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Am J Physiol Heart Circ Physiol 291: H570-H580, 2006. First published February 24, 2006; doi:10.1152/ajpheart.01288.2005
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Sex-based transmural differences in cardiac repolarization and ionic-current properties in canine left ventricles

Ling Xiao,1 Liming Zhang,3 Wei Han,2 Zhiguo Wang,3 and Stanley Nattel1

1Departments of Pharmacology and Therapeutics and 2Physiology, McGill University; and 3Department of Medicine and Research Center, Montreal Heart Institute and University of Montreal, Montreal, Quebec, Canada

Submitted 7 December 2005 ; accepted in final form 20 February 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The female sex is associated with longer electrocardiographic QT intervals and increased proarrhythmic risks of QT-prolonging drugs. This study examined the hypothesis that sex differences in repolarization may be associated with differential transmural ion-current distribution. Whole cell patch-clamp and current-clamp were used to study ionic currents and action potentials (APs) in isolated canine left ventricular cells from epicardium, midmyocardium, and endocardium. No sex differences in AP duration (APD) were found in cells from epicardium versus endocardium. In midmyocardium, APD was significantly longer in female dogs (e.g., at 1 Hz, female vs. male: 288 ± 21 vs. 237 ± 8 ms; P < 0.05), resulting in greater transmural APD heterogeneity in females. No sex differences in inward rectifier K+ current (IK1) were observed. Transient outward K+ current (Ito) densities in epicardium and midmyocardium also showed no sex differences. In endocardium, female dogs had significantly smaller Ito (e.g., at +30 mV, female vs. male: 2.5 ± 0.2 vs. 3.5 ± 0.3 pA/pF; P < 0.05). Rapid delayed-rectifier K+ current (IKr) density and activation voltage-dependence showed no sex differences. Female dogs had significantly larger slow delayed-rectifier K+ current (IKs) in epicardium and endocardium (e.g., at +40 mV; tail densities, female vs. male; epicardium: 1.3 ± 0.1 vs. 0.8 ± 0.1 pA/pF; P < 0.001; endocardium: 1.2 ± 0.1 vs. 0.7 ± 0.1 pA/pF; P < 0.05), but there were no sex differences in midmyocardial IKs. Female dogs had larger L-type Ca2+ current (ICa,L) densities in all layers than male dogs (e.g., at –20 mV, female vs. male, epicardium: –4.2 ± 0.4 vs. –3.2 ± 0.2 pA/pF; midmyocardium: –4.5 ± 0.5 vs. –3.3 ± 0.3 pA/pF; endocarium: –4.5 ± 0.4 vs. –3.2 ± 0.3 pA/pF; P < 0.05 for each). We conclude that there are sex-based transmural differences in ionic currents that may underlie sex differences in transmural cardiac repolarization.

sex difference; transmural dispersion of repolarization; ion channels; action potential

WOMEN HAVE A LONGER RATE-CORRECTED QT interval than men (4, 24), and compared with men, women respond differently to QT-prolonging drugs (class IA and III antiarrhythmic drugs, antianginal drugs, antihistamines, antibiotics, and antimalarials, etc.) and have an increased risk of acquired Torsades de Pointes (TdP) arrhythmias (11, 22). The mechanisms underlying these sex differences have been investigated in human subjects and in animal models (mouse, guinea pig, and rabbit) (14, 20, 29, 34, 36) but are still incompletely defined.

The crucial importance of transmural heterogeneity in action potential (AP) and ionic current properties is well recognized (2, 27). Female adult mice (34, 36), guinea pigs (14), and rabbits (10) have longer ventricular AP durations (APDs) than their male counterparts. Ionic mechanisms that may account for these differences have been reported, including differences in transient (Ito) and sustained (Isus) outward K+ current (36), ultrarapid delayed-rectifier current (IKur) (34), inward-rectifier current (IK1), delayed-rectifier K+ current (IK) (14, 20), rapid delayed-rectifier current (IKr) (20), and L-type Ca2+ current (ICa,L) (14, 23). Sex hormone manipulations in rabbits (10, 13, 19, 25, 26) or mice (7) can affect QT intervals, APDs, ionic currents, and arrhythmia incidence. Early afterdepolarizations (EADs) and increased transmural dispersion of repolarization have been proposed to underlie TdP (3). Previous studies suggest that female rabbit hearts have a greater susceptibility to EADs (21). Transmural ion-channel differences are known to be important in TdP susceptibility (2, 27). However, we were unable to identify studies that assess male/female differences for a broad range of ionic currents across the ventricular wall. This study was designed to assess transmural cardiac repolarization and ion-channel function in female and male adult canine left ventricular myocytes, with a view to elucidating male/female differences.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell preparation. All animal care and handling procedures were approved by the animal research ethics committee of the Montreal Heart Institute and followed the Guidelines of the Canadian Council for Animal Care. Adult mongrel dogs of both sexes [female, 26.4 ± 5.2 kg, n = 67; and male, 26.9 ± 5.9 kg, n = 68; P = not significant (NS)] were anesthetized with pentobarbital sodium (30 mg/kg iv) and ventilated with room air. A left lateral thoracotomy was performed, and hearts were quickly excised and immersed in oxygenated Tyrode solution at room temperature. The transmural free wall (~3 x 5 cm) of the lateral left ventricle, which was irrigated by a coronary artery branching from the left circumflex coronary artery, was quickly dissected, and the artery was cannulated. Cell isolation was performed as previously described (38) by perfusion with a solution containing collagenase (120–150 U/ml, Worthington type II). When the tissue was well digested, small tissue blocks were removed from the epicardial surface (1–1.5 mm thick), midmyocardial layer (2–5 mm thick) and endocardial surface (1–1.5 mm thick). Cells were dispersed by gentle trituration with a Pasteur pipette and were kept in a high-K+ storage solution (see Solutions) at 4°C.

Solutions. The standard Tyrode solution contained (in mM) 136 NaCl, 5.4 KCl, 1 MgCl2, 1 CaCl2, 0.33 NaH2PO4, 5 HEPES, and 10 dextrose (pH 7.35 with NaOH). The high-K+ storage solution contained (in mM) 20 KCl, 10 KH2PO4, 10 dextrose, 40 mannitol, 70 L-glutamic acid, 10 beta-OH-butyric acid, 20 taurine, and 10 EGTA and 0.1% BSA (pH 7.3 with KOH). The standard pipette solution used in most experiments contained (in mM) 110 K-aspartate, 20 KCl, 1 MgCl2, 5 MgATP, 0.1 GTP, 10 HEPES, 5 Na-phosphocreatine, and 5 EGTA (for current recording) or 0.025 (for AP recording), with pH adjusted to 7.3 with KOH.

For AP recordings, external solutions contained 2 mM CaCl2. For K+-current recordings, atropine (1 µM) and CdCl2 (200 µM) or nimodipine (5 µM, for IK) was added to external solutions to eliminate muscarinic K+ currents and to block Ca2+ currents. Na+ current contamination was avoided by using a holding potential of –50 mV or by substitution of equimolar Tris·HCl for external NaCl. For currents other than Ito, 1 mM 4-aminopyridine was used to block Ito. IK1 was studied as 1 mM Ba2+-sensitive current. For studies of IKr and slow delayed-rectifier K+ current (IKs), chromanol 293B (50 µM) was added to record IKr (6), and E-4031 (5 µM) was used to record IKs, after verification that chromanol 293B-resistant tail current was strongly blocked by E-4031 and that E-4031-resistant current was blocked by 50 µM chromanol 293B.

For ICa,L studies, the external solution contained (in mM) 136 tetraethylammonium chloride, 5.4 CsCl, 2 CaCl2, 0.8 MgCl2, 10 HEPES, and 10 dextrose (pH 7.4 with CsOH). Niflumic acid (50 µM) was added to inhibit Ca2+-dependent Cl– current (ICl,Ca). The pipette solution contained (in mM) 20 CsCl, 110 Cs-aspartate, 1 MgCl2, 5 MgATP, 0.1 GTP, 5 Na2 phosphocreatine, 10 EGTA, and 10 HEPES (pH 7.2 with CsOH).

Data acquisition and analysis. The whole cell patch-clamp technique was applied to record ionic currents and APs at 36°C, as previously described in detail (37, 38). Ionic currents were recorded in the voltage-clamp mode, and APs were recorded in current-clamp mode. Compensated series resistance and capacitive time constant ({tau}) values averaged 2.3 ± 0.1 M{Omega} and 294 ± 10 µs. Junction potentials (~10 mV) were corrected for AP recordings only. Leakage compensation was not used. Cell capacitance averaged as follows: epicardium, 118.1 ± 3.4 in female (n = 87) and 126.4 ± 3.9 pF in male (n = 86) cells, P = NS; midmyocardium, 123.2 ± 3.1 in female (n = 88) and 131.6 ± 3.2 pF in male (n = 93) cells, P = NS; and endocardium, 113 ± 2.9 in female (n = 77) and 116.7 ± 3.4 pF in male cells (n = 76), P = NS. Currents are expressed in terms of density (normalized to capacitance).

To obtain QT-interval data, 14 female dogs (weight, 25 ± 5 kg) and 14 male dogs (weight, 26 ± 4 kg) were studied in vivo. On study days, dogs were anesthetized with acepromazine (0.07 mg/kg iv), ketamine (5.3 mg/kg iv), Valium (6.25 mg/kg iv), and isoflurane (2%) and were mechanically ventilated with room air. Radiofrequency ablation of the atrioventricular node was performed to study the QT interval over a range of controlled basic cycle lengths (BCLs). Body temperature was maintained at 37°C. A median sternotomy was performed, and a bipolar Teflon-coated stainless steel electrode was hooked into the right ventricular free wall for stimulation. A programmable stimulator was used to deliver 2-ms twice-threshold pulses, and surface ECGs were recorded at BCLs of 300, 400, 600, and 1,000 ms. QT interval was measured from lead 2, and the mean of three QT interval measurements at each BCL for each dog was used for analysis.

Nonlinear least-square curve-fitting algorithms were performed for curve fitting. Unpaired Student's t-tests were used for comparisons between female and male groups. P < 0.05 was taken to indicate statistical significance, and group data are expressed as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
APs. Examples of APs recorded in isolated cells from various layers of female and male canine left ventricles (at 1 and 0.5 Hz) are shown in Fig. 1, A–C. In both female and male left ventricular cardiomyocytes, APs showed a prominent phase 1 and "spike-and-dome" configuration, with an intervening notch, in epicardial (Fig. 1A) and midmyocardial (Fig. 1B) cells. Endocardial cells showed more limited phase 1 repolarization and virtually no notch (Fig. 1C). APD was consistently longer in midmyocardial cells than in epicardial and endocardial cells. This pattern of transmural AP heterogeneity is consistent with the results of previous studies (2, 27).


Figure 1
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  		Fig. 1. A–C: typical action potential (AP) recordings from female (left) and male (right) canine left ventricular myocytes in epicardium (Epi; A), midmyocardium (Mid; B), and endocardium (Endo; C) at 1 and 0.5 Hz. D: means ± SE of AP duration (APD) at 90% repolarization (APD90) at 1 Hz in female (Epi, n = 17; Mid, n = 11; and Endo, n = 16) and male (epicardium, n = 16; midmyocardium, n = 21; endocardium, n = 16) transmural ventricular cells. E: means ± SE QT interval over a range of basic cycle lengths (BCLs) in male vs. female dogs. *P < 0.05, **P < 0.01, female vs. male.

 
APD was similar for female and male canine left ventricular cardiomyocytes in both epicardium and endocardium (1 Hz, Fig. 1D). In midmyocardium, female dogs had significantly longer APDs compared with male dogs (Fig. 1D). For example, at 1 Hz, APD to 90% repolarization averaged 292 ± 17 in females (n = 11) versus 235 ± 7 ms in males (n = 21, P < 0.05), whereas APD to 50% repolarization was 249 ± 19 in females (n = 11) vs. 186 ± 6 ms in males (n = 21, P < 0.05). In agreement with the APD data, QT interval at matched cycle lengths was significantly larger in female versus male dogs (Fig. 1E, n = 14, for male and female dogs, respectively).

No sex differences were observed in resting membrane potentials. Resting potentials averaged –78.4 ± 0.7 (female, n = 20) versus –79.9 ± 0.7 mV (male, n = 19) in epicardium, –80.0 ± 0.6 (female, n = 15) versus –81.3 ± 0.6 mV (male, n = 21) in midmyocardium, and –79.8 ± 0.8 (female, n = 28) vs. –81.2 ± 0.6 mV (male, n = 20) in endocardium (P = NS for all male/female comparisons).

IK1. IK1 was studied as 1 mM Ba2+-sensitive current. Figure 2A shows representative IK1 recordings from female and male epicardial cells. IK1 density was similar between female and male dogs for epicardium, midmyocardium, and endocardium as shown in Fig. 2, B–D, respectively.


Figure 2
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  		Fig. 2. A: representative 1 mM Ba2+-sensitive inward rectifier K+ current (IK1) recordings from female and male Epi cells by using voltage protocol (inset) at 0.1 Hz. B–D: means ± SE IK1 density in female and male Epi, Mid, and Endo. TP, test potential.

 
Ito. A typical transmural gradient in Ito was present in both female and male left ventricles (Fig. 3, A, C, and E). Mean Ito density was similar for epicardial and midmyocardial cells in female and male dogs (Fig. 3, B and D). In endocardial cells, mean Ito density was significantly larger in male dogs than in female dogs (Fig. 3F), with mean current density at +30 mV, averaging 2.5 ± 0.2 (female, n = 24 cells) and 3.5 ± 0.3 pA/pF (male, n = 28 cells, P < 0.05). No sex differences in the form of the current-voltage (Ito-V) relations were found for all transmural levels.


Figure 3
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  		Fig. 3. A, C, and E: typical transient outward K+ current (Ito) recordings from female (left) and male (right) canine Epi (A), Mid (C), and Endo (E) cells. Ito was obtained with 100-ms test pulses at 0.1 Hz (voltage protocol in inset). B, D, and F: means ± SE Ito density from female and male Epi, Mid, and Endo, respectively. *P < 0.05 and **P < 0.01, female vs. male.

 
The voltage dependence of Ito inactivation was studied with a two-pulse protocol, providing the results illustrated in Fig. 4, A, D, and G. Voltage for 50% activation and inactivation (V1/2) and slope factors were not significantly different between cells from males versus females for any myocardial layer (Table 1). Ito inactivation kinetics were well fitted by biexponential relations, and inactivation time constants were similar between female and male left ventricular cells (Fig. 4, B, E, and H). Ito reactivation was assessed with the two-pulse protocol shown in Fig. 4C. Reactivation kinetics were well fitted by biexponential relations (Fig. 4, C, F, and I), with no apparent dependency from sex difference. A detailed presentation of Ito recovery kinetics for each layer is shown in Table 1 and indicates no significant between-sex differences. Ito frequency dependence, as determined by steady-state current at 0.1, 0.5, 1, 2, and 5 Hz upon 100-ms pulses from –80 to +50 mV, also showed no sex differences within any regions of the left ventricle (data not shown).


Figure 4
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  		Fig. 4. Ito voltage dependence and kinetics. A (Endo), D (Mid), and G (Epi): Ito inactivation and activation voltage dependence. Inactivation was evaluated with 1,000-ms prepulses followed by 200-ms test pulse to +50 mV (at 0.1 Hz). Activation voltage dependence was analyzed from data obtained with protocol in Fig. 3 (see equation in RESULTS, ICa,L). Data are means ± SE (n = 6 cells/group, and inactivation; n = 6 cells/group, activation); curves are best-fit Boltzmann relations. B (Endo), E (Mid), and H (Epi): data are means ± SE inactivation {tau} values (n = 10 cells/group). C (Endo), F (Mid), and I (Epi): Ito reactivation time course evaluated by ratio of current (I2) during a 100-ms test pulse (P2, identical to P1) to current (I1) during a conditioning pulse (P1) with varying P1-to-P2 interval [holding potential (HP) = –80 mV, step to +50 mV at 0.07 Hz]. Data are means ± SE (n = 6 cells/group); curves are biexponential fits.

 

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  		Table 1. Ito voltage dependence and kinetics

 
IK. Figure 5A shows representative recordings of chromanol 293B (50 µM)-resistant IKr in epicardial cells from female and male dogs. IKr activated with half-activation voltages of –1.9 (male) and +2.0 mV (female, n = 8 cells/group, P = NS), based on tail currents at –40 mV after 4-s depolarizations to various test voltages. There were no significant sex differences in IKr tail-current density (Fig. 5, C, D, and E) and activation voltage dependencies in any regions. The kinetics of IKr showed no differences between male and female dogs. Activation was well-fitted by biexponential kinetics. Detailed activation time-constant data upon depolarization to +40 mV are provided in Table 2 and indicate no significant sex differences for any layer. Deactivation was similarly biexponential, and detailed results obtained on repolarizing from +40 to –40 mV (Table 2) show no significant sex-based differences.


Figure 5
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  		Fig. 5. A: representative chromanol 293B (50 µM)-resistant IKr recordings in female (left) and male (right) Epi cells. B: means ± SE normalized IKr tail currents (n = 8 cells/group from Epi) and best-fit Boltzmanna relations. C, D, and E: data are means ± SE IKr density-voltage relations in female and male Epi (C), Mid (D), and Endo (E) cells.

 

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  		Table 2. IKr kinetics

 
Representative recordings of E-4031 (5 µM)-resistant IKs from epicardial cells of female and male dogs are shown in Fig. 6A. IKs activation voltage dependence was assessed by normalizing tail-current amplitudes (obtained with the pulse protocol shown in Fig. 6A) to tail-current amplitude after depolarization to +70 mV. There were no differences in activation voltage dependence between female and male dogs in any regions, as illustrated by the mean data for epicardial cells shown in Fig. 6B. A transmural IKs gradient has been implicated in the important and well-known transmural APD gradient (2, 27). IKs showed a greater transmural density gradient in female than in male dogs (Fig. 6, C, D, and E). The IKs density was larger in female than in male dog cells in both epicardium (Fig. 6C) and endocardium (Fig. 6D), e.g., at +40 mV, female vs. male IKs tail-current density averaged as follows: epicardium, 1.3 ± 0.1 vs. 0.8 ± 0.1 pA/pF, n = 25 (female) and 20 (male) cells, P < 0.001; and endocardium, 1.1 ± 0.2 vs. 0.7 ± 0.1 pA/pF, n = 16 cells/group, P < 0.05. By contrast, IKs density in midmyocardial cells was comparable for both female (0.5 ± 0.05 pA/pF, n = 21 cells) and male (0.4 ± 0.02 pA/pF, n = 24, P = NS) cells. As in the case of IKr, the kinetics of IKs were similar for male and female dogs. IKs activation was monoexponential and showed no sex-dependent differences (Table 3). Upon repolarization from +40 to –30 mV, deactivation was biexponential, and once again there were no significant sex differences (Table 3).


Figure 6
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  		Fig. 6. A: representative E-4031 (5 µM)-resistant slow delayed-rectifier K+ current (IKs) recordings in female (left) and male (right) Epi cells, with 4-s depolarizing pulses (0.1 Hz) and 2-s repolarizations to –40 mV. B: means ± SE normalized IKs tail currents (n = 5 cells/group from Epi) and best-fit Boltzmanna relations. C (Epi), D (Mid), and E (Endo): data are means ± SE IKs density-voltage relations in female and male left ventricular cells. *P < 0.05, **P < 0.01, and ***P < 0.001, female vs. male.

 

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  		Table 3. IKs kinetics

 
ICa,L. Examples of representative ICa,L recordings from epicardial cells are shown in Fig. 7, A (female) and B (male). No transmural ICa,L density gradient was observed in either female or male left ventricles. Female dogs had significantly larger ICa,L density than male dogs for each transmural level (Fig. 7, C, E, and G). For example, at +20 mV, in epicardium, ICa,L density averaged –4.2 ± 0.4 in females and –3.2 ± 0.2 pA/pF in male cells, respectively (P < 0.05, n = 15 cells/group); in midmyocardium, mean ICa,L density was –4.5 ± 0.5 in females and –3.3 ± 0.3 pA/pF in males [P < 0.05, n = 15 (female), 18 (male) cells]; and in endocardium, current density was –4.5 ± 0.4 in females and –3.2 ± 0.3 pA/pF in males [P < 0.05, n = 16 (female) or 8 (male) cells/group]. The normalized ICa,L I-V relations were similar for female and male results in all regions (Fig. 7, D, F, and H).


Figure 7
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  		Fig. 7. Typical recordings of L-type Ca2+ current (ICa,L) from Epi of canine left ventricles in female (A) and male (B) dogs, obtained with 250-ms pulses (0.1 Hz) and a HP at –50 mV. C (Epi), E (Mid), and G (Endo): data are means ± SE ICa,L density recorded by the protocol shown in A between female and male dogs. D (Epi), F (Mid), and H (Endo): ICa,L voltage-current (I-V) relations (data are means ± SE; current was normalized to maximum current at +20 mV for each cell). *P < 0.05, female vs. male.

 
ICa,L inactivation was fitted by monoexponential relations and showed no sex differences, as illustrated by the mean data for midmyocardim shown in Fig. 8A. The voltage dependence of ICa,L activation and inactivation was evaluated as illustrated in Fig. 8B and showed no sex-dependent differences. Activation voltage dependence was assessed according to the relation ITP = aTP x Gmax(VTP – VR), where ITP is current at given test potential; aTP is activation variable a at test potential; Gmax is maximun conductants; VTP is voltage of test potential; and VR is reversal potential, which was obtained from a linear fit to the ascending portion of the I-V relation. There were no significant sex-based differences in mean activation or inactivation V1/2 values or slope factors in any layer (Table 4). Figure 8C shows ICa,L reactivation kinetics at a holding potential of –80 mV in midmyocardium. Reactivation time constants were similar between cells from female and male dogs in all layers (Table 4). Similarly, no sex differences in ICa,L frequency dependence were found. Figure 8D shows mean data for midmyocardium. Similar results were obtained for epicardium and endocardium.


Figure 8
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  		Fig. 8. ICa,L kinetics and voltage dependence from Mid (means ± SE). Similar results were obtained in all regions. A: data are means ± SE ICa,L inactivation time constant (n = 10 cells/group), obtained with the protocol in Fig. 7A. B: voltage dependence of ICa,L inactivation and activation. Steady-state inactivation was assessed with 1,000-ms conditioning pulses, followed by a 300-ms test pulse to +10 mV (0.1 Hz). Activation was assessed from data obtained with the protocol in Fig. 7A (see equation in RESULTS, ICa,L). Data are means ± SE (n = 6 cells/group); curves are best-fit Boltzmann relations. C: ICa,L reactivation time course, studied with paired 100-ms pulses delivered with varying interpulse intervals at 0.1 Hz. Curves are monoexponential fits (n = 6 cells/group). D: ICa,L frequency dependence, determined from the ratio of current during the 15th pulse to current during the first pulse of a train of 100-ms depolarizations from –80 mV to +10 mV at frequencies indicated (n = 6 cells/group).

 

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  		Table 4. ICa,L voltage dependence and kinetics

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we analyzed in detail transmural ionic current function in left ventricular cardiomyocytes from male versus female dogs. We found male/female differences in three ionic-current systems: Ito, IKs, and ICa,L. For Ito and IKs, the male/female differences varied transmurally, in a way that may have functional significance. In addition, we observed statistically significant longer APDs in females for M cells only, increasing transmural APD heterogeneity.

Relation to previous findings regarding sex-related ionic current differences in the literature. A variety of differences in ionic current properties have been described between male and female animals, and there are numerous discrepancies in the literature, possibly related to interspecies and interstrain differences. Trepanier-Boulay et al. (34) showed similar Ito and reduced IKur in female mice, whereas Wu and Anderson (36) reported that female mice have smaller Ito and larger sustained depolarization-induced outward current, which has a major contribution from IKur. In contrast to Trepanier-Boulay et al. (34), who observed longer APDs and smaller Ito in female mice, Brunet et al. (8) could not identify sex differences in K+ currents and ventricular repolarization in mice. Previous studies (20) on rabbit hearts pointed to smaller IKr in female rabbits, associated with longer QT intervals. We found no sex differences in IKr at any level of the canine left ventricle. Liu et al. (20) also reported smaller outward, but not inward, IK1 in female rabbits, whereas James et al. (14) observed smaller inward, but not outward, IK1 in female guinea pigs. We did not observe any sex-dependent IK1 differences in dogs. Unlike our observation of larger ICa,L in females, Trepanier-Boulay et al. (34) did not note an ICa,L difference between male and female mice. We were unable to identify previous studies of sex-dependent differences in IKs.

Potential relevance to sex differences in electrophysiology. We noted rather complex sex-based differences in ionic currents across the canine ventricular wall. A smaller Ito was found in females only in the endocardial layer. Females had larger ICa,L across the ventricular wall, but IKs was larger in females only in the epicardium and endocardium but not midmyocardium. These differences would be expected to cause transmurally based differences in APD, which we observed. The absence of significant male/female APD differences in the endocardium and epicardium could be due to offsetting differences in inward ICa,L and outward IKs. The larger female ICa,L in the face of similar IKs in the midmyocardium could account for the larger midmyocardial APD in females. The larger APD observed in female midmyocardium relative to male, in the face of similar endocardial and epicardial APD, increased the transmural dispersion of repolarization in females versus males. Despite smaller Ito in female endocardium, we did not observe any associated APD differences. The lack of appreciable impact of the endocardial male/female Ito difference in overall APD may have been due to the fact that Ito flows primarily during the very early phases of the AP and inactivates well before the onset of phase 3. Variations in Ito over the range that we observed (3–6 pA/pF) had no effect on canine endocardial APD in a recently published study (32) using an elegant dynamic clamp technique.

The presence of significant sex-based differences in cardiac repolarization is well recognized. The QT interval is longer in women (4, 24), and women clearly have an increased sensitivity to drug-induced QT-interval prolongation and TdP (5, 11, 16, 17, 22). The basis for these differences remains poorly understood. The present results point to male/female differences in transmural ion-channel function. The transmural distribution of cardiac ion channels is complex, and this complex distribution plays a key role in cardiac electrophysiology (1, 2, 27, 30). There is evidence for greater repolarization heterogeneity in women compared with men (31). Pham et al. (25) observed greater transmural repolarization heterogeneity in female dogs upon exposure to IKr blockers. Despite the importance of transmural ion-channel function in repolarization heterogeneity and arrhythmias, we were unable to find detailed studies of transmural ion-current properties in female compared with male subjects. Pham et al. (23) found somewhat larger ICa densities in the epicardium of female versus male rabbits, in accordance with our results, but no substantial endocardial differences. Midmyocardial cells were not studied, nor were other ionic currents. Greater midmyocardial APD has been attributed to a variety of factors, including smaller IKs density in the midmyocardium (1, 12, 18). Consistent with this notion, we observed smaller and similar IKs in midmyocardial versus endocardial or epicardial cells for both male and female dogs. Because IKs was larger in female than male dogs in the epicardium and endocardium, female dogs had a larger transmural IKs gradient compared with male dogs, potentially contributing to longer midmyocardial APDs and a larger repolarization gradient in females. These in turn may contribute to increased QT intervals and greater risks of TdP.

Sex differences in currents governing transmural repolarization might be expected to produce differences in QT interval and T-wave morphology, as well as in arrhythmia susceptibility. Women do have longer corrected QT intervals than men (4, 24). Young men have larger T-wave offset dispersion than young and old women, whereas women have greater T-wave complexity after exercise and with autonomic blockade (33). Susceptibility to drug-induced TdP is clearly greater in women than in men (11, 22). Women might be less prone to reentrant arrhythmias for which M-cell repolarization is limiting because of longer APDs; however, the enhanced transmural repolarization gradient we observed could promote reentry in women by favoring the establishment of unidirectional block at the M-cell border. This complex area clearly requires further study and analysis.

The principal arrhythmic risk known to be enhanced in women is drug-induced long QT syndrome, almost uniformly by IKr blocking drugs. Our data suggest reduced repolarization reserve in the M-cell layer, because unlike the other two transmural layers, the larger ICa,L in females was not offset by larger IKs. The notion of repolarization reserve implies an ability of the heart to minimize the effects of agents impairing repolarization, in particular IKr blocking drugs, by enhancing outward current carried by other channels, in particular IKs (28). When repolarization reserve is reduced, as in female M cells in the present study, the effect of IKr blockers would be expected to be enhanced, leading to excessive delay and destabilization of M-cell repolarization and potentially early afterdepolarizations, transmural reentry, and TdP.

Potential limitations. We performed detailed studies of a wide range of K+ currents and of L-type Ca2+ currents at three transmural levels of male and female canine myocardium. This is, to our knowledge, a much more broad and detailed comparison than in previous comparisons between male and female cardiomyocytes in the literature. However, other relevant currents and transport systems that could contribute to male/female differences, such as various Cl– currents, the Na+-current system, the Na+,Ca2+ exchanger, and Na+,K+-ATPase, were not assessed and should be in future studies. In addition, we compared ionic currents transmurally at one ventricular site, but AP properties are known to differ among cardiac regions, with the potential for distinct transmural properties in different right and left ventricular areas (8, 9, 15, 30, 35). This issue, too, would be worthy of attention in further studies. It would be interesting to quantify potential differences between males and females in the transmural expression of subunits like Kir2.1–2.3, Kv4.3, Kv4.2, Kv1.4, KChIP2, Cav1.2, Kv1.5, ERG, minK, and KvLQT1 to define potential molecular bases for the current differences we observed. In addition, it would be interesting to study regulatory differences (e.g., in protein kinase A and C phosphorylation and phosphatase-mediated dephosphorylation) and differences in membrane trafficking of ion-channel subunits. However, the investigations required would be extensive and go well beyond the scope of the present study.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Canadian Institutes for Health Research, the Quebec Heart and Stroke Foundation, and the Mathematics of Information Technology and Complex Systems Network of Centers of Excellence.


   ACKNOWLEDGMENTS
 
The authors thank Evelyn Landry, Nathalie L'Heureux, Chantal Maltais, and Chantal St.-Cyr for technical assistance and France Thériault for secretarial help with the manuscript. We also express our gratitude to Sanofi-Aventis for supplying chromanol 293B.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Nattel, Research Center, Montreal Heart Institute, 5000 Belanger Est., Montreal, Quebec, H1T 1C8, Canada (e-mail: stanley.nattel{at}icm-mhi.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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