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Departments of Medicine and of Molecular Pharmacology and Biological Chemistry and the Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, Chicago, Illinois
Submitted 6 May 2008 ; accepted in final form 29 August 2008
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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calcium transients; intercellular heterogeneity; confocal microscopy; T-wave alternans; calcium alternans; restitution
Ca2+ cycling kinetics have an important influence on AP characteristics, through direct effects on several Ca2+-sensitive current components, especially through the Na+/Ca2+ exchange (NCX) current (INCX). The result is that large transients contribute to a long APD. It is therefore possible that differences in Ca2+ transient characteristics, particularly duration, between males and females could play a part in the electrophysiological differences in APD and repolarization properties that separate the sexes and might contribute to their differing arrhythmia sensitivities. Furthermore, heterogeneities in Ca2+ transient magnitude and duration will have profound effects on cell-to-cell variability in ionic currents on a beat-to-beat basis. In addition, the extent of variability in Ca2+ cycling between adjoining myocytes is not known but is likely to have a major impact on electrical activity. Because of the importance of heterogeneity at all levels in the heart to arrhythmogenesis, the overall goal of the current study was to investigate cellular differences in Ca2+ signaling in individual myocytes in intact male and female hearts.
To accomplish this, we used laser-scanning confocal microscopy to measure cellular Ca2+ transients in intact rat left ventricular (LV) epicardium. This experimental approach has several major advantages over isolated myocytes. First, transients from large numbers of myocytes can be measured simultaneously in whole heart, thus giving a much more complete picture of where and how much heterogeneity is present in intact myocardium. Second, heterogeneity in Ca2+ cycling cannot easily be compared between isolated myocytes because of uncertainties in the site of origin of isolated myocytes compared with the known LV midepicardial recording sites in whole heart. Third, we have found dramatic differences in Ca2+-transient characteristics between cells in situ compared with isolated myocytes, which presumably arise from the isolation procedure itself, alterations in cell surface proteins after cell isolation, or perhaps simply the isolation of a myocyte from its neighbors. For example, isolated cells have longer Ca2+ transients; they cannot be stimulated over the same range of rapid rates as in intact heart (<2 hz near room temperature), and they are unable to develop sustained Ca2+ alternans (unpublished observations). Therefore, in situ measurements of intracellular Ca2+ cycling are likely to be much more representative of cellular behavior in whole heart, thus allowing us to measure more accurately the differences in cellular Ca2+ handling among myocytes in intact myocardium.
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METHODS |
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Because of the lack of a distinct T-wave in rat heart in a pseudo-ECG configuration, the duration of repolarization was measured as the time from the peak of activation of the QRS complex to 95% of terminal repolarization. Also, since rats do not have a distinct T-wave using this approach, repolarization alternans was measured as the voltage difference at the termination of repolarization between two successive cycles recorded in the ECG at the end of the rapid pacing train. The magnitude of repolarization alternans was measured at all pacing rates and plotted as a function of BCL up to the point where pacing failed to capture a ventricular response or where activation interfered with repolarization. This relationship was treated as sigmoidal over the range of cycle lengths tested (400 to 140 ms) to fit the curves for the calculation of midpoint and threshold similar to that for Ca2+ alternans. This approach allowed comparisons between hearts for calculated values of rate-dependent vulnerability to repolarization alternans.
Confocal fluorescence images were measured using Zeiss and ImageJ software. Fluorescence intensities for each cell were cut from the original images, and intensity profiles were analyzed using Matlab and pCLAMP8 software. The edges of individual myocytes are identified by regions that lack fluorescence (intercellular spaces) or regions of high-fluorescence intensity that do not change during stimulation, identifying them as capillaries (1). Mean data were compared using paired and unpaired Student's t-tests. Heterogeneity indexes were calculated to compare the overall intercellular heterogeneity for each parameter within a site and were based on the standard deviation around the mean for that parameter (14). Pairwise comparisons for intercellular differences (gradient index) were measured as the standard deviation for the difference for each parameter between cell pairs within a site.
The rate sensitivity of Ca2+ alternans was measured as the magnitude of AR (1 – small/large) as a function of BCL as described elsewhere (1). The results were sigmoidal since AR magnitude increased with increasing rate. These sigmoidal relationships were fitted (Matlab), allowing the calculation of the estimated cycle length (ECL) at 20% and 50% of maximal alternans (ECL20 and ECL50), representing the threshold and midpoint of Ca2+ alternans development, respectively.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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We compared the heterogeneity for these characteristics of Ca2+ transients within each recording site (Fig. 4). The heterogeneity index refers to the variability around the mean for each characteristic of Ca2+ release among all myocytes within a recording site. Cell-to-cell variability was pronounced for all parameters in both males and females. However, there were significant sex differences in heterogeneity indexes only for the parameters related to duration and decay times, which were greater for females compared with males. In contrast, the intercellular variability within each site was no different between males and females for amplitude, maximum rise and decay rates, total Ca2+ released, rise time, and peak width. Furthermore, when cell-pair comparisons were made to investigate further the source of Ca2+-transient heterogeneities (gradient indexes), we found once again that the significant differences lie only in those characteristics related to transient duration, including TD50 (in 
ms) (males, 5.7 ± 0.42 compared with females, 10.4 ± 0.98, P < 0.01), TD90 (10.1 ± 0.81 compared with 20.9 ± 1.65, P < 0.01), peak width (7.4 ± 0.60 compared with 19.4 ± 1.22, P < 0.01), rise time (6.4 ± 0.75 compared with 9.7 ± 1.02, P < 0.02), and decay time (13.7 ± 1.12 compared with 22.9 ± 1.79, P < 0.01). Therefore, the intercellular differences within a site are random and are not a result of cells acting in pairs or groups in either sex. Moreover, these data suggest that the differences between cells are greater in females than in males, establishing greater Ca2+ gradients between adjoining myocytes throughout the transient duration.
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Sex differences in sensitivity to the development of cellular Ca2+ alternans. Another important characteristic of Ca2+ dynamics in cardiac tissue is the development of Ca2+ alternans. The role of intracellular Ca2+ cycling in the development of APD alternans has received a great deal of attention recently because of its possible role in the development of Ca2+ alternans, the dispersion of repolarization, and the development of repolarization gradients, all of which are thought to play an important role as a substrate for reentrant arrhythmias.
We measured the rate sensitivity of Ca2+ alternans development in individual myocytes from both female and male hearts. When the rate was increased from basal pacing to progressively shorter BCLs during the test train, the myocytes in both the female and male sites (Fig. 5A) demonstrated increasing Ca2+ alternans. In addition, some myocytes in both sites demonstrated alternans, while others did not, demonstrating extensive heterogeneity as we have reported previously (1). However, there was a greater sensitivity to pacing-induced alternans development in myocytes from the female heart (250 compared with 239 ms, Fig. 5A). Furthermore, the variability between myocytes (heterogeneity index) is greater in the female heart than in the male heart. When all sites were compared, TD50 was again significantly longer in females (Fig. 5B). The ECL50 was greater in females than in males (not shown), although the difference was only about 11 ms, which raises the issue of whether or not it has important physiological significance. However, one consequence of this difference is that the magnitude of the AR at a BCL = 280 ms was greater in females than in males (Fig. 5C). In addition, the intercellular variability was much greater in the female heart, where the heterogeneity index within each site was greater than for males (Fig. 5D). Finally, the cell-to-cell gradient indexes for ECL50 were greater in females than in males (Fig. 5E), indicating that the adjoining myocytes varied more in females than in males. These results demonstrate that the average sensitivity of a given LV epicardial site to Ca2+ alternans is greater in females and that there is a greater variability within each site and between cell pairs in females than in males.
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Restitution was measured as the magnitude of recovery of an extrasystole normalized to the full recovery during basal pacing. For example, an extrasystole 300 ms after the last basal beat was not early enough to cause much change in Ca2+ release in any cells in the site of a male heart (Fig. 6A, top). When the test interval of the interpolated beat was shortened to 260 ms, there was less time for recovery and Ca2+ transient magnitude in most, but not all, cells decreased. As the test interval was further decreased, all cells showed progressively less recovery. The result is that the relationship between the coupling interval and Ca2+ transient recovery is sigmoidal. Similar results were obtained in female hearts, except that, in general, longer test intervals were required to permit recovery of the transients (Fig. 6A, bottom). Furthermore, there was greater variability between cells in the site than was observed in the male heart.
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Given the difference in transient duration in females and males and the growing evidence that Ca2+ alternans development is also related to Ca2+ transient duration (1, 3, 21), we investigated the possibility that sex differences in duration might be responsible for both the recovery of sarcoplasmic reticulum (SR) Ca2+ release and of the vulnerability to alternans development. The relationships between the cycle length dependencies of transient duration are linearly related to both that of restitution and of alternans development (Fig. 7, A and B). Furthermore, there was a similarly close relationship between restitution and alternans development (Fig. 7C), and when all three parameters were plotted (Fig. 7D), there is a singular relationship between these three parameters. Note also that males and females demonstrate the same interrelationship and are aligned with females clustering at longer TD50, R50, and ECL50 and males at the shorter end of the same relationship.
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Repolarization alternans in female and male rat heart. We also measured the magnitude and rate dependence of repolarization alternans in both female and male rat hearts during periods of rapid pacing. Since rats do not have a distinct T-wave, we measured the difference in voltage at terminal repolarization in successive beats at the end of rapid pacing. Figure 8A shows that at a BCL of 220 ms, there was greater repolarization alternans in the female heart than in a male heart. The summary data demonstrate that not only is repolarization alternans greater at this representative heart rate (Fig. 8B) but that the rate-dependence of repolarization alternans is slower in females than in males, with both the midpoint (Fig. 8C) and threshold (Fig. 8D) for alternans development occurring at longer cycle lengths in females. These data demonstrate that female rats have a greater vulnerability to repolarization alternans than male rats, which could contribute to sex-based differences in susceptibility to certain types of arrhythmias.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Sex differences in Ca2+ cycling and rate-dependent Ca2+ dynamics. Differences in transmural AP, current density, and ion channel expression have been reported, which probably contribute to sex differences in electrophysiology of the LV (23). In a study pertinent to our results, no differences were found between the sexes in APD in either 3- or 9-mo-old rats (12). However, these ruptured patch experiments included EGTA in the pipette so that Ca2+ buffering prevented any differences in Ca2+ release from affecting Ca2+-sensitive conductances and therefore AP configuration. It is well known that intracellular Ca2+ affects both inward and outward conductances, including the chloride component of the transient outward current and the inactivation rate and magnitude of L-type Ca2+ channel current (ICa) (18). Most importantly, Ca2+ removal via the NCX during the transient activates inward Na+ current carried by the NCX, which contributes to the plateau and APD.
Consequently, Ca2+ cycling characteristics are extremely important in determining APD, particularly at rapid pacing rates and in disease states where APD alternans and arrhythmias can develop. We and others have postulated that Ca2+ alternans develops when a large release depletes the SR so that an early cycle (during rapid pacing) finds insufficient Ca2+ available for release (1, 5, 11, 21). The resulting small release then permits greater SR refilling so that the next cycle produces a large release. This also explains why endocardial cells develop Ca2+ alternans at longer BCLs than endocardial cells with a shorter transient (3, 21). In fact, our previous work in intact LV suggested that, of all the characteristics of basal Ca2+ transients studied, only those related to transient duration could serve as the predictors of cellular vulnerability of individual myocytes to develop Ca2+ alternans (1). We have now extended this observation here to include the fact that the sensitivity to alternans development is also dependent on the rate of Ca2+ restitution, which is in turn a function of Ca2+ transient duration. Therefore, any changes in Ca2+ cycling that alter the transient duration are likely to alter the kinetics of Ca2+ recovery, which will in turn alter the rate vulnerability of the alternans development. We found that there is a unique relationship between these variables in both male and female hearts. The data suggest that the relationship between duration, recovery, and alternans is the same for both sexes and that the only difference is that females have longer transients than males. This explains why restitution is reduced at longer intervals, which in turn explains why Ca2+ alternans develops at slower heart rates in females.
Relationship between long Ca2+ transients and triggered activity in females. One of the most important implications of our study is that longer Ca2+ transients could contribute to the longer APD reported in female myocytes. This may have relevance for why women are more susceptible to triggered arrhythmias than men. Longer Ca2+ transients could be responsible for the longer APD and therefore for the total duration of depolarization. Delayed repolarization allows recovery of ICa from inactivation and the recovery of membrane potential to ICa threshold combine to produce early afterdepolarizations (EADs) (4, 10), whether or not Ca2+ release is the trigger or the result of the EAD. Finally, previous work has demonstrated that the Purkinje fibers are more vulnerable to EAD induction in response to APD prolongation than myocardium because of their longer basal APD (7). It is likely that the same is true for female ventricular myocytes compared with those in males, where longer Ca2+ transients and longer repolarization suggest longer APD in intact heart. Therefore, any additional influence that prolongs APD further (including bradycardia or a compensatory pause) is likely to have an exaggerated effect to promote ICa repriming in females, possibly explaining their greater propensity for developing triggered arrhythmias.
Macroscopic and microscopic heterogeneities and cardiac electrophysiology. Because of the differences in the expression patterns of ion channel and excitation-contraction (E-C) coupling proteins in different cardiac regions, it is not surprising that repolarization gradients develop in the heart. The basis for sex differences in repolarization gradients is less certain, however. There is some evidence for different patterns of the regional expression of certain E-C coupling proteins (2), although little is known about sex differences at the cellular level. In addition, mRNA levels of connexins 40 and 43 (Cx43) were found to be identical in male and female mouse hearts (6), even though there is some evidence that estrogen might increase (24) and testosterone might decrease (16) Cx43 expression. The heterogeneity of Cx43 expression is unknown and could also have very important influences on the spread of depolarization and for establishing microscopic repolarization gradients. Thus there is some molecular basis for considering the possibility that sex differences in both the passive and active electrical properties might be involved in establishing differences in APD and repolarization gradients during rapid pacing or disease.
Our data demonstrate not only that there is extensive microscopic heterogeneity in Ca2+ cycling in the heart but that there are distinct sex differences in their magnitude and distribution. Most notably, greater variability exists in the characteristics related to Ca2+ transient duration within a microscopic region in females, and the gradients between adjoining myocytes are steeper than in males. However, it is difficult to extrapolate these new observations to cardiac electrophysiology at the cellular or organ level. Heterogeneities in Ca2+ cycling could affect APD in isolated myocytes but are not likely to affect tissue APD because of the passive electrical properties of cardiac tissue. The space constant will eliminate cell-to-cell differences in APD and integrate the electrical signal over a region of 
1 to 2 mm3. The low electrical resistance between myocytes effectively eliminates the influence of intercellular heterogeneity of Ca2+ cycling on APD, maintaining uniformity of activation at the microscopic level. Thus it may not matter at all electrophysiologically that these heterogeneities exist or that they are greater in females than in males.
Theoretically, however, a reduced variability in Ca2+ cycling in males might be expected to produce sharper regional demarcations in transient and APD and the establishment of steep gradients in AP repolarization. The greater variability in females could have the opposite effect of smoothing regional differences in APD but promoting triggered arrhythmias. These differences could contribute to the sex-based sensitivities to the development of specific arrhythmias in males and females. It was therefore surprising that female hearts were more sensitive to repolarization alternans than males, which was consistent with our observations of longer transient durations in females but not with the expected repolarization gradients. Therefore, it is unlikely that intercellular heterogeneity alone can account for APD alternans and repolarization gradients. It may be that the regional differences in Ca2+ transients and APDs are greater between apex and base in males, thus making them more susceptible to discordant alternans and reentry. We would also predict that, despite an increased vulnerability to repolarization alternans, females would be more likely to respond to bradycardia with triggered activity if their long Ca2+ transients and APDs increase the likelihood of ICa repriming than in male hearts (where both are short), thus providing a greater safety margin for cardiac slowing that would allow ICa repriming and EAD activation. The basis for differential sex sensitivities to specific arrhythmias is clearly quite complex and may or may not be related to a differential intercellular heterogeneity in Ca2+ cycling.
It should be noted, however, that there is some evidence that sex-specific heterogeneities might contribute to the substrate for arrhythmias by generating qualitatively different gradients of repolarization and refractoriness. A recent analysis of the surface ECG has demonstrated that women more than men demonstrate a pattern of inverse sequence depolarization and repolarization in which cells that depolarize first are also the first to repolarize (19). The result is less global heterogeneity but greater local heterogeneity in women than in men. Furthermore, the local heterogeneities in women are exaggerated at higher heart rates (19), suggesting a rate sensitivity component to local heterogeneities that is greater in women. Although it is tempting to speculate about a role for intercellular heterogeneities in repolarization gradients, at this point there is no clear way to know whether the microscopic heterogeneities reported here contribute to these global and regional differences in whole heart electrophysiology.
Limitations of the study. It is not known whether female rats develop different types of arrhythmias than male rats in a manner analogous to humans. This is a serious limitation of the study because, if the differences in Ca2+ cycling reported here are indeed important to the sex-based differences in the development of Ca2+ and APD alternans and of repolarization gradients, then there would be a strong rationale for extrapolating these results in rat heart to sex differences in arrhythmia sensitivity in patients. However, we did find that the repolarization alternans occurs at slower heart rates in female rats compared with male rats, which is consistent with a greater susceptibility to the development of repolarization gradients in female rats compared with males. Although these differences do not directly address the issue of greater arrhythmia risk in female rats than in males, this finding provides compelling evidence in support of the use of the rat model in sex-based sensitivity to different arrhythmia mechanisms. However, it should be noted that, given the limitations in the resolution of the pseudo-ECG recording, it is unlikely that we could distinguish between triggered and reentrant arrhythmias using our methodologies. Any distinction would also probably require a different means of inducing arrhythmias than simply pacing-induced tachycardia. However, sex differences were reported in sensitivity to halothane-induced polymorphic ventricular tachycardias despite the fact that there were no changes in expression of proteins involved in cardiac conduction studied (including KCNQ1, KV1.5, and CX40 and 43) with the exception of KCNE1 (6). The authors concluded that KCNE1 alone could be responsible for the differences in arrhythmia sensitivity although our data suggest that other mechanisms could also be involved. To the best of our knowledge, the results actually demonstrating differential mechanisms of arrhythmias according to sex difference in rats have not been reported previously.
Finally, it is possible that the low temperature used in these studies could influence the results, yielding the apparent differences between males and females that might not exist at physiological temperatures. It has been known for many years that alternans is exaggerated at a low temperature (20), so most studies of this phenomenon are performed under conditions in which alternans is optimized and experimental preparations are known to be stable. However, it is highly unlikely that the sex-based differences in alternans rate sensitivity that we found could be a result only of temperature since all experiments were performed at the same temperature. Thus we would expect that any differences should represent actual sex-based differences rather than an experimental artifact. Furthermore, there is no reason to think that other characteristics studied here would be affected differently at low temperature according to sex since experimental conditions were identical.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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