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(Circulation Research. 2001;89:437.)
© 2001 American Heart Association, Inc.

Cellular Biology

Gender-Based Differences in Cardiac Repolarization in Mouse Ventricle

Véronique Trépanier-Boulay, Chantale St-Michel, Annie Tremblay, Céline Fiset

From the Research Center, Montreal Heart Institute, and Faculty of Pharmacy, University of Montreal, Montréal, Québec, Canada.

Correspondence to Dr Céline Fiset, Research Center, Montreal Heart Institute 5000, Bélanger est, Montréal, Québec, Canada, H1T 1C8. E-mail: fiset{at}icm.umontreal.ca

	Abstract

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The mouse heart has become a widely used model for genetic studies of heart diseases. Thus, understanding gender differences in mouse cardiac repolarization is crucial to the interpretation of such studies. The objective of this study was to evaluate whether there are gender differences in cardiac repolarization in mouse ventricle and to gain insights into the ionic and molecular mechanisms underlying these differences. Action potential durations (APDs) and K⁺ currents in male and female ventricular myocytes were compared using a patch-clamp technique. APD₂₀, APD₅₀, and APD₉₀ were found to be significantly longer in females than males. Examination of the different K⁺ currents revealed that a significantly lower current density exists in female ventricular myocytes compared with male myocytes for the ultrarapid delayed rectifier K⁺ current, I_Kur (at +30 mV, male, 33.2±2.9 pA/pF [n= 22]; female, 20.9±1.73 pA/pF [n= 19], P<0.001). Consistent with these findings were the results of the ribonuclease protection assay, Western blots, and confocal analysis that showed a significantly lower expression level of Kv1.5 (coding for I_Kur) in female compared with male ventricle. The additional K⁺ currents present in mouse ventricle exhibited no gender differences. In agreement with these electrophysiological data, no differences in the expression levels for the K⁺ channels underlying these currents were detected between both sexes. This study demonstrates that adult mice exhibit gender differences in cardiac repolarization. The expression of Kv1.5 and of its corresponding K⁺ current, I_Kur, is significantly lower in female mouse ventricle, and as a result, the APD is lengthened.

Key Words: K⁺ channel • electrophysiology • mouse ventricle • heart • gender

	Introduction

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Transgenic mouse models provide a powerful tool for the investigation of cellular and molecular physiology of the heart and cardiovascular abnormalities.^1–4 The use of transgenic mouse might ultimately allow the molecular identification of various factors leading to heart diseases, and it may serve as a guide to appropriate therapeutic strategies. It is therefore not surprising that the mouse heart is now extensively used in mechanical, biochemical, molecular/genetic, and electrophysiological studies.^2,3

Heart diseases are often associated with action potential prolongation.^5,6 K⁺ currents are key determinants of cardiac action potential duration (APD) and, therefore, of prime importance in controlling repolarization and duration of refractory period.^7–10 For all these reasons, there is great interest in defining the ionic and molecular basis of mouse cardiac K⁺ currents. As a result, there have been several recent studies describing the mouse ventricular K⁺ currents.^11–21

Despite the importance of understanding gender differences in cardiac repolarization for the interpretation of the work done using transgenic mice, no attempts have been made to determine whether there are sex-related differences in the ionic or molecular basis of cardiac repolarization in mouse heart. Accordingly, the main motivation for the present study was to provide a comprehensive description of the types, densities, and kinetics of voltage-gated K⁺ currents that define the action potential in adult male and female mouse ventricle. A combination of electrophysiological protocols together with ribonuclease (RNase) protection assays, Western blot analysis, immunofluorescence, and confocal imaging were used to characterize and compare the ionic and molecular basis of the cardiac K⁺ currents present in adult male and female mouse ventricle. Results presented in this study clearly demonstrate that adult mice exhibit significant gender differences in cardiac repolarization. Preliminary data were recently reported in abstract form.²²

	Materials and Methods

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Isolation of Ventricular Myocytes
Single ventricular myocytes were obtained from the right and left epicardium of adult CD1 mice of both sexes (2 to 3 months, weighing 25 to 30 g). The enzymatic dispersion technique used in these experiments has been previously described.¹¹ We recorded K⁺ currents and action potentials from the right and left ventricular epicardium on the basis of preliminary data demonstrating no interventricular differences in K⁺ current density and APD between ventricular myocytes isolated from right and left epicardium in male and female mice.

Electrophysiological Recordings
Whole-cell voltage and current-clamp recording methods and data analysis were identical to those previously described.¹¹ All experiments were carried out at room temperature (20°C to 22°C) to achieve a good voltage-clamp control. Details on the electrophysiological protocols and myocytes isolation are available in the online data supplement at http://www.circresaha.org.

RNase Protection Assay
An RNase protection assay was used to assess the mRNA levels of Kv1.5, Kv4.2, Kv4.3, and Kir2.1 K⁺ channels in mouse ventricles obtained from mice of both genders. Briefly, cDNA fragments used to generate antisense RNA probes were synthesized and cloned by reverse transcription–polymerase chain reaction. Transcription was carried out using the MAXIscript In Vitro transcription kit (Ambion). The RNase protection assays were performed using the RPA III kit (Ambion). The total RNA samples and the probes were hybridized at 48°C overnight. Mouse ß-actin was included in all reactions as an internal control. The mRNA results were quantified on a Bio-Rad PhosphorImager.

Western Blot, Immunofluorescence, and Confocal Microscopy
Western blot analyses were performed on male and female mouse ventricles using standard techniques as described in the online supplement. Immunofluorescence analysis was carried out on ventricular myocytes isolated from mice of both genders (3 mice/group) using the protocol described in details in the online data supplement.

Statistics
Results are expressed as mean±SEM. An unpaired Student t test was used to compare the differences in mean values. If probability value were <0.05, the results were considered statistically significant.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

	Results

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Action Potential Duration
Current-clamp experiments at different stimulation frequencies (0.1 to 4 Hz) revealed significant changes in APD between both genders. Figure 1 shows typical examples of action potentials recorded at 4 Hz in adult female (Figure 1A) and male (Figure 1B) ventricular myocytes. Figure 1A illustrates two examples of action potentials recorded from myocytes dissociated from two different female mice. These recordings show the range in duration of the action potentials in the female group. The action potentials were significantly longer in female myocytes compared with their male counterparts as illustrated by the example in Figure 1B. Mean APD from 18 cells in both groups obtained in 15 female and 14 male mice are summarized in Figure 1C. These bar graphs compare the APD recorded in male and female cells measured at 20% (APD₂₀), 50% (APD₅₀), and 90% (APD₉₀) of the repolarization. APD₂₀ (male, 2.7±0.2 ms, versus female, 4.2±0.4 ms), APD₅₀ (male, 4.4±0.3 ms, versus female, 9.2±1.3 ms), and APD₉₀ (male, 14.8±1 ms, versus female, 22.7±2.1 ms) were all significantly longer in the female group compared with the males (P<0.01).

View larger version (12K): [in this window] [in a new window]   Figure 1. Comparison of action potential in male and female mouse ventricular myocytes. A, Typical examples of action potential recorded from 2 different female ventricular myocytes. Stimulus current was 1 ms, 0.7 nA, and frequency rate was 4 Hz. B, Typical example of action potential recording obtained using the same conditions in male ventricular cell. C, Bar graphs showing mean APD (±SEM) at 20%, 50%, and 90% repolarization in male and female mouse myocytes.

Comparison of K⁺ Currents in Male and Female Mouse Ventricular Myocytes
In the adult mouse ventricle, several K⁺ currents are responsible for cardiac repolarization. These include the inward rectifier K⁺ current (I_K1), the Ca²⁺-independent transient outward K⁺ current (I_to), the ultrarapid delayed rectifier K⁺ current (I_Kur), and the steady-state K⁺ current (I_ss). Xu et al¹⁹ have recently characterized the outward K⁺ currents in mouse ventricle. They reported the presence of four distinct mouse ventricular K⁺ currents. A current sensitive to low-concentration 4-aminopyridine (4-AP) (I_K,slow), a tetraethylammonium-sensitive noninactivating current (I_ss), and a transient outward current that comprises a fast and a slow component (I_to,f and I_to,s). I_to,s was reported to be found exclusively in cells originating from the septum.¹⁹ In the present study, the current designated I_to corresponds to I_to,f. The steady-state component is referred to as I_ss, and I_Kur corresponds to the 4-AP–sensitive component of I_K,slow reported by Xu et al.¹⁹ Given that the 4-AP–sensitive current present in human atrium and mouse ventricle results from the expression of the same K⁺ channel isoform (Kv1.5),^21,23 we chose to keep the same nomenclature as the one used with human atrium and designated this current I_Kur.

To explore the possible ionic basis accounting for the differences in APD noticed between the two genders, macroscopic K⁺ currents were recorded from freshly isolated ventricular myocytes using the whole-cell patch-clamp technique in the voltage-clamp configuration. Figure 2A illustrates typical examples of a family of total K⁺ currents in male and female ventricular myocytes. All current amplitudes were normalized to the cell capacitance and expressed as densities (pA/pF). Cell membrane capacitances of ventricular myocytes isolated from male (84.0±2.4 pF, n=30) and female (84.7±2.5 pF, n=30) animals were indistinguishable.

View larger version (33K): [in this window] [in a new window]   Figure 2. Comparison of Ipeak and of the K+ current after inactivation of Ito in male and female mouse ventricular myocytes. A, Superimposed current records of total K+ current activated by voltage protocol shown in inset, in male and female mouse ventricular myocytes. B, Mean I-V relationships for Ipeak obtained in male () and female ( cells. C, Family of K+ currents in male and female myocytes obtained from the same cells as those in panel A, but voltage-clamp steps were each preceded by an inactivating prepulse, 100 ms at -40 mV (protocol shown in inset). D, I-V relationships for the K+ current after Ito has been removed by the inactivating protocol in male () and female ( myocytes.

Inward Rectifier K⁺ Current: I_K1
To evaluate the role of I_K1 in the gender difference seen in ventricular APD, we compared both the resting membrane potential and the current density of I_K1 in males and females. The resting membrane potentials were identical between male and female ventricular myocytes (male, -73.6±1 mV, n=30; female, -73.0±1 mV, n=30). Consistent with these data were the results of voltage-clamp experiments, which showed that the superimposed inward current traces corresponding to I_K1 were similar between male and female ventricular myocytes (Figure 2A). The mean I-V relationships for I_K1 were determined and compared between both groups in Figure 2B. I_K1 was measured at -110 mV and its density did not differ between males (-14.4±1.5 pA/pF, n=32) and females (-14.6±1.3 pA/pF, n=34) (P=NS). Depolarizing voltage steps in the range -80 to -40 mV produced very small outward current. For I_K1, this small outward current is critical for the late repolarization phase of the action potential. Values measured at -60 mV that correspond to the maximum outward component for I_K1 were not different between male (0.8±0.2 pA/pF) and female (0.8±0.14 pA/pF) mice. These results demonstrate that the longer APD in female mice is not due to gender differences in the density of either the inward or outward portions of I_K1.

Outward K⁺ Currents
The peak outward I-V relationships for the two genders show that the outward K⁺ current was significantly smaller in the female group (Figure 2B). Over a broad range of voltages (from -10 to +50 mV), the density of I_peak was significantly smaller in female compared with male ventricular myocytes. For instance, the mean current densities of the peak outward current measured at +30 mV were 58.9±2.9 pA/pF (n=34) in females and 66.6±3.3 pA/pF (n=32) in males (P<0.01). These data were obtained from 16 male and 20 female mice. The outward K⁺ currents in adult mouse epicardial myocytes consist of three distinct components (I_to, I_Kur, and I_ss), based on their different time and voltage dependence, and different sensitivities to pharmacological agents.^11,19,20 To determine whether one or more of these currents exhibited gender difference, a combination of pharmacological and voltage-clamp protocols was used to separate the three components of outward K⁺ current in these myocytes. Current recordings presented in Figure 2C show that after I_to has been removed by an inactivating protocol (100 ms at -40 mV), the remaining outward K⁺ currents were significantly larger in the male compared with the female ventricular myocytes. This is also illustrated in Figure 2D, which compares the corresponding I-V plot for the peak current (+30 mV, males, 51.3±3.7 pA/pF [n=32]; females, 35.1±2.0 pA/pF [n=34], P<0.001).

Transient Outward K⁺ Current: I_to
To investigate the possible role of I_to in lengthening the female mouse action potential, we compared the density of I_to in male and female cells. Figure 3A shows that I_to obtained by subtracting the pairs of currents (with and without the inactivating prepulse) was identical in male and female ventricular myocytes. This was confirmed on 32 male and 34 female cells isolated from 16 male and 20 female mice. The I-V relationships for I_to (Figure 3B) clearly show that the density of I_to was similar between males (at +30 mV, 28.9±2.2 pA/pF) and females (at +30 mV, 23.6±1.7 pA/pF, P=NS).

View larger version (25K): [in this window] [in a new window]   Figure 3. Transient outward K+ current (Ito; A and B) and the 4-AP–resistant current denoted as steady-state K+ current (Iss; C and D). A, Family of K+ currents in male and female myocytes by subtracting corresponding current records with (Figure 2C) and without (Figure 2A) the inactivating prepulse. B, Mean I-V relationships for Ito obtained in male () and female ( ventricles. C, Family of currents after application of 200 µmol/L 4-AP in male and female ventricular myocytes using inactivating prepulse voltage-clamp protocol shown in inset. D, Mean I-V relationships for Iss in male () and female ( myocytes.

Steady-State Outward K⁺ Current: I_ss
In mouse ventricular myocytes, there is a steady-state K⁺ current, I_ss, which is distinctly different from either I_to or I_Kur and which is not affected by concentrations of 4-AP (200 µmol/L) that completely block I_Kur.¹¹ Thus, combining the inactivating protocol with the use of 4-AP that selectively blocks I_Kur, we compared the magnitude of I_ss between male and female mouse ventricular myocytes (Figure 3C). As already mentioned, in the absence of 4-AP the prepulse abolished the I_to component. The waveform in the presence of 4-AP consisted of a slowly activating, very slowly inactivating component, I_ss (Figure 3C). The I-V relationships for I_ss (Figure 3D) indicate that the density of I_ss is similar in male and female ventricular myocytes (at +30 mV, male, 12.4±1.0 pA/pF [n=22], and female, 10.6±0.8 pA/pF [n=19], P=NS).

Ultrarapid Delayed Rectifier K⁺ Current: I_Kur
The family of superimposed current records presented in Figure 4 corresponds to the 4-AP–sensitive current, I_Kur, in male and female ventricular myocytes. These current traces were obtained by subtraction of the corresponding current records before (Figure 2C) and after application of 4-AP (Figure 3C). The 4-AP–sensitive current (I_Kur) was significantly smaller in female myocytes compared with males (Figure 4A). Pooled data in Figure 4B, plotted as I-V relationships, show that I_Kur in cells from female animals was significantly smaller than that in male myocytes over a broad range of membrane voltages (from -30 to +50 mV). In the female group, the current density of I_Kur measured at +30 mV was 20.9±1.7 pA/pF (n=19) compared with 33.2±2.9 pA/pF (n=22) (P<0.001) in the male cells. This lower current density (37%) was specific to I_Kur because the density of the three additional K⁺ currents examined in these cells did not differ between males and females. Figures 4C and 4D show the application of low concentration of 4-AP that selectively blocks I_Kur, on APD in male and female ventricular myocytes. In the presence of 4-AP, the APDs recorded in males were markedly lengthened and no longer different from those measured in female mouse ventricular myocytes.

View larger version (22K): [in this window] [in a new window]   Figure 4. Ultrarapid delayed rectifier K+ current (IKur) and action potential prolongation by 4-AP in male and female ventricular myocytes. A, Difference currents obtained by substracting corresponding current records with (Figure 3C) and without (Figure 2C) 4-AP in male and female cells. B, Mean I-V relationship for IKur in male () and female ( myocytes. C and D, Typical examples of action potentials recorded in male and female ventricular myocytes under control conditions (CTL) and with 50 µmol/L 4-AP. Dashed lines correspond to 0-mV level.

Voltage Dependence of Steady-State Inactivation of I_Kur
Figures 5A and 5B compare the voltage dependence of steady-state inactivation of I_Kur in male and female ventricular cells. Figures 5B presents the Boltzmann function fitted to the data from 15 male and 6 female myocytes. In male cells, V_1/2 was -44.7±1 mV compared with -47.6±2.7 mV for the female group. The slope factor was 5.5±0.3 mV for the male and 5.4±0.7 mV for the female group. No significant gender difference was found in the steady-state inactivation of I_Kur.

View larger version (29K): [in this window] [in a new window]   Figure 5. Steady-state inactivation and reactivation of IKur in male and female ventricular myocytes. A, Family of currents showing voltage dependence of steady-state inactivation for IKur in male and female cells. B, Graph comparing voltage dependence of steady-state inactivation for IKur in males () and females (. Smooth curves are best-fit Boltzmann functions. I/Imax indicates normalized current to the maximal amplitude of the current. C, Superimposed current records showing time-course recovery from inactivation of IKur in male and female cells. D, Graph showing reactivation of IKur in male () and female ( cells. P2/P1 is the ratio of test pulse current/prepulse current amplitudes. Smooth lines are best-fit exponential functions.

Recovery From Steady-State Inactivation of I_Kur
Figures 5C and 5D summarize recovery from inactivation of I_Kur in male and female mouse ventricular myocytes. Single exponential equations were fit to these data with time constants at -80 mV of 289.6±71 ms in the males compared with 312.7±31 ms in the female group (P=NS). These data indicate that I_Kur recovered from inactivation at a similar rate in both genders.

Inward Currents: I_Na and I_Ca-L
Although the main focus of this study was to compare ventricular repolarization between male and female mice, we also examined whether there are gender-based differences in the inward currents, I_Na and I_Ca-L. Current traces of I_Na in ventricular myocytes of both genders are presented in Figure 6A. No significant difference in the peak density of I_Na was observed between the two groups (at -50 mV, male, -27.4±2.4 pA/pF [n=7], and female, -28.2±5.8 pA/pF, [n=9], P=NS). Superimposed current traces of I_Ca-L obtained from male and female ventricular myocytes are presented in Figure 6C. Mean I-V relationships for I_Ca-L are presented in Figure 6D. The peak current density of I_Ca-L was not significantly different between male and female cells (at 0 mV, -9.96±0.9 pA/pF [n=9] and -8.71±1.3 pA/pF [n=9], respectively, P=NS).

View larger version (26K): [in this window] [in a new window]   Figure 6. Comparison of Na+ and Ca2+ currents in male and female mouse ventricular myocytes. A, Superimposed current records of INa in male and female obtained from the protocol shown in inset. B, Mean I-V relationships of INa in males () and females (. C, Superimposed current records of ICa-L obtained from protocol shown in inset in male and female ventricular myocytes. D, Mean I-V relationships of ICa-L in male () and female ( cells.

mRNA Expression of Different K⁺ Channels in Mouse Ventricle
To establish whether the reduction of I_Kur reflects a lower expression of Kv1.5 mRNA in female ventricle, we analyzed mRNA expression of selected K⁺ channels with RNase protection assay using RNA harvested from the intact ventricle of male and female mice. The K⁺ channels examined included Kv1.5 (coding for I_Kur),^21,23 Kv4.2, Kv4.3 (both underlying I_to),^{12,18,24–26} and Kir2.1 (corresponding to I_K1).²⁷ Whereas the transcript levels of Kv4.2, Kv4.3, and Kir2.1 showed no significant difference, there was a significantly lower expression (36%, P=0.01) of Kv1.5 in the ventricles of female mice relative to the males (Figures 7A and 7B). These mRNA data confirm our electrophysiological data showing that only I_Kur was decreased in female mouse ventricular myocytes compared with their male counterparts. Results were repeated using at least four mice in each group. During the course of a separate study on FVB/N mice, a similar difference in Kv1.5 mRNA levels was also noted (40% lower in female ventricle, P=0.03). Also in line with the current study, no sex-related differences were found in the message levels for the other K⁺ channels studied in this genetic strain of mice.

View larger version (45K): [in this window] [in a new window]   Figure 7. Comparison of different K+ channels expression in male and female mouse ventricle. A, Bar graphs comparing relative abundance of Kv1.5, Kv4.2, Kv4.3, and Kir2.1 mRNA transcripts in male and female mice, determined by RNase protection assay. Values were normalized to ß-actin signal. Relative abundance was calculated with value from male mice as a reference of 100%. B, Representative autoradiographs of mRNA levels for 4 K+ channel isoforms measured in 2 male and 2 female RNA samples (2.5 µg of total RNA/sample). Yeast represents negative control (yeast total RNA). C, Western blot analysis of Kv1.5, Kv4.2, Kv4.3, Kir2.1, and Kv2.1 on proteins (100 µg/lane) isolated from male and female mouse ventricle. Fractions were separated on 10% SDS-PAGE and incubated with different K+ channels antibodies. Equal protein loading was confirmed by Ponceau S–stained membranes. Results were repeated using 3 mice from each group.

Protein Expression of K⁺ Channels and Immunolocalization of Kv1.5 in Mouse Ventricle
Experiments were undertaken to determine the protein level expression of Kv1.5 as well as that of the other K⁺ channels, Kv4.2, Kv4.3, and Kir2.1, in male and female ventricular myocytes. Barry et al¹² have shown that a 4-AP–resistant, slowly inactivating K⁺ current is encoded by Kv2.1 in mouse heart. Therefore, in addition to the other K⁺ channels studied, we also examined the protein expression of Kv2.1. Western blot analysis revealed that the protein expression level of Kv2.1, Kv4.2, Kv4.3, and Kir2.1 was similar between male and female ventricle, confirming our electrophysiological data, which show no gender difference in their corresponding K⁺ currents in mouse ventricular myocytes (Figure 7C). However, for Kv1.5, there was a significantly lower protein expression in female mouse ventricle as compared with male ventricle (Figure 7C). Moreover, immunolocalization of Kv1.5 by confocal microscopy showed a significant difference in the expression of Kv1.5 proteins between male and female myocytes (Figure 8A). A reduction of 37% in the relative fluorescence intensity of Kv1.5 was observed on the plasma membrane of female ventricular myocytes when compared with their male counterparts (Figure 8C).

View larger version (60K): [in this window] [in a new window]   Figure 8. Detection and localization by immunofluorescent labeling of Kv1.5 in male and female mouse ventricular myocytes. A, Bottom, Isolated cells from both sexes were stained by exposure to primary antibody and then to TRITC-conjugated donkey anti-rabbit secondary antibody. Red fluorescent staining indicates presence of Kv1.5 in male and female myocytes. Left panels show corresponding phase images. Top, Negative controls (without primary antibody) showing a signal that is not different from background in both genders. B, Same cells as those seen on panel A (bottom) in male and female cells at a higher magnitude. C, Bar graphs showing that relative fluorescence intensity of Kv1.5 was 37% lower in female than in male ventricular myocytes. Individual measures of relative intensity of the K+ channel Kv1.5 were taken on 5 different sites above the plasma membrane of 10 male and 10 female ventricular myocytes. These values were obtained with the laser scanning microscopy examiner software using an indicator that recorded fluorescence intensity on a specific point of the cell surface.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we demonstrated that adult mice clearly exhibit gender differences in cardiac repolarization. In females, a lengthening of APD was accompanied by a significant and specific lower expression of Kv1.5 and of its corresponding K⁺ current, I_Kur, despite similarities in voltage dependence and kinetics properties of this current in both genders. In contrast, we did not detect any gender difference in the additional K⁺ currents present in mouse ventricle or in the expression level of their underlying K⁺ channel isoforms.

To add further evidence that the gender difference in action potential is mediated by the difference in I_Kur density, we applied low concentrations of 4-AP to myocytes isolated from male mice. The results show that block of I_Kur in male myocytes results in a dramatic prolongation of the action potential (Figure 4C). Furthermore, APD equalized in male and female myocytes in the presence of 4-AP concentrations that selectively eliminated I_Kur. In line with these results, we and others have previously reported that addition of 4-AP at concentrations selective for block of I_Kur markedly prolonged APD in ventricular myocytes isolated from male mice.^11,20 Taken together, these observations provide strong evidence that the gender differences in I_Kur density underlie the observed differences in action potential waveforms presented in Figure 1.

Relation to Previous Studies
The question of gender differences in cardiac repolarization is important. However, to our knowledge, only a few publications have focused on that important issue. Of these, no studies were performed using the mouse heart as an experimental model. Most of the previous research on gender-related differences in repolarization has been performed on rabbits. Liu et al first reported that female rabbit hearts had a longer baseline and drug-induced changes in QT interval than male hearts.²⁸ In the same study, they found a small but statistically significant difference for the rapidly activating delayed rectifier K⁺ current (I_Kr) and for I_K1. For both currents, the density was lower in females than in males. In their study, I_Kr currents were very atypical and activated at very positive potentials (threshold activation around +10 mV). In addition, its current density was statistically different only at test potentials positive to +30 mV where the physiological relevance remains uncertain. With regard to I_K1, they noticed a reduction only in the outward component of I_K1 in the females. In the present study, we did not notice any significant differences in the current density of I_K1 between male and female mouse ventricles. These discrepancies may be due to species differences and experimental conditions. Consistent with our results, they did not observe any significant gender differences in I_to.²⁸ In a separate study, they showed that despite a similar QT prolongation and K⁺ channel block by 4-AP, the female rabbit hearts had a higher incidence of torsades de pointe compared with males.²⁹ However, they did not provide any explanation for these results. Recently, Pham et al³⁰ reported that the APD₃₀ was longer in papillary muscles of female than male rabbits but not the APD₉₀. They also found that the I_Kr blocker dofetilide induced greater APD₉₀ prolongation in females than males whereas the I_Ks blocker chromanol had no effect on APD. These findings are interesting, but unfortunately the relationships between the APD and the K⁺ currents were not examined. Using the rat as a model system, Leblanc et al³¹ have reported that the action potential configurations, the I_Ca-L as well as the three K⁺ currents present in rat ventricle (I_K1, I_to, and I_ss), were not different between male and female animals. These results are consistent with our findings showing that I_Ca-L and the three K⁺ currents I_K1, I_to, and I_ss were not different between both genders, whereas I_Kur, which is absent from rat ventricle, was significantly smaller in females.

Potential Role for Sex Steroid Hormones in These Gender Differences
The mechanisms underlying gender-specific differences in cardiac repolarization are still largely unknown. Hormonal regulation of cardiac K⁺ channel gene expression may affect basal electrical activity. The issue of the role of sex steroid hormones in the regulation of K⁺ channel expression is an interesting question that has been the focus of previous studies.^30,32–36 However, the results of animal studies regarding the electrophysiological effects of estrogen or androgen have been divergent, and it is not clear from previous data whether sex steroid hormones consistently alter cardiac repolarization.^37,38

A major finding of this study is that gender difference in Kv1.5 K⁺ channel expression exists at the transcriptional level. The heart is a potential target not only for estrogen but also for androgen action given that both types of receptors are present in this organ.³⁹ Thus conceivably, androgen might be a candidate factor that shortens the action potential in male by upregulation of Kv1.5 expression in ventricle. Alternatively, estrogen might decrease the level of transcription and reduce the expression level of Kv1.5. Then again, both hormones may potentially contribute to these gender-related differences.

Clearly, additional experimental studies are required to delineate the mechanism(s) of male-female differences in cardiac repolarization. Ongoing studies in our laboratory are aimed at exploring the role of sex steroid hormones on K⁺ channel gene expression. Given the observed difference in APD between male and female mouse ventricles, a logical extension of this work will also be to compare QT interval in male and female mice.

Potential Significance
As the use of murine modeling of human heart diseases increases, understanding sex-based differences in mouse cardiac repolarization is critical. This work improves our understanding of the fundamental mechanisms by which gender differences in cardiac repolarization occur and enhances our awareness of gender difference in the control of K⁺ channel gene expression. The importance of this study is also that it validates the mouse model for further investigations of gender differences in cardiac electrophysiology.

	Acknowledgments

 
This study was supported by operating grants and personnel awards from the Canadian Institute of Health Research, the Heart and Stroke Foundation of Canada and Quebec, the Fonds de la Recherche en Santé du Québec, and the Fonds de la Recherche de l’Institut de Cardiologie de Montréal. We thank Louis-Robert Villeneuve for his expert assistance with the immunofluorescence and confocal experiments.

Received February 2, 2001; accepted July 6, 2001.

	References

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