Estrogen Contributes to Gender Differences in Mouse Ventricular Repolarization
Rationale: Fast-transient outward K+ (Ito,f) and ultrarapid delayed rectifier K+ currents (IK,slow, also known as IKur) contribute to mouse cardiac repolarization. Gender studies on these currents have reported conflicting results.
Objective: Key missing information in these studies is the estral stage of the animals. We revisited gender-related differences in K+ currents, taking into consideration the females’ estral stage. We hypothesized that changes in estrogen levels during the estral cycle could play a role in determining the densities of K+ currents underlying ventricular repolarization.
Methods and Results: Peak total K+ current (IK,total) densities (pA/pF, at +40 mV) were much higher in males (48.6±3.0) versus females at estrus (27.2±2.3) but not at diestrus-2 (39.1±3.4). Underlying this change, Ito,f and IK,slow were lower in females at estrus versus males and diestrus-2 (IK,slow: male 21.9±1.8, estrus 14.6±0.6, diestrus-2 20.3±1.4; Ito,f: male 26.8±1.9, estrus 14.9±1.6, diestrus-2 22.1±2.1). Lower IK,slow in estrus was attributable to only IK,slow1 reduction, without changes in IK,slow2. Estrogen treatment of ovariectomized mice decreased IK,total (46.4±3.0 to 28.4±1.6), Ito,f (26.6±1.6 to 12.8±1.0) and IK,slow (22.2±1.6 to 17.2±1.4). Transcript levels of Kv4.3 and Kv1.5 (underlying Ito,f and IK,slow, respectively) were lower in estrus versus diestrus-2 and male. In ovariectomized mice, estrogen treatment resulted in downregulation of Kv4.3 and Kv1.5 but not Kv4.2, KChIP2, or Kv2.1 transcripts. K+ current reduction in high estrogenic conditions were associated with prolongation of the action potential duration and corrected QT interval.
Conclusion: Downregulation of Kv4.3 and Kv1.5 transcripts by estrogen are one mechanism defining gender-related differences in mouse ventricular repolarization.
Gender-based differences in cardiac repolarization have been previously shown in humans, with females having longer action potentials and higher susceptibility to arrhythmias than age-matched males.1 In the mouse, however, studies on gender-related differences in right and left ventricular myocytes have yielded different results when examining fast transient outward K+ current (Ito,f) and ultrarapid delayed rectifier K+ current (IKur or IK,slow), which play an important role in cardiac repolarization.2,3 Reduction of IK,slow but not Ito,f4, reduction of Ito,f5, or no change at all6 in females versus males were reported. One common feature in all these studies was the lack of explicit report of the stage of females’ estral cycle. During the estral cycle, there are changes in plasma hormone levels, especially of estrogen (E2),7 that have been shown to downregulate cardiac Kv4.3 transcript,8 which is among the molecular correlates of Ito,f. In rats, the estral cycle lasts ≈4 days with an estrogen peak at proestrus, hours before estrus; hence, the estrus stage reflects the effects of the high E2 surge at proestrus. These effects decay toward diestrus-2, when the animal has been exposed to low E2 levels7 for the longest time (≈3 days). Here, we revisited in detail gender differences and E2-mediated changes in K+ repolarizing currents by initially measuring E2 levels during the mouse estral cycle, and correlated E2 levels with outward K+ currents, action potential duration (APD) and corrected QT (QTc) interval. We measured total outward K+ current (IK,total) densities, as well as their main components, IK,slow and Ito,f, in males and in females at estrus (under conditions of high estrogen of the preceding proestrus), diestrus-2 (under conditions of a prolonged exposure to low estrogen levels), and in E2-treated ovariectomized (OVX) mice. Our data revealed that IK,total, Ito,f, and IK,slow densities were lower in animals under conditions of high E2 (estrus and E2-treated OVX) when compared with low E2 levels (diestrus-2, males and OVX sham). Lower K+ current densities in high estrogenic conditions were associated with downregulation of Kv4.3 and Kv1.5 transcript levels, providing a molecular correlate for the prolongation of both APD and QTc interval in female mice under conditions of high estrogen.
Animals and Hormonal Treatment
Male, female and ovariectomized (OVX) mice were used (C57BL/6). Protocols received institutional review and committee approval. The female estral cycle and the treatment of OVX mice with E2 are described in the Online Data Supplement, available at http://circres.ahajournals.org.
Tetrodotoxin, tetraethylammonium (TEA), and 4-aminopyridine (4-AP) were from Sigma, and estrogen pellets were from Innovative Research of America.
Isolation of Ventricular Myocytes, Electrophysiology and Electrocardiograms
Whole-cell and voltage- and current-clamp recordings were obtained at room temperature from right ventricular myocytes. Cell isolation, recording solutions, the pulse protocols and the details of the ECG recordings and analysis are provided in the Online Data Supplement.
Real-Time Polymerase Chain Reaction
Total RNA from mouse heart right ventricle was isolated using TRIzol (Invitrogen) and was reverse-transcribed with gene-specific primers using Omniscript RT kit (Qiagen). See the Online Data Supplement for details.
ANOVA followed by Tukey–Kramer multiple comparison test was used for statistical analysis. Repeated Measures ANOVA was used for current–voltage curves. Probability values of <0.05 were considered statistically significant. Values are mean±SE.
Plasma E2 Concentration Is Highest in Females at Proestrus, Which Is Mimicked in E2-Treated OVX Mice
Similar to rats,7 female mice have an estrous cycle of ≈4 days: proestrus, estrus, metaestrus/diestrus-1, and diestrus-2. E2 plasma concentrations [E2] were measured in male and female mice (proestrus, estrus, and diestrus-2 identified from vaginal smears) and in OVX mice control (sham) and E2-treated (Figure 1). Similar to rats,7 [E2] is maximal in the proestrus stage, reaching ≈60 pg/mL, and is reduced to ≈20 pg/mL at estrus and diestrus-2. [E2] in male mice is ≈12 pg/mL, which is significantly lower than [E2] in dietrus-2 (P<0.001). As expected, ovariectomy decreased [E2] to ≈7 pg/mL, which increased to ≈120 pg/mL after E2 treatment. Note that although [E2] is low in estrus and diestrus-2, the estrus stage is primed by a preceding (0.5 to 1 day) surge of estrogen in proestrus, whereas diestrus-2 is marked by a prolonged exposure (≈3 days) to low estrogen levels. Therefore, in this work, we used mice under conditions of high estrogen (estrus and E2-treated OVX) and low estrogen (diestrus-2, OVX sham, and males).
Gender and Estral Cycle Determine Outward K+ Current Densities
To test our hypothesis that estrogen levels determine outward K+ current densities, IK,total was recorded in right ventricular myocytes isolated from males, estrus and diestrus-2 (Figure 2A). Whereas inward rectifier K+ current (IKir) remained constant, IK,total at potentials higher than −10 mV in male mice and in diestrus-2 was significantly higher than in estrus (Figure 2B). The steady-state current (Iss) at the end of the pulse is somewhat smaller in estrus than in males, but similar to diestrus-2 (Figure 2C). For comparative purposes, the underlying components of IK,total were isolated by two methods: (1) based on exponential fitting6 (Figure 2D) and (2) based on short depolarizing prepulses as to inactivate Ito,f4 (Figure 3). Both methods yielded virtually identical results (Table 1). The amplitudes and decay of IK,total are well described by the sum of fast (≈100 ms) and slow (≈1 second) exponential components (corresponding to Ito,f and IK,slow) and a steady-state current, Iss (Figure 2D). Using this method, Figure 2E demonstrates gender differences in Ito,f, IK,slow and Iss.
Figure 3A shows IK,slow (in the same cells as in Figure 2A), elicited after inactivation of Ito,f with a 100-ms prepulse to 0 mV. Ito,f (Figure 3D) was thus obtained from IK,total (Figure 2A) minus IK,slow (Figure 3A). Although there were no gender differences in the decay time constant (τdecay) of IK,slow (Figure 3C) and Ito,f (Figure 3F), their densities showed exactly the same tendency as in gender differences where the currents were separated by exponential fitting (Figure 3C and 3F versus Figure 2E).6 The kinetics of activation was similar for both IK,slow and Ito,f in males, estrus and diestrus-2 (Figure 3C and 3F). These results indicate that the gender differences found in current densities are independent of the method used.
To identify which component(s) of IK,slow underlies lower IK,slow in estrus, we used 4-AP (50 μm) to isolate IK,slow1 (Kv1.5) from IK,slow2 (Kv2.1).9,10 We first recorded IK,slow using a prepulse to inactivate Ito,f, then IKslow2 in the presence of 50 μmol/L 4-AP, which blocks IK,slow1 and IK,slow1=IK,slow−IK,slow2 (Figure 3G). We validated this approach by directly recording IKslow1 after blocking IKslow2 with TEA (25 mmol/L) and calculated IK,slow2= IK,slow−IK,slow1 (data not shown). IK,slow1, but not IK,slow2, was significantly lower in estrus (under conditions of high estrogen peak at proestrus) when compared to diestrus-2 and male (Figure 3C and 3G). These data demonstrate that gender differences in IK,slow are mediated by the differences only in IK,slow1 but not IK,slow2.
Inhibition of Outward K+ Currents by E2 Treatment in OVX Animals
To further test the hypothesis of E2-induced inhibition of outward K+ currents as being one mechanism underlying gender differences, we directly tested the effect of E2 on OVX mice. E2 treatment significantly reduced IK,total at potentials more depolarized than −10 mV, without any change in IKir (Figure 4A and 4B). IK,total densities in OVX sham were similar to males and diestrus-2, whereas IK,total densities in E2-treated OVX mice were similar to estrus (Figure 4C). Similar results were obtained for IK,slow and Ito,f, and the inhibitory action of E2 was even more pronounced for Ito,f than IK,slow (Figure 4D through 4I).
Kv4.3 and Kv1.5 Transcript Levels Are Lower in Estrus and Are Downregulated by E2 Treatment in OVX Mice
To identify the molecular correlates underlying the lower Ito,f and IK,slow in estrus compared to male and diestrus-2, we used real-time PCR to quantify relative transcript levels of Kv4.3, Kv4.2, and KChIP2 (molecular correlates of Ito,f) and of Kv1.5 and Kv2.1 (molecular correlates of IK,slow). Kv4.3 transcript levels were ≈2-fold lower in estrus compared to male and diestrus-2, whereas Kv4.2 and KChIP2 transcript levels were similar in the three conditions (Figure 5A through 5C), thus making Kv4.3 as a strong candidate to contribute to the gender differences of Ito,f. Regarding the molecular correlates of IK,slow, Kv1.5 (Figure 5D) but not Kv2.1 (Figure 5E) transcript levels were ≈2-fold lower in estrus compared to male and diestrus-2, which is in agreement with our electrophysiological data showing that gender differences in IK,slow are mediated by the differences in IK,slow1 (encoded by Kv1.5) but not IK,slow2 (encoded by Kv2.1).
We have previously shown that cardiac Kv4.3 transcript levels were downregulated by E2.8 We show now that cardiac Kv1.5 transcript levels are also downregulated by E2 treatment (Figure 5I). Kv4.2, KChIP2, and Kv2.1 (Figure 5G through 5J) transcripts were not affected by E2 treatment. These results support the view that only Kv4.3 and Kv1.5 are regulated by E2.
APD and QTc Interval Are Longer in Estrus Compared to Diestrus-2 and in E2–Treated OVX Mice
We further investigated whether changes in K+ currents in estrus versus male and diestrus-2 are associated with altered APD and QTc interval. Figure 6 shows that APDs at 50% (APD50) and 90% (APD90) of the depolarization were similar in males and in diestrus-2 (≈3.1 and 12 ms, respectively), whereas they were significantly longer in estrus (≈5 and 30 ms, respectively). E2 treatment of OVX mice increased both APD50 and APD90 (from 2.4 to 5.1 ms and from 9.28 to 34 ms, respectively) to values similar to estrus (Table 1). These data support the role of E2 in gender related differences of cardiac repolarization.
Analyses of ECG recordings revealed no differences in the shape of P waves or the QRS complexes in estrus versus diestrus-2 and in sham versus E2-treated OVX mice (Figure 6H through 6K). The heart rate and RR and QRS durations were also similar between estrus, diestrus-2, sham, and E2-treated mice (Table 1). Interestingly, QTc interval was significantly longer in estrus (51 ms) versus diestrus-2 (41 ms) and in E2-treated OVX (47 ms) versus sham (40 ms), indicating that changes in APDs in females are associated with altered ventricular repolarization. In male mice, however, although the APD was similar to diestrus-2, the QTc interval was significantly longer than diestrus-2, possibly because of the higher body weight and larger cardiac size compared to same-aged female mice (Table 1).
In summary, in female mice, changes in outward K+ current densities were associated with changes in APD and ventricular repolarization.
Previous studies have shown conflicting data regarding gender related differences in K+ current densities and ventricular repolarization in mice.4–6 These conflicting data may arise from the use of female mice at random without establishing their estral stage. Taking into consideration that estrogen levels vary during the female estral cycle (Figure 1), we revisited gender-related differences in K+ current densities and cardiac repolarization in male and female mice at estrus and diestrus-2, and in E2-treated OVX mice. We show for the first time that plasma E2 concentration is one of the key players in determining outward K+ current densities as well as affecting ventricular repolarization in mouse heart.
Higher Estrogen Levels Correlate With Reduced Outward K+ Current Densities
In agreement with the role of E2 in downregulating K+ current densities, IK,total densities were lower in animals under conditions of high plasma E2 (estrus and E2-treated OVX) compared with low plasma E2 levels (diestrus-2, males and OVX; Figures 2 and 4⇑A). The lower IK,total densities in animals under conditions of high plasma E2 were attributable to lower densities of both IK,slow and Ito,f (Figures 3 and 4⇑). Supporting a role of E2 in downregulating Ito,f, we found that males, which have lower levels of [E2] compared to females (Figure 1), have significantly higher levels of Ito,f densities than females. We also show that Ito,f density in females is regulated during the estral cycle and by E2 treatment. These findings complement our previous work which shows that Ito,f is downregulated at the end of pregnancy,8 where levels of plasma E2 are high.
Table 2 summarizes 32 previously reported IK,total, IK,slow, and Ito,f current densities, as well as our present work, in male and female mice. Lower IK,total and IK,slow but not Ito,f densities in female versus male were reported by Trepanier-Boulay et al using the CD-1 strain.4 In contrast to our findings and those of Trepanier-Boulay et al for IK,total and IK,slow,4 Brunet et al6 reported no gender differences in IK,total, IK,slow, of Ito,f densities in C57BL/6 mice; thus differences in strain is not a suitable explanation for the inconsistency in the results. Similar to our results, and opposite to Trepanier-Boulay et al4 and Brunet et al,6 higher Ito,f densities in males versus females was previously reported by Wu and Anderson.5
How can we explain the differences in these findings? One explanation is that if one uses animals at random, there is a lower probability to sample animals in estrus, where the changes are more pronounced. In addition, one can argue that the lack of significant differences in the Ito,f densities reported by Trepanier-Boulay et al4 in males and females could be attributable to recording currents from cells isolated from the whole ventricle and that the regional heterogeneity in the expression levels of Ito,f could have masked gender differences. In this work, we maximized homogeneity of our myocyte population by comparing only currents from cells isolated from the right ventricle, which also have the highest density of Ito,f.6 However, the regional differences cannot explain the discrepancy between our data and Brunet et al,6 who demonstrated no significant differences in the IK,total, Ito,f, and IK,slow densities in right ventricular myocytes between male and female mice.2 The contradiction between our present findings and Brunet et al6 in comparing Ito,f from the same ventricular regions could lie in the experimental method. Brunet et al6 used cardiomyocytes maintained in serum-free medium for up to 24 hours, by which time, they can undergo remodeling. In our study, we only used freshly dissociated cardiomyocytes within 4 to 6 hours after isolation.
Higher Estrogen Levels Correlate With Prolongation of APD and QTc Interval
Here, we show that estrogen plays a key role in regulating the ventricular repolarization in mouse heart. Lower Ito,f and IK,slow densities in female mice in estrus compared to male and dietrus-2 were associated with longer APD in estrus (Figure 6). E2 treatment of OVX mice resulted also in the prolongation of APD and QTc interval to a comparable level as in estrus, underscoring the role of E2 in the regulation of APD and ventricular repolarization. Supporting a role of E2, in larger animals E2 treatment can downregulate cardiac Kv1.5 (HK2) and KCNE1 transcript levels of rabbit hearts, which is correlated with a prolonged QTc interval.11 In addition, E2 can also influence the expression as well as conductance of the L-type calcium channel (ICa,L), which plays an important role in the cardiac repolarization phase and in arrhythmogenesis. ICa,L channels of cardiomyocytes differ significantly between day 0 (peak of plasma estrogen, proestrus) and day 4 (peak of progesterone-to-estrogen ratio, diestrus-2) of the estral cycle of female guinea pigs, suggesting that ICa,L density varies according to estral hormonal changes (reviewed elsewhere1). Moreover, E2 treatment reduced ICa,L density of OVX rat,12 and ICa,L density was increased in estrogen receptor (ER)α-deficient mice (reviewed elsewhere1), thus demonstrating that the inhibitory action of E2 was mediated mainly via the ERα. Here, we speculate that the large variability in the duration of APD50 and APD90 in estrus and in E2-treated OVX mice (Figure 6) is attributable to different expression levels of ERα in different cardiomyocytes, and the variability is more evident under conditions of high estrogen (Figure 6).
Supporting the role of sex hormones in regulating APD and cardiac repolarization, testosterone has been previously proposed to play a role in gender-based cardiac repolarization differences, because androgen deficiency in castrated mice produced a longer APD and prolongation of QTc interval (reviewed elsewhere1). Similar to mice, ventricular repolarization was significantly altered by testosterone treatment in dogs13 and rabbits (reviewed elsewhere1).
Mechanism of Estrogen Action: Regulation of Kv4.3 and Kv1.5 Transcript Levels
Several reports suggest that the expression of Kv4.3 and Kv1.5 are regulated by sex hormones.8,14 Our data demonstrate that Kv4.3 was the only molecular correlate of Ito,f; the transcript levels of which were lower under conditions of high estrogen (estrus and E2-primed OVX) compared to low estrogen (diestrus-2, male, and OVX sham). E2-induced decreased expression of Kv4.3 was not accompanied with significant alteration in Kv4.2 and KChIP2 transcripts (Figure 5). The lower Kv4.3 transcripts in estrus versus male and diestrus-2 and in E2-treated OVX mice versus sham coincided with reduced Ito,f densities. Regarding the molecular correlates of IK,slow, Kv1.5 but not Kv2.1 transcript levels were lower in estrus compared to male and diestrus-2 and were downregulated by E2 treatment. The fact that IK,slow2 peak current densities were similar (encoded by Kv2.1) in male, estrus, and dietrus-2 (Figure 3C and 3D), whereas IK,slow1 (encoded by Kv1.5) peak current densities was significantly lower in estrus compared to male and diestrus-2 further, supports that the transcriptional changes in Kv1.5 underlie changes in IK,slow, ruling out the possibility of estrogen-induced posttranslational modification of Kv1.5 protein. Higher Kv1.5 transcripts in males than in females has been reported, and testosterone has been suggested to be the main player in this regulation.14 Our data suggest that transcriptional regulation of Kv4.3 and Kv1.5 by E2 is at least one of the mechanisms responsible for the action of estrogen in remodeling cardiac repolarization.
How can estrogen regulate the transcriptional activity of Kv4.3 and Kv1.5 genes? Estrogen can regulate the transcriptional activity of a gene via a direct genomic classical pathway, which requires the presence of estrogen response elements (ERE) in the promoter of the gene. Alternatively, estrogen can stimulate the transcription through the DNA-associated transcription factors known as indirect genomic pathway via estrogen receptors.15 Analysis of 10 kb upstream of Kv4.3 and Kv1.5 transcription start sites failed to identify an ERE but showed several half-EREs, as well as Sp1 and Ap1 elements, that could be involved in reducing expression of Kv4.3 and Kv1.5 by E2. There is also an increasing body of evidence that direct application of estrogens can acutely regulate different types of K+ and ICa,L currents, affecting APDs through nongenomic pathways.1,16 For example, direct application of raloxifene (a selective estrogen receptor modulator) to human atrial myocytes inhibited Ito,f and IK,slow within minutes in a concentration-dependent manner, as well as prolonged APD.16 Although our real-time PCR data support a genomic action of E2, a nongenomic contribution cannot be excluded.
Influence of Estrogen on QTc Interval and Arrhythmia Susceptibility During the Menstrual Cycle and Estrogen Replacement Therapy in Women
Here, we showed that in female mice, the changes in plasma E2 levels during the estral cycle, as well as by E2 treatment, affect the QTc interval. The QTc is significantly longer in high estrogenic conditions (estrus and E2-treated OVX) in comparison to low estrogenic conditions (diestrus-2 and OVX). In premenopausal women, circulating levels of estrogen and progesterone change dramatically during the menstrual cycle. The early follicular phase (menses) is characterized by a low level of estrogen (≈100 pmol/L). Estrogen levels steadily increase after onset of menses and reach a peak (≈750 pmol/L) at approximately day 14 of the ovulatory phase.17 Estrogen levels fall during the luteal phase, when progesterone levels begin to increase. Changes in QTc interval during the menstrual cycle has been cited with conflicting results. Several investigators found no change in the QTc interval at different times of the menstrual cycle,17–19 whereas Nakagawa et al reported a shorter QTc interval during the luteal phase compared to the follicular phase.20 The shorter QTc interval in the luteal phase has been attributed to higher levels of plasma progesterone concentration (≈5-fold) compared to the follicular phase, because plasma estrogen and testosterone concentrations were similar in both phases.20 It is well accepted that women are at a higher risk for developing drug-induced torsades de pointes than men. How the changes in QTc interval during the menstrual cycle might influence the arrhythmogenesis in female patients with ventricular arrhythmia or paroxysmal supraventricular tachycardia has been of great interest. The prolongation of QTc duration induced by ibutilide, an antiarrhythmic agent, was significantly greater at menses and the ovulatory phase than in the luteal phase.19 The drug-induced prolongation of QTc was inversely correlated with higher levels of progesterone and progesterone-to-estrogen ratio, rather than estrogen alone.19 The occurrence and the duration of supraventricular tachycardia episodes in women has been suggested to be affected by the menstrual cycle, being higher in day 28 when progesterone levels are higher, as opposed to day 7 of the menstrual cycle, when estrogen predominates.21 The incidence of arrhythmia in supraventricular tachycardia patients was found to be slowed down by estrogen and facilitated by progesterone.21
The effect of estrogen and progesterone replacement therapy on ventricular repolarization has also been investigated in healthy postmenopausal women. Hormone replacement therapy with estrogen alone significantly prolonged QTc interval,22,23 whereas combined estrogen and progestin therapy had no effect24 or reduced the QTc interval.23,25 These findings suggest that sex hormones may directly modulate ventricular repolarization; estrogen is reported to account for the prolongation of QTc interval similar to our results in mice, whereas progesterone tends to reduce QTc interval. A detailed study to correlate plasma estrogen and progesterone to the QTc interval during the menstrual cycle and hormone replacement therapy should provide key information to set the appropriate timing of the treatment or electrophysiological procedure in premenopausal women more prone to arrhythmias.
Sources of Funding
Supported by NIH grants HL089876 (to M.E.); HL080111 and HL088640 (to E.S.); and HL54970 (to L.T.).
Original received July 27, 2007; resubmission received October 27, 2008; revised resubmission received July 1, 2009; accepted July 2, 2009.
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