(Hypertension. 1997;30:461.)
© 1997 American Heart Association, Inc.
Articles |
Intracellular Ca2+ Handling and Vulnerability to Ventricular Fibrillation in Spontaneously Hypertensive Rats
From the Department of Medicine and Cardiovascular Research Institute, University of California San Francisco (C.E.Z., S.T.W., R.J.L., J.W.-C., W.W.P.), and Division of Cardiology and Cardiovascular Research Group, Departments of Internal Medicine and Research, University Hospital Basel (C.E.Z.), Switzerland.
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Abstract |
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Abstract Spontaneously hypertensive rats (SHR) with ventricular hypertrophy show an increased vulnerability for the development of potentially lethal ventricular arrhythmias such as ventricular fibrillation (VF). The mechanisms of this increased vulnerability are not fully understood but may be related to abnormal intracellular Ca2+ ([Ca2+]i) handling under stress conditions. We therefore investigated whether [Ca2+]i handling is abnormal in hypertrophied hearts of SHR without heart failure during stimulation stress, and if so whether abnormal [Ca2+]i handling is a determinant of the increased vulnerability to VF in SHR. [Ca2+]i was measured by indo-1 surface fluorescence in perfused hearts of 8- to 10-month-old control Wistar-Kyoto rats (WKY) and age-matched SHR. The state of [Ca2+]i handling was analyzed during three different forms of stimulation stress: rapid pacing, the long rest period after cessation of rapid pacing, and preprogrammed ventricular stimulation that was simultaneously used for the determination of VF threshold. The pulse number VF threshold was used as an index to determine vulnerability to VF and to analyze the relationship of [Ca2+]i handling to vulnerability. Although VF thresholds were lower in SHR than in WKY, we found that both demonstrated similar [Ca2+]i handling during stimulation stress. The extent and rate of [Ca2+]i accumulation during rapid pacing and those of the [Ca2+]i decline after cessation of pacing were similar in SHR and WKY. In addition, the relationship between [Ca2+]i and VF threshold was unaltered in SHR. Thus, we conclude that [Ca2+]i handling is normal in hypertrophied hearts of SHR without heart failure during stimulation stress and that it is not a determinant of the increased vulnerability to VF in SHR.
Key Words: hypertrophy • arrhythmia • intracellular calcium • homeostasis • hypertension, experimental • rats, inbred SHR • ventricular fibrillation
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Introduction |
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Spontaneously hypertensive rats have been widely used as a model of essential hypertension and cardiac hypertrophy. Comparable to clinical hypertension with hypertrophy in patients, SHR are vulnerable to develop potentially lethal ventricular arrhythmias such as VF.1 2 Even though the electrophysiological mechanisms of this high vulnerability are not fully understood, an important determinant may be tissue anisotropy that arises from myocardial remodeling after myocyte cell loss, which facilitates reentry circuits.2 In addition, the prolonged duration of repolarization, concomitant ionic alterations, and altered ion handling in hypertrophied myocardium may also increase vulnerability.3 Specifically, alterations in [Ca2+]i handling may impair the ability of hypertrophied myocytes to handle elevations in diastolic [Ca2+]i. Consequently, hypertrophied myocytes may develop delayed afterdepolarizations causing triggered activity during conditions that induce an increase in [Ca2+]i.3 4
Alterations in [Ca2+]i handling, however, are not seen in all SHR5 6 and may be a function of age7 and/or of progression of heart failure that develops in older animals. While SHR with heart failure (>18 months of age) presented lower diastolic [Ca2+]i and prolonged [Ca2+]i transients, age-matched SHR without heart failure presented diastolic [Ca2+]i and [Ca2+]i transients similar to those in normotensive WKY.5 6 Indeed, it is uncertain whether altered [Ca2+]i handling is an early abnormality in the development of heart failure or a result of heart failure per se. Nevertheless, vulnerability to VF in SHR increases at an early age,8 long before the transition to heart failure.5
This temporal divergence between [Ca2+]i handling and vulnerability has two possible explanations. Either altered [Ca2+]i handling is not a primary determinant of increased vulnerability or the study of diastolic [Ca2+]i and single [Ca2+]i transients is an incomplete analysis of [Ca2+]i handling. Altered [Ca2+]i handling might not always be detectable in individual [Ca2+]i transients during resting conditions. However, it may appear during stress conditions that induce an increase in [Ca2+]i such as rapid pacing or preprogrammed ventricular stimulation used to quantify vulnerability by VF threshold.9 During such stimulation stress, [Ca2+]i may increase more rapidly in hypertrophied myocytes and consequently increase the vulnerability to VF.
On the basis of these considerations, we designed this study to answer the following questions: Is [Ca2+]i handling abnormal in hypertrophied hearts of SHR without heart failure during stimulation stress, and if so is abnormal [Ca2+]i handling a determinant of the increased vulnerability to VF in SHR? To determine the state of [Ca2+]i handling during stimulation stress we analyzed [Ca2+]i transients in perfused hearts of WKY and age-matched SHR during rapid pacing, during the long rest interval after cessation of rapid pacing, and during preprogrammed ventricular stimulation. To avoid heart failure after the age of 18 months,5 6 all rats were studied at the age of 8 to 10 months when the cardiac pump function of SHR was still preserved.5 [Ca2+]i was assessed using fluorescence ratios of the [Ca2+]i indicator indo-1 at the interventricular septum. To investigate whether abnormal [Ca2+]i handling is a determinant of vulnerability to VF in these hearts, we took advantage of the pulse number VF threshold to quantify the vulnerability to VF.9 The pulse number VF threshold reflects the number of premature pulses that is tolerated without inducing VF and depends on both premature ventricular stimulation and [Ca2+]i.
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Methods |
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Choice of Model and Animals
The isolated perfused rat heart was used as a model for the simultaneous study of [Ca2+]i handling and the vulnerability to VF. Changes in [Ca2+]i can be measured accurately in these hearts at a high time resolution even during VF using indo-1 fluorescence.9 10 11 In addition, VF threshold as an index of vulnerability can be determined accurately in this preparation.9 12 13 14 15 Because isolated rat hearts often return spontaneously to sinus rhythm after electrically induced VF, this preparation permits repeated estimations of VF threshold12 without using defibrillation shocks, which may interfere with subsequent threshold determinations because of changed electrical resistance caused by tissue necrosis.
Male WKY 8 to 10 months of age and age-matched male SHR were held at the animal facilities of the University of California San Francisco in cages with free access to water and standard rat chow. The protocol was approved by the Animal Research Committee of the University of California San Francisco, and all animals were treated humanely in compliance with approved guidelines (NIH publication No. 85-23, revised in 1985).
Perfused Heart Model and Electrode Arrangement
After ether anesthesia, hearts were excised rapidly
through a midline sternotomy. The ascending aorta was cannulated within
30 seconds, and retrograde perfusion was performed at 36°C at a
perfusion pressure of 103 mm Hg. All hearts were perfused with a
filtered (0.65 µm filter), nonrecirculating modified
Krebs-Henseleit solution containing (in mmol/L) NaCl 117.0,
KCl 4.3, CaCl2 2.8 or 3.8, MgSO4 1.2,
NaHCO3 25.0, EDTA 0.25, and glucose 15.0 at a pH of
7.4.9 10 Free extracellular Ca2+
concentrations ([Ca2+]o) were estimated from
perfusate Ca2+ concentrations at 2.55
mmol/L and 3.55 mmol/L according to a
multiequilibrium calculation.16 As in other reports, VF
induction9 10 11 17 and detection of VF
thresholds9 were facilitated at this relatively high
[Ca2+]o. The perfusate was saturated
with a gas mixture of 95% O2 and 5% CO2,
reaching an oxygen tension of 500 to 600 mm Hg.18
Temperature was monitored by in-line thermometers and controlled with
specially designed heat exchangers.19
To record a bipolar epicardial electrocardiogram (ECG) a pair of electrodes (0.28 mm diameter, 2 to 3 mm contact length) was placed on the right atrial appendage and apex. Ventricular stimulation was carried out using a pair of platinum wire electrodes (0.28 mm diameter) connected to a pulse generator (Grass S9, Grass Instruments). The stimulation electrodes were implanted in the right ventricular free wall above the circular cut for the fiberoptic cable used for [Ca2+]i measurement (described later). To reduce VF threshold variability, the pacemaker electrodes were coated and held in position by polyethylene tubes to ensure constant implantation depth of 2 mm (uncoated electrode) and consistent distance of 5 mm from each other.9 Thereby, the spatial separation and anatomic position of electrodes on the heart were kept consistent, as recommended for reproducible VF threshold determination.20 After instrumentation, the hearts were paced at 4 Hz, and stimulation threshold was measured at 0.62±0.14 V (WKY) and 0.64±0.07 V (SHR), respectively, determined by slowly increasing the voltage until consistent pacing occurred.
Experimental Protocols
Stable baseline values of LVP and
[Ca2+]i were recorded 15 minutes after
instrumentation of the perfused rat hearts at 240 beats per minute.
Subsequently, [Ca2+]i handling was
analyzed during three different forms of
ventricular stimulation: rapid pacing, the long rest period
after cessation of rapid pacing, and preprogrammed
ventricular stimulation that was simultaneously
used for determination of VF threshold. VF threshold as an index of
vulnerability to VF was determined at two different
[Ca2+]i by increasing the perfusate
Ca2+ concentration from 2.8 to 3.8 mmol/L, thus
increasing [Ca2+]o from 2.55 to 3.55
mmol/L. All recordings were performed at a sampling rate
of 200 Hz on MacLab/8e (AD Instruments).
Analysis of [Ca2+]i
Handling
Three approaches were chosen to analyze
[Ca2+]i handling in intact perfused rat
hearts during stimulation stress. In the first approach, the
accumulation of [Ca2+]i during rapid pacing
was analyzed. This accumulation was analyzed by the
asymptote A (accumulation extent) and the time constant 
(accumulation rate) of the monoexponential increase of
systolic [Ca2+]i during 100
sequential extrasystolic beats at 150-ms intervals (6.67 Hz)
according to Equation 1:
 | (1) |
In the second approach, the nearly complete decline of
[Ca2+]i during the long rest period after
cessation of rapid pacing was analyzed. This decline was
characterized by parameters 
(decline extent) and ß
(decline rate) of Gompertz’s asymmetrical double-exponential function
modified according to Equation 2, adapted from Tamiya et
al,22
 | (2) |
0 and relate to upper asymptote and
amplitude of F400/F510. Parameter 
relates to the phasic
delay of decline onset, and ß principally relates to the rate of
[Ca2+]i decline.22 For curve
fitting according to equation 2, the data between the maximal and
minimal values of declining [Ca2+]i were
considered (
60 to 70 data points). While the first approach to
analyze [Ca2+]i handling reflected
the balance between delivering and removing processes of
Ca2+ to and from the cytosol, the second approach reflected
Ca2+ removal exclusively. In the third approach, the relationship between VF triggering [Ca2+]i and VF thresholds was analyzed to investigate [Ca2+]i handling during preprogrammed ventricular stimulation for VF threshold determination (described below). This aspect of [Ca2+]i handling reflected the delivery and removal of Ca2+ to and from the cytosol during sequential premature ventricular stimulation. Moreover, this analysis could detect potential differences between WKY and SHR in the relationship between [Ca2+]i and vulnerability to VF.
Determination of VF Threshold
VF threshold was determined according to the pulse number method
that induces VF depending on both premature ventricular
stimulation and [Ca2+]i.9 In
this determination, increasing numbers of sequential pulses at
increasing prematurity but constant intensity were used to scan the
vulnerable period. Heart rate was held constant at a 250-ms pulse
interval (4 Hz) by the stimulator generating 1-ms monophasic square
wave pulses at 500% of the stimulation threshold (thus, about 3 V).
After every eighth regular pulse, increasing numbers of sequential
constant intensity-pulses were generated at increasing prematurity
(10-ms increments starting at a 100-ms interval). The number of
premature pulses was increased in the sequence 1, 2, 3, 4, 8, 16, 32,
64, and 128, until VF occurred. The VF threshold was defined as the
mean number of premature pulses (after log2-transformation;
see "Evaluation and Statistical Analysis") of at least two
successive measurements that were reproducible to within one pulse
sequence (for example 8 and 16 pulses but not 8 and 32 pulses). VF was
detected as ECG waves of irregular morphology without corresponding
effective LVP12 for longer than 1 second. After each pulse
sequence, LVP and [Ca2+]i returned to
baseline before a new pulse sequence was introduced.
VF persisting longer than 30 seconds was terminated by a bolus of 0.25 mg lidocaine hydrochloride (Elkins-Sinn, Inc) followed by a 5-minute washout. Lidocaine has been shown to effectively terminate VF and reduce [Ca2+]i during VF in this preparation,23 and 5 minutes after administration of a lidocaine bolus reproducibility of VF thresholds was not affected.9
Measurement of LVP and Coronary Flow
LVP was measured by a polyethylene catheter inserted
through the left atrial appendage into the left
ventricle.9 The catheter was connected to a Statham P23 Db
pressure transducer (Gould). Left ventricular developed
pressure was defined as the difference between systolic and
diastolic values of LVP.
Coronary flow was measured by collecting the effluent from the right ventricular outflow tract in graduated cylinders. All flow measurements were performed at a pacing rate of 4 Hz.
Measurement of [Ca2+]i
Measurement of [Ca2+]i was performed
by surface fluorometry at the interventricular septum
containing the Ca2+-sensitive fluorescent dye
indo-1 as previously described.9 10 11 24 25 26 Briefly,
fluorescence excitation of hearts loaded with intracellular
indo-1 (Molecular Probes Inc), was provided by ultraviolet light at
365±10 nm generated by a mercury vapor lamp and directed through a
custom-made silica fiberoptic cable (Welch Allyn Inc), designed to
assess excitation and emission simultaneously. The emitted
fluorescence was filtered at 400±5 and 510±12.5 nm before
reaching photomultiplier tubes. Photomultiplier output at 400 nm
(Ca2+-bound indo-1) and 510 nm (free indo-1) and its ratio
F400/F510, an index of [Ca2+]i,
simultaneously with the LVP and the epicardial ECG were
recorded digitally at a sampling rate of 200 Hz on MacLab/8e.
To place the fiberoptic cable on the interventricular septum, the tip of the cable was inserted through a circular cut in the inferior apical portion of the right ventricular free wall and fixed firmly on the right ventricular side of the interventricular septum. The approach of the fiberoptic cable through a cut in this portion of the right ventricular free wall does not interfere with septal or left ventricular perfusion of rat hearts.26 27 The interventricular septum, functionally part of the left ventricle, was preferred to epicardial sites to avoid motion artifact and contribution of endothelial cells or vasculature.
Limitations with the surface fluorometry technique with indo-1 were
discussed previously.10 26 Most importantly,
compartmentalization of indo-1, especially into mitochondria, and
binding of the dye to cellular components may lead to interference with
cytosolic fluorescence and spatial
heterogeneity of fluorescence, limiting
quantitative measurements of cytosolic Ca2+. Therefore,
we9 10 11 24 25 26 and other investigators28 have
expressed [Ca2+]i as a percentage of the
baseline amplitude of F400/F510 instead of estimating an absolute
cytosolic Ca2+ concentration. Other possible limitations
(such as reduced fluorescence by ultraviolet bleaching or
indo-1 leakage, artifact from cardiac motion, and variation of
autofluorescence between hearts or over the experimental
period) are negligible in our experiments.26 Similarly,
fluorescence from the endothelium or the
vasculature, as observed at the left ventricular surface of
rabbit hearts,28 was shown to be negligible in this
preparation.29 Finally, the dissociation rate of indo-1
from [Ca2+]i was considered sufficiently fast
(=8 ms in vitro30 ) to allow analysis of the
[Ca2+]i transient decline (70
ms).
Evaluation and Statistical Analysis
Characterization of WKY and SHR was performed by body weight,
wet and dry heart weight, ratio of dry heart weight to body weight,
coronary flow, coronary resistance, and left
ventricular developed pressure. Coronary resistance
was estimated as perfusion pressure minus left ventricular
end-diastolic pressure divided by coronary
flow.31 [Ca2+]i transients under
resting conditions were characterized by the time to peak
fluorescence, the time to 90% decline from peak
fluorescence, and the sum of these two times6 at a
pacing rate of 4 Hz. Statistical analysis of all these
characteristics was performed by unpaired Student’s t
test.
[Ca2+]i handling in WKY and SHR was assessed
by the parameters characterizing the
[Ca2+]i increase during rapid pacing (A and
) and those characterizing the [Ca2+]i
decline during the long rest period after cessation of rapid pacing
( and ß). Statistical analysis of these
parameters was performed by unpaired Student’s
t test. Additionally, [Ca2+]i
handling was analyzed during preprogrammed
ventricular stimulation by correlating VF triggering
[Ca2+]i to VF thresholds (described
below).
As previously reported,9 VF thresholds were mathematically transformed before evaluation because of skewed distribution (P<.01, rejecting normality by Shapiro-Wilk test). Because the number of introduced pulses followed approximately 2n, individual thresholds were transformed by base-2 logarithm rendering normal distribution (P=.70, thus accepting normality). Transformed VF thresholds were statistically analyzed by unpaired Student’s t test (for comparison between WKY and SHR) as well as by paired Student’s t test (for comparison between 2.55 and 3.55 mmol/L [Ca2+]o). For better expression of actual numbers of pulses introduced, log2-transformed VF thresholds were reconverted by 2n and given as the median with interquartile distance (distance between 25th and 75th percentile). Testing for far outliers was performed according to the method of Velleman and Hoaglin,32 excluding one experiment per group in the analysis of VF thresholds.
The "VF triggering interval" was defined as the prematurity at which VF was induced and thus reflects the position of the vulnerable period in the cardiac cycle of the isolated rat heart.9 This interval was compared between WKY and SHR as described for transformed VF thresholds. The "VF triggering [Ca2+]i" was defined as the increase in [Ca2+]i induced by a sequence of preprogrammed ventricular stimulation and measured as the increase of systolic [Ca2+]i from the beginning of a stimulation sequence to the cardiac cycle preceding VF.9 This VF triggering [Ca2+]i was used to determine the relationship between the rise in [Ca2+]i and VF thresholds and was therefore plotted against corresponding log2-transformed VF thresholds and tested by linear regression.
All results are expressed as mean±SEM except reconverted VF thresholds of the pulse number method, which were expressed as median with interquartile distance (distance between the 25th and 75th percentiles). Curve fitting was performed by a nonlinear regression procedure, the Marquardt-Levenberg algorithm.33 34 For all statistical analyses, the null hypothesis was rejected at the 95% level, with P<.05 considered significant. The use of nonparametric tests for the analysis of [Ca2+]i was not required because F400/F510 ratios between 0.55 and 1.05 are linearly related to [Ca2+]i in the system used.35 During rapid pacing and preprogrammed ventricular stimulation, F400/F510 ratios increased but were seldom higher than 1.05 in the present study.
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Results |
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As indicators of hypertrophy, the ratio of dry heart weight to body weight, the coronary flow, and the estimated coronary resistance of SHR were significantly different from those of WKY (Table 1). Coronary flow and resistance values were similar to those in other studies of ventricular hypertrophy in which SHR and WKY were used36 37 or pressure-overload hypertrophy after aortic banding in rats31 under similar conditions. Because of the partial dissection of the right ventricular wall for the approach of the fiberoptic to the septum, a differentiation between left and right ventricular hypertrophy was not performed. Also because of this dissection, the hypertrophy indicators involving the dry or wet heart weight may be different from other reports. Left ventricular developed pressure of SHR was not significantly higher than that of WKY (P=.42). Furthermore, [Ca2+]i transients, assessed as fluorescence ratio F400/F510, were not prolonged in SHR under resting conditions. Specifically, neither the time to peak fluorescence (68±5 versus 62±2 ms, P=.34), the time to 90% decline from peak fluorescence (158±5 versus 162±5 ms, P=.59), nor the sum of these two times (226±4 versus 225±3 ms, P=.59) differed between SHR and WKY at a pacing rate of 4 Hz.
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Similarly, none of the three approaches used to analyze
[Ca2+]i handling during stimulation stress
revealed significant differences between SHR and WKY. In the first
approach, neither the extent nor the rate of the
[Ca2+]i accumulation during rapid pacing
differed between SHR and WKY (Fig 1).
Specifically, neither the asymptote A (accumulation extent) nor
the time constant (accumulation rate) of the
monoexponential increase of systolic
[Ca2+]i during 100 sequential
extrasystolic beats differed significantly between SHR and WKY
(P=.58 and .59, respectively). Similarly, in the second
approach, neither the extent nor the rate of the
[Ca2+]i decline during the long rest period
after cessation of rapid pacing differed between SHR and WKY (Fig 2). Specifically, neither the
parameter (decline extent) nor the
parameter ß (decline rate) of Gompertz’s
double-exponential function differed significantly between SHR and WKY
(P=.31 and .43, respectively). Finally, in the third
approach, [Ca2+]i handling in SHR appeared
normal during preprogrammed ventricular pacing because the
relationship between VF triggering [Ca2+]i
and VF thresholds did not differ between SHR and WKY (Fig 3). Specifically, the slopes and the
y-intercepts of the regression lines of VF triggering
[Ca2+]i and log2-transformed VF
thresholds did not differ between WKY and SHR because they were within
each other’s 95% confidence limits.
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) and at
3.55 mmol/L [Ca2+]o (•) in WKY
(top) and SHR (bottom). This relationship was tested by linear
regression (bold lines); WKY) y=13.9+8.2x,
r=.76, P<.05, sY.X=13.5; SHR)
y=12.4+11.0x, r=.66,
P<.05, sY.X=14.9; thin lines
indicate 95% confidence limits.
[Ca2+]i was assessed as
fluorescence ratio F400/F510; VF triggering
[Ca2+]i, increase in
[Ca2+]i induced by a sequence of
prepro-grammed ventricular stimulation, measured as
increase of systolic [Ca2+]i from the
beginning of a stimulation sequence to the cardiac cycle preceding VF;
and transformed VF threshold, VF threshold after transformation by
base-2 logarithm to achieve normal distribution.Despite similar [Ca2+]i handling, SHR were more vulnerable to VF than WKY (Table 2). SHR tolerated only 3.5 premature pulses (reconverted VF threshold), whereas WKY tolerated 19.3 pulses before VF was induced at 2.55 mmol/L [Ca2+]o. Also, SHR tolerated less [Ca2+]i increase (VF triggering [Ca2+]i) than WKY before VF was induced by premature stimulation at 2.55 mmol/L [Ca2+]o. Furthermore, increasing [Ca2+]i by increasing [Ca2+]o significantly lowered VF thresholds of both SHR and WKY. At this high [Ca2+]i, few premature pulses could induce VF, and VF thresholds and VF triggering [Ca2+]i no longer differed between SHR and WKY. Furthermore, at both [Ca2+]o, the VF triggering intervals did not differ between SHR and WKY.
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Discussion |
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In the present study, in hypertrophied hearts of SHR without heart failure, we showed that [Ca2+]i handling is normal during stimulation stress and not a determinant of the increased vulnerability to VF in SHR. Although VF thresholds were lower in SHR than in WKY at 8 to 10 months of age, both presented similar [Ca2+]i handling during rapid ventricular pacing or preprogrammed ventricular stimulation. Specifically, the extent and rate of the [Ca2+]i accumulation during rapid pacing and those of the [Ca2+]i decline after cessation of pacing were similar in SHR and WKY. In addition, the relationship between VF triggering [Ca2+]i and VF threshold was unaltered in SHR, indicating that the vulnerability in SHR was increased without any changes in [Ca2+]i handling.
Our finding of increased vulnerability to VF in hypertrophied hearts of SHR is consistent with other studies.1 2 8 38 39 However, the literature about the state of [Ca2+]i handling in hypertrophied myocardium is more controversial. Part of this controversy may arise from the use of different models to analyze [Ca2+]i handling in myocardial hypertrophy, including SHR5 6 and pressure-overload hypertrophy after aortic banding in guinea pigs,40 ferrets,41 42 43 and dogs.44 In these hypertrophy models, prolonged [Ca2+]i transients may be absent5 or present6 41 42 43 44 and diastolic [Ca2+]i may be decreased5 or normal5 6 40 41 42 43 44 during resting conditions. Consistent with our finding of unaltered [Ca2+]i handling during stimulation stress, [Ca2+]i handling in response to treppe appeared normal in nonfailing SHR.6 Similarly, stress by elevated [Ca2+]o challenge on nonfailing SHR cardiomyocytes provided no functional evidence of uncompensated "Ca2+ leakiness."45 In another report, the duration of hypertrophy appeared to determine the degree of [Ca2+]i handling alterations,46 ultimately contributing to compromised pump function in the failing heart.6
However, before this report, no study had experimentally linked altered [Ca2+]i handling in hypertrophied myocardium to increased vulnerability. As suggested by Carré et al,47 prolonged [Ca2+]i transients are unlikely to cause arrhythmias under ordinary environmental conditions. Nevertheless, under stress conditions, [Ca2+]i handling could be easily disturbed by an additional Ca2+ influx and consequently increase the vulnerability to VF. Contradicting this hypothesis, the present study provides the first evidence that [Ca2+]i handling in hypertrophied hearts of SHR under stress conditions like rapid pacing or preprogrammed ventricular stimulation is normal and not a determinant of their increased vulnerability to VF.
Alternative explanations for the increased vulnerability in SHR may arise from factors that facilitate reentry circuits such as myocardial tissue anisotropy,2 [Ca2+]i-induced inhomogeneities in the action potential duration, and [Ca2+]i-induced cell-to-cell uncoupling.48 Further explanations may arise from higher absolute [Ca2+]i in SHR and from delayed afterdepolarizations caused by spontaneous Ca2+ release from the sarcoplasmic reticulum.49 Because this Ca2+ release occurs asynchronously among cells,49 it may not be detected by our method.
To correctly interpret our findings, it should be noted that we studied the kinetics of Ca2+ delivery and removal to and from the cytosol as well as their relation to vulnerability during ventricular stimulation. Thus, we limited our conclusions to the rate and extent of [Ca2+]i accumulation and decline relative to the baseline transient before stimulation stress. However, since we did not measure absolute [Ca2+]i, we cannot exclude that absolute [Ca2+]i was higher in SHR than in WKY during stimulation stress. Indeed, higher [Ca2+]i may be an explanation for the lower VF threshold in SHR since the pulse number VF threshold depends on [Ca2+]i.9 In this way, [Ca2+]i may modulate the VF-inducing potential of sequential premature stimulation.9 However, during both resting conditions and stimulation stress by treppe, peak [Ca2+]i in nonfailing SHR was not significantly higher than that in WKY.6 Furthermore, we could not detect particular alterations in membrane proteins and processes involved in [Ca2+]i handling like sarcolemmal Ca2+ channels, sarcoplasmic reticular Ca2+ release/uptake, and Na+/Ca2+ exchange. For example, the ratio of the sarcolemmal Ca2+ channel to the sarcoplasmic reticular Ca2+-release channel may be increased in hypertrophied myocardium.47 Importantly, sarcoplasmic reticular Ca2+ uptake and binding as well as Ca2+-ATPase activity were shown to be decreased in SHR starting after 10 weeks of age.7 Interestingly, activity of the alternative Ca2+ extrusion system, Na+/Ca2+ exchange, was shown to be increased in the hearts of young SHR.50 Additionally, expression of cardiac Na+/Ca2+ exchanger mRNA was increased in response to pressure overload in cats51 and in failing human myocardium.52 In light of these reports and our findings, it is possible that decreased sarcoplasmic reticulum function in hypertrophied hearts could be compensated for by increased activity of Na+/Ca2+ exchange because these mechanisms are similarly rapid Ca2+ extrusion systems and compete for Ca2+ removal from the cytosol.21 This compensation might explain why the overall [Ca2+]i handling in hypertrophied hearts of SHR during stimulation stress remained unaltered as detected by our method.
In summary, the present study carried out in hypertrophied hearts of SHR without heart failure showed that [Ca2+]i handling is normal during stimulation stress and not a determinant of the increased vulnerability to VF in SHR.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by the George D. Smith Fund. Christian E. Zaugg was supported by the Swiss Heart Foundation. Randall J. Lee was supported by the Program of Excellence in Molecular Biology, NIH/NHLBIPO-1 HL 43821.
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Footnotes |
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Reprint requests to Christian E. Zaugg, PhD, University Hospital Basel, Division of Cardiology, Departments of Internal Medicine and Research, Petersgraben 4, CH-4031 Basel, Switzerland.
Received December 3, 1996; first decision January 16, 1997; accepted March 25, 1997.
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