases are greatly amplified. These spontaneous contractions can limit the
inotropic effect of increasing Ca load (e.g. by cardioactive steroids, reduced [NaJo, elevated [CaJo
etc.). This also emphasizes the importance of the normal syncytial behavior of cardiac muscle.
While twitch force falls in Fig 161B at high [ACS], RCC amplitude is not reduced. This
may be due to synchronous SR Ca release at an RCC and the fact that RCCs reflect the sum of
SR Ca released plus cytosolic Ca. The plateau RCC amplitude at high [ACS] in Fig 161B may
be because mean cellular Ca (SR + cytosolic) does not decrease. Progressive Ca loading may be
expected, but could be limited by Ca extrusion during local Ca releases when [Cali is high.
As discussed in Chapter 8 (pg 230-2), the mechanism for these spontaneous SR Ca
releases and waves in cardiac myocytes is probably via propagated Ca-induced Ca-release
(Mulder et aI., 1989; Backx et aI., 1989; Takamatsu & Wier, 1990; Lukyanenko et aI., 1999).
Both elevated [Cali and high [Ca]sR contribute to the propagation of waves. Ca waves in single
cells can readily propagate through the whole cell (at -100 fLm/S), but in multicellular
preparations they generally do not propagate through gap junctions from one cell to the next
(Wier et al., 1997; Lamont et aI., 1998; Kaneko et aI., 2000). In intact beating rat heart, Kaneko
et al. (2000) found no diastolic Ca waves, unless the heart rate was slowed. Then sporadic Ca
waves at low frequency were seen (4 /cell/min), with only 6% propagating to a neighboring cell.
Raising [Ca]o from 2 to 4 or 6 mM increased wave frequency, even during the diastolic interval
during I Hz pacing. Some regions with higher average [Cali produced "Ca-overload" waves with
much higher wave frequency (28 /cell/min) and 23% propagated to neighboring cells. This may
especially enhance arrhythmogenesis (see below). A third variety (agonal waves) occurred in
severe Ca overload regions (133 waves/cell/sec). These appeared to immediately precede cell
death, and the wave propagation to other cells was again lower (9%), perhaps due to reduced gap
junction conductance at high local [Cali and low pHi. Thus, three types of Ca waves were
observed (sporadic, Ca-overload and agonal), and they may contribute differently to
arrhythmogenesis and contractile dysfunction. All of these waves propagated at 80-] 20 fLm/S.
Higher rates of wave propagation (200-8000 fLm/S) have been seen in trabeculae, but these rates
may well require a concomitant mechanical or electrophysiological component of propagation (in
addition to CICR).
Spontaneous SR Ca releases can be relatively synchronized among cells right after
electrical stimulation and can produce the aftercontractions and delayed afterdepolarizations
(DADs) which are commonly associated with Ca overload and digitalis toxicity (see Fig ]66).
Synchronization is probably because the Ca release events are initiated by SR Ca uptake during
twitch relaxation, when a point is reached where the combination of SR Ca load, [Cali and
recovery from refractoriness initiate SR Ca release (which can be similarly timed in many cells).
Thus, the initial Ca release and wave occurrence can be relatively synchronous in many cells.
However, at subsequent spontaneous releases the cells vary as to the timing, amount of SR Ca
release, wave duration and the amount of Ca that was extruded by Na/Ca exchange. Then
whether a second wave occurs (and at what time) will be much more variable from cell to cell.
The result is that a second aftercontraction is usually smaller and broader in time course.
This progressive decline in amplitude of oscillatory aftercontractions (see Fig 166) may
be partly due to desynchronization. It may also be due to the progressive decrease in SR Ca load.
That is, fewer cells continue to oscillate and those that do will produce smaller Ca transients. If
Na/Ca exchange is prevented from extruding Ca, oscillatory aftercontractions can continue with
minimal decrement, and this same situation can be seen in skinned cells where Ca efflux cannot
OmY- -OmY
t---f f----l
100 msec
r-::--
100 msec
101'mI~V r 1oo~msec
101'~r-:-
"1 V 100' ';'sec
Spontaneous Twitch Stimulated Twitch

Figure 167. Membrane potential and cell length during a twitch resulting from a multifocal spontaneous
Ca release (left) and an electrically driven twitch (right) in a rat ventricular myocyte (at 3rC). These
contractions are considered to be multifocal whenever two waves are moving in one cell, even if they both
originated from a single site near the middle of the cell. Multifocal contractions more often depolarize the
cell sufficiently to trigger an action potential (from Capogrossi et aI., 1987, with permission).
occur (Fabiato & Fabiato, 1978b). In addition to mechanical problems, there are also important
electrophysiological consequences associated with these aftercontractions.
Aflerdepolarizations and Triggered Arrhythmias

Aftercontractions are associated with delayed afterdepolarizations (DADs), and it is clear
that SR Ca release is responsible for both. That is, SR Ca release activates the myofilaments
(aftercontraction), but also activates a transient inward current (lti) which is responsible for
DADs (Lederer & Tsien, 1976; Kass et al., 1978). These Iti and DADs are responsible for the
oscillatory afterdepolarizations observed with Ca overload and these can, in turn, lead to
triggered arrhythmias in the heart (Ferrier & Moe, 1973; Rosen et al., 1973a,b; Ferrier, 1977;
Wit & Rosen, 1992). The process of Ca overload can lead to triggered arrhythmias via a fairly
well defined sequence of events.
Three different Ca-activated currents have been proposed to contribute to Iti : 1) Na/Ca
exchange current (lNa/Ca), 2) Ca-activated chloride current (IC1(Ca)) and 3) a nonselective cationic
current (INS(Ca), Colqu,houn et al., 1981, Cannell & Lederer, 1986; Wit & Rosen, 1992; Zygmunt
et al., 1998; Schlotthauer & Bers, 2000). Most recent work has not supported a significant role
for INS(c.) in Iti or DADs of ventricular myocytes, favoring instead key roles for ICI(Ca) and INa/ca
(Fedida et aI., 1987b; Papp et al., 1995; Laflamme & Becker, 1996; Szigeti et aI., 1998; Zygmunt
et al., 1998; Egdell & MacLeod, 2000; Schlotthauer & Bers, 2000). In dog ventricular myocytes
Zygmunt et al. (1998) attributed 60% of Iti to INa/ca and 40% to Icl(c.). There is clearly Icl(c.) in
rabbit ventricular myocytes at 37°C (Zygmunt & Gibbons, 1991; Laflamme & Becker, 1996;
Puglisi et aI., 1999), but we find that >90% of the Ca-activated current in rabbit ventricular
myocytes at 23 or 37°C is INa/ca and not ICJ(ca) (Delbridge et al., 1996; Schlotthauer & Bers, 2000;
Pogwizd et al., 2001, but see also Szigeti et al., 1998). INa/c, also seems to dominate in guinea-
pig ventricular myocytes (Fedida et aI., 1987b; Kimura, 1988). In human ventricular myocytes
the [Cali-dependent inward current is almost entirely INa/C., although atrial myocytes exhibit some
INS(Ca) (Koster et aI., 1999; Schlotthauer et al., 2000). Thus, I'i is mainly INa/ca, but IC1(Ca) (and
even INS(Ca») may playa small variable role in certain tissues or conditions.
The contributions of aforementioned currents to DAD generation may differ from those
during an I'i (where Em is clamped constant), because Em changes dynamically during DADs and
alters the electrochemical driving force (most notably for IC1(Ca))' Consider that ifEcl = -60 mY,
a DAD from -80 to -70 mV reduces the CI driving force by 50%. For INa/ca the ENa/ca may be-
40 mV at rest, but as [Cali rises ENa/ca rises to about +10 mV (see Fig 140), thereby more than
offsetting the effect of a 10 mV depolarization. Thus, voltage clamp studies will overestimate
the role of ICl(Ca) in DADs. Figure 168 shows caffeine-induced DADs (or cDADs), used to
measure the quantitative relationship between SR Ca release and depolarization (Schlotthauer &
Bers, 2000; Pogwizd et al., 2001). This sort of quantification would not be practical with
spatially inhomogeneous spontaneous Ca waves. APs were activated at different frequencies (to
vary SR Ca load), and then caffeine was rapidly applied to release SR Ca and trigger a cDAD.
With increasing SR Ca load (and ""[Ca]i) larger cDADs were observed until the point where SR
Ca release triggered an AP. Figure 168B shows how ""Em increases with ""[Ca]i and in rabbit
cells APs are triggered at ""[Ca]i ~500 nM (requiring ""[Caho, ~80 /lmollL cytosol).
This amount of Ca might be released during a spontaneous Ca release, but its ability to
trigger an AP may be limited by several factors. First, spontaneous SR Ca release normally
occurs in waves, which are not spatially homogeneous, such that the Ca-activated I'i would have a
lower peak, but be of longer duration. Even if the integrated I'i is the same, spreading it out over
a longer time reduces the cDAD amplitude and increases the amount of I'i required to trigger an
AP (Schlotthauer & Bers, 2000). This effect may nearly double the amount of Ca release needed
to trigger an AP (requiring ~ 100% of SR Ca content). Second, during a spontaneous SR Ca
release, Ca reuptake by the SR is not prevented by caffeine. Thus the inward INa/ca in rabbit or
human will only remove 30% of the released Ca at negative Em (Table 20, and less than this in rat
& mouse). This would affect the integrated I'i more than its peak, but could provide an additional
safety margin in a normal ventricular myocyte. Thus it may be difficult for spontaneous SR Ca
release to trigger an AP, but it can occur, especially when more than one Ca wave occurs
simultaneously in a cell (Fig 167; Capogrossi et al., 1987).
In the whole heart there is an additional stabilizing effect because neighboring cells will
act as current sinks, limiting the ""Em produced by a given local INa/Ca' However, the cellular
changes which cause either more SR Ca release (Ca overload) or greater local depolarization for
a given ""[Ca]i (below) would tend to increase the propensity for triggered arrhythmias. That is,
there would be a greater chance for a cell cluster that is local enough, synchronous enough and
large enough to overcome the 3-dimensional current sink limitation and trigger a propagating
arrhythmia.
In heart failure (HF) there is ~2-fold increase in the expression and function of Na/Ca
exchange (Hasenfuss et aI., 1999; Pogwizd et aI., 1999), which means that any given [Cali will
produce about twice as much inward INa/ca' In addition, if there is also reduced SR Ca-ATPase
function in HF, the Na/Ca exchanger will extrude a larger fraction of the released Ca (further
increasing I'i)' There is also a 50% reduction in the inward rectifier K current (IKI) which is
responsible for stabilizing diastolic Em (Beuckelman et al., 1993; Kiiiib et aI., 1996; Puglisi et al.,
40 SSAPs cDADs
20
:;;- 0 18 tNaCaX & J.IK1
E
-; ·20 in HF~tAEm
w -40
for any t.[Cali
-60
-80
1300
9 tl.Em Doubles for each
1100 59~M6[caJ'
..
~
S
~
900
700
500 O~~::::::=:-.---,.~-r-..---.~.....,
300
o 100 200 300 400 500 600
A[Cali (nM)
100
Figure 168. Caffeine-induced Ca transients and delayed afterdepolarizations (cDADs). Rabbit ventricular
myocytes were studied at 37°C under current clamp. Steady state (SS) APs were induced at different
frequencies, resulting in altered twitch and caffeine-induced L'>.[CaJi' Larger L'>.[CaJi caused larger cDADs and
at some point trigger APs. Mean subthreshold data are fit by 0.4exp(kL'>.[CaJi), such that L'>.E m doubles for
each L'>.[CaJi of In(2)/k (as indicated). Large squares indicate threshold for triggering APs. Data at left are
redrawn from Schlotthauer & Bers (2000) and at right from Schlotthauer et al. (2000).
2000; Pogwizd et al., 2001). Reduced lKi would also mean that any given Iti would produce a
greater depolarization, and be more likely to trigger an AP. These factors greatly increase the
propensity for arrhythmias to be triggered by Ca overload and DADs in HF.
Indeed, in a non-ischemic rabbit model of HF with INa/C. and IKi changes as above, we
find that the threshold L'>.[Ca]i during a cDAD required to trigger an AP is reduced by ~50% from
control (Fig 168B; Pogwizd et aI., 1999,2001). Moreover, these HF rabbits develop runs of non-
sustained ventricular tachycardia (not seen in control) which initiate by a non-reentrant
mechanism (e.g. DADs) based on 3-dimensional mapping studies (Pogwizd, 1995). Mapping
studies in nonischemic human HF have shown that almost all arrhythmias initiate by non-
reentrant mechanisms, whereas -50% of those in ischemic HF initiate by non-reentrant
mechanisms (Pogwizd et al., 1992, 1998). These arrhythmias in the failing rabbit model above
are inducible by infusion of ~-AR agonists, and aftercontractions (like those in Fig 166) were
readily induced by isoproterenol in isolated HF myocytes as well (Pogwizd et al., 1999,2001).
Our working hypothesis for arrhythmogenesis in HF is the following (see Fig 175). In HF SR Ca
content is typically reduced (due to increased Na/Ca exchange and/or reduced SR Ca-ATPase
function), and this by itself would make spontaneous SR Ca release less likely. However, when
SR Ca load is increased by ~-AR activation (via phospholamban phosphorylation) spontaneous
SR Ca release can occur and produce more Iti (due to greater INa/ca) and that Iti , in turn causes
much greater depolarization (due to reduced IKI)' On a related note, sudden cardiac death due to
arrhythmias occurs commonly in moderately severe HF, but much less commonly in the latest
stages of HF when ~-AR responsiveness is lost, but ventricular function continues to decline
(Bristow et al., 1982; Kjelshus, 1990). Thus, without the ~-AR-induced boost in SR Ca loading,
spontaneous SR Ca release and DADs may not occur, but contractile function may be severely
depressed by the low SR Ca content.
While Ca overload and aftercontractions or DADs secondary to Na-pump inhibition are

perhaps the most extensively studied, the same sequelae occur with other causes of cellular Ca
overload. These may include reduced [Na]o, increased Na permeability (e.g. monensin), elevated
[Ca]o, Ca channel agonists, large/long depolarizations, high frequency stimulation, decreased
membrane Ca or Na permeability barrier or decrease in energy supply required to maintain
normal ionic gradients. In summary, Ca overload causes spontaneous SR Ca release which can
contribute to:
I) High mean resting rCa]; (basal and also average from spontaneous SR Ca releases),
2) Increased diastolic force,
3) Greater inactivation of Ca-induced Ca-release at a normal pulse,
4) Asynchrony of Ca release (due to refractoriness in areas of recent Ca release),
5) Reduced twitch force
6) Partial synchronization of spontaneous release after systole
(due to the bolus of Ca influx and maybe synchrony of RyR inactivation & recovery),
7) Added series compliance (refractory cells) that contracting cells must stretch,
8) Ca;-dependent increase of inward It; (mainly INa/Ca ),
9) Depolarization (afterdepolarization),
10) Triggered arrhythmias
Acidosis
For more than 100 years acidosis has been known to depress myocardial contractility
(Gaskell, 1880), and it is important to consider because acidosis is a major consequence of
myocardial ischemia and contributes to the ischemic decline in force. The situation is potentially
complicated by the fact that changing pH can modify virtually every cellular system involved in
Ca regulation and force development. Nevertheless, some important conclusions can be drawn
from experimental work aimed at evaluating this complex problem with respect to rCa]; and force
(see Orchard & Kentish, 1990; Hulme & Orchard, 1998; Choi et a!., 2000).
Respiratory acidosis (increasing extracellular CO 2 ) produces more rapid decline in pHi
and contraction than does metabolic acidosis which is induced by decreasing [HC0 3]o or
applying weak acids such as acetic or butyric acids (Fry & Poole-Wilson, 1981). This has been
taken as evidence that the major negative inotropic effect is due to intracellular (rather than
extracellular) acidosis. Thus, even though low pHo can inhibit Ica (Irisawa & Sato, 1986; Krafte
& Kass, 1988), which would decrease contraction, that effect must be minor. Reduced pH; can
also decrease Ica (Sato et al., 1985; Irisawa & Sato, 1986; Kaibara & Kameyama, 1988), although
Hulme & Orchard (1998) found unchanged ICa with acidosis.
Ca transport by several systems is depressed at low pHi, including SR Ca-ATPase
(Fabiato & Fabiato, 1978a; Mandel et al., 1982), RyR gating (Xu et al., 1996; Kentish & Xiang,
1997) and Na/Ca exchange (Philipson et a!., 1982; Doering & Lederer, 1993). It is therefore
somewhat surprising that acidosis increases (or fails to depress) Ca transient amplitude, but
decreases contractile force (see Fig 169, Allen & Orchard, 1983; Orchard, 1987; Allen et al.,
1989; Hulme & Orchard, 1998). This indicates that the negative inotropic effect of acidosis is
mainly at the level of myofilament responsiveness to [Ca]; (vs. reduction of Ca transients).
Acidosis does indeed decrease both myofilament Ca sensitivity and maximum force
production (Fig 170, Donaldson & Hermansen, 1978; Fabiato & Fabiato, 1978a; Kentish &
30% CO 2
A
[Cal,
20 nA
'J Ill1liiWfillll
Force l b
10mN/mm~
"
1 min
B
[Ca J,10nA] ~
Force ] /\
10 mN/mm 2 --l "'---
i 0.5 sec I
Figure 169, Respiratory acidosis depresses force, but not Ca transients, A. In a ferret papillary muscle pH o
was reduced from 7.4 to 6.7 by increasing [C0 2] at 30°C and 0.33 Hz). Tension falls rapidly, but partially
recovers. [Cali was measured using aequorin, which may exagerate changes in peak [Cali and is not highly
sensitive to diastolic [Cali (due to exponential dependence of light on [Cali)' B, Averaged aequorin and
force traceS before (a) and after 1,5 min of acidosis (b), and with those traces scaled and superimposed (c
where. denotes the trace during acidosis, from Orchard and Kentish, 1990, with permission),
Nayler, 1979; Blanchard & Solaro, 1984). Blanchard & Solaro (1984) concluded that much of
the shift in myofilament Ca sensitivity could be attributed to a decrease in the affinity of 45Ca
binding to cardiac TnC. The effect of pH on Ca binding to troponin C is also amplified by a pH-
sensitive change in the affinity of TnT for TnC (El-Saleh & Solaro, 1988; Solaro et aI" 1989).
The reduction in maximum force appears to be a separate effect and also amplifies the depressant
effect of acidosis on force development (Orchard & Kentish, 1990). Acidosis decreases
maximum force much more than it does myofibrillar ATPase activity (Kentish & Nayler, 1979;
Blanchard & Solaro, 1984). This may mean that acidosis decreases the maximum force (and
efficiency) of each crossbridge as well as the turnover rate of the ATPase. These myofilament
effects are the major cause of the negative inotropic effect of acidosis.
Neonatal ventricular (and skeletal) muscle is less sensitive to the depressant effects of
acidosis than is adult ventricular muscle, and this is due primarily to a much smaller shift in
myofilament Ca sensitivity (Solaro et aI" 1988). Reducing pH from 7 to 6,5 produced a 4-fold
decrease in Ca affinity in adult rat (K e• increased from 1.5 to 4 /-tM), but only a 1.8-fold decrease
in neonate rat myofilaments (Ke• increased from 0.7 to 1.2 /-tM), Neonatal myofilament Ca
sensitivity is also higher than adult, even at pH 7.0 (see Fig 21D). The smaller pH dependence
in neonatal hearts appears to result, in part, from the different Tn1 isoform expressed in the fetal
and newborn heart (slow skeletal TnI; see pg 32-33). This may help to protect the perinatal heart
from ischemic stresses during development and at birth.
Ca transient amplitude during abrupt acidosis ean be initially increased, unchanged or
decreased (e.g. Orchard & Kentish, 1990; Hulme & Orchard, 1998; Choi et aI" 2000). However,
in almost all reports there is then a progressive increase in twitch .:i[Ca];, and this causes a partial
recovery of contractions, Acidosis also leads to a gradual increase of diastolic [Ca]; (Bers &
120 pH 7.2
r::-
i
-
100 pH 7.0
ell 80 pH 6.8
><
ell
E 60 pH 6.4
~
e..... 40 ____ -43.0&______
...0
Ql
U
u. 20
0
0.1 10 100
[Cal (IJM)
Figure 170. The effect of pH on the myofilament Ca sensitivity in triton-skinned guinea-pig ventricular
muscle at 30°C. Note that a [Cal; that produces 43% activation at pH 7.0 produces much less force at pH
6.8 and 6.4 (vertical broken line and numbers). Curves are redrawn, based on Orchard & Kentish (1990).
Ellis, 1982; Kohmoto et al., 1990, Nakanishi et al., 1990). As presented in Fig 160, this could
contribute to the progressive force recovery and increase in peak Ca transient during acidosis.
The increased diastolic [Cali may be partly due to proton competition at intracellular Ca
buffering sites, such as TnC (Bers & Ellis, 1982; Vaughan-Jones et aI., 1983; Blanchard &
Solaro, 1984). This reduced Ca buffering could result in a greater L'J.[Ca]i for a given addition of
total Ca. To simulate a pH change from 7.0 to 6.5, 1 reduced TnC affinity in Table 10 and Fig 26
by 4-fold (based on Fig 170). Then the same increment in total Ca (75 flmollL cytosol) would
increase peak [Cali from 500 to 640 nM. Looked at another way, this reduces the amount of total
Ca required to raise [Cal; to I flM (from 117 to 101 flmollL cytosol). This is a modest decrease
in overall Ca buffering, but is consistent with the non-significant decrease (from 121 to 109) in
buffering power measured by Choi et al. (2000). More importantly, for the same L'J.[Ca]i (500 nM)
developed force would decrease almost 1O-fold (from 50% of maximum at pH 7 to 6%).
Low intracellular pH also stimulates proton extrusion via Na/H exchange, especially
when pH o is relatively normal. Indeed, for acid loads, cardiac cells appear to rely mainly on
Na/H exchange for pHi regulation, while for alkali loads the ClIHC0 3 exchange appears to be
more important (Vaughan-Jones, 1982; Piwnica-Worms et aI., 1985; Ellis & MacLeod, 1985).
Thus, extrusion of protons via Na/H exchange increases [Na];, particularly at normal pHo
(Deitmer & Ellis, 1980; Bountra & Vaughan-Jones, 1989). While the Na/K-ATPase might be
expected to reduce [Na]i back toward the control level, decreasing pH below 7.5 also inhibits the
Na-pump (e.g. Sperelakis & Lee, 1971). The result is that [Na]; increases during respiratory
acidosis with a time course similar to the slow recovery phase of contraction (Fig 171, Harrison
et al., 1992b). Thus, the increase in [Na]i may contribute to the slow recovery of contractile
force via a shift in Na/Ca exchange and increase in [Cali' Under conditions where extracellular
pH was held constant, Bountra & Vaughan-Jones (1989) showed that reduced pHi could lead to
an increase in [Na]; and twitch tension in guinea-pig papillary muscle. They attribute this
positive inotropic effect to shifts of Na/Ca exchange which were sufficient to overcome the
A
Cell
Length
(~m)
123
87 1
15 % C02
..---
1 r:
Ratio 0.40
a~a
(340/380 nm)
(mM)
0.36
B
pHi
Ratio 28
(440/495 nm)18 1 ~,-~ -,r~--~._~
60 s
Figure 171. Respiratory acidosis decreases contraction (A), and pH' (B, assessed by BCECF
fluorescence) in a rat ventricular myocyte at 26°C, stimulated at I Hz. Intracellular Na activity (assessed by
SBFI fluorescence) gradually increases during acidosis. This increase aNai may contribute to the
contractile recovery (via a shift in Na/Ca exchange). The initial decrease in the SBFI fluorescence trace
was attributed to a pH effect on SBFI (from McCall et al., 1990, with permission).
largely depressant effects of intracellular acidosis. Thus, it seems likely that the gain in cellular
Na (and consequently Ca) contributes to the increased Ca transient observed with acidosis.
Na/Ca exchange is also inhibited by low pH (Philipson et aI., 1982). Together with the
increased [Na]i discussed above, the ability of the cell to extrude Ca via Na/Ca exchange may be
severely compromised. As with cardiac glycosides, this may also contribute to increased SR Ca
loading (see below) and larger Ca transients seen during acidosis, but could also lead to Ca
overload and consequent arrhythmias (Coraboeuf et al., 1976; Kurachi, 1982). If spontaneous
SR Ca release occurs during acidosis, negative inotropic effects would be exacerbated (as
discussed on pages 295-300).
Acidosis also decreases cardiac SR Ca uptake in both isolated SR vesicles and skinned
myocytes (see Chapter 7, Shigekawa, 1976; Mandel et al., 1982; Fabiato & Fabiato, 1978a;
Fabiato, 1985e). Indeed, the rate of the twitch [Ca]i decline in intact cells and muscle is slowed
by acidosis (Fig 169, Hulme & Orchard, 1998). However, the increased diastolic [Ca]i and
cellular Ca loading described above more than compensate for the slower SR Ca uptake at low
pHi. This explains the increased SR Ca content, which has been clearly documented during
acidosis (Harrison et aI., 1992b; Hulme & Orchard, 1998; Choi et al., 2000). Ca sparks in
myocytes and RyR channel gating in bilayers are inhibited by acidosis (Ma et al., 1988;
Rousseau & Pinkos, 1990; Xu et al., 1996; Balnave & Vaughan-Jones, 2000). Fabiato (l985e)
showed that the optimal free [Ca] trigger for CICR was higher at low pH, and Hulme & Orchard
(1998) showed that acidosis reduced fractional SR Ca release. Indeed, variability in the initial
6[Ca]i response to abrupt acidosis might depend on how extensively E-C coupling is depressed
(this effect may be more dramatic in cases where there is an initial decrease in Ca transients
(Choi et al., 2000). Moreover, if SR Ca release in inhibited, this will cause further gain in
cellular and SR Ca (as in the case of partial block of the RyR by tetracaine, pg 267). As SR Ca
load rises, there is a progressive increase of SR Ca release during the twitch. Thus the increase
in SR Ca load (due to the combination ofRyR block and elevated [Na]i & [Ca];) would gradually
increase twitch Li[Ca]; and contraction during acidosis. The result is that there is less fractional
release of Ca from the SR at low pH, but this can be more than offset by a larger SR Ca content.
As mentioned above, during acidosis the time constant of twitch [Cali decline is slowed.
However, as diastolic [Cali and Li[Ca]i increase during sustained acidosis, Nomura et aZ. (2000)
showed that the time constant of [Cali decline recovers significantly. Moreover, they found that
this recovery could be prevented by the CaMKII inhibitor KN-93. This is consistent with work
by Vittone et aZ. (1998). They showed that in the absence ofPKA activation, CaMKII-dependent
phospholamban phosphorylation at Thr 17 occurred with elevated Ca transients, but only at acidic
pH (possibly due to phosphatase inhibition, pg 167). Thus, accelerated SR Ca-pumping could
explain the faster [Cali decline and partially overcome the direct inhibitory effect of acidosis.
This CaMKII-dependent acceleration of [Cali decline is reminiscent of the frequency-dependent
acceleration of relaxation (FDAR) discussed on pg 270-272. While PLB is not required for
FDAR, it is unknown whether PLB is required for this secondary regulation during acidosis.
The increase in [Na]i via Na/H exchange at low pHi can be limited if extracellular pH is
low, as in ischemia (Bountra & Vaughan-Jones, 1989). If pH o is suddenly returned to normal
while pHi is still low (as during reperfusion after ischemia), there is a large outwardly directed
[W] gradient. This is precisely the condition where Na/H exchange can extrude protons rapidly,
but with the consequence that [Na]; rises rapidly (Bountra & Vaughan-Jones, 1989). The high
[Na]i increases cell and SR Ca content via Na/Ca exchange and can be arrhythmogenic.
Lazdunski et aZ. (1985) hypothesized that this mechanism is responsible for the large cellular Ca
accumulation associated with reperfusion after ischemia, and there is much evidence supporting
this view (see Ischemia below, and Karmazyn et aI., 1999).
In conclusion, the effects of acidosis on cellular Ca and force production are complex,
but some important aspects seem clear. Reduced myofilament Ca sensitivity and maximum force
are the main factors responsible for the negative inotropic effect of acidosis. Acidosis also
depresses RyR sensitivity to Ca, but this is offset (progressively) by an increase in cellular and
SR Ca content. This gain in cell Ca is attributable mainly to proton extrusion via Na/H exchange
& Na/K-ATPase inhibition, which together elevate [Na]i, and this consequently raises Ca due to
shifts in Na/Ca exchange (or simply depressed Ca extrusion via Na/Ca exchange). Decreased
myofilament Ca binding may also reduce Ca buffering slightly. The increased cellular and SR
Ca load can contribute to the progressively larger Ca transients (but still with reduced force), and
can also lead to cellular Ca overload.
Before leaving this subject it is worth briefly reviewing pH buffering and the transporters
which are responsible for regulating pH; (e.g. Leem & Vaughan-Jones, 1998; Leem et aZ., 1999:
Puceat, 1999). As mentioned on pg 46, cardiac intracellular pH buffering is very high (~=20-90
roM/pH unit). In the absence ofC02 /HC0 3 Leem et aZ. (1999) described intrinsic pHi buffering
with two classes of buffers: Bmaxl =84 roM, pK al = 6.03 and B maxl =29 roM, pK a2 = 7.57. Thus
intrinsic pHi buffering power falls biphasically from ~50 at pH 6 to ~20 at pH 7.6. In contrast,
buffering by CO 2/HC03 increases with increasing pH (from ~ 11 at pH 6.9 to 48 at pH 7.3), and
the overall buffering power is about doubled in the presence of CO 2/HC0 3• So at a normal pHi
of7.04 in COz/HC03 buffer (vs. 7.07 in Hepes buffer), total cellular buffering power is ~45.
12
Integrated pHi Regulation
10
C 8
'E I I
:::J 6
: perm issive .:
:::.
0 :.. range :
E 4
I I
.§. I I
..,
:I:
2 I
I 7.0 7.2 I
I
7.4
0
pH; cr/oH- Exchange
-2
cr/Hco3 Exchange
-4
Figure 172. Acid transporters involved in pH; regulation. Acid efflux (positive) or influx (negative) on
each of the four transport systems is indicated for different pH. The range for nonnal pH; is indicated as the
pennissive range. Curves were drawn from equations in Leem el al. (1999).
There are also four ion transport mechanisms involved in regulating pH;. Two are acid
extruders, Na/H exchange (which removes H) and Na-HCO j cotransport (which brings in HCO j ).
These are activated at low pH; (Fig 172). The other two bring acid into the cell, CrIHCO j -
exchange (or anion exchanger, which extrudes HCO j -) and cnow exchange (which extrudes
OH} Figure 172 shows the relative acid flux mediated by each of these systems in a guinea-pig
ventricular myocyte (Leem et at., 1999). In the presence of an acid load, the Na/H exchange is
more strongly activated than is Na-HC03 cotransport, and during alkalosis CnHCO j - exchange
is more strongly activated than CrIHCOj - exchange. At normal resting pH; all four systems may
produce similar non-zero flux (-0.15 mM/min).
Hypoxia and Ischemia

The effects of hypoxia and ischemia on Ca and force development wi II only be discussed
briefly here (see Downey, 1990; Reimer & Jennings, 1992; Jennings et al., 1995; Goldhaber,
1997; Opie, 1998). In a simplistic sense acidosis can be considered a stepping stone toward
understanding pathological changes associated with the more clinically relevant situations of
hypoxia and ischemia. Indeed, acidosis is an early consequence of hypoxia (due to shifting of
metabolism to glycolysis and lactic acid production) and ischemia (where lactic acid and other
metabolites are also not washed away). In this context much of the acidosis discussion above
may be extended to hypoxia and ischemia. To simplify, we can consider 4 main attributes of
ischemia: I) acidosis (as above), 2) hypoxia, 3) altered high energy phosphates
([ATP]/[ADP][P;D and 4) elevated [K]o. While ischemia creates dire conditions by itself,
reperfusion after ischemia can suddenly make things even worse. I will briefly discuss a few
key issues which are most related to the theme of this book, but cannot review in any
comprehensive way here the extensive field of ischemia and reperfusion.
As discussed above, acidosis decreases myofilament Ca sensitivity and maximum force.
During ischemia (or hypoxia with inhibition of glycolysis) high energy phosphates are gradually
depleted and inorganic phosphate (P;) can increase from -I mM to 20 mM (Allen & Orchard,
1987). This high [Pil by itself depresses the Ca sensitivity of the myofilaments (Herzig & Riiegg,
1977; Kentish, 1986). In combination with intracellular acidosis, myofilament Ca sensitivity can
be profoundly depressed. The combination may work synergistically if, as in skeletal muscle, the
inhibitory form of Pi is diprotonated (H 2 P04 , Nosek et al., 1987). This form would make up a
higher fraction of the total Pi pool at lower pH and could exert a more powerful depressant effect.
Thus, the decline in force generation in ischemia, as in acidosis, is probably largely attributable
to decreased myofilament activation. Again, as with acidosis, force can be severely depressed
while Ca transients remain large (Lee et al., 1988; Allen et al., 1989).
While we typically think of [ATP] when considering energetic limitations, L'.G ATP also
depends on [ADP] and [Pi], as L'.GATP=L'.Go + RT In {[ADP][Pil/[ATP]}, where L'.Go=-30 kllmo\.
So, even though ATP is well buffered and a decline of [ATP] from II to 10 mM would seem
inconsequential, this may result in an increase of [ADP] from 50 flM to 350 flM, and a IO-fold
change in [ADP][Pil/[ATP] (for [P i]=4-5 mM). This reduces the energy available from ATP
hydrolysis by 10%. While this may contribute to dysfunction at the myofilament level, it will
also affect ion transport. As pointed out on pg 62, the Na/K-ATPase and SR- and sarcolemmal
Ca-ATPases all function at relatively high energetic efficiency. However, when the L'.GATP
declines, the maximal [Na] and [Ca] gradients that can be established by these ATPases will be
lower. This can be appreciated by inspection of Fig 89 (pg 174). For the above 10% reduction
of L'.GAT?, the maximal [Ca]sR/[Ca]j gradient would be reduced by 50%. This would lower both
the maximal SR Ca content achievable and also slow the terminal phase of [Ca]i decline
mediated by the SR Ca-pump (which would elevate diastolic [Ca]i, for both thermodynamic and
kinetic reasons). This energetic issue can limit cardiac contractile reserve (Tian & Ingwall,
1996; Tian et al., 1997, 1998). The lower [Ca]sR decreases both the Ca available for release and
the fraction released during E-C coupling (Fig 119). This L'.GATP effect on Na/K-ATPase could
also contribute to the increase in [Na]i, with its array of consequences discussed above.
As ischemia progresses and creatine phosphate becomes depleted, [ATP] declines more
rapidly, [Pi] continues to rise and [ADP] rises more steeply (Fig 173). This can dramatically
reduce the energy available from ATP (L'.GATP ) and that can affect all ATP dependent processes,
including myofilament ATPase as well as ion transport ATPases. This can depress intrinsic
myofilament contractile parameters and reduce the maximal [Ca] gradient across the SR and
[Ca], [Na] and [K] gradients across the sarcolemma. In addition, since cytosolic Mg is strongly
buffered by ATP, the decline in [ATP] also results in an increase in free [Mg]i (Murphy et al.,
1989b). The higher [Mg]i (along with the lower pHi) will also affect numerous Ca-dependent
systems and ion channels. For example elevated [Mg]i itself depresses myofilament Ca
sensitivity, Ca-ATPases, ryanodine receptor gating and outward current carried by inwardly
rectifying K channels (such as l K1 and IK(ATP), see chapters 2, 4 & 7). These effects can contribute
to both contractile and electrophysiological dysfunction during ischemia.
As high energy phosphates are depleted, [ATP]i declines and [ADP]i rises, both of which
contribute to the activation of ATP-sensitive K current (IK(ATP), Noma, 1983, see pg 82). IK(ATP) is
inhibited by flM ATP (K i for ATP=20 flM, Nichols & Lederer, 1990) and appears to be
preferentially inhibited by ATP produced by glycolysis (Weiss & Lamp, 1987, 1989). The ATP
sensitivity of IK(ATP) is shifted by increasing ADP in the context of ischemia (to K i 100-300 flM,
Weiss et al., 1992; Deutsch & Weiss, 1993). So, perhaps appropriately, this channel responds
Chapter 10 Cardiac Jnotropy & Ca Mismanagement 307
.s:? 30
l:
,g 20 [PJl. Ischemia
~ \
C /'
/'
..... .....
~. leK_
Cl>
g 10-1----
/'
..... .....
o
U
--==--:=-[,X.ill>i;"
I ftm~
7.4
M!WumiE
------ __
_ Reperfusion w Na Na
W W [t[H]" IINa);, t[Cal" IIKI" 1
7.0
:f
Q.
6.6
Reperfusion
6.2
w Ca K
~
Potential
Arrhythmias
[Cal; 1] f\JTri-
Ww
w Na Na
(11M)
iINa]" ilCal,"" Ca overload
0.1 t
I excitability..., arrhythmias
Force or
Pressure
Figure 173. Schematic diagram of some changes which occur during ischemia and reperfusion. Points
were extrapolated from a wide variety of sources and are not intended to be an accurate quantitative
description for any specific ischemic condition.
more directly to .1GATP (i.e. [ATP]/[ADP]) rather than just [ATP]. Moreover, these channels are
numerous in heart cells and they have high unitary conductance. This means that activation of
less than I% of maximal IK(ATP) can shorten AP duration by 50%, and this can happen with only a
small change in [ATP] (Nichols et al., 1991; Weiss et aI., 1992). Put simply, [ATP] declining
from 10 to 9 mM would not change IK(ATP), but if ADP shifts the K i from 20 to 100 11M, that
would produce I % of maximal IK(ATP). There is experimental evidence that IK(ATP) activation
shortens AP duration during the early contractile failure and then prevents the AP altogether in
rat ventricular myocytes under complete metabolic blockade (Lederer et al., 1989). Thus, IK(ATP)
may protect energetically compromised cells by preventing depolarization and Ca transients.
This allows the cell to conserve energy, since the myofilaments and Ca pumps should have
minimal energy consumption in this state. However, this could create islands of non-excitable
cells, and alter the normal conduction pathway in the heart. This might then contribute to setting
the stage for reentrant arrhythmias.
During early ischemia and hypoxia there is also a large increase in 42K efflux which
elevates [K]o during ischemia (to as high as 20 mM), and could be partially prevented by
glibenclamide, a selective IK(ATP) blocker (Weiss & Lamp, 1989). While IK(ATP) activation
seemed an initially plausible explanation of the K loss in hypoxia and ischemia, the same group
demonstrated that this is not the case (Shivkumar et al., 1997). One concern was that K loss via
IK(ATP) would be self-limiting. That is, increasing K conductance drives Em toward E K, and that
would limit the driving force for K efflux (Em - E K). Moreover, when they activated IK(ATP) with
cromakalim, they found AP duration shortening comparable 10 that in hypoxia, but there was no
significant 42K loss. They found that if they prevented the Na gain in hypoxia, they could also
prevent the K loss. They concluded that the K loss is largely a passive consequence ofNa gain.
Thus, Na gain is centrally involved in pH regulation, Ca gain and also elevated [K]o in ischemia.
During severe hypoxia (associated with ischemia) there is a large rise in (Na]; which can
reach 40 mM in prolonged anoxia. An elegant study by Eigel & Hadley (1999) evaluated the
cellular basis of the Na gain. While the two main pathways for normal Na influx are INa and
inward INa/c. (- IO and 30 Ilmol/L cytosollbeat respectively), the main mechanisms of net Na gain
during anoxia are via TTX sensitive Na channels and HOE-642-sensitive Na/H exchange. TTX
and HOE-642 each blocked -50% of the Na gain during simple anoxia. For anoxia with acidosis
the Na gain was blocked by TTX, but not by HOE-642. However, when anoxia was coupled
with high [K]o (10 mM) or high [K]o plus acidosis (pH o 6.85 to simulate ischemia), the Na gain
was suppressed almost completely by HOE-642, but not appreciably altered by TTX. This may
be most relevant to ischemia and accounts for the dramatic protective effect of Na/H exchange
blockers in ischemia and reperfusion (below). It is not clear why TTX-sensitive Na influx would
occur in the two cases with normal [K]o (while cells are quiescent), but anoxia and ischemic
metabolites have been shown to produce persistent Na channel opening in ventricular myocytes
(Ju et ai., 1996; Undrovinas et ai., 1992). Since 10 mM [K]o prevented the TTX-sensitive Na
gain, it is possible that anoxia allows a tiny window I a at resting Em, but inactivation prevails
upon depolarization in high [K]o. Thus, during ischemia, the large rise in [NaJ may be mediated
mainly by Na/H exchange, but TTX sensitive Na entry may also occur (Xiao & Allen, 1999).
Reperfusion-Acute effects. Ischemia or metabolic blockade produces acidosis and progressive
gain in cellular Na and Ca (Fig 173, as discussed above for acidosis, but now with additional
energetic limitations and extracellular effects). In ischemia, the increase in [Na]; via Na/H
exchange is limited by low pH o (Bountra & Vaughan-Jones, 1989). Indeed, Yan & Kleber
(1992) showed that pH o declines more than pH; during ischemia (presumably because the cell is
struggling to maintain normal pH;). Upon reperfusion, pH o is quickly restored, creating a large
outward [W] gradient. This causes rapid Na gain via Na/H exchange, which in turn induces
rapid Ca gain via Na/Ca exchange (which becomes more active as pH; recovers). Indeed, this
large bolus of Ca entry upon reperfusion can produce Ca overload, and with the variable
recovery of excitability and the electrophysiological substrate, there is an enhanced propensity
for arrhythmias. This mechanistic scenario (Fig 173) now has overwhelming experimental
support (reviewed by Karmazyn et ai., 1999). Moreover, Na/H exchange inhibitors, including
amiloride, ethylisopropylamiloride (EIPA), HOE-642 (cariporide), HOE-694 and EMD-8513 I,
all prevent (or limit) hypercontracture and arrhythmias associated with reperfusion after ischemia
(Karmazyn, 1988, Murphy et ai., 1991; Scholz et ai., 1993, 1995; Hendrikx et ai., 1994;
Yasutake et ai., 1994; Xue et ai., 1996; Gumina et ai., 1998; Karmazyn et ai., 1999; Stromer et
ai., 2000). Inhibition of Ca entry via Na/Ca exchange (KB-R7943) also limited post-ischemic
arrhythmias and improved mechanical recovery (Mukai et ai., 2000).
It is worth considering the quantity of protons that are extruded upon reperfusion. IfpH j
must recover -0.5 pH units, this would require extrusion of -23 mmol protons/L cytosol from the
cell (based on pH buffering, pg 304) and for a I: I Na/H exchange this would require 23 mM Na
influx. If all of this were to exchange for extracellular Ca via Na/Ca exchange, the total Ca gain
would be 7-8 mM! Clearly this is an extreme upper limit, because the Na/K-ATPase will be
expected to deal with most of Na extrusion, but this takes time. Even if the Na/Ca exchange
brings in only 5% of this Ca (350 Ilmol/L cytosol) is a large Ca bolus for the cell to deal with.
This is especially the case while [Na]; is high, which limits the ability of the Na/Ca exchanger to
extrude Ca. Moreover, this explains the large, potentially lethal rise in rCa]; during reperfusion.
Another major problem which occurs upon reperfusion (or reoxygenation after hypoxia)
is a burst of free oxygen radical generation (Goldhaber, 1997). This is partly because of a
decrease in free radical scavengers (e.g. glutathione peroxidase and superoxide dismutase, SOD),
but it is also due to infiltration of activated neutrophils which produce superoxide, hydrogen
peroxide and hypochlorous anion (Ferrari et at., 1985; Shlafer et at., 1987; McCord, 1974; Rowe
et al.1984). These neutrophils can also produce cytokines (e.g. interleukins and TNF-<x) that can
further stimulate nitric oxide and free radical production, and may also exert direct effects on
myocytes. Several studies have shown that post-ischemic recovery can be improved when free
radical production and/or neutrophil effects are inhibited (Werns et at., 1985, Ambrosio et at.,
1986; Hearse & Tosaki, 1987; Puett et at., 1987; Mak & Weglicki, 1988; Przyklenk & Kloner,
1989; Lefer et at., 1990; Flaherty et aI., 1994).
While details of how free radicals and cytokines worsen recovery are complex and not
entirely understood, cellular Ca mismanagement appears to be a common endpoint. For
example, oxygen free radical production can cause myocyte Ca overload, transient inward
currents (It;) and triggered action potentials (Barrington et at. 1988; Matsuura & Shattock, 1991;
Josephson et at., 1991). Indeed, many Ca transporters appear to be affected by free radicals,
including the SR Ca-ATPase (Hess et at., 1983,1984b; Goldhaber & Liu, 1994), RyR (Boraso &
Williams, 1994), Na/Ca exchanger (Reeves et al., 1986; Coetzee et at., 1994; Goldhaber, 1996),
mitochondria (Harris et at., 1982) and sarcolemmal Ca-ATPase (Kaneko et aI., 1989). Na/K-
ATPase is also inhibited by free radicals (Kim & Akera, 1987; Shattock & Matsuura, 1993),
which would contribute to Na gain. Since Ca leak and Na window current may also be increased
by free radicals (Wang et at., 1995b; Bhatnagar et aI., 1990), cellular Na and Ca gain may
mediate Ca overload as discussed earlier. Moreover, the activation of Na/Ca exchange by free
radicals may worsen the Ca overload in response to increased [Na]; (especially during
reperfusion, see Fig 173). Thus, it appears that this free radical pathway may exacerbate the
Na/H exchange-Na/Ca exchange problem with respect to reperfusion after ischemia.
Hibernation. Hibernating myocardium is a term used to describe temporary depressed
contractile function upon reduction of regional blood flow (Feigl, 1983; Braunwald & Ruterford,
1986; Marban, 1991). This decrease is completely and reversible upon resumption of perfusion
pressure and occurs without change in pH;, [Pi] or high energy phosphates. It had been supposed
that this might be due to the "garden hose effect" where local vascular stretch increased
sarcomere length and increased contractility via Starling's law (Fig 19). However, Kitakaze &
Marban (1989) showed that higher perfusion pressure increased contraction, even though end-
diastolic length was already optimal. Moreover, they found reversible changes in Ca transients
that may explain the altered contraction with altered coronary perfusion. Heusch (1998) noted
that reduction of 6G ATP or Ca responsiveness might also be involved, but the mechanism remains
unclear. While such hypoperfusion and function can be chronic, key features of hibernation are
that there is no necrosis and function is promptly restored when flow is restored. Of course,
during prolonged hypoperfusion secondary changes can occur which complicates things further.
Stunning. Myocardial stunning is quite distinct from hibernation (Braunwald & Kloner, 1982;
Bolli, 1990; Marban, 1991; Bolli & Marban, 1999). Stunning is post-ischemic contractile
dysfunction which persists for hours or days after restoration of perfusion, but is not associated
with either reduced coronary flow or irreversible damage (i.e. no necrosis). Bolli & Marban
(1999) consider that there are two plausible contributing pathways to myocardial stunning and
they are not mutually exclusive: 1) oxygen free radicals and 2) Ca-overload/myofilament
dysfunction. Indeed, the contractile dysfunction can persist when energetic status is completely
restored and even with normal action potentials and Ca transients (Hanich et al., 1993; Gao et
al., 1995). It is now clear that myofilament maximal force and/or Ca sensitivity are reduced in
the stunned heart (Westfall & Solaro, 1992; Hoffman et al., 1993; McDonald et al., 1995b; Gao
et al., 1995). Troponin I (TnI) appears to be selectively degraded in stunned heart, although (1.-
actinin and other myofilament proteins may also degraded after longer ischemic episodes
(Westfall & Solaro, 1992; Gao et ai., 1995, 1997d; Van Eyk et al., 1998). These studies also
showed that reperfusion is required for both TnI degradation and for the myofilament
dysfunction (Miller et ai., 1996). Replacing endogenous troponin with troponin from stunned
hearts also reduces myofilament Ca sensitivity (McDonald et ai., 1998). Murphy et ai. (2000)
created transgenic mice overexpressing a truncated TnI (1-193, which matches the major
degradation product in stunned hearts). This truncated TnI replaced 9-17% of the endogenous
TnI and caused a similar decrease in contractile function and myofilament responsiveness to Ca,
without altered cellular Ca transients. Moreover, these transgenic hearts were hypocontractile
and developed ventricular dilatation. Thus TnI degradation during reperfusion is likely to be a
major factor in contractile dysfunction in myocardial stunning. The slow functional recovery
(hours-days) may reflect the time required to synthesize replacement Tn!.
So how does reperfusion cause TnI degradation? The leading theory is that Ca-activated
proteases (calpains) activated by high [Cal during reperfusion are responsible (Bolli & Marban,
1999). I have already discussed the basis of Ca overload upon reperfusion, and calpain I (present
in the ventricle) is activated by [Cal in the 1-20 !!M range in vitro (Suzuki, 1990) and by
ischemia/reperfusion (Yoshida et ai., 1995). TnI has also been shown to be a substrate for
Calpain I (DeLisa et ai.1995). This also brings us back to the role of oxygen free radicals in the
genesis of stunning. As described above, the production of these free radicals upon reperfusion
can exacerbate Na and Ca overload and this can add to the likelihood of both TnI degradation as
well as other potentially reversible dysfunction. Thus, while stunning is complex and there will
surely be additional explanations identified for its many etiologies, some first steps in molecular
understanding are becoming clearer.
Preconditioning. Ischemic preconditioning is an intriguing aspect of the ischemic response.
Preconditioning refers to the result that brief periods (e.g. 5-10 min) of ischemia, which do not
create any dysfunction after reperfusion per se, can protect the heart from damage during a
subsequent prolonged ischemic episode (Murry et ai., 1986; Bolli, 2000; Cohen et ai., 2000).
There is an initial acute phase of preconditioning «3 hr after the initial ischemic episode) and
also a delayed or late phase (developing over 24 hr) sometimes called the second window of
protection (see reviews by Okubo et ai., 1999; Bolli, 2000; O'Rourke, 2000). Several factors
may contribute to ischemic preconditioning, including adenosine, nitric oxide, protein kinase C,
l K(ATP) and heat shock proteins.
Adenosine is produced during ischemia and administration of adenosine receptor
agonists can simulate preconditioning, while blocking adenosine (AI) receptors can inhibit
preconditioning (Van Wylen et al., 1990; Baxter et al., 1994). Some other G-protein coupled
receptors such as bradykinin and endothelin can also activate this pathway. Blocking nitric oxide
synthase (eNOS) can inhibit the protective effects of brief ischemic episodes, while exogenous
nitric oxide can reproduce the effects of preconditioning (Bolli et al., 1997; Takano et al., 1998;
Banerjee et aI., 1999). The effect of nitric oxide is not blocked by guanylyl cyclase inhibitors
(suggesting that cGMP and PKG are not involved; Kodani et al., 2000), but is blocked by
antioxidants (suggesting peroxynitrite or hydroxyl free radical involvement, Takano et aI., 1998).
Protein kinase C isoforms PKCE and PKCr1 are activated by ischemia, but only PKCE activation
is required to simulate preconditioning (Qiu et al., 1998; Ping et al., 1999a). The PKCE
activation can be blocked by blocking nitric oxide production, linking these pathways (Ping et
al., 1999b). Moreover, transgenic mice expressing a constitutively active PKCE recapitulate the
preconditioning phenotype (Ping et al., 2000). PKCE may ~Iso work through a downstream
activation of the mitogen activated protein kinase (MAPK) known as ERKIIERK2 or p38 (Ping
et al., 1999c; Nakano et al., 2000).
IK(ATP) has also been implicated in ischemic preconditioning (O'Rourke, 2000; Grover &
Garlid, 2000). The sarcolemmal KATP channel may contribute to action potential shortening and
abortion during ischemia, and was thought to playa role in preconditioning. IK(ATP) activators
(pinacidil & cromakalim) could simulate preconditioning, while IK(ATP) blockers (glibenclamide)
inhibited the protective effect (Grover et al., 1989, 1990). However, it became clear that there
were discrepancies between effects on sarcolemmal IK(ATP) or AP duration and protective effects
of these K-channel openers (Yao & Gross, 1994; Grover et al., I995a,b). There was early
evidence of a novel K ATP channel on the inner mitochondrial membrane (mito-KATP , Inoue et al.,
1991), but characterization of mito-IK(ATP) has developed slowly, because it is not as amenable to
patch clamp studies as are sarcolemmal channels (see reviews by O'Rourke, 2000 and Grover &
Garlid,2000). Garlid et al. (1997) showed that diazoxide opened mito-IK(ATP) with a K m of -0.5
/-lM, (vs. 840 /-lM for sarcolemmal IK(ATP» and that 5-hydroxydecanoate (5-HD) could selectively
block mito-IK(ATP) (K; -50 /-lM). They went on to show that selective activation of mito-IK(ATP)
protected hearts from ischemic contracture and that this could be blocked by a mito-1 K(ATP)-
selective concentration of 5-HD. Note that pinacidil & cromakalim activate both sarcolemmal
and mitochondrial IK(ATP) and glibenclamide is also a non-selective blocker. Another group (Liu
et aI., 1998; Sato et al., 2000a) found parallel results with different methods, and both groups
concluded that the protective effect of IK(ATP) activation was attributable to mitochondrial, rather
than sarcolemmal K ATP channels. Sato et al. (1998a, 2000b) and Takashi et al. (1999) have gone
on to show that both adenosine and PKC activation are upstream modulators of mito-IK(ATPl
activation, and Sasaki et al. (2000) showed that nitric oxide facilitates activation by diazoxide.
This connects the mito-IK(ATPl pathway to the aforementioned roles of adenosine, NO and PKC in
ischemic preconditioning. What is less clear at this stage is why opening mito-K ATP channels
elicits protection. Indeed, opening a K channel may be expected to dissipate the mitochondrial
membrane potential (E mito ) and proton gradient. This would waste the energy required to rebuild
the mitochondrial [H] gradient and Emi,o' Three possible mechanistic hypotheses discussed by
O'Rourke (2000) are that mito-IK(ATP) activation: 1) causes mitochondrial swelling and a
consequent higher rate of energy production (Halestrap, 1989). 2) lowers Emilo which might limit
mitochondrial Ca overload (Holmuhamedov et ai., 1999). 3) alters mitochondrial production of
oxygen free radicals, somehow conferring protection. At this point it is too early to speculate as
to which pathway (if any of these) is important in the mito-IK(ATP) induced cardioprotection.
Finally, there is evidence that heat shock proteins (HSP) may be involved in ischemic
preconditioning (Benjamin & McMillan, 1998). These stress-activated proteins are molecular
chaperones, and they can facilitate protein folding and translocation. HSPs can confer cross-
tolerance to different stressors, and their function may be to aid refolding of proteins damaged by
these noxious stimuli (e.g. hyperthermia or ischemia). HSP-70 is rapidly induced during
ischemia (Knowlton et ai., 1991), and HSP-70, HSP-27 and HSP-22 (aB-crystallin) all exhibit
cardioprotective effects (Marber et ai., 1995; Mestril et ai., 1996; Martin et ai., 1997). How
these HSP effects interdigitate with the other mechanisms above is not clear at present. Thus,
there may be several factors involved in ischemic preconditioning and additional work will be
required to claritY which mechanisms are most dominant. However, it would not be surprising to
find that these systems constitute an array of partially redundant protective mechanisms.
Ischemic preconditioning and Na/H exchange inhibition are generally similar with
respect to cardioprotection during ischemia and reperfusion (Shipolini et al., 1997; Gumina et
al.,1999; Avkiran, 1999; Xiao & Allen, 1999; Mosca & Cingolani, 2000). However, with longer
ischemia (>60 min) Na/H exchange inhibition may be even more protective (Gumina et al.,
1999). NaIH exchange inhibition and preconditioning are also additive and provide greater
protection when combined. It is controversial whether preconditioning works partly by
inhibiting NaIH exchange (Shipolini et ai., 1997; Xiao & Allen, 1999). In conclusion, ischemia
and reperfusion are very complex. While much work is needed to claritY the basis of post-
ischemic dysfunction and how it might be improved, clearly multiple mechanisms are involved.
Hypertrophy
Ventricular hypertrophy occurs in response to numerous physiological and patho-
physiological stimuli. In all cases the increase in ventricular mass is paralleled by myocyte
hypertrophy (i.e. most investigators agree that myocyte hyperplasia does not occur; Soonpaa &
Field, 1998, but see Anversa & Kajstura, 1998). The classic physiological hypertrophy example
is where endurance training causes a "healthy" hypertrophy with increased cardiac function and
stroke volume (and lower resting heart rate). Pathophysiological stimuli include hemodynamic
load (pressure or volume) and neurohumoral stimuli (e.g. renin-angiotensin n, endothelin,
adrenergic). In general then, hypertrophy can be viewed initially as an adaptive process, to help
the heart deal with an increase in work load. To the extent that the hypertrophy allows the heart
to meet the needs of the body, this can be considered compensatory hypertrophy. At some point,
the hypertrophic response may be inadequate, and further changes can become decompensatory
and contribute to the genesis of heart failure (HF). Thus the transition or progression from
hypertrophy to HF is functionally quite important, but is not unidirectional (Lorell, 1997; Katz,
2000). Ventricular hypertrophy can be either concentric or eccentric. Concentric hypertrophy,
where the walls & myocytes thicken, but the chamber does not dilate, is especially common with
pressure overload (i.e. when the heart cannot generate sufficient pressure to adequately overcome
elevated afterload). Eccentric hypertrophy, where the chamber becomes dilated and myocytes
become elongated, is most common in volume overload (e.g. valvular insufficiencies).
The heart and myocytes seem somehow tuned to produce optimal function, a variety of
factors can disturb this balance. In addition to hemodynamic load and neurohumoral factors,
hypertrophy is induced by many transgenic or gene knockouts that may unbalance the tuning of
excitation and contraction processes in the heart, or in more overt ways undermine cardiac
function. An example is familial hypertrophic cardiomyopathy (FHC) caused by congenital
defects in myofilament proteins (Seidman & Seidman, 1998). This somewhat generalized
response has led some investigators to suggest a relatively stereotypical pathway to hypertrophy.
In addition, some gene expression changes commonly observed in hypertrophy seem to
recapitulate a neonatal or fetal phenotype (e.g. switches from a-MHC to ~-MHC reexpression of
skeletal a-actin, reduced SERCA2 and increased Na/Ca exchange). These 2 points support the
idea that hypertrophy is a programmed reversion toward a more fetal phenotype. While there is
surely truth in this, it is increasingly clear that multiple and complexly interacting pathways and
phenotypes are involved in the etiology of hypertrophy (see Fig 174). Moreover, the pathways
which cause changes in gene expression (Fig 174) are not yet well connected to the resulting
molecular and functional phenotypes, especially with respect to contractile proteins, Ca
transporters, ion channels and modulators (e.g. kinases & phosphatases).
The signaling pathways in hypertrophic remodeling are very complex (for references see
reviews by Sadoshima & Izumo, 1997; Hefti et al., 1997; Sugden, 1999; Molkentin & Dorn,
2001). The most detailed analysis of the central molecular signaling cascades has been from
work in primary cultures of neonatal rat ventricular myocytes (NRVM). One important set of
pathways are the mitogen-activated protein kinase (MAPK) pathways, illustrated in Fig 174
(Sugden & Clerk, 1998,2000; Sugden, 1999). There are three main MAPKs (Ser-Thr kinases):
1) ERKI and ERK2 (extracellular regulated protein kinase, also known as p44-MAPK and p42-
MAPK), 2) JNKs (the c-Jun N-terminal kinases) and 3) the p38-MAPK. The MAPKs are focal
points of this complex cascade and they phosphorylation numerous substrates, including nuclear
transcription factors (which activate expression of different groups of genes, Fig 174). Some of
the early response genes used as indicators of hypertrophic gene expression are c-Jun, c-Fos, c-
Myc, Egr-1, ANP (atrial natriuretic peptide) and BNP (brain natriuretic peptide).
The MAPK cascades are activated by several Gq-protein coupled receptors, such as the
al-AR, endothelin (ETA), and angiotensin II receptor (AT 1). Indeed, ET-l and to a less complete
degree phenylephrine and angiotensin II have been shown to activate all 3 MAPKs and be a
potent stimulator of cellular hypertrophy (see Sugden, 1999 for references). The receptors above
all activate a Gq to release its ~y subunit, activating phospholipase C to produce diacylglycerol
(DAG) and IP 3 . DAG activates PKC, and PKCo and PKCE seem to be the key isoforms in
hypertrophic signaling. PKC and several other signals can activate Ras a small G-protein (21
kDa) and other related members of this group (Rae, Rho). These other Ras-activating pathways
include receptor tyrosine kinases activated by growth factors (e.g. fibroblast growth factor
{FGF}, insulin-like growth factor-l {IGF-l} and platelet-derived growth factor {PDGF}) and
mechanical stretch (via activation of the focal adhesion kinase FAK, and also via an autocrine/
paracrine release of ET-l or angiotensin-II). Ras activates Raf and some other upstream kinases
which are collectively referred to as MKKKs (MAPK kinase kinases) because they
phosphorylate MKKs (MAPK kinases) which in turn phosphorylate the MAPKs themselves.
While some key elements and interactions in this cascade are well worked out, there are
constantly new players and interactions being uncovered in this very active field. These
pathways studied in NRVM undoubtedly contribute to hypertrophic signaling in the intact animal
and man, but this obviously adds additional layers of complexity.
Stretch is an important physiological hypertrophic stimulus that may work independently
or in concert with neurohumoral factors above. Mechanical stretch of isolated myocytes can
rapidly activate all three MAPK cascade at multiple sites from phospholipase C to ERK1/2,
JNKs and c-fos (Komuro et at., 1991; Sadoshima & Immo, 1993, 1997). Candidates for the
mechanosensor have included stretch-activated ion channels (e.g. letting Ca in), integrins (via
activation of FAK, a tyrosine kinase) and other tyrosine kinases. Blocking some stretch-
activated channels by Gadolinium or some integrin signaling by an RGD peptide does not block
stretch-induced c-fos expression, while tyrosine kinase blockade can (Sadoshima et of., 1992,
1993). However, this does not rule out other stretch-activated channels or integrin signaling.
Indeed, FAK and mechanical stretch appear to be important in ET-I induced hypertrophy (Eble
et of., 2000). Thus, all three of these pathways might contribute to hypertrophic signaling.
Mechanical stretch can also cause autocrine/paracrine release of angiotensin II and ET-I
(Sadoshima et 01., 1993; Lin et 01., 1995; Yamazaki et 01., 1996; Cingolani et 01., 1998). In this
case stretch can induce release of angiotensin II and/or ET-1 from ventricular myocytes as well
as nonmyocyte cells. Thus, angiotensin II (and ET-1) may work both globally (e.g. in reno-
vascular hypertrophy) and locally via this autocrine/paracrine pathway. Indeed, angiotensin
converting enzyme (ACE) inhibitors and AT 1 receptor blockers can prevent or revert ventricular
hypertrophy induced by pressure overload (Baker et 01., 1990, 1992; Kojima et 01., 1994). The
mechanisms by which stretch causes angiotensin II or ET-I secretion is still unknown.
Elevation of cellular Ca has been implicated in cardiac hypertrophic signaling, and
Ramirez et 01. (1997) showed that activation of cardiac nuclear CaMKII-&B was important in
hypertrophic gene expression. The next year it became clear that calcineurin (a Ca-calmodulin
activated phosphatase, phosphatase 2b) may also be involved in cardiac hypertrophy (Molkentin
et 01., 1998; Sussman et at., 1998; Olson & Molkentin, 1998; reviewed by Mo1kentin, 2000).
Calcineurin binds to the nuclear transcription factor NF-AT3, which is basally phosphorylated
and cytoplasmic. When calcineurin is activated by Ca-CaM it dephosphorylates NF-AT3, which
is then translocated to the nucleus where it promotes gene expression. Transgenic over-
expression of calcineurin or NF-AT3 in mice caused hypertrophy and HF. Furthermore,
inhibitors of calcineurin (cyclosporin and FK-506) could ameliorate the hypertrophy induced by
overexpression of either calcineurin or 3 different sarcomeric protein mutants, or by pressure
overload (but not that due to overexpression of the retinoic acid receptor). Molkentin & Dorn
(2001) reviewed 7 other studies in different hypertrophic models which confirmed this effect, but
also 4 studies that did not find significant prevention of hypertrophy with calcineurin blockers.
More recently cardiac myocyte-targeted expression of 3 different calcineurin inhibitory proteins
was shown to attenuate hypertrophy in vivo (DeWindt et 01.,2001; Rothermel et 01., 2001). The
latter study showed that the myocyte-enriched calcineurin interacting protein (MCIPI) resulted
in a 5-10% smaller heart and inhibited exercise-induced hypertrophy as well. This means that it
--------Mechanical
----_, stretch C
__,-__-_,_-__-"'__"'/-7,-""-'------i.~
-===-\---e::....!:.:~=s.:::....:-=~:::::..:::.:...::::....:....::....:.'...-=..:..i'>'...::...:..:-lJ-------""--_:::-
Ca ~Ca/CaM FAK :s
MKKKs
MKKs MKK1/2
MAPKs
Figure 174. Hypertrophic signaling cascades. Gq-protein (G-pn) coupled receptors bind agonists above,
activating phospholipase C to produce diacylglycerides (DAG) that activate PKC, which can also be
activated by membrane permeant phorbol esters like PMA. Mechanical stretch induced activation of focal
adhesion kinase (FAK) and receptor- and nonreceptor tyorine kinases (TyrK) along with PKC can activate
the small G-protein Ras or its relatives (Rae, Rho). This stimulates the three MAPK cascades (see text and
Sugden, 1999, Molkentin & Dorn, 200] for abbreviations). Two Ca/calmodulin (CaM) activated pathways
are also indicated. These work via calcineurin which dephosphorylates a nuclear factor of activated T cells
(NF-AT3), and via Ca-Calmodulin dependent protein kinase (CaMK). All of these pathways converge on
various targets which are mostly nuclear transcription factors that alter gene expression. Diagram is based
on related ones by Sugden (1999), Passier et al. (2000) and Molkentin & Dorn (200]).
may be hard, at this point in the pathway, to selectively inhibit pathophysiological vs.
physiological hypertrophy. Thus, while Ca/CaM and calcineurin may not be a unique pathway
to hypertrophy, it seems clear that it is an important one.
An interesting side point in the calcineurin story (from the E-C coupling standpoint) is
that the FK-506 receptor which blocks calcineurin activation is the same FKBP (12 & 12.6) that
binds to the ryanodine receptor and is thought to modulate E-C coupling (see pg 198-200). That
is, the inhibition of calcineurin occurs when the FK-506-FKBP (or cyclosporin-cyclophilin)
complex binds to calcineurin and prevents its activation by Ca-CaM.
Passier et at. (2000) showed that CaMK can activate a Ca-dependent hypertrophic
pathway that is parallel to, and additive with, the calcineurin pathway in producing hypertrophy.
Zhu et at. (2000) also showed that CaMKIl activation may be critical in the hypertrophy induced
by endothelin-L Thus Ca-dependent signaling may playa prominent role in hypertrophy. An
unanswered question is how these signaling systems can decipher the relevant Ca signal from the
dynamically complex and modulated Ca transients associated with E-C coupling. Presumably
this is via either some sort of integration of Ca signals or localized signaling (see pg 243 & 284).
PKC activation is directly involved in the MAPK cascade, but it can additionally activate
NaiR exchange either directly (Wallert & Frolich, 1992) or via downstream ERK activation
(Snabaitis et al., 2000). This Na/H exchange activation causes proton extrusion and intracellular
alkalosis which can alter [Na]; and rCa]; (see pg 284-285). However, alkalosis can be a
hypertrophic stimulus itself. One possible explanation is that high pHi may increase Ca binding
to calmodulin just as it does for troponin C (a related Ca-binding protein) where high pH
increases myofilament Ca sensitivity. This increased Ca affinity of CaM may enhance signaling
through the Ca-CaM, CaMKII and calcineurin pathways.
An unusual Ca link to this story is that enhancing Ca transients and SR Ca-ATPase by
SERCA2a gene transfer or phospholamban gene ablation can inhibit hypertrophy and HF due to
aortic banding or hypertrophy/HF where the cause is an apparently unrelated structural gene
(Miyamoto et aI., 2000; Minamisawa et aI., 1999). This emphasizes that while there are many
pathways which lead to hypertrophy and HF, cellular Ca regulation and its ability to regulate
contractility playa central and interactive role in determining the ultimate cardiac phenotype.
So what are the results of these hypertrophic changes in gene regulation on Ca transport
proteins, Ca transients, contractions and APs? The extensive literature is really quite variable,
and phenotype may depend on the stage of hypertrophy and progression to HF. Thus, while there
are reports that may, in majority, suggest that SR Ca-ATPase is downregulated and Na/Ca
exchange is upregulated in hypertrophy (Arai et al., 1993; Kent et aI., 1993; Hasenfuss, 1998b),
there is tremendous variation in the effects of hypertrophy on cellular Ca transients, ionic
currents and contractions (increased, decreased & unaltered). This may reflect real biological
differences in relative states of compensation-decompensation during hypertrophy, but it makes
summarizing results complicated. Thus, I will reserve this type of discussion to the following
section on HF (where there is somewhat broader, if still incomplete agreement).
Heart Failure (HF)

HF is an increasing health problem, affecting >2 million people in the U.S. alone. In the
simplest terms HF means that the heart is unable to provide sufficient cardiac output to supply
the metabolic demands of the organism. Thus the bottom line is that contractile function of the
heart is depressed. The diagnosis ofHF carries a very high 5-yr mortality rate (-50%), and there
are two fatal pathways: 1) progressive decline in contractile function (pump failure) and 2)
sudden cardiac death due to arrhythmias. While a certain component of mechanical dysfunction
may be due to geometric factors, with respect to the law of LaPlace (Tension =Pressure x radius /
(2 x wall thickness)), it is clear that intrinsic defects in contraction occur at the myocyte and
trabecular level. For arrhythmias, there are also important tissue factors (e.g. pg 99-100)
involved in propagation of arrhythmias, and the initiation of some. However, mar.y arrhythmias
also have their basis at the cellular level and may be related to both Ca handling and cellular
ionic currents. Therefore, this section will focus on the cellular basis of contractile dysfunction
and arrhythmogenesis.
So what is the basis of the contractile dysfunction? As for hypertrophy, there are a
plethora of different HF model studies and some disagree about whether particular factors (e.g.
Ie" SR Ca-ATPase, Na/Ca exchange, myofilament Ca sensitivity) are increased, decreased or
unchanged (see reviews by Hasenfuss, 1998b; Richard et al., 1998; Mukherjee & Spinale, 1998;
Wickenden et aI., 1998; Niibauer & Kiiiib, 1998; Phillips et al., 1998, de Tombe, 1998; Houser et
al., 2000). I will not try to review the entire area here, but Table 26 provides a summary of some
results in human HF. I have tried to indicate the most usual findings, which are also largely
consistent with animal HF models. Choosing one working phenotypic hypothesis is hazardous in
that it unfairly discounts some discordant findings that are genuine, but it makes it much easier to
integrate. The integration below will reflect my working hypothesis and some balance as well.
Twitch contraction. Ca transients and APD. Almost by definition, reduced twitch force and cell
contraction in HF are expected, and this is seen in almost all HF models. However, at very low
heart rates HF may have little effect on force (Pieske et aI., 1995). In general, there is a less
positive (or even negative) force-frequency relationship in failing vs. nonfailing heart (see Fig
148B). Thus, the lower force in HF becomes increasingly apparent at physiological heart rates.
These force changes are usually paralleled by changes in Ca transient amplitudes. In addition,
there is a slowing of the rate of relaxation and [Ca]; decline in HF in most cases. This is related
to changes in Ca transport discussed below. Another ubiquitous finding in HF is a prolongation
of AP duration (Beuckelmann et al., 1992, 1993; Kiiiib et al., 1996, Niibauer & Kiiiib, 1998).
This is particularly prominent at very low heart rates, but as frequency increases the AP duration
shortens and there is a smaller difference between failing and nonfailing hearts (Vermeulen et
al., 1994; Pieske et al., 1995).
Myofilament Ca sensitivity. Most studies have found unaltered myofilament Ca sensitivity in
HF (Gwathmey & Hajjar, 1990; Perrault et al., 1990; D'Agnolo et aI., 1992; Wolff et aI., 1995b,
1996; Hajjar et aI., 2000), although the Wolff papers found increased myofilament Ca sensitivity
until they corrected for the lower phosphorylation state of the HF myofilaments. In two rat HF
studies there was a reduction in maximal force and Ca sensitivity (Fan et aI., 1997; Perez et aI.,
1999). It should be noted that rats (and mice) shift from fast to slow myosin heavy chain (ex to ~
MHC) during hypertrophy and HF, whereas larger adult mammals (rabbit, dog and human) are
predominantly ~-MHC to begin with and don't change much. Even Miyata et al. (2000), whose
data challenged the prevailing dogma that human ventricle is all ~-MHC, found ex-MHC to drop
from only 7% to 0% of total MHC in human HF. There are also reports of changes in other
myofilament proteins that could alter force development (TnT, Tnl and myosin light chains, see
deTombe, 1998 for review). However, there are clearly decreases in contraction in HF which are
more likely to be ascribable to altered Ca transients. Indeed, many HF studies that have
measured both contraction and [Ca]i have found roughly proportional decreases in both,
consistent with unaltered myofilament Ca sensitivity in HF. Thus, while myofilament changes
may occur in HF, it is probably not the most central factor in explaining reduced function.
Ca current. Many reports show no change in peak lea density in HF, but decreases have also
been reported (see Table 26 & Mukherjee & Spinale, 1998). There was no HF-associated change
in lea at any voltage in human (Beuckelmann & Erdmann et aI., 1992), rat (Gomez et al., 1997),
canine (Kiiiib et al., 1996; O'Rourke et al., 1999), rabbit (Pogwizd et al., 1999) or guinea-pig
ventricular myocytes (Ahmmed et al., 2000), despite strong depression of both contractions and
Ca transients. This demonstrates that reduced Ca transients occur in many HF models with
unchanged lea. Thus, while lea may be reduced in some HF models, I conclude that lea is not
primarily responsible for the reduced Ca transient in HF. Indeed, it is remarkable that with
nearly doubling of cell size, in HF the density of lea (in NF) can keep pace exactly with this
cellular hypertrophy (Pogwizd et al., 1999).
SR Ca-ATPase & phospholamban (PLB). A tremendous number of studies have measured

cardiac SR Ca-ATPase expression and function, since de la Bastie et at. (1990) first reported
down-regulation of SERCA2 expression in pressure-overloaded rat heart. Feldman et at. (1993)
showed data suggesting that down-regulation of SERCA2a expression may mark the transition
from hypertrophy to HF. It seems clear that SR Ca-ATPase is functionally decreased in almost
all HF models (despite a few reports to the contrary; Table 26). PLB also appears to be down-
regulated in HF, roughly in proportion to SERCA2. This should not alter the [Cali-dependence
of SR Ca transport, but of course would decrease Ca transport at all [Cali. There are also data to
suggest that the phosphorylation state of PLB may be reduced in HF (Huang et at., 1999;
Schwinger et at., 1999c, but see Currie & Smith, 1999). This would reduce the [Cal-sensitivity
of SR Ca uptake and further slow Ca transport at physiological [Cali. FurtheIIDore, if SERCA2
were down-regulated without altered PLB, the depressant effect on SR Ca transport could be
disproportionately high (since more SR Ca-pumps would be PLB-inhibited, see pg 165). An
example where this SERCA2:PLB ratio changes dramatically is in response to thyroid hormone
state, which may decline in HF (Hamilton et at., 1990; Kiss et at., 1994; Ojamaa et at., 2000).
Increases in thyroid hormone increase SERCA2 expression and decrease PLB expression. This
greatly increases the SERCA2:PLB ratio, and disproportionately stimulates SR Ca transport.
The hypothyroid state results in the converse and the greatly reduced SERCA2:PLB ratio,
profoundly depresses SR Ca transport.
Reduced SR Ca-ATPase function fits well with the characteristic slowed relaxation and
[Cali decline of HF. Moreover, when SERCA2 expression in myocytes is increased by
adenoviral gene transfer, relaxation and [Ca]i decline can be accelerated (del Monte et at., 1999;
Miyamoto et at., 2000). Thus, it seems clear that reduced SERCA expression and function are
important in the slowed relaxation and [Cali decline characteristic ofHF.
RyR and SR Ca content. RyR mRNA seems to be reduced in human HF, but Western blot and
ryanodine binding indicate that RyR protein levels are unchanged (Go et at., 1995; Schillinger et
at., 1996; Sainte Beuve et aI., 1997). In the pacing-induced dog HF model there seems to be
down-regulation of RyR (Vatner et at., 1994; Yano et at., 2000), but not in spontaneous
hypertensive HF rat (Gomez et at., 1997). Thus, results are somewhat mixed for RyR number,
but intriguing new results have also suggested that RyR regulation may be altered in HF. Marx
et at. (2000) showed that in HF the RyR2 can be hyper-phosphorylated by PKA, causing
displacement of FKBPI2.6 from the RyR (see pg 189-190 & 199-200). The hyperphos-
phorylation may be due to their finding of less phosphatase associated with the RyR complex,
despite a generalized increase in phosphatase expression in HF (Neumann et at., 1997). Without
FKBP 12.6 the RyR open probability is higher at rest, but shows less coordinated gating. This
could increase diastolic SR Ca leak, as seen in intact cells treated with FK-506, which also
displaces FKBPI2.6 from the RyR (McCall et at., 1996a). This may contribute to a lower SR Ca
content in HF and might also alter how the RyR responds to lea during E-C coupling. Yano et at.
(2000) also found reduced FKBP:RyR stoichiometry in HF and greater Ca leak from SR vesicles.
On the surface, the Marx et at. (2000) story seems consistent with an intrinsic depression
ofE-C coupling in hypertrophy or HF in rats (Gomez et at., 1997; McCall et at., 1998). In those
studies there was reduced SR Ca release in hypertrophy or HF for the same lea and SR Ca
content. On the other hand, removal of FKBP from RyR by FK-506 enhances (rather than
depresses) fractional SR Ca release (McCall et al., 1996a) and also sensitIzes the RyR to
activator Ca (Marx et al., 2000). This would imply increased Ca transients in HF. Eisner et at.
(1998, 2000) also argued that an alteration in RyR gating alone cannot produce long-lasting
depression of Ca transients, because of the sort of autoregulation discussed on pg 266-267.
Thus, it may be that the diastolic SR Ca leak induced by the PKAlFKBP effect in HF is the more
functionally important aspect of this RyR modification in HF.
Table 26
Alterations of Expression and Function in Human Heart Failure.
Change in HF* Prevalence Data type+ & Ref (Counter-refl
Twitch Contraction lower/slowed -all F-1,2,3
Twitch L'.[Ca]; lower/slowed -all F-1,2,4,5
Force-frequency from + to more - -all F-2,3,6,7; (see also Fig 148)
MF Ca sensitivity unaltered (higher) -all F-8,9,1O,11,12 (/3)
lea/ DHPR unaltered (lower) most F-4,16,17,18 (19,20); R-14 (/5);
SR CaATPase lower (unaltered) most F-1 ,2,30,31; R-15,21,22,23,24,25;
P-26,27,28 (22,25,29)
SR Ca content lower -all F-10,30,32
Phospholamban lower (unaltered) most R-22,24,25,33; P-27 (22,25,29)
Calsequestrin unaltered -all R-15,24; P-27,29
Calreticulin unaltered -all P-27
RyR unaltered (lower) mixed R-24 (34,35); P-27,36,37;
F-38 (8,39)
Na/Ca exchange higher (unaltered) -all F-30,41,42; R-28,40;
P-28,40,41,42 (43);
Na/K-ATPase lower -all P-43,44,45
AP duration higher -all F-4,46,47
Ito lower -all F-46,48,49
IK1 lower -all F-46,47
Gj higher -all F-53; R-50; P-51,52,53
Gs unaltered (higher) -all R-50 (54)
*where groups disagree, I have weighed evidence in an attempt to provide more useful information. In
cases of disagreement, there may be true differences in etiology or direct cause of dysfunction.
+supporting data are from F (functional tests in cells, hearts or transport assays), R (mRNA measurements)
or P (protein measurements; Western blot or ligand binding). Prepared with the help of L.S Maier.
I Gwathmey et al., 1987 19 Piot et al., 1996 37 Sainte Beuve et al., 1997
2 Pieske et al., 1995 20 Ouadid et al., 1995 38 Holmberg & Williams, 1989
3 Davies et al., 1995 21 Mercadier et al., 1990 39 Nimer et al., 1995
4 Beuckelmann et al., 1992 22 Schwinger et al., 1995 40 Flesch et al., 1996
5 Sipido et al., 1998b 23 Limas et al., 1987 41 Reinecke et al., 1996
6 Mulieri et al., 1992 24 Arai et al., 1993 42 Hasenfuss et al., 1999
7 Hasenfuss et al., 1992 25 Linck et al., 1996 43 Schwinger et al., 1999b
8 D'Agnolo et al., 1992 26 Hasenfuss et al., 1994 44 Schmidt et al., 1993
9 Hajjar et al., 1992 27 Meyer et aI., 1995 45 Norgaard et al., 1988
10 Denvir et al., 1995 28 Studer et al., 1994 46 Beuckelmann et al., 1993
11 Gwathmey & Hajjar, 1990 29 Movsesian et al., 1994 47 Koumi et al., 1995
12 Hajjar et al., 2000 30 Pieske et al., 1999a 48 Wettwer et al., 1994
13 Wolff et al., 1996 3 I Schmidt et al., 1998 49 Nabauer et al., 1996
14 Schwinger et al., 1999a 32 Lindner et al., 1998 50 Eschenhagen et al., 1992
IS Takahashi et al., 1992 33 Feldman et al., 1991 51 Neumann et al., 1988
16 Beuckelmann & Erdmann, 1992 34 Brillantes et al., 1992 52 Feldman et al., 1988
17 Rasmussen et al., 1990 35 Go et al., 1995 53 Bohm et al., 1990
18 Mewes & Ravens, 1994 36 Schillinger et al., 1996 54 Feldman et al., 1989
Intra-SR Ca buffering capacity is probably unchanged, since calsequestrin (and

calreticulin) does not seem to be altered in HF (Hasenfuss, 1998b). This means that if SR Ca
content is lower in HF, the free [Ca]sR is also lower. There are few measures of SR Ca under
relatively physiological conditions. Nevertheless, in HF the SR Ca content does seems to be
reduced in human (Lindner et al., 1998), rabbit (Pogwizd et al., 1999; 2001) and dog (Hobai &
O'Rourke, 2001), based on caffeine-induced Ca transients. Moreover, the reduced SR Ca content
is completely consistent with the reduced SR Ca-ATPase activity, the increased RyR leak above
and also the upregulation of Na/Ca exchange (below). A reduced SR Ca content would also
explain the reduced twitch ~[Ca]; and contractile function.
Na/Ca exchange. Studer et al. (1994) first showed an increase in Na/Ca exchange mRNA in
human HF. This seems to be a rather consistent finding in HF in human (Flesch et al., 1996;
Hasenfuss et al., 1999) and most rabbit, guinea-pig and dog HF models (e.g. Pogwizd et al.,
1999; Hobai & O'Rourke, 2000; Sipido et al., 2000; Ahmrned et aI., 2000), as well as in rabbit
myocytes from the peri-infarct zone during post-infarct HF (Litwin & Bridge, 1997). Indeed, in
our non-ischemic rabbit HF model, we find that Na/Ca exchange is consistently upregulated by
~100% at the level of mRNA, protein, [Cali decline of caffeine-induced contractures and INa/c.
(with either [Cali clamped or dynamically changing, Pogwizd, 1999,2001). To the extent that
Na/Ca exchange is primarily engaged in Ca extrusion, higher Na/Ca exchange will be expected
to compete better with the SR Ca-ATPase during relaxation (and diastole). This would tend to
reduce SR Ca content (as above). This interpretation is supported by overexpression of Na/Ca
exchange in rabbit ventricular myocytes, where the phenotype was depressed contractility,
blunted force-frequency relationship and reduced SR Ca content (Schillinger, et al., 2000).
As we have seen (pg 147-150 & 290-294), the effects of Na/Ca exchange are
complicated by its bi-directional nature and its dependence on Em and gradients for [Na] and
[Ca]. Indeed, if Ca transients in HF are of low amplitude, greater Ca influx via Na/Ca exchange
could occur, even early in the AP (see Figs 72 & 74). Also with low [Cali and long AP duration
in HF, there can be an extended period of Ca influx via Na/Ca exchange during the AP (Dipla et
aI., 1999), which would not be expected for large Ca transients and short AP duration. It seems
that this Ca influx enhancement via Na/Ca exchange would be most likely at low frequency in
HF, where AP duration is especially prolonged. This may also explain why the Ca transients and
contractile force are less depressed compared to control at low heart rates. Thus it seems likely
that Na/Ca exchange upregulation is an important factor in altered Ca handling in HF.
Na/K-ATPase and [Nal). The way that Na/Ca exchange functions is critically dependent on the
level of[Na];, which is in turn dependent on the activity of the Na/K-ATPase (see Chapter 6 and
pg 286-294). There are several reports which indicate reduced Na/K-ATPase expression in HF
(Dixon et al., 1992; Semb et aI., 1998; Schwinger et al., 1999b). This would be expected to
elevate [Na]; and be inotropic. While only preliminary data are available, there does seem to be
higher [Na]; in HF vs. control ventricle from human (Maier et al., 1997b), dog (Verdonck et aI.,
2001) and rabbit (S Despa, M Islam, SP Pogwizd & DM Bers, unpublished). It is premature to
evaluate how this perturbs Ca regulation, but one would expect a shift toward less Ca extrusion
and greater Ca influx via Na/Ca exchange (as in glycoside inotropy). It is possible that this
elevated [Na]; partly offsets an even greater depression of Ca transients and contractile function
that might otherwise be observed in HF. While lower Na/K-ATPase could explain higher [Na];
in HF, there is also evidence to suggest a more prominent contribution of a very slowly
inactivating INa component (INa.Slow) in HF (Saint et al., 1992; Maltsev et al., 1998; Undrovinas et
al., 1999). INa.Slow could also contribute significantly to the AP prolongation observed in HF.
Other ion currents. Other ventricular ion currents may also be altered in HF. The most
consistent findings so far are reductions in Ito and IK1 in HF (Table 26, reviewed by Wickenden et
al., 1998 and Niibauer & Kiiiib, 1998). Reduction in Ito can reduce the early repolarization during
the AP (phase 1). This notch in the AP may normally serve to enhance the driving force for Ca
entry once Ca channels are activated. Hence, in HF reduced Ito could decrease early Ca influx
and triggering of SR Ca release, but this has not been tested. However, reducing Ito is expected
to have little effect on overall AP duration in human, dog, rabbit or guinea-pig ventricle (e.g.
Priebe & Beuckelmann, 1998). Exceptions to this are rat and mouse ventricle, where the very
large Ito is a predominant cause of repolarization of the very short AP that is observed in those
species. The decrease in IKI may contribute to AP prolongation, but this would be mainly in the
very late phases of final repolarization, because of its inward rectification (see Figs 41 & 45).
An even more important aspect of the 40-50% IKI reduction in human, dog and rabbit HF
(Beuckelmann et al., 1993; Kiiiib et al., 1996; Pogwizd et al., 2001) is that it destabilizes the
diastolic Em. This may increase the propensity for arrhythmogenesis (see pg 298-299 & below).
There is some data to suggest that delayed rectifier K currents (IK" IKs , IKur) may also be
reduced in HF (VoIders et al., 1999), but there is not enough data available to provide definitive
conclusions about this. This is partly because these currents vary among species and are also
somewhat more tricky to dissect electrophysiologically. On the other hand, changes in these
delayed rectifier K currents can strongly alter AP duration (Priebe & Beuckelmann, 1998), so
additional information on delayed rectifiers would be particularly valuable.
So which currents contribute to APD prolongation in HF? Reductions in the outward
currents Ito, IK1 (and possibly other K currents) and Na/K-ATPase could all contribute. In
addition increases in inward currents (Ic., INa,Slow & INa/ca) could also contribute. If peak Ica is
unchanged in HF, but SR Ca release is reduced, we might expect less Ca-dependent inactivation
of Ica and hence more integrated Ca influx (see Fig 60; Ahmrned et al., 2000). This would
increase the total inward current during the AP. It would also mean that there must be more
overall inward INa/ca during the cardiac cycle to extrude all of the Ca which entered via Ica . Thus,
greater inward INa/ca may result from upregulation of Na/Ca exchange expression and the slower
rCa]; decline in HF (which would drive more inward INa/ca). Of course an elevated [Na]; would
also tend to shift INa/ca in the outward direction, such that the exact mode of INa/ca during the AP
is hard to predict. Thus several factors probably contribute to the AP prolongation typically
observed in HF. In addition, it should be appreciated that the AP prolongation itself has a
salutary effect on Ca transients (Kaprielian et al., 1999), since it increases Ca influx (by Ica and
possibly by INa/ca) and slows Ca extrusion via Na/Ca exchange. Thus, the longer AP may partly
offset other negative inotropic effects ofHF.
The contractile dysfunction in HF (Fig 175) is probably largely due to a reduction of SR
Ca content during steady state physiological conditions. Three factors may contribute to the
lower SR Ca content: 1) reduced SR Ca-ATPase function, 2) increased Ca efflux via Na/Ca
Sarcolemma
Ca Ca
.Lt>[Cal,-' .Ll ea inact.

ilnward INaeaX il NalCa -. ii, for any t>[Cal, Ca
.L1,o' IK ,. IK1 - . i APD .LI K1 - . t>i Em for any I"
El IIII:'
1111111
illiITEJ
illiITEJ L-_=-,---_--' (espec. at low HR) p.AR-.iSR [Cal & spont rei
!SR Ca Load !6[Ca]1 & ~

Mechanical Dysfunction
Other possible factors
JAP~
i Diastolic RyR Ca leak -..LSR Ca
.Ll ca or RyR could also .LFrac Release i Propensity for
.LMF Ca response could .LContraction Triggered Arrhythmias
Figure 175. Contractile dysfunction and arrhythmogenesis in HF. The contractile dysfunction probably
results from a reduction in SR Ca content (due to reduced SR Ca-ATPase, increased Na/Ca exchange, and
increased diastolic SR Ca leak). Other possible contributors are indicated. Arrhythmogenesis may be
triggered by either early or delayed afterdepolarizations (EADs or DADs). EADs are more likely at longer
AP duration and factors which may contribute to this are shown. DADs are due to spontaneous SR Ca
release and there may be three key factors which increase propensity for EADs in HF (see text).
exchange (by competing with the SR Ca-ATPase for Ca) and 3) possible diastolic Ca leak from
the SR. Furthermore, a lower SR Ca content reduces SR Ca release both because there is less Ca
available for release and also because fractional release is reduced for a given lea trigger (pg 224-
226). In some HF cases there might also be decreases in trigger Ie" reduced intrinsic RyR
responsiveness during E-C coupling, or reduced myofilament Ca sensitivity. However, I find the
reduced SR Ca content explanation a more simple and unifying working model right now.
Reduced SR Ca-ATPase and increased Na/Ca exchange can both tend to lower SR Ca
content, but exert opposite effects on relaxation rate and [Cali decline. This combination can
thus result in unchanged relaxation rate, as we found in our rabbit HF model where a doubling of
Na/Ca exchange appeared to offset ~24% decrease in SR Ca-pump function (Pogwizd et ai.,
1999). Hasenfuss et al. (1999) found this same situation in 44% of human HF (nearly doubled
Na/Ca exchanger with modest SERCA reduction). These muscles did not show elevated
diastolic force at 3 Hz (vs. 0.5 Hz). However, another group of HF muscles (25%) that showed
slower relaxation and elevated diastolic force at 3 Hz, had a greater reduction in SERCA2
expression, but unaltered Na/Ca exchange. Thus, there may be a real heterogeneity of diastolic
dysfunction in HF that depends on the balance of SR Ca-ATPase and Na/Ca exchange function.
With respect to arrhythmogenesis in HF, I will restrict discussion to triggered

arrhythmias (i.e. DADs and EADs). Electrical reentry (pg 99-100) contributes to ventricular
tachycardia (VT), but 3-dimensional mapping studies have shown that most VTs in HF initiate
by a non-reentrant mechanism such as DADs and EADs (Pogwizd, 1994, 1995; Wit & Rosen,
1992). In human HF this is true for 100% of VT in nonischemic HF and 50% in post-ischemic
HF (Pogwizd et at., 1992, 1998). Figure 175 indicates possible reasons for the enhanced
arrhythmogenesis observed in HF. EADs are more likely when AP duration is prolonged (as
occurs in HF, especially at low heart rates). The same factors described above may prolong the
AP and increase the likelihood that Ic• has recovered sufficiently during the late plateau phase to
induce the type of EAD discussed on pg 97-98. Thus, EADs may be most common in brady-
arrhythmias. However, as noted above, AP duration is not always so prolonged in HF, especially
at physiological frequencies, and tachyarrhythmias occur at short cycle lengths. DADs would
initially seem unlikely in HF because spontaneous SR Ca release is less likely to occur at low SR
Ca content (as seen in HF). However, as discussed on pg 299, bursts of I3-AR activation can
readily drive SR Ca content to the point where spontaneous SR Ca release occurs in HF
(Pogwizd et at., 1999, 2001). Moreover, any given SR Ca release causes greater Iti in HF
(because Na/Ca exchange is increased), and any given Iti causes a greater DAD in HF (largely
because IK1 is decreased). If, during very long APs, there is Ca loading occurring via Na/Ca
exchange, one could even get a spontaneous SR Ca release, triggering what looks like an EAD,
but is more mechanistically related to a DAD. Thus the key facets ofHF that contribute to DAD-
induced triggered arrhythmias are: 1) increased INa/C., 2) reduced IK1 and 3) residual I3-AR
activity (to cause SR Ca load to sufficiently for spontaneous Ca release). Indeed, with loss of 13-
AR responsiveness in very late stage HF, arrhythmias are less common (see pg 299).
In hypertrophy and HF there can also be energetic limitations. This may not be apparent
under low work conditions, but may manifest as a limited cardiac reserve, when cells, muscles or
hearts are challenged with higher work loads (Neubauer et at., 1995; Tian et at., 1997; Brandes
et at., 1998; Ito et aI., 2000). This may also contribute somewhat to the blunted force-frequency
relationship seen in human HF (Pieske et at., 1995). The limiting factor could be the alteration
in ~GATP as discussed above for ischemia (which could alter SR Ca transport, Na/K-ATPase and
myofilament function). This may also result, in part from a reduction in the ratio of cell volume
occupied mitochondria vs. myofilaments (Lund & Tomaneck, 1978; Anversa et at., 1979). This
value is 16-37% higher in control than hypertrophied rat hearts.
In conclusion, there are several interdependent factors that may contribute to both the
mechanical dysfunction and the propensity for arrhythmias in the failing heart. There is much
still to clarify in this area, but a tremendous amount of research effort is targeted in this direction.
The coming years ShOldd make this complex HF story increasingly clear.
SITES FOR INDUCTION OF CARDIAC INOTROPY

Since contractility is depressed during HF, it is important to consider mechanisms that
can improve contractility. The two main inotropic mechanisms are: 1) increasing the amplitude
or time course of the Ca transient, so that more Ca is supplied to the myofilaments, and 2)
enhancing myofilament Ca sensitivity, by either increasing the Ca affinity oftroponin C (TnC) or
increasing force for a given degree of Ca occupancy of TnC. I will focus below on some of the
EMD-57033 Ca Sensitizing Enantiomer

20 £mMCa
A. B.
a Normalized
traces
~l]._ _
~~
N
Figure 176. The effect of EMD-57033 on contractions and Ca transients (assessed by aequorin
luminescence, a non-linear [Cal; indicator) in ferret ventricle at 30°C, 0.33 Hz. A. [EMD-57033] was
progressively increased and [Ca]o was raised from I to 4 mM as indicated to verify that [Cal; and force
could still be increased. B. Normalized traces from points a and b are superimposed to compare the kinetics
of Ca transients and contractions. Figure is modified from White et aI., 1993, with permission).
advantages and disadvantages of specific inotropic strategies or targets, rather than review results
with many individual agents. While there are certainly many ways to increase myofilament Ca
sensitivity and Ca transients, one must always be mindful of key limitations, such as energetics,
diastolic force and arrhythmogenesis. Anything we do to increase Ca transients and myofilament
ATPase will consume more energy, and the heart (especially in pathophysiological conditions)
may have trouble matching increased energy demands with oxygen/energy supply. Increased
myofilament Ca sensitivity may result in elevated diastolic force, which can require more
diastolic ATP consumption, and also mechanically limit ventricular filling and coronary blood
flow during diastole. If we bring more Ca into the cell, the necessary Ca transport will use ATP,
and can also lead to diastolic dysfunction and arrhythmias, as discussed above. Thus, there are
very practical limitations that overlay this entire discussion of inotropic mechanisms.
Modulation ofMyofilament Ca Sensitivity

Several agents can increase the myofilament Ca sensitivity (e.g. caffeine, theophylline,
pimobendan, sulmazole, isomazole, adibendan, perhexiline, bepridil, CGP-48506, EMD-57033,
MCI-154, levosimendan, see pg 35). This is a rather direct strategy to increase contractility.
That is, increasing myofilament Ca sensitivity will lead to greater force for the same amount of
activating Ca (SR Ca release + Ca influx). If the sensitization works by increasing Ca affinity of
TnC, then peak [Ca]; during the contraction is expected to be lower. This is a consequence of
TnC being a major [Ca]; buffer and the fact that >95% of the activating Ca is buffered during the
Ca transient (pg 41-47). Thus .he peak rCa]; is determined by both the amount of activating Ca
and the amount of intracellular Ca buffering. Sulmazole (AR-L 115BS) is a fairly typical early
drug which increases myofilament Ca binding and sensitivity and showed these characteristics
(Solaro & Ruegg, 1982; Blinks & Endoh, 1984). However, sulmazole, like many other inotropic
drugs, is not a pure Ca sensitizer. It also inhibits a cyclic nucleotide phosphodiesterase (POE-III)
which raises cAMP levels and increases PKA activity (Endoh et al., 1985). Sulmazole also has a
caffeine-like action to open SR Ca release channels (Williams & Holmberg, 1990).
EMD-57439 PDE Inhibitor Enantiomer

4 mMCa
A. B. C.
E
'0
Q) '
traces .!:l
"iii
~57439
~<i]
ro c
E
0
a
U
~~
O
b
-S
Q) ~
a b ~ 57033
0
~
LL
Q)
E] 0
u.€ 0.4 0.• 0.8 1.0
N
~z
o E 500 ms [Cali (normalized)
LL.~ '10 min'
Figure 177. The effect of EMD-57439 on contractions and Ca transients (assessed by aequorin) in ferret
ventricle at 30°C, 0.33 Hz. A. [EMD-57439] was progressively increased and [Ca]o was raised from I to 4
mM as indicated. B. Nonnalized traces from points a and b. C. [Cali-dependence of force (peak values)
for data in A and Fig 176A, showing that with EMD-57033 force increases as peak [Cali declines, while for
EMD-57439 they increase in parallel. Figure is modified from White et aI., 1993, with permission).
EMD-53998 is another cardiotonic agent which shows both Ca sensitizing effects and
PDE inhibition (Allen & Lee, 1989; Beier et at., ]991). An intriguing aspect of this drug is that
the two effects seem to be separately attributable to the two optical stereoisomers of the
compound ((+)EMD-57033 & (-)EMD-57439, Lues et aI., 1993; Solaro et aI., 1993; White et
at., 1993). Figure 176 shows the effect of the pure Ca sensitizer (EMD-57033). There is a
progressive increase in force and decrease in the peak of Ca transients (as expected due to
increased Ca binding by TnC, see Fig 177C). In Fig 176A there is only a slight increase in
diastolic force at 10-20 J.lM EMD-57033, although Solaro et al. (1993) found greater diastolic
contracture at lower [EMD-57033]. The normalized traces show that Ca transient kinetics were
little changed, but that relaxation was slowed. This is an intrinsic limitation of the myofilament
sensitization approach, where relaxation is slowed and diastolic filling is reduced (Hgashiyama et
aI., 1995; Hajjar et aI., 1997). On the other hand, in vivo studies have shown that EMD-57033
can produce strong inotropic effects (at relatively lower energy cost), without slowing ventricular
relaxation or filling (Senzaki et at., 2000). EMD-57033 and CGP-48506 (Neumann et at., 1996;
Wolska et aI., 1996a) seem to be Ca sensitizers which most clearly lack PDE inhibitory activity,
although CGP-48506 may increase Ie" (Herzig, 1996).
Figure 177 shows the effects of the other EMD-53998 isomer (EMO-57439) which
exhibits primarily POE inhibitory effects (White et at., 1993). In this case Ca transients and
contractions increase roughly in parallel, and there is faster relaxation and [Cali decline. These
effects mainly reflect PDE inhibition, which can increase cAMP levels and thereby activate the
same cascade of effects as P-AR activation (pg 275-282). The net results expected are: I)
increased lea, 2) more rapid SR Ca-ATPase and [Cali decline, 3) increased SR Ca release (due to
higher trigger and load) and 4) an a decrease in myofilament Ca sensitivity. For drugs which are
both Ca sensitizers and POE inhibitors, the net effect on myofilament Ca sensitivity will be a
compromise between the direct increase and the decrease due to the PKA-dependent TnI
phosphorylation. Indeed, for these mixed Ca sensitizer-POE inhibitors, the PKA-dependent
Myofilament Ca Sensitivity
_---iMax Force
E::l
E 100
'xC1l
E iCooper-
~
~ erath/ity
c: 50
0
'iii
c:
Ql
iCa sensi-
I- tivity
0
0.01 0.1 10
[Ca] (IJM)
Figure 178. Increasing myofilament Ca sensitivity can limit relaxation. For Normal, a Hill curve was
used with Km=630 nM [Cal and n=2. Increased maximal force used 127% of Normal, but the same other
parameters. A parallel shift of myofilament Ca sensitivity is assumed to change Km to 280 nM [Cal (with
n=2). Increased myofilament cooperativity used Km= 400 nM [Cal and an Hill slope n=4.
stimulation of SR Ca uptake may offset the problem of slower relaxation caused by increased
myofilament Ca sensitivity. Thus, while both Ca sensitization and PDE inhibition have pluses
and minuses, they may synergize. There may even be an optimal balance in the combination of
these two attributes.
The rate of Ca binding to TnC is fast (nearly diffusion-limited, Table 11). If this is
correct, then changes in Ca affinity would depend primarily on changes in the rate of dissociation
of Ca from Tne. Smith & England (1990) found that the rate of Ca dissociation from the bovine
cardiac troponin-tropomyosin complex was unaffected by sulmazole or pimobendan, but reduced
by isomazole, perhexiline and bepridil. These results are consistent with an increased Ca binding
with isomazole, perhexiline and bepridil (Solaro et aI., 1986), but not with results which showed
that Ca binding to dog myofilaments was also increased by sulmazole and pimobendan (Solaro &
Riiegg, 1982; Jaquet & Heilmeyer, 1987). It should also be noted that myofilament Ca
sensitivity can be increased without a change in Ca binding (as with caffeine, Powers & Solaro,
1995). For example, alterations in cross-bridge cycling or the coupling between contractile
proteins downstream from Ca binding could increase the Hill coefficient of activation by Ca (see
pg 23-25 and Solaro, 2001). Along these lines, peptides derived from the myosin head can alter
Tnl-actin interaction and thereby increase myofilament Ca sensitivity (Riiegg et al., 1989).
A pure increase in myofilament Ca sensitivity can increase the amount of force for a
given amount of activating Ca. A key advantage is that the balance of transsarcolemmal Ca
fluxes need not be altered. Thus, the cells are not subject to additional Ca load (and the potential
negative inotropic, energetic and arrhy1hmogenic consequences). Figure 178 shows a key dis-
advantage of Ca sensitizers which produce parallel shifts of the [Cal-force relationship (typical
of many Ca sensitizers). At diastolic [Cali (~150 nM) there can be incomplete relaxation, and
this may be of particular concern in pathological conditions where diastolic [Cali is elevated or
[Cal; decline is slowed. Thus, it is crucial to know what diastolic [Cali is with respect to the
threshold for contractile activation (and this may change with conditions). The dotted curve in
Fig 178 shows an increase in the steepness of the [Cal-force relationship (with a smaller shift in
K JI2 ). This type of change could be especially beneficial, because the threshold for contractile
activation is not shifted to lower [Cal;, but myofilament Ca sensitivity is greatly increased.
However, as a practical matter it should be kept in mind that >95% of the Ca released from the
SR or entering the cell is bound (for a t>[Ca]; of I 11M a total flux of -60 Ilmol/L cytosol is
required, Chapter 3). The steeper [Cal-force relationship may allow a greater fraction of
activating Ca to bind to TnC (vs. other Ca buffers), but the absolute amount of activating Ca may
still be limiting. That is, if most of the activating Ca is already bound to TnC, there may be little
Ca available for further saturation ofTnC (without increasing the amount of activating Ca). The
other inotropic mechanisms discussed below all deal with changing the amount of activating Ca.
On balance, enhancing myofilament Ca sensitivity seems an appropriate inotropic target.
This is because it does not require additional Ca transport, does not have intrinsic electrophysio-
logical complications, and the main extra energy can go directly toward the work output of the
heart. The key challenges may be to obtain sufficient selectivity and avoid diastolic dysfunction.
Phosphodiesterase Inhibition
Many inotropic agents are PDE inhibitors. These include caffeine, theophylline, amri-
none, milrinone, enoximone, piroximone, saterinone, pimobendan, adibendan, sulmazole, EMD-
5998, MCI-154 and levosimendan (Butcher & Sutherland, 1962; Alousi et al., 1983; Endoh et
al., 1985; Weishaar et aI., 1988; von der Leyen et aI., 1988). There are at least 11 classes of
PDE, with 6 expressed in the heart (I-V and VII, see Table 27, Beavo, 1988; Beavo &
Reifsnyder, 1990; Schmitz et al., 1989; Francis et aI., 2000). Caffeine, theophylline, isobutyl-
Imethylxanthine (IBMX) and other methylxanthines are relatively non-selective PDE inhibitors.
Most of the inotropic drugs above are relatively selective inhibitors of PDE III, whereas agents
which inhibit PDE-I, II & IV do not appear to be particularly good inotropes. Bode et al. (1991)
showed that the most abundant PDE in heart (PDE-I) was absent in isolated cardiac myocytes,
suggesting that PDE-I is restricted to other cells in the heart.
These PDE-I1I inhibitors prevent the breakdown of cAMP to 5' -AMP and can thereby
increase cellular cAMP levels. This, of course, activates the same cascade as ~-AR agonists (see
pages 275-282). An advantage over ~-AR agonists is that these agents can still inhibit PDE-III,
when ~-ARs are down-regulated and cells are unresponsive to catecholamines. However, if the
Table 27
Classification of Cardiac Phosphodiesterases and Inhibitors
Class Substrates Key Regulator Inhibitors
I cAMP, cGMP Ca-CaM-stimulated KS-505
II cAMP, cGMP cGMP-stimulated EHNA
III cAMP> cGMP cGMP-inhibited Milrinone, amrinone,
pimobendan, sulmazole...
IV cAMP cAMP-specific Rolipram, RO 201724
cGMP-insensitive
V cGMP Zaprinast, sildenafil (Viagra)
VII cAMP ??
rate of cAMP production by adenylyl cyclase is too low, PDE inhibition may not greatly elevate
[cAMP];. This limitation can occur in human HF, where reduced ~-AR numbers and elevated G;
levels may limit cAMP formation (Schmitz et aI., 1989; Bristow et al., 1982, 1986). While one
could circumvent this by direct activation of adenylyl cyclase with forskolin, the side effects of
forskolin (including excessive vasodilation) are prohibitive.
When cAMP is increased by PDE-III inhibition, we expect an increase in Ica , an increase
of SR Ca pumping, and a decrease in myofilament Ca sensitivity. While in some cases these
effects have been reported, there are again multiple effects of most of these agents (e.g.
myofilament Ca sensitization or ion channel effects). The myofilament sensitizing effect might
more than offset the Ca desensitizing effect expected from cAMP-dependent phosphorylation of
Tnl. Ohte et al. (1997) found that in HF the response to a simple PDE-III inhibitor (arnrinone)
was blunted, while pimobendan (a Ca sensitizer with some PDE-III inhibitory activity) was still
robust. Sulmazole and milrinone also exhibit caffeine-like actions on the SR Ca release channel,
which may offset the PKA-dependent phosphorylation of phospholamban and stimulation of the
SR Ca-pump (Rapundalo et aI., 1986; Holmberg et al., 1990; Williams & Holmberg, 1990).
Thus the net results on Ca fluxes and contractile force can be rather complicated.
A major advantage with PDE-III inhibitors compared to Ca channel activators (and
catecholamines) is that the inotropy is accompanied by vasodilation. This effect is a conse-
quence of the relaxant effect of cAMP in vascular smooth muscle (Somlyo & Himpens, 1989).
This combination of effects is desirable in a cardiac inotrope and the emphasis on development
of PDE-III inhibitors as inotropic agents seems justified. Disadvantages with PDE-III inhibitors
are side effects, that they are not effective if the cAMP pool is low, and importantly, that they
increase Ca cycling and overall energy consumption just as ~-AR activation does. This makes
POE-III inhibitors expensive inotropes energetically, and the ~-AR-like effects may also increase
the propensity for arrhythmogenesis (as discussed on pg 299 & 323).
Ca Current Modulation
Dihydropyridine Ca channel agonists such as Bay K 8644 can increase lea and produce
dramatic positive inotropic effects (Schramm et al., 1983). The potential advantage with Ca
channel activators is that Ca influx is increased precisely when it can best contribute to inotropy.
That is, Ca influx is increased during the AP, when it can increase: 1) the fraction of SR Ca
release (via CICR), 2) the amount of Ca supplied directly to the myofilaments and 3) SR Ca
loading. During diastole, Ca extrusion via Na/Ca exchange should not be compromised (as it is
with Na-pump inhibition). Thus, during diastole the cell may be able to extrude the larger
amount of Ca which enters during a steady state AP in the presence of Bay K 8644. This
interpretation is consistent with measurements of net Ca fluxes in rabbit ventricle using
extracellular Ca selective microelectrodes (Bers & MacLeod, 1986). Bay K 8644 did not
increase the net Ca uptake nearly as much as did increased frequency (where Ca efflux may be
compromised). Thus, Ca channel activators may produce a large inotropic effect, with less of the
Ca overload problems which limit cardiac glycoside action.
The main disadvantage with Ca channel activators is the major effects of these agents on
other tissues (e.g. causing smooth muscle vasoconstriction and central nervous system effects).
Modulating Ca channels in this way might also increase the likelihood of EADs, caused by
reopening of Ca channels. Nevertheless, if a highly cardiac-muscle-selective Ca channel

activator could be found, it might be a good inotropic agent, but this goal has so far been elusive.
Compared to Bay K 8644, Sandoz 202-791 has less vasoconstricting action for a given cardiac
inotropic effect (Bechem et al., 1988; Hof et al., 1985) and the Lilly compound (LY249933)
produced modest cardiac inotropy and vasodilation (Holland et al., 1989), but these drugs are not
nearly selective enough. Bay Y 5959 appeared to have particular initial promise, and increased
cardiac efficiency in dogs and cardiac function in patients (Bechem et al., 1997; Sato et al.,
1997; Rousseau et aI., 1997), but CNS side effects may have been too great. Given the
molecular and functional diversity of Ca channels (Chapter 5), there is certainly reason to hope
that a highly cardiac muscle selective compound can be found. As we learn more about the
molecular structure of Ca channels and how they differ, progress in this area could accelerate.
Na/Ca Exchange and [Na]i Modulation

Cardioactive steroids were prototypical inotropes which function via Na/Ca exchange,
where the Ca movements are secondary to [Na]; elevation (see pg 286-294). I will simply point
out that any agent which elevates [Na]; (or reduces the transsarcolemmal [Na] gradient) will have
the same consequences. This includes Na-ionophores (e.g. monensin), or agents which increase
Na channel open time (e.g. veratridine, batrachotoxin, grayanotoxin, DPI 201-106, SDZ 210-921,
see Scholtysik et al., 1989). Direct inhibitors of Na/Ca exchange may also be expected to
produce the same type of effects (e.g. benzamil, dichlorobenzamil). Unfortunately, all of the
Na/Ca exchange inhibitors identified so far are not particularly selective (see Chapter 6). DPI
201-106 is a Na channel activator which has been extensively studied as an inotropic agent. It
does increase contractility and prolong AP duration (Buggisch et al., 1985; Scholtysik et al.,
1985; Kihara et al., 1989), and this AP prolongation will limit Ca extrusion via Na/Ca exchange.
Like many other inotropes, DPI 201-106 has multiple effects. It also increases myofilament Ca
sensitivity, and may also have Ca channel blocking effects (Scholtysik et al., 1985; Siegl et al.,
1988; Kihara et al., 1989).
Na channel activation might have some functional advantage over Na-pump inhibition.
If Na entry via INa can activate Ca entry via Na/Ca exchange, due to the higher [Na]; near the
sarcolemma. (as suggested by Leblanc & Hume, 1990; see pg 232-234), then this might be a way
to boost SR Ca release right when it matters, but without loading the cells with Na and Ca. That
is, Ca influx via Na/Ca exchange may increase phasically during contraction, but the cell may be
able to extrude much of the extra Na and Ca gain during diastole. Anything that increases AP
duration would tend to reduce Ca extrusion and possibly favor Ca influx. This was discussed in
the context of HF (pg 320), where the contractile dysfunction could be even worse without this
Ca loading effect. Again, this is still shifting Ca into the cell in a relatively general way.
As discussed earlier, Ca overload and the negative inotropic and arrhythmogenic
consequences are a general disadvantage for digitalis or any inotropic strategy which shifts the
Na/Ca exchange system to be less effective at Ca extrusion. Since Na/Ca exchange seems to be
the main means by which Ca is extruded from the cell (see Chapter 5), prevention of Ca
extrusion can increase cellular Ca load and force. Indeed, from a historical perspective, this has
clearly been the most successful type of inotropic agent. However, the consequences of Ca
overload are a serious limitation (see pg 294-300). KB-R7943 is the novel Na/Ca exchange
blocker which seems to preferentially inhibit Ca influx vs. efflux (see pg 144 & 294). This might
help to limit Ca overload associated with Na-pump inhibition and reperfusion, but precisely how
this drug works is not known (although it may compete with [Ca]o). The lack of specificity of
KB-R7943 is also likely to limit its overall usefulness.
SR Ca Uptake and Release

Ryanodine receptors may not be an ideal inotropic target, at least from the perspective
that their systolic function is subject to autoregulatory influences (pg 267, Eisner et al., 2000).
That is, increasing SR Ca release would increase Ca extrusion via Na/Ca exchange, tending to
lower SR Ca content, and thereby reducing SR Ca release at subsequent beats. This may limit
how much intrinsic gain-of-function might be obtainable by manipulating RyR gating. On the
other hand, diastolic Ca leak from the SR via Ca sparks may limit the SR Ca content, both under
normal conditions and in HF (pg 175, 192, 199 & 318). If this is the case, then reducing this
diastolic leak may raise SR Ca content and enhance Ca transients (due to both increased SR Ca
availability and fractional release). If this could be accomplished by a shift to slightly lower Ca-
sensitivity (such that fewer diastolic Ca sparks and waves occur), but so that normal E-C
coupling still occurred with a good margin for safety (e.g. analogous to the dashed curve for
myofilament Ca sensitivity in Fig 178), then this might increase systolic cardiac function,
without being arrhythmogenic. This may be a role that FKBP normally serves (pg 199 & 318).
Thus, enhancing this effect of FKBP might be beneficial, as suggested by FKBP-12.6
overexpression in rabbit myocytes (Prestle et al., 2001). It is also important that the RyR turns
off promptly during the cellular Ca transient. While late RyR openings would prolong the active
state, they would also delay relaxation and increase futile Ca cycling during pump-release fluxes
(thereby using more ATP). Indeed, desynchronized SR Ca release and late Ca sparks during
twitches have been reported in myocytes from the peri-infarct zone in post-infarction HF rabbit
hearts (Litwin et al., 2000). Overall goals with the RyR would be to I) minimize leak during
diastole (to maximize SR Ca load and not waste ATP in futile Ca cycling), and 2) ensure that
RyRs open and close with high fidelity, synchrony and Ca flux during systole.
The SR Ca-ATPase and phospholamban (PLB) are also important inotropic targets.
Results with the PLB knockout (KO) mouse, where PLB cannot inhibit the SR Ca-ATPase,
directly attest to this (Luo et al., 1994; Hoit et al., 1995; Wolska et al., 1996b; Li et aI., 1998;
reviewed by Kiriazis & Kranias, 2000). The PLB-KO mouse exhibits hyperdynamic cardiac
function, with larger Ca transients, and SR Ca content. Surprisingly, there is little down side,
since the PLB-KO mice do not seem to develop cardiovascular disease, exercise as well as wild
type mice and do not die prematurely. That is, there is neither evidence of detrimental effects of
higher energy consumption by the heart, nor of arrhythmias which might have been expected
based on the high SR Ca content. While there are more Ca sparks and waves in isolated
myocytes from PLB-KO mice (Santana et al., 1997; Huser et al., 1998b) this does not appear to
translate into triggered or fatal arrhythmias in the mice. In general, these sorts of Ca overload
and spontaneous SR Ca release events (pg 294-300) are the principal potential disadvantage of
strategies which increase SR Ca-pump function.
Transgenic overexpression of SERCA2a can produce similar, but less dramatic effects
compared to the PLB-KO (He et al., 1997; Baker et al., 1998). Notably, the increase in SERCA2
protein expression is only 20-50%, despite mRNA increases of 160-700%. It has been
speculated that, because the SR membrane is already crowded with Ca-ATPase molecules, there
may be little room to incorporate more of the protein. Nevertheless, SERCA2 overexpression
can enhance cardiac contraction and relaxation (del Monte et at., 1999; Miyamoto et ai., 2000).
A particular advantage of the PLB-KO compared to ~-AR activation is that the effect is
isolated to the SR Ca-pump function. None of the other PKA-mediated effects should occur.
These include the decrease in myofilament Ca sensitivity (due to TnI phosphorylation) and the
disproportionate increase in oxygen consumption vs. cardiac output. If PLB also decreases the
energetic efficiency of the SR Ca-ATPase (Frank et ai., 2000; Shannon et at., 2001), PLB
inhibition could have direct energetic benefits, in terms of either the amount of ATP needed for a
given SR Ca load or the maximal [Ca]sR that can be attained for a given [Ca]i. Some other novel
agent which interferes specifically with the PLB-SERCA interaction could have the same
remarkable benefits that are observed in the PLB-KO.
CONCLUSION
In the end, the normal ventricular myocyte (and heart) is a remarkably well tuned system,
which can rapidly vary its contractile output by changing ion currents, Ca handling and
myofilament properties in response to a wide variety of physiological stimuli. On one hand there
is a dynamic, yet delicate balance of Ca fluxes, electrophysiological & mechanical properties.
On the other hand, this system is extremely robust in short- and long-term adaptation. In this
context there are some redundancies to assure this robustness. For example, ~-AR activation is
still inotropic in the absence of phospholamban and there are multiple overlapping hypertrophic
signaling systems. Nevertheless, if there are major perturbations in any system, it may throw off
the normal tuning of the system (and its ability to respond to stress). Moreover, when there are
major problems in one aspect with little redundancy (e.g. key myosin mutations in FHC or
drastic reductions in SR function), it is not surprising that a generalized hypertrophic signal
results. That is, if the short term cellular regulation can't cope with the mechanical demands on
the heart, the logical next line of defense would be to make more muscle mass to deal with that
load. Somewhat surprisingly, even changes which appear to be unrelated to the causative nature
of mechanical dysfunction, but which enhance Ca transients and function (e.g. phospholamban
knockout) can take the heart out of this maladaptive mode. Clearly there is much more to learn
about how the heart cell modulates Ca and contraction. A clear understanding of how the
cellular and molecular processes regulate Ca movements and contractile force should help make
it possible to design inotropic agents to act specifically at strategic locations in the heart. 1 hope
that this chapter and book help the reader develop a greater understanding of the dynamic
regulation of Ca in cardiac muscle cells, particularly as it relates to the control of contraction.
333
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INDEX
Bold entries refer to key illustrations & tables
A61603,282 ischemia, 307
A-band, 16. See also Angiotensin II IK (Ach),81
ACE inhibitors, 314 IK1 ,77-78
Acetylcholine, 69, 81, 87,128-130,280. Ito, 78-79
See also Muscarinic effects K channels, 76-83
phosphatase stimulation, 128 model, 93, 96
Acetylstrophanthidin, 286-94. See also Na channels, 76
Cardiac glycoside Na-pump, 89, 96
slows ryanodine-induced SR Ca loss, 292 Na/Ca exchange, 88, 95, 147-51,261-
Acidosis, 300-305. See also Ischemia, 63,264
Reperfusion (acute), Ischemia and notch,92
reperfusion, Free radicals, pH; regulation, phase 0,92
Na; accumulation phase 1, 92
Ca buffering, 47, 48, 302 phase 2, 92-94
Ca current, 300 phase 3, 94
Ca sparks, 303 phase 4, 94-97
Ca transients, 300-4, 301 plateau, 92-94
CaM kinase, 304 propagation, 64-66, 67
fractional SR Ca release, 303 rapid upstroke, 74
gap junction permeability, 9 refractory,. 82, 99
SR Ca release channel, 193, 197, 300, regional variation, 64
303 resting Em, 64-67
myofilament activation, 48 sino-atrial node, 95, 96
myofilament Ca sensitivity, 32-33, 300- species dependent differences, 120,261
305,301,302 temperature dependence, 273
Na/Ca exchange, 135, 143, 300, 303 Activator Ca, 47-48, 39-62, 289, 245-254
Na/H exchange, 302-303 species dependent differences, 250
Na-pump, 302 frequency dependence, 254
respiratory, 301-3 Acto-myosin ATPase, 23, 24, 26, 35, 239
respiratory VS. metabolic, 300 acidosis, 301
RyR open probability, 193, 300 Adenosine, 131,287,311
SR Ca-pump, 170,300,303 Adenosine receptors
SR load, 303-4 density, 287
Actin, 6,16-17,19-38 species dependent differences, 287
F-actin, 17, 20 Adenylyl cyclase, 87, 128-130,276,279-
G-actin,20 280. See also PKA (cAMP-dependent),
Actinin,17 Adrenergic (~) effects
Action potential, 39, 63-100. See also Adhalin, 18
Afterdepolarizations Adibendan, 324, 327-28
adrenergic (<x) effects, 282-85 ADP/ATP translocase, 59
adrenergic (~) effects, 278 ADP-ribose,196
Ca channels, 76, 92-94,101,116,118, Adrenergic (<x) effects, 282-85. See also
120,121,122,131 PKC effects, Adrenergic (<x) receptors
CI channels, 83-85 action potential, 282-85
DADs, 97-99 Ca transients, 282, 283
delayed rectifier K currents, 79-81 hypertrophy, 282
developmental changes, 249 Ica , 130, 283
duration, 63-64 IP3 production, 242
EADs,97 K channels, 282-85
heart failure, 317, 319, 321 multiphasic effects, 285
heterogeneity, 63-64 myofilament Ca sensitivity, 282-85, 284
If, 86-87, 94-97 Na channels, 75
Na/H exchange (sarcolemma), 282-85 Afterload, 36

Na-pump, 90, 285 Ag
pharmacology, 282 8R Ca release, 197,237
phosphodiesterase stimulation, 283 Agatoxin (w), 102
Adrenergic (~) effects, 275-82. See also AKAP-79,128
PKA effects, Phosphodiesterase Aldolase, 210
inhibitors; Adrenergic (a) receptors Alkalosis, 236, 239, 282-85
action potential, 278 myofilament Ca sensitivity, 239
Ca transients, 276, 283 Almokalant, 79
delayed rectifier K channels, 79-81 Alternans. See Mechanical alternans
heart failure, 119,281,322-23 Amiloride, 308
heart rate, 278 Aminopyridine, 79, 81,264
Ica , 76, 127-30, 129, 255,275-82 Amphiphiles, charged, 143
Icl(cAMP), 84, 278 AMP-PCP, 197
If ,278 Amrinone, 33, 327-28
IKs stimulation, 80 Angiotensin 11,131,282-85,312
inotropy, 34, 275-82 ACE inhibitors, 314
K conductance in pacemaker cells, 278 Ca channels, 131
lusitropy, 34, 277 hypertrophy, 313-16
metabolism, 279 receptor density, 287
myofilament Ca sensitivity, 33-34, 275- species dependent differences, 287
82,285 Anion exchanger, 305
Na channels, 75, 235 Ankyrin,17
Na-pump, 90, 279 Anomalous mole fraction effect, 109
phospholamban, 34, 165-166,236,275- ANP. See Atrial natriuretic peptide
82,277 Antiarrhythmic agents
protein C, 280 class I, 74
8R Ca release channel, 189, 198-200, class II, 74
275-82 class III, 79
8R Ca-pump, 166,267 class IV, 102, 104, 123-127,206,291
Adrenergic (a) receptors. See also Antioxidants, 311
Adrenergic (a) effects Antisense mRNA, 159
density, 287 Aortic banding, 316
inotropy vs. hypertrophy, 282 AP clamp, 120, 227, 254
isoforms, 287 Aprikalim, 83
species dependent differences, 287 Arachadonic acid, 131
subtypes, 282 AR-L 11588, 197
Adrenergic (~) receptors. See also Arrestin, 279, 281
Adrenergic (~) effects Arrhythmias
density, 287 afterdepolarizations, 97-99, 297-300,
heart failure, 279-281 322-23
isoforms, 287 cardiac glycosides, 288, 294
sensitivity to norepinephrine, 280 heart failure, 322-23
species dependent differences, 287, 282 ischemia and reperfusion, 308
subtypes, 279-82, 281 reentry, 99-100
Adrenergic agonists. See specific agents AT 1 blockers, 314
Aequorin, 49, 283, 295, 301, 324-325 Atomic absorption spectroscopy, 179, 185
Aftercontractions, 231,294-97. See also ATP. See also Hypoxia, Ischemia,
Arrhythmias Mitochondria, IK(ATP), Acto-myosin
Afterdepolarizations, 63, 97, 98, 294-300, ATPase, Crossbridge cycling
322. See also Arrhythmias, Action Ca channels, 130
potential 68,62,88-89,173-174,176,306
caffeine induced (cDAD), 298-299 ion transport, 306
delayed (DAD), 97, 98, 99,138,258,295 Mg-ATP, 48,82,130,193
early (EAD), 74, 97, 98 8R load, 306
heart failure, 322-23 Atrial natriuretic peptide, 130-131, 313-16
Index 409
Atrial vs. ventricular differences mitochondria, 51-52

corbular SR, 14 measuring, 181
E-C coupling, 3 rapid and slow components, 45, 46
IP3 R density, 242 sarcolemma, 43, 51
spatial [Cali gradients, 248 SR, 14,49, 171-73
T-tubules, 248 Ca calmodulin dependent protein kinase.
Atrio-ventricular node, 63-64 See CaM kinase
block,95 Ca channels (sarcolemma), 39, 75-76, 101-
If ,86 32. See also Ca current, T-type Ca
IK(Ach),81 channels
propagation rate, 67 acetylcholine effects, 128
Autocrine, 314 activation and deactivation, 73, 105
AV node. See Atrio-ventricular node adenosine effects, 131
Azidopine, 123, 126 adrenergic (~) agents, 127-30
Background inward current. See IBk adrenergic (~) effects, 76, 127-130, 275-
BAPTA, 44,116-117,119,173,226 282
Barium, 104 agonists, 123-27, 328-29
~ARK, 279, 281 angiotensin II effects, 131
Bastadin 10, 197,208 anomalous mole fraction effect, 109
Batrachotoxin, 329 ANP effects, 130
Bay K, 104,123-124, 167,212-213,328- action potential, 76, 92-94, 101, 116, 118,
29 120-22,121,131
selectivity, 329 arachadonic acid effects, 131
Bay Y, 124,329 block, 104, 123-27, 125, 126,206,223,
BCECF,303 236, 291
Benzamil, 329. Ca near inner mouth, 221
Benzohydroquinone, 169 CaM kinase, 119, 286
Benzothiazepines, 104, 108, 123-27, 126 cloning, 108
Bepridil, 33, 324, 326 conductance, 105, 112,113
Big conductance K channels (BK ca ), 238- current in absence of CaD' 218
240 density, 2, 114, 287
Biphasic contractions, 254-55 different classes of, 101--4
Birefringence, 203, 204 dihydropyridines, 101, 104-105, 108,
Blockers. See specific currents 123-27, 126, 328-329
Blood flow. See Ischemia, Hypoxia, distribution, 5,15
Hibernation, Capillaries EADs, 97
BM 14.478, 33 endothelin effects, 131
BMY-7378,282 gating, 114-20
Boltzmann relation, 70, 87 histamine effects, 130
Bradycardia, 81, 97, 280-281 hypertrophy, 102
Bradykinin, 311 inactivation, 76,104,105,108,116-19,
Bromo-eudistomin D, 197 117
Buffering. See Ca buffering, Total cytosolic interaction with SR Ca release channel,
[Cal, Mg, specific buffers 189,209-13,211
Ca activated CI channels. See ICI(ca) ions supporting charge movement, 208
Ca activated K channels (SKCa), 240 IP3 activated, 237
in smooth muscle, 238 localization, 15, 104
Ca activated nonselective channel. See loop peptides (II-III), 210-13
Ins(Ca) Mg-ATP, 130
Ca buffering, 40-47. See also Total cytosolic modeling, 109, 111
Ca modes, 123
acidosis, 47--48, 302 molecular characterization, 104-8
cardiac muscle, 41--47, 43, 45 open channel probability, 114
developmental changes, 250 permeation, 110, 111, 114
effects of Mg, 44--45 pore-mutant skeletal Ca channel, 208
heart failure, 319, 320 rundown, 130
selectivity, 108, 109,114 Na/Ca exchanger triggered, 15, 223-224,

skeletal VS. cardiac muscle, 104, 106, 232-35,233,234,291
112,204-209 INalCavs. Ica , 233, 234
species dependent differences, 103, 121, photolysis of caged Ca, 218, 220, 224,
204, 287 230
structure and subunits, 104-8,107 refractory period, 229, 231
surface potential effects, 115, 116 skeletal muscle, 215
temperature, 122, 273 smooth muscle, 238, 239
tissue dependent differences, 102, 105- species dependent differences, 247
106 timecourse, 226-27
Hype VS. L-type, 76, 93, 95,101-8,105, turned off by repolarization, 220
232 voltage dependence, 220
tyrosine kinase effects, 131 Caleak, 39-40,48, 173-77,309
voltage dependence, 104, 116-19, 204 Ca microelectrodes, 49, 183-85, 261-262,
Ca current, 75. See a/so Ca channels 268,291,298
acidosis, 300 Ca overload, 225, 230-32, 294-97
activation, 105, 114-16 arrhythmias, 309
as sensor of local [Cal;, 122 cardiac glycosides, 286-94
Bay K, 104, 123-124, 167, 212-213, free radicals, 309
328-29 gap junction permeability, 9
block, 104, 123-27,223,206,236,291 MTP opening, 60
Ca buffering titration, 45 role of mitochondria, 61
Ca dependent facilitation, 119, 257 role of Na/Ca exchange, 160
Ca depletion/accumulation, 112 stunning, 310
Ca induced inactivation, 256 Ca paradox, 9
cardiac glycosides, 293 Ca pump (sarcolemmal), 39, 133-35
contribution to activator Ca, 55, 56, 245- Ca affinity, 157
54 Ca-calmodulin affinity, 134
current-voltage relationship, 104 calmodulin, 133-134
developmental changes, 102, 249 carboxyeosin, 134
facilitation, 119-20, 286, 293 cloning of, 133
heart failure, 317, 319 competition with Na/Ca exchange, 270
integration, 120-22 contribution to Ca removal fluxes, 53, 54,
recovery from inactivation, 229 152-59,250-53
reversal potential, 113 eosin, 134
sino-atrial node, 95, 96 free radicals, 309
staircase, 119-20 tlG,62
tail current, 124,217, 228 homology, 133
turned off by repolarization, 221 O2 consumption, 62
window current, 255 pharmacology, 134
Ca flux analysis, 251, 252 PKA effects, 133-134
Ca fluxes during cardiac cycle (general rapid cooling contractures, 158
scheme), 39-41, 40 species differences, 134, 260
Ca induced Ca release, 215-32, 217. See splice variants, 133
a/so SR stoichiometry, 133
alternative triggers, 232-35 temperature, 274
at constant voltage, 219 transport rate, 133, 134
axial propagation, 248 Vmax , 134
Ca dependent inactivation, 215 Ca pump (SR). See SR Ca-pump
cAMP effects, 236 Ca quarks, 224
dependence on Ca current, 219, 220 Ca release activated current (Icrac), 240
fractional release, 173, 186, 224-26, 245, Ca sensitizers. 35, 324-327, 326. See a/so
253-260,267,270 Myofilament Ca sensitivity
gain, 223, 224-26, 225 Ca sparks, 190-92, 191, 223, 225-227
local control theory, 223, 224 acidosis, 303
modulation by SR Calcium, 225 activation of lateral neighbor, 224
Index 411
Bay K, 212, 213 Cai. See Diastolic Ca, Systolic Ca, Ca

developmental changes, 249 buffering, Total cytosolic [Cal, Uniformity
FK-506 effects, 199 of [Ca)i, Ca transients, Model Ca
rrequency,225,230,258 transient, specific [Ca)i indicators
IP3 effects, 243 Cai accumulation
latency of activation, 226 reperfusion (acute), 308-9
removal of [Na)a, 293 Calcineurin, 198,313-16
resolution, 223 Calmodulin. See CaM
rest potentiation, 258 Calpain, 310
resting frequency, 192 Calreticulin, 173,319,320
skeletal muscle, 208 Calsequestrin, 14, 171-73, 172
smooth muscle, 240 heart failure, 318-320
SR load dependence, 230 CaM, 197,239. See also CaM kinase
summation, 223 CaM kinase, 243, 285-86, 313-16
triggering of Ca waves, 230 acidosis, 304
Ca spike, 226, 228 activation near nucleus, 284
Ca transients. See also Inotropy, Adrenergic auto-phosphorylation, 286
(~) effects frequency dependent acceleration of
acidosis, 300-4 relaxation, 270, 271, 272
adrenergic (a) effects, 282, 283 hypertrophy, 284
adrenergic (~) effects, 276, 283 Ica facilitation, 119, 286
autoregulation, 267 inhibitors, 271, 286
developmental changes, 249 MLC2 phosphorylation, 34
heart failure, 317, 319 phospholamban, 166-167, 271,286
isoproterenol, 276 phosphorylation of KV1.4, 79
kinetic model, 48-50, 49,50,92,228 SR Ca release channel, 190, 198, 286
temperature, 274 SR Ca-pump, 133-134, 166-167,286
thapsigargin acute effects, 247 cAMP. See PKA, Adrenergic (~) effects
Ca waves, 224-225, 229-32, 231, 239, cAMP-Activated CI Channel. See IC1(cAMP)
294-300. See also Spontaneous SR Ca Capacitance
release cell surface area and volume, 5, 6
agonal vs. sporatic vs. Ca-overload, 296 Capacitative Ca entry, 240-41
annihilation of, 230 Capillaries
intact heart, 296 proximity to cardiac cells, 10
propagation velocity, 296 Carboxyeosin, 53, 134
threshold load, 294 Cardiac contractile reserve, 306
Cacodylate, 210 Cardiac glycosides, 60, 74, 90, 135, 162,
Cadmium. See Cd 235, 254, 286-94, 288. See also Nai
Caffeine, 33, 35, 52, 194,229,267, 290- accumulation, Inotropy
291 Ca current, 293
Ca buffering measurement, 180, 181 induction of DADs, 98
contractures, 178-81 low concentrations, 288
increased Ca influx, 245 Na channels, 293
myofilament Ca sensitivity, 35, 245, 324 Na-free conditions, 293
Na/Ca exchange, 35, 53,149,150,180 smooth muscle, 293
phosphodiesterase inhibition, 245, 327- SR Ca content, 289, 290, 292
28 SR Ca release channel, 293
SR Ca release channel, 52, 193-194, toxicity and arrhythmias, 288, 294-97
232,267 Cardiac infarction, 330
SR load measurement, 181, 253 Cardiac output, 275-82
vs. ryanodine, 245, 246, 247 Cardiac reserve, 323
Caged Ca. See Photolysis of caged Ca Cardioactive steroids. See Cardiac
Caged IP3 . See Photolysis of caged IP3 glycosides
Caged phenylephrine. See Photolysis of Cardiomyopathy. See also Heart failure
caged phenylephrine dilated, 286, 287
familial hypertrophic, 313
FK-506 treatment, 199 Contractile proteins. See Myofilaments,

ischemic, 286, 287 individual proteins
Carnosine, 31, 35 Contractility. See Adrenergic (~) effects,
Catecholamines. See also Inotropy, Inotropy, Lusitropy, Frequency dependent
Adrenergic effects, Phosphorylation, acceleration of relaxation, PKA
Isoproterenol Cooling. See Hypothermia, Rapid cooling
Caveolae,6 contractures
Caveolin-3, 6 Copper. See Cu
Cd Corbular SR, 4, 7,14,15,171,214
blockade of Ica , 223 Couplons, 13, 14
CICR,236 C-protein. See Protein C
INa, 74 Creatine phosphate, 306
Na/Ca exchange, 141, 145 Creep currents, 138
SR Ca release, 237 Cromakalim, 83, 311
cDAD, 298, 299 Crossbridge cycling, 25, 26, 239
Central core disease, 189-190, 196 Crystallin (aB), 312
c-Fos, 313-16 Cu
CFTR. See ICI(cAMP) SR Ca release, 197,237
cGMP. See PKG (cGMP-dependent), Current sinks, 298
CGP 28392, 123 Cyclic ADP-ribose, 196, 197
CGP 48506, 35, 324, 325 Cyclophilin D, 59
Chaperone, 312 Cyclopiazonic acid, 169
Charge-coupled Ca release. See Voltage- Cyclosporin, 59, 313-16
dependent Ca release Cytokines, 309
Chimeric DHPR studies, 210 Cytoskeleton, 17, 18
Chloride channels. See CI channels Cytosol
Chloroethylclonidine, 282 Ca buffering, 41-47, 43, 45
Chloro-m-cresol, 196, 197 volume, 41
Cholinergic stimulation. See Muscarinic D600, 102, 125, 139, 145,206,223
effects, Acetylcholine D888, 125
Chromaffin cells, 58 D890, 125
Chronotropy. See Heart rate DADs, See Afterdepolarizations
Cirazoline, 282 DAG, See Diacylglycerol.
c-Jun, 313-16 Dantrolene, 196, 197
CI channels, 83-85 Debye length, 116
ICI (Ca),84 Delayed rectification, 77, 79-81
IC1(cAMP), 84 Density. See specific protein
IC1(swell), 85 Depolarization-induced Ca release. See
CI-/HC0 3- exchange, 305 Voltage-dependent Ca release
CI-IOW exchange, 305 Desmin,17
Clathrin, 279 Desmosome, 6, 17
Clofilium, 79 Developmental changes
c-Myc, 313-16 AP duration, 249
Cobalt, 108 Ca buffering, 250
Collagen, 10, 17 Ca current (SL), 102, 249
Colloidal iron stain, 10 Ca sparks, 249
Competition between efflux pathways Ca transients, 249
(general scheme), 39. See also specific myofilament Ca sensitivity, 301
fluxes Na/Ca exchanger current, 249
Confocal microscopy, 190. See also Ca sensitivity to acidosis, 301
sparks, Ca waves spatial [Cali gradients, 249
Congenital defects, 313 SR,248
Connexin, 8, 9 surface to volume ratio, 5, 249, 250
Connexon,8 Tnl isoforms, 301
Conotoxin (Ol), 101 DHPR. See Ca channels (sarcolemma)
Index 413
DHPR-RyR Interaction, 210-13. See also smooth muscle, 239

Ca channels (sarcolemma) sources of activator Ca, 39-62
Diacylglycerol, 238-239, 242, 284-285, SR Ca content, 177-86
313-16., See also IP 3 metabolism, PKA, SR Ca release channel, 186-202
Adrenergic (a) effects. See DAG ultrastructure, 1-18
Diastolic Ca, 230, 289 EEEE locus, 110
frequency dependence, 268 EF-hand, 117
hypertrophy, 316 EGTA, 44,117, 119
Diazo-2,34 Electrical restitution, 256
Diazoxide, 311 Electrodiffusion, 222
Dibutyryl-cAMP, 129 Electron Probe Microanalysis, 178
Dichlorobenzamil, 138, 144,329 EMD 53998, 33, 324, 325
DIDS, 83, 85 EMD 57033, 33, 35, 324, 325
Diffusion coefficient, 112 EMD 57439,33,325
Diffusion equation, 221 EMD 5998, 327-28
Diffusion of Ca, 222, 229 EMD 85131,308
Diffusive vs. local signaling, 280 Encainide, 74
Digitalis, 286-94. See Cardiac glycosides Endogenous ouabain, 288
Digitonin permeabilized myocytes, 43, 56 Endothelin, 131,243,282-85,311
Digoxin, 235 hypertrophy, 313-16
Dihydropyridine receptors. See Ca channels receptor density, 287
(sarcolemma) species dependent differences, 287
Dihydropyridines, 3,101-105,108,126, Energy wells, 109
123-27,192,206,209,212,328-29 Enoximone,327-28
Dilantin,74 Eosin, 134
Diltiazem, 102, 125 Erg-1,313-16
Dimethonium, 143 ERK, 311, 313-16
Diphenylbutylpiperidine neuroleptics, 126 Ethylisopropylamiloride, 308
DM-nitrophen, 218. See also Photolysis Excitation-contraction coupling. See E-C
DNDS,83 coupling
Dofetilide, 79 Exercise, 312
Doxorubicin, 197 Extended junctional SR, 8, 14, 214
Dropsy, 286 External lamina, 9, 10
Dyadic cleft Extracellular Ca depletions, 185, 268, 298
Ca ions in, 222 Extracellular matrix, 17-18
Na channels, 15 Extracellular space, 9-10
Na/Ca exchanger, 15 as a source of Ca, 50-51
Dyads, 6 proximity to capillaries, 10, 11
Dysgenic myotubes, 210 Extrasystole, 256, 264
Dyspedic myotubes, 210 Failing heart. See Heart failure
Dystroglycan, 18 FAK,17
Dystrophin,17 Familial hypertrophic cardiomyopathy, 313
E-4031, 79 Fascia adherens, 6, 17
EADs. See Afterdepolarizations FCCP,53
E-C coupling, 40, 203-44, 205. See Ca FDAR. See Frequency-dependent
induced Ca release, Voltage-dependent acceleration of relaxation
Ca release, IP3 induced Ca release Feet structures, 12, 13, 14. See also SR Ca
cardiac muscle, 243 release channel
action potential, 63-100 FGF,313-16
Ca channels (sarcolemma), 101-32 Finch cardiac muscle, 8
general scheme, 41 FK binding protein, 188, 198- 201, 315, 318,
inotropy, 273-94, 273-331 330
inotropy (negative), 294-323 FK-506, 188, 198, 199,201, 313-16
myofilaments, 16-17, 19-38 FKBP. See FK binding protein
Na/Ca exchange, 135-50 Flecanide,74
skeletal muscle, 243 Flippase, 142
Fluorescent indicators. See also specific Glycosylation, 137

indicators Goldman-Hodgkin-Katz current equation,
acetoxymethylester (AM), 50 112
calibration, 50 Goldman-Hodgkin-Katz equation, 65
Fluspiriline, 126 Golgi apparatus, 18
Focal adhesion kinase. See FAK Gouy-Chapman theory, 115
Foot protein. See SR Ca release channel Grayanotoxin, 329
Force. See Length-tension, Inotropy GRK, 279, 281
crossbridge re-attachment, 38 Guanylyl cyclase, 131,281,311. See also
Force-frequency relationship, 268, 269-71 PKG (cGMP-dependent)
heart failure, 319 Glyceraldehyde 3-phosphate
Force-pCa relationship, 216, 289. See dehydrogenase, 210
Myofilament Ca sensitivity H-89,129
Force-velocity relationship, 35-38, 36 Halothane, 196, 197
Forskolin, 128, 129, 130 H-band, 16
FPL-64176, 124, 126,228 Heart failure, 316-23. See also
Fractional SR Ca release, 173, 186,224-26, Cardiomyopathy, Antiarrhythmics.
225,245,253-260,267,270,274,278 adrenergic (~) effects, 119, 281, 322-23
acidosis, 303 action potential, 317, 319, 321
Frank-Starling law, 27, 28, 309. See also acute therapy, 279
Length-tension relationship adrenergic receptor (~3), 281
post extra-systole, 256 animal models, 299, 316-23
reduced diastolic filling time, 270 arrhythmias, 97, 99, 297-300, 322-23
Free radicals, 309-311 Ca buffering, 319, 320
antioxidants, 311 Ca current, 317, 319
Ca pump (sarcolemmal), 309 Ca removal fluxes, 253
Na-pump 309 Ca transients, 317, 319, 318-20
stunning, 310 calreticulin, 319
Frequency-dependent acceleration of calsequestrin, 318-20
relaxation, 270, 271, 272, 304. See also cardiac glycosides, 90
CaM Kinase contraction, 317
Fura-2,49 downregulation of ~-ARs, 279-281
"Fuzzy space", 223 early VS. late, 299
G protein, 82, 279-85, 281. See specific energetics, 323
kinase pathways, Phosphorylation force-frequency relationship, 269, 270,
heart failure, 319 319, 323
Gabapentin, 105 G proteins, 319
Gadolinium, 314 hyperphosphorylation of RyR2, 199
Gap junction, 6-9 ICI(sWell), 85
acidosis, 9 IK1 , 99, 298, 319,321,323
AP propagation, 66 INa.Slow, 320-21
Ca overload, 9 inotropic agents, 323-31
pore diameter, 9 110 ,79, 321
Gating current, 70, 71, 73, 114, 130,205-7 K currents, 79, 321
Gel permeation chromatography, 188 myofilament Ca sensitivity, 22, 317, 319
Genestein, 85, 131 Na/Ca exchange, 99, 253, 261, 298, 319-
Gentamycin, 197 22
Giant excised patch technique, 139,140 Nai,320-21
Glibenclamide, 83, 85, 307, 311 Na-pump, 91, 320-21
Glutathione peroxidase, 309 phospholamban, 318, 319
Glycocalyx, 9 PKA (cAMP-dependent), 119,322-23
Glycogen granules, 18 protein expression, 319
Glycogenolysis, 279 rapid cooling contractures, 270
Glycolysis, 305, 306 rest decay and rest potentiation, 261
Glycoprotein, 18, 173 SR Ca content, 177,318-20,321-22
Glycosides. See Cardiac glycosides SR Ca release channel, 199,318-20
Index 415
SR Ca-pump, 253, 261, 298, 318, 319 ICI(Ca), 75, 83-85, 87-88, 92, 98, 120, 181,
thyroid hormone, 318 240
Heart rate. See also Frequency-dependent [Ca]submembrane, 84
acceleration of relaxation, Force- delayed afterdepolarizations, 99
frequency relationship, Rest potentiation pharmacology, 85
adrenergic (~) effects, 278 reversal potential, 298
bradycardia, 81, 97, 280 ICI(eAMP), 83, 84, 278
endurance training, 312 ICI(swell)' 75, 83, 85
If, 87 lerae, 240-41
IKIACh), 81, 87 If, 75, 87, 86-87, 95, 96
parasympathetic effects, 280 aderenergic (~) effects, 278
tachycardia, 74, 99 atrial VS, ventricular differences, 86
variability, 82 AP phase 4, 94-97
Heat shock proteins, 312 cyclic nucleotide activation, 87
HeavySR, 161, 186, 192, 214 heart rate, 87
Heparin, 238, 241 hyperpolarization activation, 86
Heteropodatoxins, 79 IGF-1,313-16
Hg IK(Ach), 75, 81, 85
SR Ca release, 197,237 heart rate, 87
Hibernation, 248, 309-10 atrial VS. ventricular differences, 81
Hill equation, 28-32, 36, 47-48, 87, 134, IK(ATP), 75, 82-83
141,170-171,174-75,251-252,288, ischemia, 306, 307
326. pharmacology, 307, 308, 311
Histamine, 130, 280 preconditioning, 311
HMR-1556,79 IKlo 75, 77-78
Hoe-642, 308 heart failure, 99, 298, 319, 321, 323
Hormone receptors table, 287 IKP ' 75
Hydroxydecanoate, 311 IKr , 75, 80, 92
Hydrogen peroxide, 309 IKs , 75, 80, 92
Hyperkalemia, 77 IK,slow, 75
Hyperthermia. See Malignant hyperthermia IKur , 75, 81,92
Hypertrophy, 312-16 Imidazoles, 31
adrenergic (0:) effects, 282, 313-16 Imperatoxin, 189, 196, 197,208
Ca channel subtypes, 102 INa' See Na channels
Caj elevation, 316 INaICa' See Na/Ca exchange
CamK activation, 284 Inactivation, 71-73. See also specific ion
concentric vs. eccentric, 312 channels
endothelin, 313-16 ball-and-chain, 72
microtubules, 18 Ca channels (sarcolemma), 76,119
models, 319 K channels, 76
myosin isoforms, 22, 37 N-type inactivation, 72
Na/H exchange (sarcolemma), 316 Indo-1,44,49, 144, 154-157, 180, 220,225
signaling pathways, 313-315, 316 Mn quench, 59
stretch,85 Infarction, 330
Hypochlorous anion, 309 Inositol triphosphate, See IP3
Hypokalemia, 77, 97 Inotropy, 272, 273-94, 323-31,273-331,
Hypothermia, 29, 30, 32, 35 See also Cardiac glycosides, Staircase,
inotropy, 273, 274, 275 Adrenergic (~) effects, Force-frequency
Hypothyroid, 22, 318 relationship, PKA (cAMP-dependent)
Hypoxia, 74, 305-12. See also Ischemia adrenergic (0:) effects, 285
I band, 2 adrenergic (~) effects, 275-82, 277
lanthella basta, 196 adrenergic (0:) VS. adrenergic (~), 283
18k , 96, 97 CamK, 285-86
Ica. See Ca current cardiac glycosides, 286-94
ICa,nx, See Tetrodotoxin-sensitive Ca hypothermic, 273-75
current inotropic agents, 323-31
Inotropy (negative), 281, 294-323. See also timecourse of changes, 307

PKG (cGMP-dependent) lsi (slow inward current), 101
acidosis, 300-305 Isocitrate dehydrogenase, 60
Ins(Ca), 87, 88, 98, 99, 181 Isoflurane, 196
Integrins, 17, 314 Isomazole, 33, 35, 324, 326
Intercalated disk, 6 Isoprenaline. See Isoproterenol
localization of IP 3 R, 242 Isoproterenol, 34, 74, 87, 128, 175, 275-82,
Interleukins, 199, 309 277,283-284,299
Intermediate filaments, 6 post-rest staircase, 276
Intermediate junction, 6 Isradipine. See PN200-11 0
Intramembrane charge movement, 203, 205, Iti (transient inward current), 88, 98, 138,
207 297-300. See also Afterdepolarizations,
Inverse agonism, 281 Na/Ca exchange, ICI(ca), Ins(ca)
Inward rectification, 76 free radicals, 309
lodipine, 123 Ito (transient outward current), 75, 78-79, 92
lodocyanopindolol, 287 heart failure, 79, 319
Ion channels. See also specific channels activation, 73
cardiac ion channels, 75 JNK,313-16
deactivation, 71 Junctin, 172, 190
gating, 70, 71-73 Junctional cleft, 209
inactivation, 70, 71-73 [Cal, 221, 222, 293
permeation, 67-68 Junctional feet, 12, 13, 14. See also SR Ca
selectivity, 67-68 release channel
structural models, 69 Junctional SR, 59
voltage sensor, 67 K channels, 76-83, 78
Ion transporters table, 287 antiarrhythmic agents, 74, 97
IP3 induced Ca release, 197, 201, 237-43, AP phase 2, 92-94
242, 284 AP phase 3, 94
CaMK activation, 284 big conductance K channels, 238
cardiac muscle, 241-43, 284 Ca activated K channels, 238
skeletal muscle, 241 delayed rectifier, 2, 77, 79-81
smooth muscle, 238, 237-40 heart failure, 319, 321
IP3 metabolism, 238, 239, 284, 313-16. IK(ACh), 75, 81,85, 87
See also Diacylglycerol, PKC, Adrenergic IK(ATP), 75, 82-83, 306-308, 311
(a) effects IK1 , 75, 77-78, 99, 298, 319, 321, 323
IP 3 receptor, 200-201,237-43 IKp , 75
atrial vs. ventricular differences, 242 IKr, 75, 80, 92
IpTx. See Imperatoxin IKs , 75, 80, 92
Ischemia, 32, 33, 48, 61,82,89, 100, 143, IK slow, 75
170, 193,305-12,307. See also IK~r, 75, 81,92
Reperfusion (acute), Acidosis, Ischemia Ito, 75, 78-79, 92, 319
and Reperfusion, Free radicals inward rectification, 76
action potential, 307 Ito, 78-79
Ca transients, 306 mitochondrial IK(ATP), 83
Ko elevation, 307 PKC (Ca/lipid-dependent), 282-85
myofilament Ca sensitivity, 306 rectification, 76-77
Na channels, 308 sino-atrial node AP, 95, 96
Na/H exchange (sarcolemmal), 308 structural model, 69
Naj,308 voltage gated vs. inward rectifiers, 76
Na-pump, 306 KB-R7943, 144, 145,294,308,329
preconditioning, 310-12 Ketoglutarate dehydrogenase, 60
reentrant arrhythmias, 307 KMD-3123,282
SR load, 306 KN-62,271
stunning, 310 KN-93, 271,286, 304
Ischemia and reperfusion L-768,673, 79
stunning, 310 Lactic acid, 305
Index 417
Laminin, 17, 18 free [Mg]j, 193

Lanthanides, 56 inward rectification, 77
Lanthanum hydroxide, 8 ischemia, 306
LaPlace law, 316 Mg-ATP, 130, 193
Leak. See Sarcolemma leak, SR leak mitochondrial uniporter, 56, 58
Lemakalin, 83 myofilament Ca sensitivity, 33
Length constant, 67 pathophysiology, 48, 61,193
Length-tension relationship, 27-28 sarcolemmal binding, 51
Frank-Starling law, 28 skinned fiber solutions, 28, 32, 215
osmotic compression, 28 SR Ca release channel, 161, 170, 188,
steric hindrance, 27 193-197
Leucine zipper, 168 SR Ca-pump, 161, 169, 170
Levosimendan, 33, 35, 324, 327-28 Twitch Ca dynamics, 48
Lidocaine, 74 VDCR,214
Light SR, 161 Mibefradil, 108
Lipid droplets, 18 Microtubules, 18
Lipofuscin granules, 18 Milrinone, 33, 255, 327-28
Local anesthetics, 124 Mitochondria, 15-16,56,57-62. See also
Local control theory, 223, 224 ATP, Ischemia, Free radicals, specific
Localization, 15. See also specific protein enzymes, NADH
Long Q-T syndrome, 74, 80,97 ADP/ATP translocase, 59
Longitudinal resistivity, 67 ATP synthase, 62
Loop peptides (II-III loop), 107, 196,210-13 [H] gradient, 312
L-type Ca channels. See Ca channels association with junctional SR, 59
(sarcolemma) Ca buffering, 51-52, 59
Lusitropy, 34, 166,272,275,277-279 Ca content, 56
adrenergic (ex vs. ~) effects, 283 Ca transport, 53, 54, 56-62
negative, 283 Ca uniporter, 39, 53, 54, 56, 274
Lysophospholipids, 74 Ca uniporter blockers, 56
Lysosomes, 18 Ca-pump, 309
Macula adherens, 6 [CalM model, 58
Magnesium. See Mg contribution to Ca removal fluxes, 53, 54,
Malignant hyperthermia, 190, 197 250-53
MAPK, 311,315,313-16 dehydrogenases,60
Mapping studies (3D), 299 electrochemical gradient, 56, 312
Maximal theoretical current, 112 electron transport chain, 56
Maximum shortening velocity, 37 energy supply meeting demands, 60
MBED,197 fluctuations in [CalM, 58
MCI 154, 33, 35, 324, 327-28 free [Cal ([Ca]M), 57, 59
MCIPI,313-16 in chromaffin cells, 58
mdg mouse, 209 matrix loading, 51
Mechanical alternans, 256, 264 mito-IK(ATP), 311
Mechanical restitution, 256, 295 Na/Ca antiporter, 57
Membrane resistance, 67 Na/H exchange, 57
Mercury. See Hg permeability transition pore, 59
Metabolic acidosis, 300 surface area, 16
Methoxamine, 282 volume, 7,41
Methylurapidil, 282 Mexiletine,74
Methylxanthines, 33, 194. See also Caffeine MLC kinase, 239
Mg myofilament Ca sensitivity, 34
binding to TnC, 20, 21, 22 M-line, 17, 19
block of Na/Ca exchange, 141, 145 Model. See also Heart failure models
blockade of INS, 109 action potential, 93, 96
Ca buffering effects, 44, 45 Ca channel gating, 124
Ca channel permeability, 109, 111, 208 Ca channel permeation, 109
E-C coupling, 193, 197 Ca synapse VS. cluster bomb, 223
Ca transient, 48, 49, 50 EMD 57439,33,325

calsequestrin & junctional SR, 173 force-pC a relationship, 216, 289
cardiac AP, 91 heart failure, 317, 319
DHPR-RyR Interaction, 210-13 imidazoles, 31, 33, 35
diffusional modeling in cleft, 222 ionic strength, 33, 35
force-frequency relationship, 229 isolazole, 324
gap junction, 8 isomazole, 33, 35, 326
ion channel structure, 69 levosimendan, 33, 324
mdg disease model, 208 magnesium, 33, 35
mitochondrial Ca, 58 MCI 154,33,324
myofilaments, 24 milrinone, 33
Na/Ca exchange, 137, 149 modulation, 35, 324-27
Na-pump,91 muscarinic effects, 34
phospholamban, 164, 165, 168 nitric oxide, 281
plunger, 206, 207 perhexiline, 33, 324, 326
Poisson-Nernst-Planck (PNP), 111 pH,32,35
rat ventricular action potential, 264 phosphate, 33, 35
SERCAIPhospholamban interaction, 167 pimobendan, 33, 324, 326
single channel Ca current, 187 post extrasystolic potentiation, 256
SR Ca load, 175 sarcomere length, 28, 31, 32, 35
SR Ca release, 217 skinned vs. intact, 32
Monensin, 300, 329 species dependent differences, 30, 31
Mouse. See Transgenic mouse stunning, 310
M-protein, 17 sulmazole, 33, 35, 324, 326
Muscarinic effects, 280. See also ~m~ffi~re,~,W,~,~,V3
Acetylcholine, Muscarinic receptors theophylline, 324
Ca channels (sarcolemma), 128 Tnl phosphorylation, 35
If ,86-87 Myofilaments, 16-17, 19-38,21. See also
IK(Ach), 81, 85 Myofilament Ca sensitivity
IP 3 production, 242 A band, 16
myofilament Ca sensitivity, 34 acidosis, 48
Muscarinic receptors, 128. See also actin, 16-17, 19-38
Acetylcholine, Muscarinic effects acto-myosin ATPase, 23, 26, 35
density, 287 contractile mechanism, 22-24
species dependent differences, 287 force-velocity relationship, 35-38, 36
Muscular dysgenesis, 208 heart failure, 317
Myofilament Ca sensitivity, 28-32, 289, 326 length-tension relationship, 28, 27-28
acidosis, 300-305, 301, 302 model,24
adibendan, 324 myosin, 16-17, 21, 38
adrenergic (a) effects, 283, 282-84, 285 O2 consumption, 62
adrenergic (~) effects, 33, 34, 275-282, overlap, 27
284 crossbridge re-attachment force, 38
alkalosis, 239 sliding filament theory, 24
ATP, 33 species dependent differences, 22
bepridil, 33, 324, 326 stunning, 310
8M 14.478, 33 subfragments, 16-17
Ca buffering, 41-47 temperature, 273
caffeine, 33, 35, 245, 324 troponin complex, 24
cAMP, 34 volume, 41
carnosine, 31,33, 35 Myomesin, 17
CGP 48506, 324, 325 Myosin, 16-17, 19-38,21
cytokines, 309 ATPase, 26
DPI-201-106,33 crossbridges,17
developmental changes, 301 heavy chain, 19
EMD 53998, 33, 324 hypertrophy, 22, 37
EMD 57033, 33, 35, 324, 325 light chain, 19
Index 419
protrusion angle, 17 Ca activation. See Na/Ca exchange

spacing, 17 allosteric regulation
Myosin light chain kinase. See MLC kinase. Ca affinity, 136, 140-141, 157
Na activity (aNa;), 263. Ca chelator, 145
frequency dependence, 270 Ca efflux, 39, 50-51, 52-53, 54, 142,
rat VS. rabbit, 261 152-59,153,250-53,294
Na channels, 73-76. See also Na; Ca induced Ca release, 15,223-224,
accumulation 232,233,234,235,291
activation, 73 Ca influx, 39, 50-51, 56, 142, 147,254,
antiarrhythmic agents, 74 291
AP phase 0, 92 Ca/Ca exchange, 138, 156
cardiac glycosides, 293 cadmium, 141, 145
Cd sensitivity, 74 caffeine, 35, 55, 149, 150, 180
cloning, 73 cardiac glycosides, 135, 254, 286-94
current during sino-atrial node AP, 95, 96 chlorpromazine, 145
density, 287 chymotrypsin, 137, 140
depolarization, 65, 74 competition with SR Ca-pump, 52-53, 54,
distribution, 2, 5 152-59,155,250-53,270
EAOs,74 creep currents, 138
"fuzzy space", 223 0600,145
gating current, 73 delayed afterdepolarizations, 98, 99
heart failure, 320-21 density, 146,287
Ica .nx , 235 developmental changes, 249
INa.Slow, 320-21 dichlorobenzamil, 145
inactivation, 71-73 diducaine, 145
ischemia, 308 doxorubicin, 145
local anesthetics, 124 driving force, 94, 147-51, 260-64
localization, 15 EGTA,144-145
long Q- T syndrome, 74 enhancers of, 145
modulation, 329-30 equation, 149
Na induced Ca release, 237 ethanol, 145
PKA effects, 75 FMRF-amide, 145
PKC effects, 75 giant excised patch technique, 139, 140
P-loop,74 harmaline, 145
refractory, 66, 75 heart failure, 253, 298, 319-322
slip mode conductance, 75, 235 inhibitors, 145
structural model, 69 inorganic cations, 145
subunits, 74 isoforms, 138
TTX sensitivity, 74 KB-R7943, 144, 145,294, 308, 329
Na current. See Na channels lipids, 145
Na induced Ca release, 237 localization, 15, 146,234
Na/Ca exchange, 135-60 methylation, 145
[Na]; accumulation, 233, 291 model, 137
acidic phospholipids, 143 modulation, 145, 329-30
acidosis, 135, 143, 300, 303 Na affinity, 136, 140
action potential, 88, 95, 147-51,261-64 Na/Na exchange, 138
activation by free radicals, 309 nickel,145
adriamycin, 145 organ differences, 138
allosteric regulation, 137-140, 145, 149, pH, 135, 143, 145
259 pharmacology, 138, 141,144-145
amphiphiles (charged), 145 phospholipase, 145
ankyrin association, 17 phospholipid translocase (flippase), 142
antisense mRNA, 159 phosphorylation, 145
ATP, 143, 145 ping-pong mechanism, 138
bepridil, 145 PIP2 , 143, 145
biphasic contractions, 255 polymyxin B, 145
proteinase, 145 acidosis, 302

quinacrine, 145 adrenergic (a) effects, 90, 285
quinidine, 145 adrenergic (~) effects, 90, 279
rapid cooling contractures, 152-54, 158 cardiac glycosides, 90
redox modification, 145 cloning, 90
reperfusion (acute), 308-9 current during AP, 89
rest decay, 157, 158, 184,259 density, 287
reversal potential, 88, 94, 98, 147-51, density of sites, 90
148,158-159,233,261-64,263,288- distribution, 2
298 free radicals, 309
sino-atrial node, 95, 96 L1G,62
snubs peak [Cal;. 251 heart failure, 91, 320-21
species differences, 147, 152,153,158, isoforms, 90, 293
252 model,91
8R Ca release effects on INalCa, 246 Nai affinity, 88
stoichiometry, 135, 151,152 O2 consumption, 62
submembrane Ca, 149-50, 151 sino-atrial node, 96
submembrane Na, 149-51 species dependent differences, 90, 287
surface charge, 143 temperature, 275
temperature, 274 topology, 90
tetracaine, 145 turnover rate, 90
thermodynamics, 147--49 NE-10064,79
transgenic mice, 159-60 NE-10133,79
turnover rate, 142 Negative inotropy. See Inotropy (negative)
unitary flux, 234 Neomycin, 197
used to measure 8R content, 55 Neonatal Myocytes. See developmental
verapamil,145 changes
vesicle studies, 135-36, 156 Nernst potential, 65, 67
Vmax , 146 N-ethylmaleimide 81 heads, 37
voltage dependence, 89, 139, 141, 156 Neuraminidase, 9
XIP, 137, 140, 143-145 Neutrophils, 309
Na/H exchange (sarcolemma), 144,284-85, Nexus, 6
305 NFAT3,313-16
acidosis, 302-303 Nicardipine, 124
adrenergic (a) effects, 282-85 Nicorandil, 83
hypertrophy, 316 Nicotinamide adenine dinucleotide. See
ischemia, 308 NAD
ischemic preconditioning, 312 Nifedipine, 104, 123,206,291
pharmacology, 308 Niflumate, 85
reperfusion (acute), 308-9 Niguldipine, 282
smooth muscle, 239 Nimodipine, 123
Na/K ATPase. See Na-pump Nisoldipine, 104, 123, 124
N-acetyl histidine, 33 Nitr-5, 218, 220
NAD,196 Nitrendipine, 104, 123, 124, 126
NADH, 60, 61 Nitric oxide, 128, 197
Na-HC0 3 cotransport, 305 Ica , 281
Naj accumulation, 51,151,237,275,279, myofilament Ca sensitivity, 281
285,308,330 preconditioning, 311
acidosis, 303 stimulation by cytokines, 309
cardiac glycosides, 286-94, 290, 291 synthase, 6, 281
hypoxia, 306 NO. See nitric oxide
reperfusion (acute), 308-9 Nonselective channel. See Ins(ca)
Naj depletion, 53, 171 Norepinephrine, 280, 281, 282, 285
Na-microelectrodes, 288, 290 Nuclear transcription factor (NFAT), 198
Na-pump, 88-91. See also Cardiac Nuclear transcription factors, 313-16
glycosides Nucleus, 7, 18
Index 421
ion channels, 243 caffeine, 327-28

volume, 7,41 classes, 327-28
Null-point titration, 49 classification, 327
O 2 consumption, 62, 279 EMD 53998, 33, 324
Okadaic acid, 167, 272 EMD 57439, 33, 325
Oligomycin, 53 EMD 5998, 327-28
Oregon green, 226 enoximone, 327, 328
Ortho- and retrograde signaling, 210 levosimendan, 327-28
Ouabain, 90, 235, 286-94 MCI154,327-28
Oxalate, 170 milrinone, 33, 327-28
Ploop, 67-68 pimobendan, 33, 35, 327-28
p38, 313-16 piroximone, 327-28
Pacemaker current. See If saterinone, 327-28
Pacemaking, 94-97 sulmazole, 33, 35, 324, 327-28
Paired-pulse stimulation, 264 theophylline, 327-28
Pandinus imperator, 196 Phosphodiesterases, 128, 327
Paracrine, 314 Phospholamban, 34, 164-69
Parasympathetic. See Acetylcholine, CaM kinase, 166, 167,271,286
Muscarinic effects complex formation, 168
Pathophysiology. See also Adrenergic density, 165
effects frequency dependent acceleration of
acidosis, 300-305 relaxation, 270-71
ADs and triggered arrhythmias, 297-300 heart failure, 318-319
Ca overload, 230-32 inhibition of SR Ca-pump, 165, 166, 168
Ca overload and spontaneous Ca model, 165, 168
release, 294-97 phosphorylation, 275-82, 275-82
heart failure, 316-23 PKA-dependent phosphorylation, 165-66,
hypertrophy, 312-16 236,275-82,277
hypoxia and ischemia, 305-12 PKC-dependent phosphorylation, 167
magnesium, 193 PKG-dependent phosphorylation, 167
stunning, 310 sequence and structure, 164
Paxillin, 17 transgenic mouse, 165,278,330
PDGF,313-16 Phospholipase C, 239, 313-16
Pentifylline, 194 DAG vs. IP3 effects, 242
Perhexiline, 33, 324, 326 Phospholipid translocase (flippase), 142
Peri-infarct zone, 330 Phosphorylase b, 279
Permeability transition pore, 59 Phosphorylation. See also individual targets
Pertussis toxin, 280, 281 and kinases, Adrenergic, PKA, PKC,
PESP. See Post extrasystolic potentiation PKG, CaMK, Tyrosine kinase, MLC
pH. See Acidosis kinase
Pharmacoelectrical coupling, 238 Ca channel (sarcolemma), 275-82
Pharmacomechanical coupling, 238, 239 Ca pump (sarcolemmal), 133, 134
Phenylalkylamines, 106, 108, 123-27 IP3 receptor, 201
Phen~ephrine, 130,237,238,282, 283,284 KV1.4,79
hypertrophy, 313-16 myofilaments,21
pH; regulation, 304-5 Na channels, 75
Phorbol ester, 131 Na/Ca exchange, 145
Phosphatase 2b, 313-16 Na-pump, 279
Phosphatase stimulation, 128 phospholamban, 34, 129, 165, 166, 236,
Phosphatidylinositol,51 271,275-82
Phosphatidylserine, 51 protein C, 34, 280
Phosphodiesterase inhibitors, 33, 35,128, sorcin, 200
255,327-28. See also PKA (cAMP- SR Ca release channel, 190, 198-200,
dependent) 275-82
adibendan, 327, 328 SR Ca-pump, 163
amrinone, 33, 327, 328 Tnl, 33, 34, 166,275-82
Photoaffinity labeling, 126 P-loop,68

Photolysis of caged IP3 Plunger model, 206, 207
cardiac muscle, 242 PMA,131
skeletal muscle, 241 PN200-110,123,206,287
smooth muscle, 237 Poisson-Nernst-Plank model, 111
Photolysis of caged phenylephrine, 237 Polyamines, 77
Photoylsis of caged Ca, 218, 220, 224, 230 Polylysine, 197
Pimobendan, 33, 35, 324, 326, 327, 328, Post extrasystolic potentiation, 256, 264
327-28 Post-ischemic recovery, 309
Pimozide, 126 Post-rest contractions, 40,183,246,247,
Pinacidil, 83, 311 255-61,269-72,292
PIP 2 ,239 Post-rest recovery, 247, 265-68
Piroximone, 327-28 Post-vagal tachycardia, 128
PKA (cAMP-dependent), 127-30,275-82. Potassium channels. See K channels
See also Adrenergic (~) effects, Power output, 37
Phosphodiesterase inhibitors, Inotropy Power stroke, 25
action potential, 278 Prazosin, 287
Ca pump (sarcolemmal), 133-34 Preconditioning, 310-12
Ca transient, 276, 283 Preload, 35, 256
delayed rectifier K channels, 79-81 Premature ventricular contraction, 256
FK-506 displacement, 199 Pressure overload, 313, 314
heart failure, 119, 322-23 Procainamide,74
Ica , 76, 127-30, 129, 255, 275-82 Procaine, 170, 197
Icl(cAMP), 84, 278 Propaferone,74
Ir,278 Protein C, 17, 19
metabolism, 279 PKA phosphorylation, 34, 280
myofilament Ca sensitivity, 33-34, 275- Protein concentration, 41
82 Protein kinase. See PKA, PKC, PKG,
Na channels, 75, 235 CaMK, Tyrosine kinase
Na-pump, 90, 279 Protein phosphatases, 128
phospholamban, 165, 166, 236, 275-82, P-type ATPase, 90, 133, 161
277 Puffer fish. See Tetrodotoxin
protein C, 34 Pump backflux, 175, 177, 252
sorcin phosphorylation, 200 Purinergic activated CI current, 83
SR Ca release channel, 198-200,275-82 Purinergic receptors, 131
PKC (Ca/lipid-dependent), 6,17,131,189, Purkinje cells, 288
239, 282-85. See also Adrenergic (a) AP phase 4, 95
effects, IP3 corbular SR, 14
action potential, 282-85 ICa ,T,95
alkalosis, 282-85 Ir,95
Ca current, 130,283 longitudinal resistivity, 5
hypertrophy, 313-16 propagation rate, 64, 67
Ito, 79 Pyruvate dehydrogenase, 60
K channels, 282-85 Qr,205
myofilament Ca sensitivity, 282-85 Q10, 273, 274, 275
Na channels, 75 Quercetin, 197
Na/H exchange (sarcolemma), 282-85 Quin2,49
Na-pump, 90, 285 Quinidine, 74
phosphodiesterase stimulation, 283 Quinuclidinyl benzilate, 287
phospholamban, 167 R-202-791, 126
preconditioning, 311 Rac,313-16
smooth muscle, 239 RACK-1,17
PKG (cGMP-dependent), 128, 281 Radiotracer techniques, 185-86
phospholamban, 167 Raf, 285, 313-16
Tnl phosphorylation, 281 RaM,58
Plant alkaloids, 186 Rapamycin, 198, 199
Index 423
Rapid cooling contractures, 52, 152-54, Restitution. See Mechanical restitution,

158,181-82,183,229,269,270,290, Electrical restitution
296 Rewarming spike, 181, 182
frequency dependence, 268 RGD peptide, 314
rest decay, 184, 259 Rho, 313-16
Ras, 17,285,313-16 Rigor, 21, 22, 25, 26, 62
Rat vs. rabbit, 261-65 RO 201724, 327
[Ca],esI> 261 Rolipram, 327
[Na];, 260, 261,294 Ru360,56
adrenergic (a) receptors, 282 Ruthenium red, 44,56,58, 161, 170, 186,
AP duration, 261, 262 187, 194, 197
Ca influx and efflux, 261, 262 Ryanodine, 186, 197,287,290. See also SR
Ca removal fluxes, 250-52, 253 Ca release channel
capacitance, 5 acceleration of rest decay, 184, 262
cell volume, 5 binding studies, 189, 192
contractile protein isoforms, 22 depression of post-rest contractions, 246
diastolic Ca entry, 6, 260 FK-506 binding, 199
force-frequency relationship, 262, 269 rapid cooling contractu res, 183, 184
fractional release, 260 specificity, 245
Ica during AP, 120 vs. caffeine, 245, 246, 247
myofilament Ca sensitivity, 31 Ryanodine receptor. See SR Ca release
Na/Ca exchange, 260 channel
Na/Ca exchanger reversal, 261, 263 S-202-791, 123, 126
post-rest recovery, 247 S4 voltage sensor, 67-68, 70-71
rest decay, 257,263 SA node. See Sino-atrial node
rest potentiation, 257 Sarcalumenin, 173
sources of activator Ca, 245-54 Sarcolemma, 2-9
spontaneous SR Ca release, 294 Ca binding, 51
SR Ca-pump, 163, 252, 260, 270 leak, 251, 259
surface area, 5 phospholipids, 51
ultrastructure, 4, 8, 247 protein distribution, 2
Recirculation fraction, 269 ultrastructure, 2-9
Reentry, 67, 99-100, 322 vesicle studies, 3, 41, 124, 135, 136, 138
Refractory. See a/so Mechanical restitution Sarcomere, 16, 24-38, 60, 146,226
action potential, 82, 99 Sarcoplasmic Reticulum. See SR
Ca induced Ca release, 229, 231 Saterinone, 327-28
Na channels, 66, 75 Saxitoxin, 287
SR Ca release channel, 206, 207, 217, SBFI,303
230,256,258,264,265 Scattered light intensity fluctuations, 294
Relative refractory period. See Refractory Scorpion toxins, 189, 196, 197,208
Relaxation, 39, 52-54. See also Lusitropy, Serotonin, 280, 282
SR Ca-pump, Na/Ca exchange Shaker K channels, 72
Relaxation rate constants, 54 Sialic acid, 9
Relaxing factor, 161 Sildenafil, 327
Reperfusion (acute), 308-9. See also Silver. See Ag
Ischemia, ATP Sino-atrial node, 63-64, 280
arrhythmias, 308 action potential, 94, 95-97
Caj accumulation, 308-9 block,95
free radicals, 309 ISk ,95-96
Na; accumulation, 308-9 Ica , 95-96
proton extrusion, 308 It, 86, 95-96
Respiratory acidosis, 300-303 IK(Ach),81
Rest decay, 157, 184,257, 258-61, 292 K current, 95-96
Rest potentiation, 256-257, 258, 260-61 Na current, 95-96
Rested-state contraction, 259 Na/Ca exchange, 95-96
Resting Ca. See Diastolic Ca Na-pump
SITS, 83 adenosine receptors, 287

Skeletal vs. cardiac muscle adrenergic (a) effects, 282
Ca channel permeation, 112 adrenergic (a) receptors, 282, 287
Ca channel subunits, 104, 106 adrenergic (~) receptors, 287
contractile protein isoforms, 22 angiotensin II receptors, 287
dependence on [Ca)o, 203 AP duration, 120,261
DHPR-RyR Interaction, 15, 210-13 Ca channel (sarcolemma),103, 121,204,
DHPR-RyR ratio, 13,15,212 287
diffusional limitations, 1 Ca fluxes, 54,152,153,245-54,252,
dyads vs. triads, 6 261-65
E-C coupling, 1, 3 capacitance, 5, 6
glycocalyx, 9 Ca-pump (sarcolemmal), 134,260
Ica activation, 204 cell volume, 4-6
II to III loop of Ca channel, 209 CICR,247
length-tension relationship, 27 contractile protein isoforms, 22
mitochondria volume, 8 delayed rectifier K currents, 79
myofilament bundles, 1, 8, 16 endothelin receptors, 287
nucleus volume, 8 force-frequency relationship, 269-70
SR Ca release channel activation, 192 frequency-dependent acceleration of
SR volume, 1, 4, 8, 11 relaxation, 271
surface area to volume ratio, 2, 4 hormone receptors, 287
tetanus, 19,64 Ica during AP, 120
TnC, 21, 30 ion transporters, 287
T-tubules, 1-4, 2, 3, 8 Iii, 297
voltage-dependent Ca release, 205, 207, Ito, 92
205-15 muscarinic receptors, 287
Skinned cells, 215, 295, 296, 303 myofilament Ca sensitivity, 30, 31
Skinned fibers, 28-31,33,34,214,215,237 Na/Ca exchange, 147, 152,253
"Skipping a beat", 256, 143 Na/Ca exchanger reversal potential, 261
Sliding filament theory, 24 Na-pump, 90, 287
Slip mode conductance, 75, 235, 293. See post-rest recovery, 247
also Cardiac glycosides rest decay and rest potentiation, 257, 259,
Slow inward current, 101 261
Smooth muscle resting [Na)i, 158
capacitative Ca entry, 240-41 SL Ca-pump vs. Na/Ca exchange, 159,
E-C coupling, 237-41 253
energy cost of contraction, 240 sources of activator Ca, 250, 245-54,
myofilament activation, 239 252-54
Na/H exchange, 239 spontaneous SR Ca release, 294
pharmacomechanical coupling, 239 SR Ca release channel, 287
phasic vs. tonic, 238 SR Ca-pump, 163,252,253
PKC effects, 239 SR dependence, 6, 245, 246-254
superficial buffer barrier hypothesis, 239 SR ultrastructure, 247
vasodilation via dihydropyridines, 125 SR volume, 3-8, 4
Sodium channels. See Na channels surface area, 5, 6
Sodium pump. See Na-pump surface to volume ratio, 3-8, 247
Sodium/Calcium exchange. Se, Na/Ca transverse tubules, 3-8, 7
exchange T-type vs. L-type Ca channels, 102
Sodium/Hydrogen exchange. See Na/H Spermidine, 77
exchange (sarcolemma), Mitochondria Spermine, 58, 77,197
Na/H exchange Spiral waves, 99, 100
Sorcin, 200, 210 Spontaneous SR Ca release, 85, 87, 88, 97,
Sotalol,79 138,169,225,230,258,294-97.See
Space constant, 99 also Ca overload
Spatial [Cali gradients, 248 intact heart, 295
Species dependent differences
Index 425
Spontaneous transient inward currents acidosis, 193, 197,300,303

(STICs),240 adaptation, 194-96,227-28
Spontaneous transient outward current alkalosis, 236
spikes, 238 ATP effects, 193
SR, 10-15, 161-202. See also SR Ca Ca activation of, 192
release channel, Ca induced Ca release, Ca dependence, 206
Ca sparks, Caffeine, Spontaneous SR Ca Ca release favors inward INaICa, 246
release, Ca overload Ca release inactivates lea, 246
"foot" processes, 161 Ca release shortens AP, 246
anion-selective channels, 202 Ca selectivity, 186
atrial VS. ventricular differences, 14 Ca sparks, 190-92
Ca buffering, 14,49, 171-73,180 cadmium, 237
Ca content, 49,51,55,177-86,179 caffeine, 52, 193, 194,232
Ca content determination, 55 CaM kinase, 190, 198,280
Ca overload, 230-32 cloning, 187
Ca refilling, 265-68 clusters, 191
Ca release, 39, 49, 51,186-202,226-27, density, 287
246 developmental differences, 249
calsequestrin, 14, 171-73 domains, 190
Ca-oxalate precipitation, 171 effects of luminal [Ca], 194, 258
cardiac glycosides, 289, 292 electron microscopy, 13
contribution to activator Ca, 55, 56, 245- flux, 227
54 free radicals, 309
corbular, 14 heart failure, 318-22
depletion of local Cai, 227-28 heavy metals, 237
developmental changes, 248 inactivation, 194-96,227-28
direct depolarization of, 213-14 interaction with calmodulin, 189
electrochemical gradient, 174, 202 interaction with FKBP, 188
extended junctional, 8, 14,214 interaction with Ca channel, 12, 189,209-
fractional release, 173, 186,224-26,245, 13,211
253-260,267,270 IP3 receptor homology, 189
frequency dependence of load, 268 isoforms, 187
heart failure, 319, 318-20, 322 isolation of, 186
IP3 induced Ca release, 237-43 junctin/triadin interactions, 172, 190
K selective channels, 202 location, 206
leak,48, 173-76, 177,259 loop peptides (II-III loop), 210-13
load modulation, 197, 330-31 magnesium, 188, 193, 195-197,214
longitudinal, 161 modulation, 197, 330-31
membrane potential, 214 modulation by SR Ca, 195, 226
post rest recovery, 265, 266-68 open probability, 192
rested-state load, 259 open time, 187
skeletal VS. cardiac muscle, 1, 4, 8, 11 ouabain, 293
species dependent differences, 179 permeability and conductance, 188
spontaneous release of Ca, 230-32, 297 phosphorylation, 198-200, 275-82
steady state load, 186,265,267 PKA anchoring protein, 190
sucrose density gradient fractions, 161 PKA effects, 198-200, 275-282
temperature, 275 pore diameter, 186
terminal cisternae, 161 purification, 187
termination of Ca release, 227-28 recovery from inactivation, 228-31, 229,
ultrastructure, 10-15,247 256
vesicle studies, 11, 161, 170, 177, 186- refractory, 206-7, 217, 230, 256, 258,
97,202,236,237,241,303 264-65
voltage-dependent Ca release, 215 ryanodine binding sites, 194
volume, 8, 3-8, 4, 41 single channel conductance, 186, 187,
SR Ca release channel, 191, 186-202. See 193
also SR, Ca induced Ca release, Caffeine skeletal VS. cardiac muscle, 192, 193
species dependent differences, 287 block of Na/Ca exchange, 145

stochastic attrition, 227 SR Ca release channel, 188
temperature, 273 Stunning, 310
three-dimensional reconstructions, 188, Submembrane space, 149-51
189 Sucrose density gradients, 161
timecourse of release, 206, 226-27 Sudden cardiac death, 299
toxins, 197 Sulfhydryl reagents, 197
ultrastructure, 187-90,209 Sulmazole, 33, 35, 197,324,326,327,328,
unitary current, 187 327-28
SR Ca-pump, 39, 161-77 Summation, 19, 190, 223, 230
acidosis, 170, 300, 303 Superoxide, 309
adrenergic (~) effects, 166, 267 Superoxide dismutase, 309
ATP effects, 169 Suramin, 197
backflux, 173-77, 175, 177 Surface area of organelles, 3-8
blockade by thapsigargin, 247 Surface density. See specific protein
CaM kinase, 133, 134, 166, 167,286 Surface potential, 114-16
cardiac vs. skeletal muscle, 164 Surface to volume ratio, 3-8
cloning, 161 Swelling-activated CI Current. See ICI(swell)
competition with Na/Ca exchange, 52-53, Sympathetic. See Adrenergic
54,166,183,250-53,259 Sympathetic stimulation. See Adrenergic
density, 11, 163, 287 effects
energy consumption, 177 Syncytium, 8, 19, 63, 295
free radicals, 309 Systolic [Calio 140
tlG,62 Tachycardia, 74, 79
heart failure, 298, 318-19, 253 Tail currents, 124,218
inhibitors, 169 Tail transients, 217, 218
isoforms, 161, 162 Talin,17
kinetic parameters, 48,175 Tamoxifen, 85
Km(Ca),165 Temperature. See also Hypothermia, Rapid
magnesium, 161, 169, 170 cooling contractures, Malignant
model, 162, 163, 164 hyperthermia
modulation, 330-31 action potential, 273
O2 consumption, 62 Ca transients, 274
pH effects, 167, 170 myofilament Ca sensitivity, 29, 30, 32, 35,
phospholamban, 164-69, 165, 166, 168, 273
318 Tensin,17
sequence, 161 Terminal cisternae, 11
snubs peak [Cali, 247, 251 calsequestrin in, 14
species dependent differences, 163, 252 formation of triads, 6
SR Ca uptake rate, 170, 171 skeletal vs. cardiac muscle, 6
SR distribution, 11 Tetanus, 19, 64
stoichiometry, 163 IP3 production, 241
temperature, 274 Tetracaine, 197, 206, 236, 267, 303
thermodynamics, 173-77 Tetrads, 208
transport reaction, 162 Tetramethylammonium, 108
turnover rate, 163 Tetramethylmurexide, 264
Staircase, 119, 120, 135, 262, 263, 265, Tetrodotoxin, 74, 75, 96, 205, 235
268, 270. See also Rest potentiation, Tetrodotoxin-sensitive Ca current, 205, 232,
Rest decay, Force-frequency 235
isoproterenol, 276 Thapsigargin, 44,169,247,251
Steady state, 54, 253-54, 265, 266-68 acutely increases Ca transient, 247
Stochastic attrition, 227 in multicellular preparations, 247
Streaming potential measurements, 186 Theobromine, 194
Stretch, 85 Theophylline, 194, 324, 327-28
Stretch activated channels, 75, 85, 314 Thick filament, 16, 17, 19. See also Myosin
Strontium, 22, 68, 109, 141, 208 Thin filament, 16, 20-22. See also Actin
Index 427
Thyroid hormone, 22, 318 Tyrosine kinase, 131

Titin, 17, 19 Ca current, 131
TNF-a, 309 hypertrophy, 313-16
Tnl Ultrastructure
phosphorylation, 275-82 cardiac, 1-18,3
Tolbutamide, 83 developmental changes, 249
Torsades de pointes, 80 extracellular matrix, 17-18
Total cytosolic [Cal, 41-50, 49, 251, 289 extracellular space, 9-10
acidosis, 302 mitochondria, 15-16
Transcriptional regulation myofilaments, 16-17, 21
Ca dependence, 243 sarcolemma, 2-9
Transgenic mouse skeletal,2
adrenergic receptor (~1) knockout, 280 SR,10-15,247
adrenergic receptor (~2) overexpression, SR Ca release channel, 12, 187-89, 190
280 surface to volume ratio, 249
calsequestrin overexpression, 173 transverse tubules, 2-9, 3, 12
constitutively active PKC, 311 Unidirectional block, 99
hypertrophy induction, 313 Uniformity of [Cali, 248
IK(Ach) knockout, 82 Valvular insufficiency, 313
Na/Ca exchanger overexpression, 159- Ventricular fibrillation, 99
60 Ventricular tachycardia, 99
phospholamban knockout, 34, 167, 271, Verapamil, 124, 125
278, 330 Veratridine, 329
phospholamban overexpression, 165 Vesicle studies. See SR vesicle studies,
RyR3 knockout, 188 Sarcolemma vesicle studies
SR Ca-pump overexpression, 330 Viagra. See Sildenafil
Tnl truncation, 310 Vinculin, 17
Transient inward current. See Iii Vmax ' See different processes
Transverse tubules, 2-10,15 Voltage-dependent anion channel, 59
species/regional differences, 8, 248 Voltage-dependent Ca release, 205-15
ultrastructure, 2-10, 12 in myocytes, 235-36
protein distribution, 2 in skeletal muscle, 205-12
spatial [Cali gradients, 248 Volume, 3-8
Triadin, 3,172,190,210 cytosolic, 41
Triads, 6, 12, 14 mitochondrial,41
Trigger[Ca),215,216,220, 223, 224 myofilament space, 41
Triggered arrhythmias, 258. See nuclear, 41
Afterdepolarizations species dependent differences, 4
Tropomodulin, 17 SR,41
Tropomyosin, 20, 21, 23 unit conventions, 41, 42
Troponin complex, 21, 24. See a/so Volume overload, 313
Phosphorylation, Myofilaments, Acidosis, Waves. See Ca waves, Spiral waves
Alkalosis, PKA, Adrenergic (~) effects WB-4101,282
developmental changes, 301 Window current, 73, 80, 86, 97, 118,238,
stunning, 310 255, 309
TnC,20-22,34 XIP, 137, 140, 143-145
Tnl, 20-22, 34, 35, 281 X-ray diffraction, 23, 24,162
TnT,20-22,23 Zaprinast, 327
T-tubules, See Transverse tubules Zinc. SeeZn
TTX. See Tetrodotoxin Zinterol, 280
T-type Ca channels, 73, 76, 92, 95, 101-8, Z-line, 2, 5, 16, 17,27
205, 232, 235. See also Ca channels Zn
activation, 73 SR Ca release, 237
Ca induced Ca release, 232
localization, 232
sino-atrial node AP, 95, 96

Excitation-Contraction Coupling and Cardiac Contractile Force

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EXCITATION-CONTRACTION COUPLING AND

CARDIAC CONTRACTILE FORCE

SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

ISBN 978-0-7923-7158-8 ISBN 978-94-010-0658-3 (eBook)

Printed an acid-tree paper

AII Rights Reserved

PREFACE TO FIRST EDITION

The main aim of this monograph is to provide an overview of calcium regulation in

PREFACE TO THE SECOND EDITION

Julio Altamirano Larry Hryshko Burkert Pieske

INTRODUCTION TO THE FIRST EDITION

INTRODUCTION TO THE SECOND EDITION

The second edition of Excitation-Contraction Coupling and Cardiac Contractile Force is

Ca influx must match Ca efflux 253

Chapter 3 Sources & Sinks of Activator Ca

Chapter 4 Cardiac Action Potential and Ion Channels

40 Em-dependent gating of ion channels 73

Chapter 5 Sarcolemmal Ca Channels

Chapter 6 Na/Ca Exchange and Sarcolemmel Ca Pump

Chapter 7 SR Ca Uptake and Release

Chapter 8 Excitation-Contraction Coupling

126 E-C coupling in smooth muscle 239

Chapter 9 Control of Cardiac Contraction by SR & Sarcolemmal Ca Fluxes

Chapter 10 Cardiac Inotropy and Ca Mismanagement

170 Acidosis decreases myofilament Ca sensitivity 302

MAJOR CELLULAR STRUCTURES INVOLVED IN

Numerous cellular structures are involved with the process of excitation-contraction

SARCOLEMMA AND TRANSVERSE TUBULES

Figure 2. Schematic diagram of T-tubules, SR and myofilaments in mammalian cardiac muscle.

Finch va 57.3 34 0 0.21 0.62 0.83

Mouse Vb 54.3 37.5 0.95 0.22 0.65 0.87 0.92

Rat V g 48.1 34 1.2 0.3 3.2 3.5 2.9

a Bossen et ai., 1978 h Page, 1978

CONNECTIONS TO THE EXTRACELLULAR MATRIX

OTHER CELLULAR CONSTITUENTS

When cytoplasmic [Cal rises, the myofilaments are activated in a [Cal-dependent

~LMM (100 nm)

MECHANISM BY WHICH Ca ACTIVATES CONTRACTION

\"'" Myosin blocked------'\

A. Rest C. Force Development

A -M~A-M-ATP---7A -M-ADP-P -4A -M-ADP*----.A-M-ADP-4. A-M

Sarcomere Length (~m)

THE LENGTH-TENSION RELATIONSHIP

THE Ca SENSITIVITY OF THE MYOFILAMENTS

Temperature and Ca Sensitivity

A. Fabiato, 1982 B. Harrison & Bers, 1989

C. Lues et aI., 1988 D. Solaro & Coworkers

FORCE-pC a RELAnON IN INTACT CARDIAC MUSCLE

><III 80 Yue et al,1986

FACTORS WHICH INFLUENCE THE FORCE-[Ca] RELATIONSHIP

a) Harrison & Bers, 1989a k) Harrison et aI., 1985

FORCE VELOCITY CURVES

Quick Release and Re-stretch

Force redevelopment ::J

SOURCES AND SINKS OF

GENERAL SCHEME OF Ca CYCLE IN CARDIAC MYOCYTE

CELLULAR VOLUME CONVENTIONS AND Ca FLUXES

Ca BUFFERING IN THE CYTOSOL