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Cardiovascular Research 2002 53(3):752-762; doi:10.1016/S0008-6363(01)00449-7
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Copyright © 2002, European Society of Cardiology

Effects of gonadal steroids on gender-related differences in transmural dispersion of L-type calcium current

Thai V Phama, Richard B Robinsona, Peter Danilo, Jra and Michael R Rosena,b,c,*

aDepartment of Pharmacology, College of Physicians and Surgeons of Columbia University, 630 West 168 St., PH7 West-321, New York, NY 10032, USA
bDepartment of Pediatrics, College of Physicians and Surgeons of Columbia University, 630 West 168 St., New York, NY 10032, USA
cPartnership for Women's Health, College of Physicians and Surgeons of Columbia University, 630 West 168 St., New York, NY 10032, USA

* Corresponding author. Tel.: +1-212-305-8754; fax: +1-212-305-8351 mrr1{at}columbia.edu

Received 25 April 2001; accepted 27 August 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Statistical analysis
 4. Results
 5. Discussion
 References
 
Objectives: Repolarization-prolonging drugs induce torsades de pointes (TdP) in females more than males. The action potential plateau and the early afterdepolarizations that induce TdP are determined, in part, by L-type calcium current (ICa,L). Therefore, we studied gender- and hormone-related differences in ICa,L in age-, and weight-matched normal male, female and hormonally-treated, castrated rabbits. Methods: Oophorectomized (OVX) or orchiectomized (ORCH) 50- to 60-day-old rabbits were subcutaneously implanted with pellets impregnated with placebo (PLA), 5{alpha}-dihydroxytestosterone (DHT), or 17β-estradiol (EST). Four to five weeks later, epicardial and endocardial myocytes were isolated from the left ventricle. Patch clamp technique was performed to assess ICa,L. Results: ICa,L density (measured as peak current density [pA/pF] at +15 mV, Vh=–40 mV), was greater in female epicardium (–7.4±0.9) than endocardium (–5.6±0.7, P<0.05), while male epicardial ICa,L density (–6.5±0.7) did not differ from endocardial (–5.9±1.0, P>0.05). OVX-female, DHT and EST-treated groups had epicardial ICa,L density (–5.6±0.6, and –5.9±0.7, respectively) greater than endocardial (–4.3±0.3, and –3.6±0.4, P<0.05). However, OVX-females had hormone levels not significantly different from female controls and EST-treated females had non-physiological levels of estradiol. There were no differences between endocardial and epicardial ICa,L activation and inactivation. In contrast, epicardial–endocardial differences in ICa,L density in EST-treated OVX-females were associated with epicardial–endocardial differences in ICa,L activation and conductance; in DHT-treated OVX-females only epicardial–endocardial activation differed. The other groups, showed no ICa,L transmural gradient, or differences in activation, inactivation or conductance. Conclusions: The greater dispersion in ICa,L density of OVX–DHT and OVX–EST than OVX–PLA suggests both hormones can modulate ICa,L density in females. That gonadal steroids had no effect on ICa,L dispersion in males suggests gender differences in mechanism of action of both hormones. The greater ICa,L dispersion in females may contribute to gender differences in repolarization.

KEYWORDS Ca-channel; Gender; Hormones; Ion channels; Ventricular arrhythmias


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Statistical analysis
 4. Results
 5. Discussion
 References
 
There are important gender-related differences in cardiac rhythm and arrhythmias. Women have longer rate-corrected QT intervals than men and are more prone to develop torsade de pointes (TdP) when taking drugs that prolong repolarization [1,2]. Similarly, in congenital long QT syndrome female gender is a risk factor for sudden death [3]. The higher propensity toward arrhythmias in females is associated with differences in ventricular repolarization in the heart such that the rate-corrected QT interval is longer in females than males.

Previous research has validated the use of a rabbit model that manifests gender-related differences in repolarization having characteristics similar to those in humans [4–9]. This research has focused mainly on outward K+ currents such as IKr and IK1 that contribute largely to phase 3 of repolarization [4,5]. Yet, clinical and basic studies suggest the inward Na+ and Ca2+ currents that contribute to the action potential plateau also determine gender differences in repolarization and possibly arrhythmogenesis. For example, men have a greater slope of the ascending limb of the T-wave, consistent with differences early in repolarization [10]. In addition, phases 2 and 3 of repolarization are shorter in male than female rabbits [9]. Finally, L-type calcium current (ICa,L) is important to the development of early afterdepolarizations (EAD) [11]. Thus, we have used the rabbit to test the hypothesis that there are gender-based differences in ICa,L which are modulated by gonadal steroids.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Statistical analysis
 4. Results
 5. Discussion
 References
 
This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US PHS NIH Pub. No. 85-23, 1996. Female and male New Zealand White rabbits were gonadectomized at age 50–60 days and implanted with 60-day sustained released pellets (Innovative Research of America) of placebo (PLA), 17β-estradiol (EST) or 5{alpha}-dihydroxytestosterone (DHT) 2 weeks after gonadectomy (as described previously [9]). Blood (2 ml) was obtained before euthanasia, and serum separated and stored at –20°C for analysis. EST immunoassay was via solid-phase chemiluminescence (Immulite Diagnostic Products, Los Angeles, CA; sensitivity 20 pg/ml) and DHT was measured by radioimmunoassay (Diagnostic Systems, Webster, TX) coupled with an oxidation/extraction procedure to remove testosterone; sensitivity 4 pg/ml. At terminal study, all rabbits (aged-matched and weighing 3.0–3.5 kg) were anesthetized with sodium pentobarbital (30 mg/kg, i.v.) and the hearts, excised.

2.1. Cell preparation
The solution for cell isolation contained (mM); NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1, Hepes 5, glucose 5.5, and pH adjusted to 7.4 with NaOH. Nominally Ca2+-free Tyrode's solution was identical, but without CaCl2, while low Ca2+ Tyrode's solution contained 180 µM CaCl2. Epicardial and endocardial myocytes were isolated by an enzymatic Langendorff perfusion method modified from Tytgat [12]. The heart was first perfused with normal Tyrode's solution (gassed with 100% O2 at 37°C for 5–10 min, followed with nominally Ca2+-free Tyrode's solution for 10 min. The heart was then perfused and recirculated with 0.6 mg/ml collagenase (type I, Worthington Biochemical, Freehold, NJ) in Ca2+-free Tyrode's solution for 15 min. Protease (0.088 mg/ml, type XIV, Sigma, St Louis, MO) was then added to the recirculating enzymatic perfusate for another 15-min perfusion. Finally, the heart was perfused with enzyme-free low Ca2+ Tyrode's solution for 5 min. Approximately 1-mm strips of epicardium (epi) and endocardium (endo) were cut from the base of the left and placed in separate flasks. Cells were dispersed from the tissue strips by gentle agitation in low Ca2+ Tyrode's solution and stored at room temperature. Experiments were performed within 12 h of cell dissociation. Only quiescent, rod-shaped myocytes with clearly defined ends, clear striations and debris-free surfaces were studied. Cells were isolated from three to six animals per group.

2.2. Solutions
The internal pipette solution contained (mM): CsOH 125, aspartic acid 125, TEACl 20, Hepes 10, ATP-Mg salt 5, EGTA 10, pH adjusted to 7.3 with CsOH. The external Na+-free, and K+-free bath solution contained (mM): CaCl2 1.8, TEACl 140, MgCl2 0.5, glucose 10, Hepes 10, and 4-AP 2, pH adjusted to 7.3 with CsOH.

2.3. Electrophysiologic studies
Experiments were conducted using standard whole-cell patch clamp techniques and an Axopatch-1D amplifier (Axon Instruments). Pipette resistances were 1–3 Mohm (when filled with internal pipette solution).

Bath temperature was maintained at 35±0.5°C. Myocytes were initially superfused (2–3 ml/min) with normal Tyrode's solution. Five minutes after membrane rupture the external solution was switched to the Na+- and K+-free solution containing Cs+. Cells stabilized for 10 min. Preliminary study demonstrated that run-down of ICa,L was minimal after 15 min of cell dialysis. Thus, data were recorded 15–20 min after membrane rupture. Membrane currents were filtered at 5 kHz and digitized at a sampling interval of 0.1 ms for whole-cell currents and 0.03 ms for capacitative transients, and stored on computer for off-line analysis. Cell capacitance was determined in the appropriate internal and external solution for the ionic current being recorded by integrating the area under the capacitive transient evoked by a 10-mV hyperpolarizing pulse and dividing this area by the voltage step. Cell capacitance did not differ among groups (range 105–144 pF). Any cell with voltage error >5 mV introduced by series resistance for a current amplitude ≥1 nA was discarded. In addition, unless a complete current–voltage and steady state inactivation protocol were recorded from the cells, the data were not included in the calculation. Voltages were not corrected for liquid junction potentials.

ICa,L current–voltage relationships were obtained using a single 250 ms depolarizing pulse to various potentials (Vts) from a holding potential VH of –40 mV at 6-s intervals. Peak ICa,L at various Vts was measured as the difference between the maximal inward peak and the level at the end of the 250-ms depolarizing voltage clamp step. The time course of ICa,L decay was characterized by fitting the current change between the inward peak and the current level at the end of the voltage step.

Steady state inactivation variables (finf) of ICa,L were determined using a double-pulse gapped protocol [13]. Potential was held at VH=–40 mV, then pulsed to a conditioning pre-pulse (VC) ranging from –70 to +20 mV for 1000 ms, returned to VH=–40 mV for 10 ms, and stepped to Vt=+20 mV (250 ms duration) at 8-s intervals.

The time course of recovery from inactivation of ICa,L was studied using a double-pulse protocol (delivered every 8 s) consisting of a 250-ms pre-pulse from a VH of –40 mV to a Vt of +20 mV, followed by a similar pulse delivered at gradually increasing inter-pulse intervals (IPI) of 10–2000 ms. Recovery at each IPI was determined by dividing peak current amplitude at each IPI by peak current of the pre-pulse. The time course of recovery for ICa,L was determined by fitting the data points to a single exponential function using a simplex method [14].


   3. Statistical analysis
Top
 Abstract
 1. Introduction
 2. Methods
 3. Statistical analysis
4. Results
 5. Discussion
 References
 
Data are reported as mean±S.E.M. One-way ANOVA was used to compare single parameters between groups. For multiple comparisons, we used a nested ANOVA. When the F-value permitted, post-hoc comparisons were performed with Bonferroni's test when variance was equal and Dunnett's test when variance was unequal [15,16]. Significance was assumed when P<0.05.

In cases where there were no significant differences among groups and the sampling size was small (n<6), power analysis [16] was performed to verify whether it was feasible to increase the n-values to test the hypothesis that the test groups are different. For example, in the determination of whether there is a difference in the epicardial and endocardial ICa,L conductance in normal male rabbits the mean variance among the groups is 0.05 and the approximate sampling size was five cells in each group. Considering the Type I error that we may reject the null hypothesis that there are no differences among groups and the Type II error of failing to reject the null hypothesis, we set {alpha}=0.05 and the power at which we can determine a difference between the groups to 95%. Under these conditions power analysis determined that with the given sampling size of five cells/group we have the power to detect a difference of 0.005 nS/pA. The difference between epicardial and endocardial ICa,L conductance in the normal male group was 0.02 nS/pA which is greater than 0.005 nS/pA. Here, sampling size is sufficient to detect a difference and statistically there is no difference in the conductance of ICa,L. In instances where power analysis was unable to detect a difference smaller than the one seen, we then ascertained the sampling size that would be necessary to test the null hypothesis. In all cases we would have had to increase the sample size by a factor of 3–4. Given that the sample distribution is normally distributed and given that the variance in the groups where n<6 is equivalent to groups in the same study where n>6 we determined that increasing the sample size by a factor of 3–4 would not decrease the variance to a level that provides us enough power to determine a significant difference between groups.


   4. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Statistical analysis
 4. Results
5. Discussion
 References
 
4.1. Hormone levels
Control male rabbits had low estradiol levels yet high levels of DHT, and control females have low estradiol and DHT levels (Table 1), consistent with reported values [17–19]. Oophorectomized females treated with placebo (OVX–PLA) had estradiol and DHT levels equivalent to control females. Placebo-treated ORCH-males (ORCH–PLA) had lower DHT levels than control males. Chronic estradiol and DHT treatment induced higher levels of estradiol in OVX–EST and ORCH–EST rabbits, and higher DHT in OVX–DHT and ORCH–DHT than in respective placebo-treated groups.


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  		Table 1 Serum levels of 17β-estradiol and 5{alpha}-dihydrotestosterone

 
4.2. L- and T-type calcium currents
ICa,L and ICa,T have been demonstrated in canine and guinea pig cardiac myocytes [20–22]. Only one Ca2+ current, ICa,L, has been reported in rabbit ventricle [23,24]. Similarly, in preliminary studies we did not detect ICa,T (data not shown).

4.3. Gender differences in peak ICa,L density current–voltage (I–V) relation
Fig. 1 shows typical peak ICa,L–voltage relationship for control female and male epicardium and endocardium, demonstrating a transmural gradient in female only. Peak ICa,L density–voltage relationships for all groups are in Fig. 2. The peak ICa,L–voltage relationship was different between epicardium and endocardium in females but not males (Fig. 2A).


Figure 1
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  		Fig. 1 Representative recordings of ICa,L from control female and male epi and endo. ICa,L was elicited by a 250-ms depolarizing step to test potentials of –20 to +15 mV from a holding potential (VH) of –40 mV. An epi-endocardial difference in ICa,L was demonstrated in females but not males. Dashes represent 0 current level.

 

Figure 2
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  		Fig. 2 Current–voltage relationship of ICa,L. (A) Transmural dispersion exists in females but not males. (B) Gonadectomy eliminates the transmural dispersion in ICa,L females and has no effect in males. (C and D) DHT and estradiol restore transmural dispersion of ICa,L in females but do not induce dispersion in males. *P<0.05 cf endo.

 
4.4. Effects of castration and chronic EST and DHT treatment on the ICa,L–voltage relationship
There was no gender difference in the ICa,L transmural gradient in castrated animals (Fig. 2B). Estradiol and DHT had no effect on the ICa,L–voltage relationship in castrated males (Fig. 2C,D). Endocardial and epicardial ICa,L–voltage relationships of estradiol- and DHT-treated ORCH-males were equivalent to placebo-treated ORCH- and control males. However, in OVX-females estradiol and DHT both restored ICa,L transmural dispersion (Fig. 2C,D), such that ICa,L density in estradiol and DHT-treated OVX-female epicardium increased relative to endocardium. Moreover, epicardial ICa,L–voltage curves from estradiol and DHT-treated OVX-animals had a slight negative shift compared to endocardium. The shift in epicardial ICa,L–voltage relationships suggests that estradiol and DHT alter activation properties of ICa,L.

Given that cell capacitances were comparable among control and castrated groups, the transmural dispersion in control females, and estradiol and DHT-treated OVX-females may result from increases in ICa,L channel density, or altered properties in the decay of peak current, steady state inactivation, steady state activation or recovery from inactivation. Hence each variable was studied.

4.5. Current decay time course
The time course of ICa,L decay was best fit by a biexponential function as previously described [13,25]. Fig. 3 shows representative tracings comparing fit of decay of peak ICa,L for control male and female epicardium and endocardium. There was no difference in ICa,L peak decay among all groups (Table 2).


Figure 3
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  		Fig. 3 Representative current tracings illustrating the biexponential fit of ICa,L decay time course for male and female endocardium and epicardium.

 

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  		Table 2 Time course of ICa,L decay and recovery from inactivation

 
4.6. Steady state inactivation of ICa,L
Steady state inactivation was obtained by a conventional two-pulse protocol (see Methods). Current elicited by the test pulse was expressed as a fraction of the maximum available current from a pre-pulse hyperpolarized to –70 mV. For each individual cell, the normalized data were fitted to the Boltzmann distribution of the form:


Formula (1)

where finf(V) is the steady state inactivation parameter, Vm is membrane voltage, V1/2 is the half-maximal inactivation potential, and k is the slope factor. Mean values for V1/2 and slope were compared (Fig. 4, inset table) and used to generate the continuous curve that fitted the average normalized data for males and females (Fig. 4, left). There were no differences in steady-state inactivation properties of ICa,L among all groups and between endocardium and epicardium of each group. Hence, steady-state inactivation properties of ICa,L do not account for the transmural dispersion in control females, and hormonally-treated OVX-females.


Figure 4
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  		Fig. 4 Steady state inactivation of ICa,L. Left: normalized steady state inactivation for female and male endocardium and epicardium. Right: half-maximal voltages (V1/2) and the slope factors (k) for steady-state inactivation of ICa,L.

 
4.7. Time course of ICa,L recovery from inactivation
Time course of ICa,L recovery was best described by a mono-exponential function, consistent with other reports [22,26]. Fig. 5A is a representative illustration of the time dependence of ICa,L recovery from inactivation. Fig. 5B shows the normalized ICa,L data plotted versus the inter-episode interval (IPI) and demonstrates no transmural difference in the time course of ICa,L recovery from inactivation in females and males. Similarly, the time course of recovery from inactivation was equivalent between endocardium and epicardium of all groups (Table 2). In addition, we did not find differences in the time course of recovery from inactivation among groups. Thus, recovery of ICa,L from inactivation does not account for the transmural differences observed.


Figure 5
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  		Fig. 5 ICa,L recovery from inactivation. (A) Representative recordings from female epicardium demonstrating time dependence of ICa,L recovery from inactivation. The protocol is shown above the record. (B) Normalized ICa,L data plotted against the inter-episode interval for males and females. There is no difference between epicardium and endocardium in females and males.

 
4.8. Steady state activation and whole cell ICa,L conductance
Based on data from current–voltage relationships, voltage dependence of ICa,L activation was estimated from the peak conductance according to Isenberg and Klockner [27] as


Formula (2)


Formula (3)

where GCa is peak conductance, ICa is peak Ca2+ current, Vrev is reversal potential of the Ca2+ current, dinf(V) is steady-state activation parameter, and GCa,max is the maximum value of GCa. Vrev was measured as the apparent zero-current potential extrapolated from the linear portion of the current–voltage relation. For each individual cell, dinf was plotted against the test voltage and was fitted to a Boltzmann distribution of the form:


Formula (4)

Average V1/2 and k values are used for comparison (inset table of Fig. 6) and to generate the continuous curve that fitted the average activation curves (left panel, Fig. 6). Steady state activation properties of ICa,L were similar among male and female endocardium and epicardium. Thus, steady state activation properties of ICa,L do not contribute to the gender difference in transmural dispersion.


Figure 6
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  		Fig. 6 Steady state activation of ICa,L. Left: normalized activation curve for OVX–EST and OVX–DHT. There is a negative shift of the activation curve in epicardium relative to endocardium. Right: half-maximal voltages (V1/2) and slope factors (k) for steady-state activation of ICa,L. *P<0.05 cf endo.

 
With respect to hormonal actions, estradiol and DHT had no effect on steady state activation in estradiol- or DHT-treated ORCH-males. However, in OVX-females treated with estradiol or DHT there was a negative shift (6–7 mV) of voltage-dependent ICa,L activation in epicardium relative to that in endocardium (Fig. 6, left panel). This shift could contribute to the greater ICa,L density of epicardium compared to endocardium.

Mean GCa,max and Vrev values are shown in Table 3. Vrev did not differ among groups. There were no differences in GCa,max among males (control, castrated and castrated treated with hormones). In addition GCa,max of ICa,L in epicardium and endocardium were equivalent in all male groups. However, maximal ICa,L conductance in female epicardium (0.18±0.02 nS/pF) was 38% greater than in female endocardium (0.13±0.02 nS/pF; P<0.05). Hence, ICa,L transmural dispersion in females is due to an increase in epicardial ICa,L conductance relative to endocardium.


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  		Table 3 Current density, reversal potential and conductance for ICa,L

 
Epicardial and endocardial whole cell ICa,L maximal conductances were equivalent in OVX–PLA. Epicardial ICa,L conductance in females was reduced after gonadectomy. DHT in OVX-females did not affect ICa,L conductance. However, chronic estradiol treatment significantly increased epicardial ICa,L conductance (0.17±0.02, nS/pF) compared to the endocardium (0.10±0.01, nS/pF; P<0.05).


   5. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Statistical analysis
 4. Results
 5. Discussion
References
 
There is transmural dispersion of ICa,L in female but not male rabbit ventricle. In the only other study of male–female differences in ICa,L LeBlanc et al. [28] used cells isolated from the whole rat ventricle rather than distinguishing epicardium and endocardium. Thus, they did not evaluate potential differences in the transmural gradient of ICa,L. Consistent with these data in the rat [28] we found that when epicardial and endocardial data are pooled there is no difference between males and females.

This transmural gradient of ICa,L is due to a greater whole-cell conductance in epicardium than in endocardium of female ventricle. This finding may explain the previously reported gender differences in the early phase of repolarization on ECG in human subjects [10]. Furthermore, the ICa,L transmural gradient in females may contribute to a greater transmural dispersion of APD in female ventricles and predispose to arrhythmogenesis [9].

5.1. Effects of gonadectomy and hormone replacement on ICa,L
Whereas gonadectomy, estradiol and DHT treatment had no effect on ICa,L density or other ICa,L properties in males, castration eliminated the transmural gradient in females. There was also a decrease in OVX-female epicardial ICa,L density that corresponded with a decrease in conductance compared to control female epicardial ICa,L conductance. This result suggests an ovarian contribution of hormonal factors important for ICa,L modulation. Assessment of hormone levels in Table 1 argues against unique involvement of estradiol or DHT because levels of these hormones in normal females are similar to those of OVX-females. Hence, additional factors contribute to the greater ICa,L dispersion in females. Nevertheless, we observed modulatory effects of DHT and of pharmacological doses of estradiol on ICa,L properties, in that chronic treatment with either hormone in OVX-females altered the voltage dependence of ICa,L activation such that epicardial ICa,L activated at more negative potentials than endocardial ICa,L. This shift in activation may determine the ICa,L transmural gradient in estradiol- and DHT-treated OVX-females. Additionally, pharmacological concentrations of estradiol increased epicardial ICa,L conductance in estradiol-treated OVX-females that may also account for the increased epicardial ICa,L density and the transmural gradient in estradiol-treated OVX-rabbits.

The epicardial conductance data concerning effects of gonadectomy and gonadal steroids are consistent with previous work in spontaneously hypertensive rat (SHR) hearts, in which gonadectomy significantly decreased the number of [3H]nitrendipine binding sites in female SHR hearts [29]. Furthermore, the number of [3H]nitrendipine binding sites in hearts from chronically estradiol-treated SHR increased compared to untreated OVX-female SHR. Chronic DHT treatment had no effect on [3H]nitrendipine binding sites compared to OVX-female SHR. Interestingly, in male SHR, gonadectomy and chronic hormone treatment did not affect [3H]nitrendipine binding sites [29].

Our data on estradiol might appear at odds with some previously reported studies of estradiol (0.27–8.1 µg/ml), which acts as an ICa,L antagonist when applied acutely to vascular smooth muscle or cardiac myocytes [30–33]. However, these concentrations substantially exceed physiologic levels of circulating estrogen (20–60 pg/ml) reported by us (Table 1) and others [18,19] in male and female rabbits. Hence, the ICa,L blockade reported is a pharmacologic effect whose relevance to the normal physiologic environment is questionable.

5.2. Mechanism of hormonal actions
Unlike the non-genomic effects of estradiol to block ICa,L rapidly and reversibly [31,32], our data, in light of results from Ishii et al. [29], suggest that chronic estradiol and DHT treatment modulate ICa,L via binding to specific intracellular receptors which in turn bind to hormone response elements and activate gene expression. Recently, Liu et al. [34] identified a functional hormone response element in the 5'-flanking sequence of the {alpha}1c-subunit gene transcription start site. The {alpha}1c-subunit gene is cardiac and vascular smooth muscle-specific, and encodes the {alpha} subunit of ICa,L. Furthermore, testosterone stimulates gene expression via this hormone response element.

Although DHT induced an increase in epicardial ICa,L density by altering the activation of ICa,L, the whole cell conductance did not increase over that of endocardium suggesting no effect on transcription. That endocardial and epicardial voltage dependence of activation differ in castrated females treated with estradiol and DHT but not in control females suggests that hormones alone can induce the gradient, but via mechanisms different from those in normal females. That epicardial and endocardial whole-cell conductance differ in castrated females treated with estradiol but not with DHT treatment suggests that estradiol and DHT may act via different mechanism to regulate ICa,L. Furthermore, that estradiol and DHT did not modulate ICa,L and that gonadectomy had no effect on the ICa,L gradient in castrated males suggest there are different ICa,L modulatory mechanisms in females and males.

Our results differ from those of an earlier study [30] in which single ventricular myocytes acutely isolated from estrogen receptor knock-out (ERKO) male mice expressed elevated dihydropyridine receptor levels with corresponding increases in ICa,L density and action potential duration. These changes correlated with an increase in QT duration in ERKO compared to wild-type mice [30]. The authors suggested that ICa,L channel density is regulated by the estrogen receptors such that a decrease in estrogen levels would increase the number of ICa,L channels, altering cardiac excitability and the risk of arrhythmia. However, these data if applied to the clinical setting suggest that premenopausal women should have shorter QT intervals and less risk of arrhythmia than men of the same age. This is not what is reported in the clinic [1–3].

5.3. Limitations
Female rabbits lack estrus cycles and their serum estradiol levels are consistently low (<100 pg/ml) [17–19]. Furthermore, gonadectomy does not alter their serum estradiol levels. Hence, the oophorectomized rabbit fails to replicate the differences in estradiol levels in menstruating women or between normal premenopausal and postmenopausal women. This restricts our ability to infer the role of variations in physiological estradiol concentrations in modulating ventricular repolarization in women. However, this model may provide insights into the role of estrogen on cardiac electrophysiology in cases where women or men have high levels of estrogen, or during post-menopausal hormone replacement therapy.

5.4. Clinical relevance
These gender-related differences in ICa,L may contribute to the longer APD and QT intervals in females which are associated with a greater propensity to drug- and congenital long QT syndrome-related arrhythmias. The ICa,L transmural gradient together with lower IKr and IK1 densities in female compared to male rabbit ventricle [5] could indeed result in prolonged repolarization and greater transmural dispersion of repolarization. These conditions in the presence of IKr-blockade could result in higher incidences of EAD in females than males. Prolonged repolarization, transmural dispersion and EAD are important for induction of TdP [35–37]. Thus, together these factors create conditions putting females at greater risk for proarrhythmic effects of drugs.

These conclusions are consistent with our previous work [9] demonstrating that female rabbits are at greater risk than males for dofetilide-induced excess APD prolongation, EAD and transmural dispersion of repolarization. The effects of this IKr-blocking drug were reversed in castrated males and females, i.e. ORCH-males were at greater risk than OVX-females for dofetilide-induced EAD and excessive APD prolongation. That ICa,L transmural dispersion in normal females was eliminated by gonadectomy suggests the ICa,L gradient in females may contribute to the proarrhythmic response to dofetilide. However, the parallelism does not follow in males in that no ICa,L gradient existed in normal males and gonadectomy had no effect on ICa,L in ORCH males. These findings also suggest gender differences in the regulatory mechanisms of repolarization.

DHT protects males against the risk for drug-induced excess APD prolongation and EAD [9]. That DHT has no effect on ICa,L in males implies that the protective action of DHT is not through changes in ICa,L but rather that DHT protective effects might be via modulation of other ionic currents that contribute to repolarization.

Time for primary review 27 days.


   Acknowledgements
 
We are grateful to Dr Michel Ferin for performing the serum steroid assays, to Dr Penelope A. Boyden for helpful discussions, to Dr Natalia Egorova for assisting with some experiments, and to Susan McMahon and Eileen Franey for their careful attention to the preparation of the manuscript. This work was supported by USPHS-NHLBI grant, HL-28958, and by funding from Procter and Gamble and Pfizer.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Statistical analysis
 4. Results
 5. Discussion
 References
 

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C. Sims, S. Reisenweber, P. C. Viswanathan, B.-R. Choi, W. H. Walker, and G. Salama
Sex, Age, and Regional Differences in L-Type Calcium Current Are Important Determinants of Arrhythmia Phenotype in Rabbit Hearts With Drug-Induced Long QT Type 2
Circ. Res., May 9, 2008; 102(9): e86 - e100.
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