Abstract
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CaMKII-dependent phosphorylation regulates basal cardiac pacemaker function via modulation of local Ca2+ releases
Abstract
Spontaneous beating of the heart pacemaker, the sinoatrial node, is generated by sinoatrial node cells (SANC) due to gradual change of the membrane potential called diastolic depolarization (DD). Spontaneous, submembrane local Ca2+ releases (LCR) from ryanodine receptors (RyR) occur during late DD and activate an inward Na+/Ca2+exchange current to boost the DD rate and fire an action potential (AP). Here we studied the extent of basal Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation and the role of basal CaMKII-dependent protein phosphorylation in generation of LCRs and regulation of normal automaticity of intact rabbit SANC. The basal level of activated (autophosphorylated) CaMKII in rabbit SANC surpassed that in ventricular myocytes (VM) by approximately twofold, and this was accompanied by high basal level of protein phosphorylation. Specifically, phosphorylation of phospholamban (PLB) at the CaMKII-dependent Thr17 site was approximately threefold greater in SANC compared with VM, and RyR phosphorylation at CaMKII-dependent Ser2815 site was ~10-fold greater in the SA node, compared with that in ventricle. CaMKII inhibition reduced phosphorylation of PLB and RyR, decreased LCR size, increased LCR periods (time from AP-induced Ca2+ transient to subsequent LCR), and suppressed spontaneous SANC firing. Graded changes in CaMKII-dependent phosphorylation (indexed by PLB phosphorylation at the Thr17site) produced by CaMKII inhibition, β-AR stimulation or phosphodiesterase inhibition were highly correlated with changes in SR Ca2+ replenishment times and LCR periods and concomitant changes in spontaneous SANC cycle lengths (R2 = 0.96). Thus high basal CaMKII activation modifies the phosphorylation state of Ca2+ cycling proteins PLB, RyR, L-type Ca2+ channels (and likely others), adjusting LCR period and characteristics, and ultimately regulates both normal and reserve cardiac pacemaker function.
NEW & NOTEWORTHY
Elevated basal level of activated CaMKII in intact rabbit sinoatrial node cells leads to high basal CaMKII-dependent protein phosphorylation that is critically important for generation of spontaneous local subsarcolemmal Ca2+ releases and normal pacemaker cell automaticity. CaMKII inhibition prevents generation of local Ca2+ releases and suppresses spontaneous beating of sinoatrial node cells.
the spontaneous beating of the heart originates in the primary pacemaker, the sinoatrial (SA) node. Sinoatrial node cells (SANC) generate spontaneous action potentials (APs) due to spontaneous gradual change in the membrane potential called spontaneous diastolic depolarization (DD) (16). Various ionic currents are involved in the generation of DD, including hyperpolarization activated current (If), delayed rectifier potassium currents IK, L- and T-type Ca2+ currents (ICa,L and ICa,T), and others (16). Spontaneous beating of SANC is also critically dependent on subsarcolemmal local Ca2+ releases (LCR) from the sarcoplasmic reticulum (SR), which appear during DD and activate an inward Na+/Ca2+ exchange current (INCX), which imparts an exponential increase to the later part of DD to regulate speed at which the membrane potential reaches the threshold to fire AP (5). The LCR period, the time between prior AP-induced Ca2+ transient and LCR occurrence, determines timing of INCX activation and thus regulates the spontaneous SANC beating rate (5, 44).
Spontaneous LCRs reflect rhythmic SR Ca2+ cycling in SANC, i.e., AP-triggered Ca2+ transient depletes SR and ryanodine receptors (RyR) become inactivated, when SR Ca2+ content is replenished and activation of RyR is recovered spontaneous LCRs begin to occur. The efficiency of Ca2+ pumping into SR both in ventricular myocytes (VM) and SANC is regulated by SR Ca2+ATPase (SERCA), which is regulated by phospholamban (PLB). In the unphosphorylated state PLB is endogenous inhibitor of SERCA, but when PLB is phosphorylated by protein kinase A (PKA) or Ca2+/calmodulin-dependent protein kinase II (CaMKII), it dissociates from SERCA and relieves SERCA inhibition, increasing Ca2+ pumping rate into SR (2, 26). The LCR period is regulated by the rate of SR Ca2+ replenishment by SERCA, and changes in the SR Ca2+ replenishment rate produced by inhibition of SERCA by cyclopiazonic acid or by changes in PLB phosphorylation at PKA-dependent Ser16 site modulate the LCR period and proportionally shift the spontaneous SANC cycle length (47).
CaMKII, a Ser/Thr protein kinase, is one of the major downstream targets of Ca2+ signaling in a variety of cells; in the heart the δ-isoform (CaMKIIδ) is predominant (14). When intracellular Ca2+ concentration is elevated, Ca2+ binds to calmodulin (CaM) and Ca2+/CaM complex binds to CaMKII leading to CaMKII phosphorylation and autophosphorylation (14). In response to varying Ca2+ signals, CaMKII can phosphorylate several Ca2+ regulatory proteins in the heart including PLB, RyR, and L-type Ca2+ channels (26, 27). Consistent with observations in VM (2, 27) inhibition of CaMKII activity in rabbit SANC by autocamtide-2 related CaMKII inhibitory peptide II (AIP) or KN-93, but not KN-92, markedly decreases L-type Ca2+ current (ICa,L) amplitude and delays its recovery from inactivation (43). Suppression of CaMKII activity decreases spontaneous beating of freshly isolated rabbit SANC, and this effect was attributed to CaMKII-dependent modulation of inactivation and reactivation of L-type Ca2+ channels (43).
Autonomic neuronal impulses to the heart modulate spontaneous firing of intact SANC and lead to an increase or decrease in the spontaneous firing and concomitant changes in [Ca2+]c, a key regulator of CaMKII-dependent protein phosphorylation in the cell (14). We recently demonstrated that intrinsic SR Ca2+ cycling in saponin-permeabilized rabbit SANC was strongly modulated by cytosolic Ca2+ concentrations ([Ca2+]c), i.e., an increase in [Ca2+]c increased LCR parameters and this effect was associated with Ca2+-induced stimulation of both CaMKII- and PKA-dependent protein phosphorylation (37). Permeabilized SANC, however, is a simplified, convenient model to permit an easy access and control of free [Ca2+]c and to probe effects of [Ca2+]c on intrinsic SR Ca2+ cycling. From our results in permeabilized SANC (37), it would be difficult to predict either the extent of basal endogenous CaMKII-dependent protein phosphorylation in intact SANC or its impact on basal LCR characteristics or establish links among alterations in CaMKII-dependent phosphorylation, changes in LCR parameters, and resultant changes in the spontaneous SANC beating rate.
Intact rabbit SANC represent a complete system, which includes not only intrinsic SR Ca2+ cycling but also ionic channels and membrane bound regulatory proteins as well as interactions between all these structures. The present study specifically focuses on the evaluation of basal CaMKII-dependent protein phosphorylation in intact spontaneously beating rabbit SANC using Western blot or immunostaining technique. We also explored functional implications of changes in endogenous CaMKII-dependent protein phosphorylation on parameters of local RyR Ca2+ releases and spontaneous beating rate of freshly isolated rabbit SANC. These experiments were performed in the absence and presence of CaMKII inhibitors to decrease endogenous CaMKII activity or during β-AR stimulation or PDE inhibition to stimulate cAMP-mediated PKA-dependent phosphorylation and study its impact on CaMKII-dependent phosphorylation in intact rabbit SANC. The specificity of modulation of LCR parameters by CaMKII inhibitors in intact SANC was also verified in permeabilized SANC, the latter lack functional ionic channels that eliminate possible nonspecific effects. Our results provide novel insights into CaMKII-dependent regulation of LCRs in intact rabbit SANC in the basal state and establish links among changes in CaMKII-dependent protein phosphorylation, modulation of LCR characteristics, and spontaneous SANC beating rate under different experimental conditions.
MATERIAL AND METHODS
SANC Isolation
Animal procedures were approved by the National Institutes of Health (NIH) Ethics Review Board and treated in accordance with NIH Guide for the Care and Use of Laboratory Animals. Single SANC were isolated according to the modified method of Ito and Ono (17). New Zealand White rabbits (Charles River Laboratories) weighing 1.8–2.5 kg were deeply anesthetized with sodium pentobarbital (50–90 mg/kg). The heart was removed quickly and placed in the Tyrode solution containing the following (in mmol/l): 130 NaCl, 24 NaHCO3, 1.2 NaH2PO4, 1.0 MgCl2, 1.8 CaCl2, 4.0 KCl, and 5.6 glucose, after continuous saturation with a mixture of 95%O2-5% CO2 the pH was maintained at 7.4; temperature was maintained at 36°C. The SA node region was cut into small strips (~1.0-mm wide) perpendicular to the crista terminalis (CT). The final SA node preparation consisted of SA node strips attached to the small portion of CT.
The SA node preparation was washed twice in Ca2+-free Tyrode solution containing the following (in mmol/l): 140 NaCl, 5.4 KCl, 0.5 MgCl2, 0.33 NaH2PO4, 5 HEPES, 5.5 glucose, pH 6.9; then, incubated at 34°C for 30 min in Ca2+- free Tyrode solution containing elastase type III (0.6 mg/ml; Sigma Chemical), collagenase type 2 (0.6 mg/ml; Worthington, NJ), and 0.1% bovine serum albumin (Sigma Chemical). Thereafter, the SA node preparation was washed in modified Kraftbruhe (KB) solution, containing the following (in mmol/l): 70 potassium glutamate, 30 KCl, 10 KH2PO4, 1 MgC12, 20 taurine, 10 glucose, 0.3 EGTA, and 10 HEPES (titrated to pH 7.4 with KOH), and kept at 4°C for 1 h in KB solution containing 50 mg/ml polyvinylpyrrolidone (PVP 40; Sigma Chemical). Finally, cells were dispersed from the SA node preparation by gentle pipetting in the KB solution and stored at 4°C for future use.
Ventricular Myocyte Isolation
The rabbit heart was perfused in Langendorff mode for 5 min with a nominally Ca2+-free modified Krebs solution containing the following in mmol/l: 120 NaCl, 5.4 KCl, 1.6 MgSO4, 1.0 NaH2PO4, and 20 NaHCO3, at 37°C; bubbled with 95% O2-5% CO2. Perfusion continued for 3–4 min with protease (0.02 mg/ml, Type XIV; Sigma-Aldrich) and collagenase (1 mg/ml; Type B, 220 to 230 U/mg; Boehringer-Mannheim, Indianapolis, IN; or Type 2, Worthington, Lakewood, NJ), and then for further 10–15 min with 50 μmol/l CaCl2 added. The ventricles were separated from the heart, chopped into chunks, and placed for a second digestion for 10–15 min in a shaker (60 to 70 rpm) at 37°C, with Tyrode solution containing 100 μmol/l CaCl2 and 1 mg/ml collagenase. Digestion was quenched by filtering the supernatant for centrifugation at 500 g and three washes with a modified Tyrode solution the following in mmol/l: 137 NaCl, 4.9 KCl, 15 glucose, 1.2 MgSO4, 1.2 NaH2PO4, and 20 HEPES, pH 7.4; Ca2+ concentrations were successively increased to 250, 500, and 1,000 μmol/l. Ventricular cells were stored at room temperature in 1 mmol/l Ca2+ Tyrode solution.
Cell Permeabilization
A subset of SANC was permeabilized with 0.01% saponin in a solution containing the following in mmol/l: 100 dl-aspartic acid potassium salt, 25 KCl, 10 NaCl, 3 MgATP, 0.81 MgCl2 (free [Mg2+] ~1 mM), 20 HEPES, 0.5 EGTA, 10 phosphocreatine, and 5 U/ml creatine phosphokinase, pH 7.2 and temperature 35 ± 0.5°C, as previously described (37). The free intracellular [Ca2+]i at a given total Ca2+, Mg2+, ATP, and EGTA concentration was calculated using a computer program (WinMAXC 2.50; Stanford University, CA). Considering that physiological free [Ca2+]i in rabbit SANC is ~150 nmol/l (44), LCR characteristics were recorded at this [Ca2+]i. The SR Ca2+ content in SANC under control conditions and in the presence of drugs was determined from the peak amplitude of Ca2+ transients produced by rapid application of 20 mmol/l caffeine onto permeabilized cells.
Confocal Imaging of Cytosolic Ca2+ Transients and LCRs
Intact or permeabilized SANC were loaded with fluo-3AM or fluo-4 pentapotassium salt (Thermo Scientific, Waltham, MA), respectively, and placed on the stage of Zeiss LSM-410 inverted confocal microscope (Carl Zeiss, Germany) with the bath temperature maintained at 35 ± 0.5°C. All images were recorded in the line-scan mode, with the scan line oriented along the long axis of SANC just beneath sarcolemma. Images were processed with customized IDL software (6.1; Research Systems, Boulder, CO). The amplitude of either LCR or AP-induced Ca2+ transient was expressed as the peak value (F) normalized to minimal fluorescence (F0). The LCR spatial size was indexed as the full width at half maximum amplitude (FWHM) and its duration characterized as the full duration at half maximum amplitude (FDHM). The number of LCRs in permeabilized SANC was normalized per 100 μm of the linescan image and 1-s time interval, as previously described (44). The rate of [Ca2+]i decline during AP-induced Ca2+ transient, which reflects the kinetics of the SR Ca2+ refilling, has been estimated using the time to 90% decay of AP-induced Ca2+ transient (T-90), as previously described (47).
Western Blots of Ca2+ Handling Proteins
Phosphorylation of PLB in SANC and VM.
The detection of site specific PLB phosphorylation was performed as previously described (21). For drug treatment the SANC suspension was equally divided into four to six parts; each part was individually treated for 5 min (at 35 ± 0.5°C) with the following: solvent control, 1 μmol/l KN-93 or 1 μmol/l KN-92, 100 μmol/l IBMX, 1 μmol/l isoproterenol (ISO), and 50 μmol/l milrinone. In the separate set of experiments, SANC were treated with 25 μM BAPTA-AM or 15 μM PKI for 30 min. To completely dissociate PLB into monomers (6.7 KDa), cells were solubilized and boiled at 95°C for 10 min. The protein amount was determined using BCA protein assay kit (Pierce Chemical, Rockford, IL), and proteins were resolved by 7.5% urea/SDS-PAGE gel and transferred (10 μg protein/lane) to polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech). To detect PLB phosphorylation at the Ser16 or Thr17 sites the site specific P-Ser16 PLB antibody 1:10,000 (Badrilla) or specific P-Thr17 PLB antibody 1:5,000 (Badrilla) were used, followed by HRP-conjugated secondary antibody 1:15,000 (Bio-Rad Laboratories, Philadelphia, PA). Total PLB was detected using anti-PLB monoclonal antibody IgG1 1:10,000 (Badrilla). PVDF membranes were exposed to the chemiluminescence reaction (ECL; Amersham Pharmacia Biotech) and quantified with a video documentation system (Bio-Rad Laboratories).
RyR Phosphorylation at CaMKII-Dependent Ser2815 Site in Rabbit SA node and Ventricular Tissues
Isolated rabbit SA nodes were perfused for 20–30 min with either regular Tyrode buffer or with Tyrode buffer containing 1 μmol/l KN-93. Ventricular tissue and treated and untreated SA node tissues were collected, snap frozen in liquid nitrogen, ground into a fine powder, and then lysed in RIPA lysis buffer with added protease and phosphatase inhibitors (Sigma-Aldrich). Proteins (5–30 μg/lane) were resolved on 7.5% TGX gel (Bio-Rad Laboratories) by SDS-PAGE, transferred to PVDF membranes, and probed with either site-specific P-Ser2815 RyR2 antibody at 1:2,000 (Badrilla) or total RyR2 antibody at 1:2,000 (Clone C3-33; Thermo Scientific), followed by HRP conjugated goat anti-rabbit IgG (H+L) or goat anti-mouse IgG (H+L) (Invitrogen) antibody at 1:10,000. α-Actinin antibody at 1:2,000 (Sigma-Aldrich) was used to verify protein loading. Blots were developed with Pierce SuperSignal West Pico or West Dura ECL substrate kits (Thermo Scientific) and quantified using Kodak MI SE software (Carestream Health, Rochester, NY).
Total and Activated (Autophosphorylated at Thr286/287) CaMKII
Rabbit SANC or VM were transferred to Ca2+ containing Tyrode solution, pelleted by centrifugation, and lysed in RIPA Lysis buffer. Protein quantity was measured by BCA Assay (Thermo Scientific). Proteins (15 μg/lane) were resolved on a 4–20% TGX gel (Bio-Rad Laboratories, Philadelphia, PA) by SDS-PAGE, transferred to PVDF membranes and probed with either site-specific (Thr286/287) P-CaMKII antibody at 1:500 (Cell Signaling Technology, Danvers, MA) or total pan-CaMKII antibody at 1:500 (Millipore), followed by HRP-conjugated goat anti-mouse IgG (H+L) or goat anti-rabbit IgG (H+L) (Invitrogen) antibody at 1:10,000. GAPDH antibody at 1:5,000 (Thermo Scientific) was used to verify protein loading. Blots were developed with Pierce SuperSignal West Pico or West Dura ECL substrate kits (Thermo Scientific) and quantified using ImageQuant TL software (GE Healthcare Life Sciences).
Immunostaining of RyR in SANC and VM
Rabbit SANC were pretreated with either Tyrode solution or PKA inhibitor peptide (15 μmol/l PKI) for 10 min or CaMKII inhibitor (3 μmol/l KN-93 or its inactive analog 3 μmol/l KN-92) for 30 min. SANC and VM were fixed with 4% paraformaldehyde, permeablized with 1% Triton, and incubated with blocking solution (1× PBS containing 2% IgG-free BSA+ 5% donkey serum + 0.02% NaN3 + 0.1% Triton) for 4 h. Cells were incubated with a primary anti-RyR2 total (mouse, 1:1,000, Thermo) antibody and anti-phosphorylated RyR2 at Ser2809 (rabbit, 1:200; Badrilla) antibody. Secondary Atto 647-conjugated anti-mouse IgG antibody (1:1,000; Sigma) was used for total RyR, and secondary Cy3-conjugated anti-rabbit IgG antibody (1:1,000; Jackson ImmunoResearch Laboratories, West Grove, PA) was used for phosphorylated RyR immunostaining. Dual confocal images of central sections of SANC and VM were obtained with a Zeiss LSM 510 (Carl Zeiss).
Considering that cells vary in size and total RyR protein density might vary from cell to cell (25), the fluorescence density of phosphorylated RyR2 in a given cell was normalized to its total RyR2 fluorescence density (the nuclear area was excluded in all cells). Only secondary antibodies were applied to the negative control, which displayed negligible fluorescence.
Drugs
Specific cell-permeable AIP; specific PKA inhibitor peptide PKI (14–22); KN-93, its inactive analog KN-92 and isoproterenol were from Calbiochem (EMD Millipore, Calbiochem); broad spectrum PDE inhibitor 3-Isobutyl-1-methylxanthine (IBMX) and PDE3 inhibitor 1,6-dihydro-2-methyl-6-oxo-(3,4-bipyridine)-5-carbonitrile (milrinone) were from Sigma (Sigma-Aldrich).
Statistical Analysis
Data were presented as means ± SE. Statistical significance was evaluated by Student t-test, ANOVA, or column statistics, where appropriate. A value of P < 0.05 was considered statistically significant.
RESULTS
Basal CaMKII-Dependent Phosphorylation Regulates SR Ca2+ Cycling in Intact SANC
A specific membrane-permeable CaMKII inhibitory peptide AIP was employed to suppress endogenous CaMKII activity in freshly isolated spontaneously beating rabbit SANC. Following a 3-min exposure to AIP, SR Ca2+ cycling in SANC was markedly suppressed, i.e., the amplitude of AP-induced Ca2+ transient was reduced by ~56% (from 1.4 ± 0.13 to 0.62 ± 0.05 ΔF/F0), the LCR size, and number per each spontaneous cycle were markedly decreased (Fig. 1, B and C), while the LCR period was prolonged. Note, that prolongation of the LCR period in response to CaMKII inhibition was highly correlated with the concurrent increase in the spontaneous cycle length (Fig. 1D). Constant exposure of SANC to AIP for 5 min abolished LCRs and stopped spontaneous beating of SANC (Fig. 1). The effects of CaMKII inhibition by AIP on SR Ca2+ cycling and spontaneous beating of SANC were largely reversible on washout (Fig. 1). Additional evidence that SR Ca2+ cycling in SANC depends on CaMKII activation was obtained employing another CaMKII inhibitor KN-93. To verify specificity of KN-93 effects to suppress CaMKII activity, we employed its inactive analog KN-92. Similar to AIP, low (0.3 μmol/l) concentration of KN-93 suppressed SR Ca2+ cycling in rabbit SANC, i.e., decreased the amplitude of AP-induced Ca2+ transient, LCR size, and number of LCRs per each spontaneous cycle (Fig. 2), while the same concentration of KN-92 had no significant effects on AP-induced Ca2+ transient or LCR characteristics (Fig. 2). Please note that during a short exposure to CaMKII inhibitors (AIP or KN-93) spontaneous beating ceased in approximately half of SANC, while another half continue to beat with markedly decreased spontaneous beating rate.
Inhibition of basal Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity in intact rabbit sinoatrial node cells (SANC) by autocamtide-2 related inhibitory peptide II (AIP) time dependently and reversibly suppressed local Ca2+ releases (LCRs) and increased the LCR period and spontaneous SANC cycle length. A: confocal line scan images of a representative SANC depicting action potential (AP)-induced Ca2+ transients and LCRs (arrowheads) during spontaneous beating of SANC in control and when CaMKII activity was inhibited by 10 μmol/l AIP. Normalized subsarcolemmal fluorescence averaged over an image width is shown in red and superimposed with the image. Insets: LCR period and the time to 90% decay of AP-induced Ca2+ transient (T-90) are defined. Inhibition of CaMKII activity markedly suppressed LCRs, increased the LCR period and spontaneous cycle length; subsequently spontaneous firing ceased. Following drug washout LCR resumed and spontaneous beating recovered. B and C: AIP markedly decreases the number of LCRs per each spontaneous cycle and the LCR size (n = 4). D: the time-dependent increase in the cycle length following AIP was linked to its effect on the LCR period. *P < 0.05.
Suppression of basal CaMKII-dependent signaling by KN-93 in intact rabbit SANC decreased amplitude of AP-induced Ca2+ transient, LCR number, and size in intact SANC. A and B: confocal linescan images and Ca2+ waveforms of a representative spontaneously beating rabbit SANC before and during superfusion with either 0.3 μmol/l KN-93 (A) or 0.3 μmol/l KN-92 (B). C–E: KN-93 (n = 5), but not KN-92 (n = 4), markedly decreased amplitude of AP-induced Ca2+ transients (C), size of LCRs (D), and number of LCRs per each spontaneous cycle (E). LCRs are indicated by arrowheads. *P < 0.05.
Basal CaMKII-Dependent Phosphorylation Regulates Intrinsic Ca2+ Cycling in Permeabilized SANC
To verify direct effects of CaMKII-dependent phosphorylation on SR Ca2+ cycling in rabbit SANC, we permeabilized SANC with saponin to avoid possible concurrent effects on ionic channels. Robust, spontaneous LCRs were present in permeabilized rabbit SANC (Fig. 3). Consistent with results in intact SANC (Figs. 1 and and2),2), inhibition of CaMKII activity in permeabilized SANC by AIP or KN-93, but not KN-92, markedly suppressed SR Ca2+ cycling and reduced the LCR size and number (Fig. 3).
Inhibition of basal CaMKII activity suppressed LCRs in permeabilized SANC. A and B: confocal line-scan images of a representative saponin-permeabilized SANC before and following exposure to 10 μmol/l AIP (A) or 6 μmol/l KN-93 (B). C and D: average changes in the LCR size (C) or number (D) produced by 2-min superfusion with AIP (n = 6), KN-93 (n = 6), or KN-92 (n = 4) and expressed as (%control), considering LCR size or number before drug application as 100%. *P < 0.05, one-way ANOVA, Newman-Keuls multiple comparison test.
To delineate mechanisms underlying CaMKII-mediated modulation of SR Ca2+ cycling in permeabilized SANC, we measured the SR Ca2+ content by application of rapid pulse of caffeine directly on the cell. Both AIP and KN-93, but not KN-92, significantly decreased the SR Ca2+ content in rabbit SANC (Fig. 4), which might be attributable to the decline of Ca2+ pumping into SR by SERCA caused by decrease of PLB phosphorylation at CaMKII-dependent the Thr17 site.
Suppression of basal CaMKII activity decreases the SR Ca2+ content in permeabilized SANC. A–D: effect of a rapid application of 20 mmol/l caffeine on representative permeabilized rabbit SANC in the absence (A) or presence of 10 μmol/l AIP (B) or 6 μmol/l KN-92 (C) or 6 μmol/l KN-93 (D). E: average effects of AIP, KN-93, or KN-92 on the initial rapid component of the caffeine-induced SR Ca2+ release: control group (n = 35); 2-min superfusion with: AIP (n = 10), KN-93 (n = 9), or KN-92 (n = 8). *P < 0.05, one-way ANOVA, Newman-Keuls multiple comparison test.
SANC Have High Basal Level of Activated CaMKII and CaMKII-Dependent Protein Phosphorylation
The necessity of basal CaMKII activation for both spontaneous firing of intact SANC and intrinsic SR Ca2+ cycling in permeabilized SANC suggests a high basal level of activated (autophosphorylated) CaMKII in cardiac pacemaker cells. To verify this hypothesis we compared basal levels of autophosphorylated at the Thr286/287 site and total CaMKII in SANC and VM. The total CaMKII was comparable in both cell types, while the ratio of phosphorylated (activated) CaMKII to total CaMKII in SANC by approximately twofold surpassed that in VM, confirming high basal level of activated CaMKII in SANC (Fig. 5A). Consistent with these results, the basal level of PLB phosphorylation at the CaMKII-dependent Thr17 site was also markedly elevated in intact rabbit SANC, compared with that in VM, i.e., the ratio of phosphorylated to total PLB in SANC exceeded that in VM by approximately threefold (Fig. 5B). Inhibition of CaMKII activity by KN-93, but not KN-92, markedly decreased phosphorylation of PLB at the Thr17 site in SANC (Fig. 5C). Since CaMKII is activated by intracellular [Ca2+]i, we used Ca2+ chelator BAPTA-AM (40) to decrease intracellular [Ca2+]i and prevent CaMKII activation to define if this would reduce PLB phosphorylation at the Thr17 site. Indeed, buffering of intracellular Ca2+ by BAPTA-AM substantially reduced PLB phosphorylation at the Thr17 site in intact SANC (Fig. 5C), confirming that phosphorylation of PLB at this site was regulated by Ca2+.
Intact rabbit SANC have high basal levels of phosphorylated CaMKII and phospholamban (PLB) phosphorylated at CaMKII-dependent Thr17 site. A, top: representative Western blots of activated (autophosphorylated at Thr286/287) CaMKII (P-CaMKII) and total CaMKII in SANC and ventricular myocytes (VM). A, bottom: average values of autophosphorylated CaMKII normalized to total CaMKII (n = 7). B, top: representative Western blots of PLB phosphorylated at Thr17 site and total PLB in SANC and VM. B, bottom: average values of phosphorylated PLB normalized to total PLB (n = 12). C, top: representative Western blots of phosphorylated PLB and total PLB at baseline and after treatment with 1 μmol/l KN-93 or 1 μmol/l KN-92. C, middle: typical Western blots of the PLB phosphorylated at Thr17 site before and following chelation of intracellular [Ca2+]i by 25 μmol/l BAPTA-AM. C, bottom: average values of phosphorylated PLB normalized to total PLB in basal conditions, after treatment with KN-93 (n = 3) or KN-92 (n = 3) or BAPTA-AM (n = 10); data are presented as %control. D, top and middle: representative Western blots of total PLB and PLB phosphorylated at PKA-dependent Ser16 site (top) or CaMKII-dependent Thr17 (middle) in response to inhibition of PKA by 15 μmol/l PKI; (bottom) average values of PLB phosphorylated at Ser16 site (n = 5) or Thr17 site (n = 4) and normalized to total PLB after treatment with 15 μmol/l PKI; data are presented as %control. *P < 0.05, one-way ANOVA, Newman-Keuls multiple comparison test.
Phosphorylation of PLB at the Thr17 site in VM can occur before or following PLB phosphorylation at Ser16 site, which depends on the balance of PKA, CaMKII, and phosphatase activities (31). Rabbit SANC have high basal level of PLB phosphorylation at PKA-dependent Ser16 site (45), but whether phosphorylation at Ser16 site is prerequisite for phosphorylation of PLB at the Thr17 site is unknown. To test this idea intact SANC were treated with the specific PKA inhibitor peptide PKI, which markedly suppressed phosphorylation of PLB at Ser16 site (Fig. 5D). However, PKI had no effect on phosphorylation of PLB at the Thr17 site, strongly suggesting that phosphorylation of PLB at the Thr17 site in SANC was not dependent on phosphorylation of PLB at Ser16 site (Fig. 5D).
Cardiac Pacemaker Cells Had High Basal Level of RyR Phosphorylation at Both Ser2815 and Ser2809 Sites
Phosphorylation of cardiac RyRs is an important modulatory mechanism of SR Ca2+ release (2, 6, 27, 35, 41, 49). There is a strong evidence that RyR phosphorylation increases sensitivity of RyRs to cytosolic Ca2+ (27, 41) and open probability of RyR (36, 49), leading to elevation of RyR Ca2+ release (2, 6, 27, 35, 36, 41, 49). In rabbit VM RyRs are phosphorylated by CaMKII at both Ser2815 and Ser2809 sites (2, 6, 27, 35, 41, 49). The former site is exclusively phosphorylated by CaMKII (6, 27, 49), while the latter is phosphorylated by both CaMKII and PKA (6, 27, 35). Up to now direct measurements of RyR phosphorylation at either Ser2815 or Ser2809 site in the cardiac pacemaker (either tissue or isolated cells) had not been implemented. The high level of basal CaMKII activation in rabbit SANC suggests that basal level of CaMKII-dependent RyR phosphorylation could also be augmented. To test this idea we compared basal phosphorylation of RyR at CaMKII-dependent Ser2815 site in the rabbit SA node and ventricle and discovered that ratio of P-Ser2815/total RyR in the SA node surpassed that in the ventricle by ~10-fold (Fig. 6).
High basal level of ryanodine receptors (RyRs) phosphorylation at CaMKII-dependent Ser2815 site in the rabbit SA node. A, top: representative Western blots of RyR phosphorylated at Ser2815 site and total RyR in SA node and ventricular tissues. A, bottom: average values of phosphorylated RyR normalized to total RyR in SA node and ventricular tissues (n = 10 rabbits) and expressed as percentage, assuming that ratio P-RyR/total RyR in ventricular tissue is 100%. B, top: representative Western blots of RyR phosphorylated at Ser2815 site and total RyR in SA node tissue at baseline and in response to CaMKII-inhibition by 1 μmol/l KN-93 (n = 3). B, bottom: average values of phosphorylated RyR normalized to total RyR in basal conditions (n = 3) and after treatment with 1 μmol/l KN-93 (n = 3); data are presented as %control. *P < 0.05, by t-test.
Double immunostaining of phosphorylated and total RyR demonstrated high phosphorylation of RyR at Ser2809 site under basal conditions in isolated intact rabbit SANC compared with VM (Fig. 7). Inhibition of either PKA-dependent phosphorylation by PKI or CaMKII-dependent phosphorylation by KN-93 markedly suppressed RyR phosphorylation at Ser2809 site, strongly suggesting that this site in SANC, similar to VM, is phosphorylated by both protein kinases (Fig. 7H).
High basal level of RyR phosphorylation at Ser2809 site in rabbit SANC is mediated by both PKA- and CaMKII-dependent phosphorylation. Representative confocal images of SANC (A–C) and VM immunolabeled for total RyR (red) and RyR phosphorylated at Ser2809 site (P-RyR, green) (E). D and F: show staining with secondary antibodies and represent negative controls in SANC and VM, respectively. G: relative amount of P-RyR/total RyR in SANC (n = 49) is shown as %control, considering that the ratio of P-RyR/total RyR in VM (n = 9) is equal to 100%. H: relative changes in the P-RyR at Ser2809 normalized to total RyR in SANC exposed to KN-93, KN-92, or PKI (n = 36–62 cells in each group). *P < 0.05.
CaMKII-Dependent Phosphorylation Regulates the LCR Period and Spontaneous SANC Cycle Length through Modulation of the SR Ca2+ Refilling Kinetics
We had established previously that SR Ca2+ refilling kinetics following AP-induced Ca2+ release is a determinant of the LCR period in SANC and that the time to 90% decay of AP-induced Ca2+ transient (T-90) could be used as a convenient estimate of the SR Ca2+ refilling rate (47). Here we studied whether SR Ca2+ refilling kinetics would provide a functional link between changes in CaMKII-dependent protein phosphorylation and SR Ca2+ cycling in intact rabbit SANC. When CaMKII activity was inhibited by either AIP or KN-93, the histograms of T-90 were shifted to longer times (Fig. 8, A and B), and average values of T-90 were increased from 256.3 ± 14.3 to 350.7 ± 38.6 ms and from 238.7 ± 14.3 to 366.9 ± 28.8 ms, respectively. The prolongation of T-90 produced by CaMKII-inhibition was consistent with a decrease in PLB phosphorylation at the Thr17 site (Fig. 5) and resultant decline in the SR Ca2+ pumping rate. The increase in T-90 produced by CaMKII inhibition was paralleled by an increase in the LCR period, and changes between these two parameters were highly correlated R2 = 0.85 (Fig. 8D). Note, that KN-92 did not shift the histogram of T-90 (Fig. 8C) and did not increase the LCR period (Fig. 8D).
CaMKII-dependent phosphorylation regulates the LCR period via modulation of SR Ca2+ refilling kinetics linked to PLB phosphorylation at Thr17 site. A, B, and C: histograms of the decay of AP-induced Ca2+ transient, indexed by T-90, before and during superfusion with AIP (A), KN-93 (B), or KN-92 (C). D: AIP or KN-93, but not KN-92, produced prolongation of T-90, which was paralleled by an increase in the LCR period (each symbol represents an individual SANC). E: representative Western blots of PLB phosphorylated at Thr17 site and total PLB in SANC in the basal state and following β-AR stimulation with isoproterenol (ISO; 1 μmol/l) or PDE inhibition with IBMX (100 μmol/l) or milrinone (50 μmol/l). F: changes in PLB phosphorylation at Thr17 site produced by (ISO, n = 10), IBMX (n = 17), or milrinone (n = 4); KN-93 (n = 3) or KN-92 (n = 3) were highly correlated with changes in SR refilling times indexed by T-90 [for ISO (n = 5), IBMX (n = 5), milrinone (n = 6), KN-93 (n = 5), KN-92 (n = 4)].
It is well-known that activation of cAMP-mediated PKA-dependent phosphorylation stimulates CaMKII-dependent phosphorylation in VM (30), but whether the same link exists between PKA- and CaMKII-dependent phosphorylation in cardiac pacemaker cells is unknown. SANC have high level of cAMP and PKA-dependent phosphorylation in the basal state (45), and both are under tight control of PDEs (46). Inhibition of PDE activity by broad-spectrum PDE inhibitor (IBMX) or PDE3 inhibitor (milrinone) markedly increases level of cAMP and cAMP-mediated PKA-dependent phosphorylation, indexed by PLB at Ser16 site (46).
We employed phosphorylation of PLB at the Thr17 site to estimate changes in CaMKII-dependent protein phosphorylation in intact rabbit SANC created by increase in cAMP-mediated PKA-dependent phosphorylation during β-AR stimulation or PDE inhibition. Both β-AR stimulation (ISO) and PDE inhibition (IBMX or milrinone) markedly increased PLB phosphorylation at the Thr17 site in SANC (Fig. 8, E and F), and there was a close correlation (R2 = 0.92) between changes in PLB phosphorylation at the Thr17 site and changes in SR refilling times indexed by T-90 (Fig. 8F). Note, that KN-92 did not change either PLB phosphorylation at the Thr17 site (Fig. 5C) or the SR refilling time (Fig. 8, C and F).
The effects of CaMKII-dependent phosphorylation on the SR refilling times, T-90, were paralleled by changes in LCR periods, i.e., an acceleration of the SR Ca2+ refilling times in response to β-AR stimulation or PDE inhibition reduced LCR periods, whereas CaMKII inhibition by AIP or KN-93 prolonged SR Ca2+ refilling times and increased LCR periods (Fig. 9, A and B). Changes in LCR periods created by β-AR stimulation, PDE inhibition, AIP, or KN-93 were highly correlated (R2 = 0.96) with changes in the spontaneous SANC cycle lengths (Fig. 9, A and C).
Graded changes in the SR Ca2+ refilling times produced proportional changes in LCR periods that were highly correlated with concurrent changes in spontaneous SANC cycle lengths. A: confocal linescan images of representative spontaneously beating SANC in control conditions, during β-AR stimulation (0.1 μmol/l ISO), or PDE inhibition with IBMX (100 μmol/l) or CaMKII inhibition with AIP (10 μmol/l). B: average changes in SR Ca2+ refilling times, indexed by decays of AP-induced Ca2+ transients (T-90), produced by β-AR stimulation (ISO, n = 5), PDE inhibition with milrinone (n = 6) or IBMX (n = 5), and CaMKII inhibition with AIP (n = 4) or 0.3 μmol/l KN-93 (n = 5) or 0.3 μmol/l KN-92 (n = 4) were paralleled by concomitant changes in average LCR periods. C: changes in LCR periods were tightly linked to concurrent changes in spontaneous SANC cycle lengths; each symbol represents an individual SANC. LCRs are shown by arrowheads.
DISCUSSION
Spontaneous beating of cardiac pacemaker cells is produced by spontaneous DD, which is generated by orchestrated work of sarcolemmal ionic channels, so called “Membrane clock,” and rhythmic SR-generated local subsarcolemmal Ca2+ releases, so called “Ca2+ clock” (22, 52). Although a critical role of LCRs for spontaneous SANC firing had been verified in multiple studies and in different species, including humans (4, 5, 34, 44, 50), novel mechanisms that regulate generation of LCR in cardiac pacemaker cells continue to emerge. This report discovered that, compared with VM, intact rabbit SANC had high basal level of activated CaMKII, i.e., ratio of autophosphorylated CaMKII to total CaMKII in cardiac pacemaker cells surpassed that in VM by approximately twofold. As a result, rabbit SANC had high basal level of CaMKII-dependent phosphorylation of Ca2+ cycling proteins PLB and RyR, i.e., PLB phosphorylation at the CaMKII-dependent Thr17 site was approximately threefold greater in intact rabbit SANC than in VM (Fig. 5); basal phosphorylation of RyR at CaMKII-dependent Ser2815 site in the SA node by ~10-fold exceeded that in ventricle (Fig. 6); and basal phosphorylation of RyR at Ser2809 site, which is phosphorylated by both CaMKII and PKA (27, 35), was approximately twofold higher in intact SANC than in VM (Fig. 7). Inhibition of CaMKII-dependent phosphorylation by AIP or KN-93 suppressed basal phosphorylation of PLB and RyR (Figs. 5, ,6,6, and and7),7), leading to a decrease in the LCR size and number per each spontaneous cycle and an increase in the LCR period, which was paralleled by an increase in the spontaneous SANC cycle length (Figs. 1, ,2,2, and and9).9). These data clearly demonstrated that generation of LCRs and normal spontaneous beating of intact rabbit SANC were critically dependent on high level of CaMKII-dependent phosphorylation in the basal state.
LCRs preferably propagate as wavelets beneath sarcolemma of rabbit SANC (5, 44), suggesting that subsarcolemmal Ca2+ diffusion is supported by specific organization of RyR and surface membrane. Indeed, the highest intensity of RyR immunolabeling in rabbit SANC, which exceeded that in VM by almost threefold, was found beneath sarcolemma of SANC (25). We have previously reported that activated CaMKII was located beneath sarcolemma, while total CaMKII was uniformly distributed in rabbit SANC (43). CaMKII is associated with RyR in cardiac myocytes (9, 27, 49), and high basal CaMKII-dependent phosphorylation of RyR in rabbit SANC could be due, at least in part, to similar distributions of RyR and active CaMKII within primary pacemaker cells (43). Endogenous CaMKIIδ directly modulates RyR2 activity in rabbit hearts (9) increasing Ca2+ sensitivity and open probability of RyR (27, 35, 36, 41, 49). The increase in open probability would increase the LCR size as well as chances that RyRs would open with a shorter latency and thus reduce the LCR period. As a result, an enlarged inward Na+/Ca2+ exchange current would be activated at earlier time, hastening DD rate and increasing the spontaneous SANC beating rate. To sustain long-lasting elevated basal RyR Ca2+ release, cardiac pacemaker cells need a rapid replenishment of SR Ca2+ provided by SERCA. High basal CaMKII-dependent phosphorylation of PLB would promote Ca2+ pumping into SR by SERCA and support robust, local RyR Ca2+ releases in SANC. A concurrent increase in basal CaMKII-dependent phosphorylation of RyRs and PLB in SANC could balance each other, maintaining the same SR Ca2+ content, i.e., while the former would increase the SR Ca2+ leak through RyRs, the latter would raise the SR Ca2+ uptake to support enlarged LCRs. Indeed, at the same physiological [Ca2+]c permeabilized rabbit SANC were able to release more Ca2+ from SR, compared with VM, despite similar SR Ca2+ content in both cell types (37). The effects of CaMKII-dependent phosphorylation were likely mediated by phosphorylation of multiple proteins that regulate cell Ca2+ balance and were beyond scope of the present study, e.g., L-type Ca2+ channels and probably others. Indeed, L-type Ca2+ channels are also targets of CaMKII-dependent regulation in rabbit SANC (43), and Ca2+ supply provided by these channels is an important part of “Ca2+ clock” (22). Our prior study in rabbit SANC showed a marked decrease of basal ICa,L amplitude and delayed recovery from inactivation in response to AIP or KN-93 (43).
CaMKII is a downstream target of the β-AR pathway in VM that activates PKA-dependent phosphorylation leading to an increase in intracellular Ca2+ and activation of CaMKII-dependent phosphorylation, i.e., phosphorylation of PLB at the Thr17 site (7). Electrical pacing of rat VM, however, increases CaMKII-dependent PLB phosphorylation at the Thr17 site, which is independent of PKA-mediated phosphorylation of PLB (12, 48). Up to now an affiliation between PKA and CaMKII-dependent phosphorylation in intact rabbit SANC either in the basal state or during β-AR receptor stimulation remained unknown. Here we demonstrated that high basal CaMKII-dependent protein phosphorylation (reflected in phosphorylation of PLB at the Thr17 site) was, likely, independent of PKA-dependent phosphorylation since suppression of PKA-dependent phosphorylation did not affect basal level of CaMKII-dependent phosphorylation in intact rabbit SANC (Fig. 5). In contrast, boost of cAMP-mediated PKA-dependent protein phosphorylation produced by β-AR stimulation or PDE inhibition in intact SANC (45, 46), similar to VM, markedly increased CaMKII-dependent protein phosphorylation and specifically PLB phosphorylation at the Thr17 site (Fig. 8).
A functional link between gradations in CaMKII-dependent protein phosphorylation (indexed by PLB phosphorylation at the Thr17 site) the LCR period and spontaneous SANC cycle length was fulfilled via the SR Ca2+ refilling time (T-90). Indeed, inhibition of CaMKII-dependent phosphorylation delayed SR Ca2+ refilling time, prolonged the LCR period (Fig. 8D), and increased the spontaneous SANC cycle length. In contrast, during β-AR stimulation or PDE inhibition, increased PLB phosphorylation at Thr17 stimulated SR Ca2+ uptake, leading to decrease in T-90 (Fig. 8, E and F), which was paralleled by a decrease of the LCR period and concurrent decrease in the spontaneous SANC cycle length (Fig. 9). The tight correlation between CaMKII-dependent effects on the LCR period and changes in the spontaneous cycle length (Fig. 9) indicated a critical role of CaMKII-dependent phosphorylation in the modulation of basal and reserved cardiac pacemaker function. It is well accepted that CaMKII-dependent activation in VM increases arrhythmogenic SR Ca2+ leak, which could be harmful. In contrast, robust LCRs in intact rabbit SANC, called “Ca2+ clock,” could not exist without efficient SR Ca2+ cycling in the basal state, which definitely requires high basal CaMKII-dependent protein phosphorylation.
Genetic manipulations of mice have enabled valuable insights in the mechanisms of CaMKII-dependent regulation of the mouse heart beating rates. Recent studies in transgenic mice (11, 50, 51, 53), except for one (53), did not find any differences in the basal heart beating rate between wild-type mice and mice with either conditional inhibition of CaMKII (AC3-I mice) (11, 50) or CaMKIIδ knockout (KO) mice (51), suggesting that basal CaMKII-dependent phosphorylation was not essential for basal pacemaker function in the mouse heart. This conclusion was consistent with a lack of CaMKII-dependent phosphorylation in mouse SANC in the basal state (50); as a result, normal cardiac pacemaker function was preserved in transgenic mice with conditional CaMKII inhibition (50, 51). Compared with control mice, cardiac myocytes isolated from either AC3-I mice or CaMKIIδ KO mice had a significant reduction of the SR Ca2+ content and a marked increase of the L-type Ca2+ current amplitude (51, 53), and the latter could not be explained by CaMKII inhibition. Additional analysis linked amplification of ICa,L in mouse cardiac myocytes to either a compensatory increase of basal PKA-mediated phosphorylation of L-type Ca2+ channels in AC3-I mice (53) or to a marked increase of Cav1.2 protein level in CaMKIIδ KO mice (51). Furthermore, several recent reports identified a number of potential shortcomings related to a genetic approach in the mouse heart as a tool to study cardiac regulatory mechanisms (8, 32). For example, a deletion of the PLB gene in mice markedly augments cardiac contractility, protecting mouse heart from the failure (38); in contrast, human families null for PLB develop lethal cardiomyopathy at a relatively young age (13).
The mouse SA node generates an extremely high beating rate 600 beats/min (~10-fold or 4-fold higher than that of the human or rabbit heart, respectively). This high beating rate of mouse SANC cannot be achieved even during high-frequency pacing of either isolated rabbit SANC (1) or isolated rabbit SA node (18). Both SANC and SA nodes of larger mammals, e.g., rabbit, are protected against high-frequency beating rates by specific defense mechanisms (which are certainly absent in the mouse cardiac pacemaker) to provide protection against tachycardias, e.g., atrial fibrillation (19). While the differences in cardiac pacemaker mechanisms between mouse and rabbit heart require further study, it is absolutely clear that such differences exist. Thus conclusions regarding cardiac pacemaker mechanisms, made from data obtained in transgenic mice, ought to be drawn with caution and relevance of these mechanisms to other species, especially humans, should be verified by employing different species and diverse experimental technique (8, 32).
The rabbit heart more resembles the human heart because of the larger size and slower beating rates. Similar to human hearts, the ''slow'' β-myosin heavy chain isoform is the predominant isoform expressed in the rabbit and human hearts, whereas α-myosin heavy chain is the predominant isoform expressed in the mouse heart (39). Compared with rabbit, mouse SANC have higher density of Na+ channels and sodium current is required for murine cardiac pacemaking (24, 28). In contrast, primary pacemaker cells of larger mammals, e.g., rabbits or dogs, rely more on L-type Ca2+ current to generate an AP upstroke, and Na+ channels are not essential for normal automaticity of adult SANC (29, 33). Ca2+ handling is also different in mouse cardiac myocytes where the SR is ~10-fold more dominant over the Na+-Ca2+ exchanger and ~90% of Ca2+ is removed from cytosol by SERCA, whereas both rabbit and human cardiac myocytes rely on the Na+-Ca2+ exchange current to remove Ca2+ from cytosol with a lesser input from SERCA (2, 3). Indeed, both rabbit and human SANC had similar amplitude of INCX, compared with the net diastolic current (42). With multiple distinctions both in ionic channels and Ca2+ handling, it is likely that modulation of Ca2+ cycling by CaMKII-dependent phosphorylation is different in mouse and rabbit SANC. This question, however, was beyond the scope of this study and will need further investigation.
Study Limitations
1) To evaluate a role of CaMKII-dependent protein phosphorylation for basal pacemaker function of rabbit SANC, we used pharmacological approach and employed available CaMKII inhibitors a specific CaMKII inhibitory peptide AIP, KN-93 and its inactive analog, KN-92. Some concerns had been raised previously regarding possible nonspecific effects of KN-93 on ionic channels (10, 23). However, nonspecific effects are often created by the extended time of exposure (10) or high concentration of the drug (10, 23). To avoid these issues we used short application times (≤5 min) and low KN-93 concentration for treatment of intact SANC. The specificity of low KN-93 concentrations had been already tested in our previous study (43), which demonstrated similar suppression of spontaneous SANC firing by KN-93 (but not KN-92) and AIP, which is not known to produce nonspecific effects on ionic channels (27). In the present study specificity of CaMKII inhibitors was additionally verified employing saponin-permeabilized rabbit SANC. A lack of functional ionic channels in permeabilized SANC would remove possible nonspecific effects of KN-93. Both CaMKII inhibitors AIP and KN-93, but not KN-92, similarly decreased LCR parameters in permeabilized and intact rabbit SANC, confirming specificity of these CaMKII inhibitors in our experimental conditions.
2) Phosphorylation of RyR is an important modulatory mechanism of the SR Ca2+ release characteristics. Here we discovered that in isolated rabbit SANC RyR at Ser2808/2809 site were phosphorylated by both PKA and CaMKII (Fig. 7). Phosphorylation of this site in VM was studied by many groups with the majority of evidence pointing to Ser2808/2809 being a target for both PKA and CaMKII; however, this issue remains unresolved (6, 15, 35, 36). Recent report in double-knockout (DKO) mice lacking the two cardiac CaMKII genes δ and γ in cardiomyocytes showed preserved basal phosphorylation of RyR at Ser2808 site, suggesting that this site in VM could be phosphorylated by PKA alone (20). However, a thorough examination of kinases responsible for basal phosphorylation at Ser2808/2809 site in VM discovered that it was not caused by either PKA or CaMKII but by another unknown kinase (15). Thus, whether both PKA and CaMKII, PKA alone or an unknown kinase phosphorylated RyR at Ser2808/2809 site in VM in the basal state is still uncertain and requires further investigation beyond the scope of the present report.
In summary, the present study showed, for the first time that, compared with VM, intact rabbit SANC had high basal levels of both activated (autophosphorylated) CaMKII and basal CaMKII-dependent protein phosphorylation, which were critically important for generation of basal spontaneous LCRs and normal spontaneous firing of rabbit SANC. Gradations in CaMKII-dependent phosphorylation, as indexed by degree of PLB phosphorylation at the Thr17 site, were closely linked to gradations in the SR Ca2+ refilling times, the LCR periods, and spontaneous cycle lengths across the wide physiological range of SANC beating rates.
GRANTS
This work was supported by the Intramural Research Program of the National Institute on Aging.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Y.L., S.S., D.R.R., D.Y., and T.M.V. performed experiments; Y.L., S.S., D.R.R., D.Y., H.A.S., and T.M.V. analyzed data; Y.L., S.S., H.A.S., E.G.L., and T.M.V. interpreted results of experiments; Y.L., S.S., D.R.R., D.Y., and T.M.V. prepared figures; Y.L., S.S., and T.M.V. drafted manuscript; Y.L., S.S., D.R.R., D.Y., H.A.S., E.G.L., and T.M.V. edited and revised manuscript; Y.L., S.S., D.R.R., D.Y., H.A.S., E.G.L., and T.M.V. approved final version of manuscript; E.G.L. and T.M.V. conception and design of research.
ACKNOWLEDGMENTS
We are deeply grateful to Dr. Su Wang, Dr. Magdalena Juhaszova, Dr. Alexey E. Lyashkov, and Bruce Ziman for excellent technical support.
Present address or Y. Li: Department of Internal Medicine, Carver College of Medicine, University of Iowa, IA.
REFERENCES
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