LETTERS
FEBS Letters 343 (1994) 151-154
ELSEVIER
FEBS 13918
Simvastatin-sodium delays cell death of anoxic cardiomyocytes
inhibition of the Na’/Ca2’ exchanger
by
E.M. Lars Bastiaanse*, Douwe E. Atsma, Marinette M.C. Kuijpers, Arnoud Van Der Laarse
Department of Cardiology, University Hospital Leiden, PO Box 9600, 2300 RC Leiden, The Netherlands
Received 24 February 1994
Abstract
When incubated under anoxic conditions, cultured neonatal cardiomyocytes undergo cell necrosis. Simvastatin-sodium, the bioactive metabolite
of simvastatin (a potent serum cholesterol-lowering drug), delayed the anoxia-induced myocyte necrosis in a dose-dependent manner. This beneficial
effect of simvastatin-sodium could not be attributed to its cholesterol-lowering properties. We found that simvastatin-sodium, at concentrations of
20 and 50pM, attenuated the rise in intracellular Ca2’ concentration ([Ca2+]Jmeasured with Fura- in anoxic cardiomyocytes. In a test of sarcolemmal
Na’/Ca*’ exchange activity, simvastatin-sodium attenuated the rise of [Ca”], upon incubation in sodium-free buffer, which normally causes a reversal
of Na+/Ca2+ exchange and cellular calcium overload. The inhibitory action of simvastatin-sodium on the sarcolemmal Na+/Ca*+ exchanger could well
explain the cardioprotective effect of the drug on myocytes subjected to anoxia.
Kev words:
Simvastatin-sodium;
Na’/Ca*‘-exchanger;
Intracellular
1. Introduction
Simvastatin-sodium,
the bioactive metabolite
of
simvastatin, is an inhibitor of the rate-determining enzyme in the biosynthesis of cholesterol, /I-hydroxy-pmethylglutaryl-coenzyme A (HMG-CoA) reductase, and
is used as an anti-hyperlipidemic drug in humans [1,2].
In addition, inhibitors of HMG-CoA reductase have
been used experimentally to lower cellular cholesterol
content of cultured cells [3].
Earlier, we reported that modulation of cellular
cholesterol content of cultured cardiomyocytes using liposomes altered the tolerance to anoxia [4,5]. Recently,
we employed simvastatin-sodium in an attempt to diminish cellular cholesterol content. We found that simvastatin-sodium had a protective effect on cardiomyocytes
during anoxia that could not be attributed to its cholesterol-lowering capabilities. The simvastatin-sodium-induced delay of cell death during anoxia was associated
with an attenuation of the rise of the intracellular Ca*’
concentration in cardiomyocytes. To elucidate the mechanism responsible for the beneficial effects of simvastatin-sodium
on anoxic myocytes, we investigated
whether simvastatin-sodium
affects the Na+/Ca*+-exchanger. The Na’/Ca*’ exchanger is considered to be
responsible, at least in part, for intracellular Ca*+ overload during anoxia [6,7].
*Corresponding
author. Fax: (31) (71) 226 567.
ICa*‘l; Anoxia;
Cell death
2. Material and methods
2.1. Neonatal cardiomyocytes
Two-day-old rats were anesthetized with diethylether. Their hearts
were excised and the ventricles dissociated using collagenase as the
dissociating enzyme [8]. Cultures were grown for 3 days in a culture
medium containing Ham’s F-10 (Flow) with 10% fetal calf serum
(Flow) and 10% horse serum (Flow) on plastic Petri dishes (diameter
30 mm, primaria coated; Falcon) and maintained at 37°C in a humidified incubator with an atmosphere of 95% air and 5% CO*. Culture
medium was refreshed after 4 h and after 48 h. At the third day after
plating the cells had formed a monolayer and were used for the experiments. Intracellular calcium ion concentration ([Ca*+],)was measured
in cultures grown on glass coverslips (10 x 32 mm) coated with laminin
(G&co) in a plastic Petri dish.
2.2. Simvastatin-sodium
The bioactive metabolite of simvastatin (Zocor; Merck, Sharp &
Dohme) was formed in vitro by heating (60°C) a 1 mM solution of
simvastatin in 0.1 N NaOH for 1 h [9]. The resulting 1 mM solution
of simvastatin-sodium was brought to pH 7.5 with 12 N HCl.
2.3. Anoxic incubation
Cultures were preincubated in a balanced salt solution (BSS) which
contained (in mM): NaCl 140, KC1 4.0, CaCl, 2.5, MgSO, 1.2, KH,PO,
0.44, Na,HPO, 0.34, NaHCO, 21; sodium pyruvate 5; pH 7.4, for 1 h
(37”C, 95% sir/5% COJ. Then the medium was poured off and replaced
by BSS equilibrated for at least 1 h with 95% NJ5% CO1 (resulting in
a pOz < 5 mmHg). Eight dishes at a time were placed in an anoxic
incubation chamber with a continuous gas flow of 95% N,/5% CO? at
37°C.
2.4. Cell death
At indicated time points during the anoxic incubations, aliquots of
the medium were taken to measure lactate dehydrogenase (LDH) activity released by the cells. After anoxic incubation the medium was removed, and the cardiomyocytes were taken up in 1.0 ml ice-cold Tris
buffer (10 mM, pH 7.4). The cells were homogenized in a glass PotterElvehjem homogenizer and then sonicated for 1 min at 30 W (Branson
Sonic Power Co.). Medium and cellular homogenate were analyzed for
LDH activity using a spectrophotometric assay (Boehringer 543047).
0014-5793/94/$7.00 0 1994 Federation of European Biochemical Societies. All rights reserved.
SSDZ 0014-5793(94)00303-D
E. M. L. Bastiaanse et al. IFEBS
152
Letters 343 (1994)
151-154
The release of LDH during metabolic inhibition was expressed as the
percentage of total (pre-anoxic) cellular LDH.
70
2.5. [CcIcIi measurement
Confluent myocyte cultures grown on glass coverslips were rinsed
with a HEPES-buffered salt solution (HBSS) containing (in mM): NaCl
125, KC1 5.0, CaCl, 2.5, MgS04 1, KH,PO, 1, NaHCO, 10, HEPES
10, glucose 5, probenecid 2.5; pH 7.4. The cells were loaded in HBSS
containing 2 ,uM Fura-2/AM (Molecular Probes) for 60 min at 37°C
[IO]. Subsequently, the coverslips with the cells were washed twice in
HBSS and fitted in a cuvette in a spectrofluorometer (Perkin-Elmer
LS-3) equipped with a thermostated cuvette holder (37°C). The fluorescence of Fura-2-loaded cells was measured at 340 and 380 nm excitation
wavelengths and 490 nm emission wavelength. The ratio of the fluorescence intensities measured at excitation wavelengths 340 nm and
380 nm, &J&s,, provides an index for [Ca”], [1 11. As the numerous
problems concerning the calibration of Fura- have not been resolved,
we have chosen not to express the ratio values in nM Ca” concentration. Instead, the F&F,,, ratio values during the experiments were
expressed as a percentage increase compared to the control value at the
start of the experiment.
2.6. Na’lCrz’ exchanger
The activity of the Na’/Ca*’ exchanger was measured by incubating
the cardiomyocytes in Na’-free HBSS. In this medium, sodium salts
were replaced by equimolar quantities of choline chloride. Incubation
in Na’-fee medium causes reversal of the Na’/Ca2’ exchange activity
[6] leading to a rapid rise in [Ca”],. Using this procedure, the effect of
simvastatin-sodium or simvastatin on the Na’/Ca” exchanger was investigated.
2.7. Statistics
All data are presented as mean values + S.E.M. Student’s t-test was
used for statistical comparison, and corrected for multiple comparison
(Bonferroni method) when necessary. A P value less than 0.05 was
considered to indicate a significant difference.
3. Results and discussion
The time-courses of LDH release from cultured cardiomyocytes incubated with or without simvastatin-sodium (20 and 50 PM) under anoxic conditions are illustrated in Fig. 1. Simvastatin-sodium delayed LDL release, which is more pronounced with 50 PM than with
20 PM simvastatin-sodium. Table 1 shows that 2 ,uM
simvastatin-sodium did not alter the time to 50% LDH
release, as compared to controls, whereas 100 ,uM
simvastatin-sodium did not prolong the time to 50%
LDH release any further compared to 50 ,uM simvastatin-sodium.
Table 1
Prolongation (positive values) or shortening (negative values) of the
time to 50% LDH release from cardiomyocytes during anoxia by the
presence of simvastatin-sodium,
as compared to control cells
GtLDH,,)
Concentration
2
20
50
100
simvastatin-sodium
(uM)
dLDH,,,
-4 f
30 +
53 f
44 f
(min)
3.9
12*
6.1*
8.5*
Mean f S.E.M., n = 5; *P c 0.05 compared to controls (no simvastatin-sodium).
60
50
40
30
20
10
J
0
0
60
90
120
150
180
210
240
Time (min)
Fig. 1. Timecourses of lactate dehydrogenase (LDH) release from
cultured neonatal cardiomyocytes incubated under anoxic conditions.
The activity of LDH release is expressed as a percentage of the LDH
activities present in the cells at t = 0. l, without simvastatin-sodium; +,
with 20 ,uM simvastatin-sodium; and n, with 50 PM simvastatin-sodium. Each point represents themean f S.E.M. (n = 5). *P c 0.05 compared to anoxia without simvastatin-sodium.
Simvastatin-sodium could exert its protective effect by
interfering with a variety of factors and pathways involved in the mechanism of cell death during anoxia [I 21.
Calcium overload is considered to play an important role
in the development of irreversible cell damage during
anoxia [6,7]. Calcium overload can lead to cell death
through several pathways, including activation of calcium-dependent phospholipases, proteases and endonucleases [ 131. Therefore, we have monitored the [Ca”], in
cultured myocytes during normoxia and anoxia. Under
normoxic conditions 20 and 50 ,uM simvastatin-sodium
increased the F3JF380ratio of Fura- (Fig. 2), which
0
OPM
20 pM
Concentration
50 pM
SN
values of Fura- measured in normoxic
Fig. 2. Increase in F,JF,,,
cardiomyocytes in the presence of 20 or 50 PM simvastatin-sodium
(SN). Ratio values are expressed relative to the ratio value measured
before addition of 0, 20 or 50 ,uM simvastatin-sodium. Indicated are
mean values f S.E.M. (n = 4). *P < 0.05 compared to controls (no
simvastatin-sodium).
153
E.M.L. Bastiaanse et al. IFEBS Letters 343 (1994) 151-154
300
s
g
2
L
-t
,
T
2.50
1
200
G
.r(
150
-
100
-
50
-
%
2
5
$2
.H
R
i
0-
0
45
30
Time
60
90
(min)
Fig. 3. Increase in FJF380 values of Fura- measured in anoxic cardiomyocytes in the absence (open bars) and presence of 50pM simvastinsodium (solid bars). Ratio values are expressed relative to the ratio
value measured at t = 0, that is, at the start of anoxia. Indicated are
mean values f S.E.M. (n = 4). *P < 0.05 compared to anoxia without
simvastatin-sodium.
indicates that simvastatin-sodium causes an increase in
[Ca2+]i.
In another series of experiments, we assessed the effect
of 50 ,uM simvastatin-sodium on [Ca*‘]; during anoxia.
As shown in Fig. 3, the F3dF38o ratio of Fura- was
increased by 60% after 45 min of anoxia without simvastatin-sodium, whereas in the presence of 50 ,uM simvastatin-sodium no significant increase in the F34,jF380ratio
was seen after 45 min of anoxia. After 90 min of anoxia,
the F&F38o ratio had increased by 248% without simvastatin-sodium, and by 162% in cultures incubated with 50
PM simvastatin-sodium. Unfortunately, we could not
measure reliable fluorescence values of Fura-loaded cells
at time points beyond 90 min of anoxia due to detachment of the myocytes from the coverslips. However, it is
clear from Fig. 3 that simvastatin-sodium attenuated the
increase in [Ca2+li during anoxia.
Recently, Ziegelstein et al. [6] showed that the antioxidant, dimethylthiourea (DMTU), increased [Ca*‘]i in
normoxic myocytes but attenuated the rise in [Ca2’li during hypoxia/reoxygenation.
In this respect DMTU has
effects quite similar to those of simvastatin-sodium presented in this study. Ziegelstein et al. [6] showed that
DMTU inhibits the Na’/Ca*’ exchanger in the sarcolemma of myocytes. Under normal conditions the
Na’/Ca2’ exchanger pumps one Ca2’ ion out of the cell
in exchange for three Na’ ions, driven by the transmembrane Na’ gradient and by the membrane potential [14].
During anoxia, membrane depolarization and an increase in intracellular Na+ concentration favors a reversal of the direction of the Na’/Ca2’ exchanger [7]. Thus,
during normoxia, an inhibitor of the Na’/Ca2’ exchanger will increase [Ca’+]i, whereas during anoxia, inhibition of the Na+/Ca” exchange will attenuate the rise
in [Ca2+]i.To determine whether a compound is capable
of inhibiting the Na’/Ca*+ exchanger, Ca” entry through
the exchanger can be challenged by Na’-free incubation
while the determination of the F3.JF380 of Fura- reflects
[Ca2+li. Upon Na’-free incubation of the cardiomyocytes, the F34dF38oratio rose immediately, reached its
maximum value within 2 min, and remained elevated for
at least 15 min. The effect of simvastatin-sodium on the
rise of F34dF380induced by Na+-free incubation is shown
in Fig. 4. 20 and 50 ,uM simvastatin-sodium attenuated
the rise in the F3JFsEoratio to 30 f 5% and 25 & 8% of
the values in untreated cells, respectively. These findings
indicate that simvastatin-sodium inhibits the Na’/Ca2’
exchanger, which could explain the attenuation by
simvastatin-sodium of the anoxia-induced rise in [Ca*‘]i,
thereby delaying cell death.
Due to the short incubation times used in our experiments (C 1 h), the effects of simvastatin-sodium can only
be explained by its acute direct action, as changes in
cellular cholesterol content by HMG-CoA reductase inhibition take much longer to develop. It is important to
note that the effects of simvastatin-sodium described in
the present study are observed at concentrations much
higher than that reached in plasma of hypercholesterolemit patients who take the anti-hyperlipidemic drug,
simvastatin [ 151.
In conclusion, we have shown that simvastatin-sodium
improves the tolerance of cultured cardiomyocytes to
anoxia. The protective effect of simvastatin-sodium is
mediated by its inhibitory effect on the Na’/Ca” exchanger, thereby attenuating the rise in [Ca’+]i during
anoxia.
120
I
100 -
80 60 40 -
20 OL
0 PM
20 pM
Concentration
50 FM
SN
Fig. 4. Increase in F3dF38,, values of Fura- measured in cardiomyocytes after 10 min of Na’-free incubation. The change in the F&F,,,
ratio observed in the absence of simvastatin-sodium (SN) is set at 100%
(control). The presence of 20 and 50 PM simvastatin-sodium reduced
the rise of the F34dF380ratio caused by Na’-free incubation by about
70%. Means f S.E.M., n = 5; *P < 0.05 compared to controls (no
simvastatin-sodium).
154
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