Males and females show distinct differences in action potential waveform, ion channel expression patterns, and ECG characteristics. However, it is not known how sex-based differences in Ca2+ cycling might contribute to these differences in electrophysiological activity. The goal of this study was to investigate the differences in cellular Ca2+ transients in males and females and to examine how these might contribute to electrophysiological function. Ca2+ transients were measured in individual myocytes within microscopic regions of the fluo-4 AM-loaded left ventricular epicardium of intact rat heart of both sexes (3 to 5 mo old). Pacing protocols were used to measure transient characteristics at a basic cycle length of 500 ms and during 10-s trains of rapid pacing delivered to the left ventricular apex. Ca2+ transients were smaller in magnitude and longer in duration in females than in males. More importantly, the variability in Ca2+ transient characteristics between myocytes in a microscopic recording site (heterogeneity index) was greater for females than males for characteristics related to transient duration. The rate sensitivity of Ca2+ alternans development in individual myocytes was greater in females than in males, but there was also a greater heterogeneity in cellular responses to the rate dependence of alternans development in females. The longer Ca2+ transients in females were also associated with slower restitution, which was likely to be responsible for the development of Ca2+ and repolarization alternans at slower heart rates. These results demonstrate that there are distinct differences in cellular Ca2+ cycling in male and female rat hearts. Not only is there slower reuptake of Ca2+ in female rats, but there is greater local variability in Ca2+ cycling at the microscopic level. These sex-based differences in Ca2+ cycling could contribute to differences in ECG morphology and in arrhythmia sensitivity in males and females.
- calcium transients
- intercellular heterogeneity
- confocal microscopy
- T-wave alternans
- calcium alternans
it is known that women are more sensitive to both genetic and acquired long QT syndrome-based arrhythmias and bradycardia-induced torsades de pointes, whereas men are more likely to develop atrial fibrillation, early repolarization, and Brugada syndromes and sudden cardiac death (9). It has been suggested that one of the reasons underlying this difference is the combination of higher heart rate and longer QT interval in women (9), although the mechanisms by which these differences in the characteristics of the electrocardiogram (ECG) could influence cardiac electrophysiological behavior at the cell and organ level have not been described. There are now many reports of sex differences in ion channel expression patterns in a number of species including dog (23), rabbit (13, 15), guinea pig (8), rat (12), and humans (9, 15) that could account for some of the electrophysiological differences in males and females, particularly in action potential (AP) duration (APD).
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.
Rats of either sex (3–5 mo old) were anesthetized with ketamine-xylazine (80:8 mg/kg ip), and the heart was removed and placed on a Langendorff apparatus for retrograde perfusion with a modified Tyrode solution. All animal use protocols were approved by the Institutional Animal Care and Use Committee in accordance with National Institutes of Health guidelines. The heart was then placed in an experimental chamber on the stage of a Zeiss LSM510 laser-scanning confocal microscope. Temperature was maintained at 25 ± 1°C. Fluo-4 AM (15 μM) was added to the solution that was then recirculated for 30 min, at which time the heart was washed with normal solution. Cytochalasin-D (50 μM) was recirculated to prevent contraction. Hook platinum electrodes were inserted into the LV apex so that basal pacing at a basic cycle length (BCL) of 500 ms could be interspersed with 10-s epochs of rapid pacing at different test BCLs. A section of LV midepicardial surface was scanned (488 nm at 10% laser power) using a 25 mW argon laser, and the heart was moved until a site in the midmyocardium was located that gave good signal-to-noise characteristics. Sites in the base and apex were excluded from this study. Measurements of intracellular Ca2+ can be made for up to several hours using this approach. The scan line was then placed across the short axis of 10–25 myocytes, and the pacing protocols were initiated. The characteristics of Ca2+ transients were recorded (1.92 ms/scan over 512 pixels with a dwell time = 1.6 μs) during basal pacing and rapid pacing. The restitution for each myocyte was measured as the fractional recovery of the first beat after the initiation of rapid pacing compared with the last basal beat. The rate sensitivity of intracellular Ca2+ alternans magnitude was measured as the alternans ratio (AR, 1 − small/large) (17, 22) at steady state (after 10 s) at each BCL. Each epoch was followed by a return to basal pacing for 1 min before the next test interval was initiated at a 10-ms shorter test BCL. Pacing BCLs were tested in the range of 400–140 ms or until a 2:1 block or tachycardia occurred. Details of these methods have been published elsewhere (1).
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.
Sex differences in repolarization in rat hearts.
It is well known that repolarization is delayed in women compared with men. We investigated whether this difference is also true in rats. There were distinct differences in ECG recordings in males and females. In particular, when the duration of repolarization (starting from the peak of the QRS wave to 95% recovery of full repolarization) was measured in rat ECG recordings (Fig. 1), females showed a significantly longer duration than males (Fig. 1, inset). This finding is similar to that in humans and supports the possibility that sex-based differences in repolarization are similar in rats and humans.
Characteristics of Ca2+ transients in female and male hearts.
Typical results from the recording sites on the LV epicardium halfway between the base and apex for both female and male hearts are shown in Fig. 2. The line-scan images were taken from multicellular sites in which the transverse recordings of many cells were made. In Fig. 2A, the basal Ca2+ transients varied markedly between all 12 myocytes both in magnitude and in duration in a female rat heart. Single cell intensity profiles are shown for three typical myocytes to the right of each image and show the extent of cell-to-cell variability within each site, whereas the mean intensity profile (Fig. 2A, top trace) shows the Ca2+ transient averaged from the entire site. Figure 2B shows typical recordings from a male heart, which are also characterized by a high degree of cell-to-cell variability between the 13 myocytes in this site. When the mean intensity profiles from these two sites are normalized for magnitude (Fig. 2C), the most profound difference between them was a marked increase in the duration of the transient from these two midmyocardial regions.
When the characteristics of basal Ca2+ transients were compared, all characteristics displayed sex differences (Fig. 3). The magnitude of Ca2+ transients, the total amount of Ca2+ released, and the maximum Ca2+ release rate (+dF/dt) were greater in males, whereas the rise time was shorter. In contrast, all characteristics relating to Ca2+ duration were prolonged in females, including transient durations at 50% and 90% (TD50 and TD90) of total recovery, maximum rate of transient decay (−dF/dt), decay time, and peak width. These data demonstrate that there are a number of significant differences in Ca2+ cycling properties in male and female hearts primarily based on a greater and more rapid release in males and a longer recovery time for females.
Heterogeneity of Ca2+ transients in female and male hearts.
One of the most striking observations in the original images (Fig. 2) is the fact that there is a great degree of variability in Ca2+ transients even in adjoining myocytes of intact heart. It is well known that isolated myocytes demonstrate modest and expected cell-to-cell variability in nearly all characteristics from current densities to Ca2+ cycling and contraction that probably represents normal biological variability. However, it is not possible to know the magnitude of this normal cell-to-cell variability since there is no way to determine the precise site of cardiac origin of isolated myocytes. Also, because of the passive membrane properties of cardiac tissue, the AP is assumed to be uniform over a distance of at least one space constant, which is about 1–1.5 mm3 in the ventricle. It was this commonality of electrical activity that led us to assume in the past that Ca2+ cycling would be similarly constant over microscopic regions of the LV.
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 Δms) (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.
These data demonstrate that, in addition to longer Ca2+ transients in females, there is a greater variability between myocytes in the duration of Ca2+ transients within microscopic regions of the LV epicardium.
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.
Differences in Ca2+ restitution in males and females.
One of the major determinants of the sensitivity to alternans development is the rate of recovery of the Ca2+ transient of each myocyte. Thus it is likely that the differences in sensitivity to alternans development between the sexes could be a result of different rates of recovery of Ca2+ release. We next investigated any sex differences in restitution in myocytes from intact LV.
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.
When restitution rates in males and females were compared from individual sites for all experiments (Fig. 6B), there was a shift in the midpoint of restitution (R50) in females to longer test intervals. In addition, the heterogeneity at the cellular level within each site was greater in females than in males (Fig. 6, A and B) as was the gradient index for cell-to-cell variability (Fig. 6C).
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.
These results demonstrate that the duration of the Ca2+ transient determines the restitution rate, which is in turn responsible for the rate sensitivity of alternans development. SR Ca2+ release recovers more slowly from long transients than from short transients so that alternans development occurs more easily (slower heart rates). This is probably the reason that myocytes in female hearts where transients are longer than in males, are more sensitive to alternans development at longer BCLs.
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.
There have been extensive studies of the electrophysiological basis for sex differences in the AP and the ECG. Nearly all have focused on differences in ion channel expression underlying the AP, which is likely to be the primary determinant of APD. However, there is increasing interest in how differences in cellular Ca2+ cycling might also contribute to AP characteristics, particularly repolarization, because of the depolarizing influences on APD of the NCX via inward current in response to Ca2+ release. The result is that regional and transmural differences in Ca2+ cycling are now thought to play a major role in establishing APD and repolarization gradients during rapid pacing. One outcome is that Ca2+ cycling dynamics may also play a critical role in the development of T-wave alternans, which is known to contribute to arrhythmogenesis, particularly in heart failure patients. The results of this study provide evidence for extensive differences in Ca2+ cycling between males and females in intact heart that might also contribute to electrophysiological differences. These are matched by the fact that, like in humans, female rats also have a prolonged duration of repolarization as well as a greater vulnerability to repolarization alternans development, making this a potentially relevant model for studies of sex differences in cardiac electrophysiology. Furthermore, the intercellular variability of Ca2+ transients was greater in females, a fact that could have important implications for understanding the basis for the electrophysiological differences at the cellular, regional, and whole heart level.
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.
This work was supported in part by National Heart, Lung, and Blood Institute Grant 5R01-HL-075382 (to A. H. Kadish).
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- Copyright © 2008 by the American Physiological Society