Oestrogen upregulates L-type Ca²⁺ channels via oestrogen-receptor-α by a regional genomic mechanism in female rabbit hearts

Abstract

Non-technical summary

Women during their child-bearing years have longer QT intervals in their electrocardiograms than men and are more susceptible to lethal arrhythmias elicited by drugs that delay repolarization. Current theories posit that women have a reduced ‘repolarization reserve’ due to reduced potassium currents resulting in longer QT and greater repolarization delays. We proposed an alternative mechanism of higher calcium currents in women which would likewise prolong QT intervals, delay repolarization while increasing the force of contractions and intracellular calcium load. Here, we show that physiological concentrations of oestrogen increase the calcium current only in cells from the base of the heart, by increasing messenger RNA and proteins levels that encode for the calcium current. Moreover, oestrogen acts by interacting with oestrogen receptors (ER)α but not ERβ which may explain why hormone replacement therapy increases the risk of arrhythmia and offers a possible protective solution of using an oestrogen mimetic that selectively binds to ERβ.

Abstract

In type-2 long QT (LQT2), adult women and adolescent boys have a higher risk of lethal arrhythmias, called Torsades de pointes (TdP), compared to the opposite sex. In rabbit hearts, similar sex- and age-dependent TdP risks were attributed to higher expression levels of L-type Ca²⁺ channels and Na⁺–Ca²⁺ exchanger, at the base of the female epicardium. Here, the effects of oestrogen and progesterone are investigated to elucidate the mechanisms whereby I_Ca,L density is upregulated in adult female rabbit hearts. I_Ca,L density was measured by the whole-cell patch-clamp technique on days 0–3 in cardiomyocytes isolated from the base and apex of adult female epicardium. Peak I_Ca,L was 28% higher at the base than apex (P < 0.01) and decreased gradually (days 0–3), becoming similar to apex myocytes, which had stable currents for 3 days. Incubation with oestrogen (E2, 0.1–1.0 nm) increased I_Ca,L (∼2-fold) in female base but not endo-, apex or male myocytes. Progesterone (0.1–10 μm) had no effect at base myocytes. An agonist of the α- (PPT, 5 nm) but not the β- (DPN, 5 nm) subtype oestrogen receptor (ERα/ERβ) upregulated I_Ca,L like E2. Western blots detected similar levels of ERα and ERβ in male and female hearts at the base and apex. E2 increased Cav1.2α (immunocytochemistry) and mRNA (RT-PCR) levels but did not change I_Ca,L kinetics. I_Ca,L upregulation by E2 was suppressed by the ER antagonist ICI 182,780 (10 μm) or by inhibition of transcription (actinomycin D, 4 μm) or protein biosynthesis (cycloheximide, 70 μm). Therefore, E2 upregulates I_Ca,L by a regional genomic mechanism involving ERα which is a known determinant of sex differences in TdP risk in LQT2.

Introduction

The congenital form of long QT type 2 (LQT2) is caused by mutations of the K⁺ channel protein HERG that result in a loss of function of the rapid component of delayed rectifying K⁺ current, I_Kr, a prolongation of the action potential duration (APD) and QT interval (Morita et al. 2008)). Although the incidence of all forms of congenital LQT is rare (<1/5000), drug-induced LQT2 remains a serious public health problem because a wide range of cardiac and non-cardiac drugs suppress I_Kr, prolong APDs and promote early afterdepolarizations (EADs) that lead to Torsade de pointes (TdP) (Splawski et al. 2000; Vincent, 2000; Drici & Clement, 2001; Levine et al. 2008; Morita et al. 2008)). Women are known to be at higher risk to congenital and acquired forms of TdP (Makkar et al. 1993; Coker, 2008)) but in adolescents (<14 years old) before the surge of sex steroids, the risk of TdP is reversed, with boys being more susceptible to TdP (Goldenberg et al. 2008)). Rabbits exhibit the same sex differences in arrhythmia risk with adult females (>8 weeks) being more prone to TdP and the arrhythmia phenotype being reversed in young rabbits (Liu et al. 2005)) (<42 days), before the surge of steroids (de Turckheim et al. 1983)). In females, ovariectomy (OVX) reduced dofetilide-induced APD prolongation and EADs, whereas 17β-oestradiol (E2) replacement promoted EADs (Drici et al. 1996; Hara et al. 1998; Pham et al. 2001)). These studies suggest that E2 promotes TdP in female hearts.

There is general agreement that TdP is initiated by EADs that are caused by the re-activation of L-type Ca²⁺ channels. However, controversies persist regarding the mechanisms that re-activate the L-type Ca²⁺ current (I_Ca,L) during long APs and whether or not an elevation of intracellular Ca²⁺ () precedes and initiates EADs. Some studies found that the re-activation of I_Ca,L occurred spontaneously, independent of Ca²⁺ release from the sarcoplasmic reticulum (SR) because in ferret hearts, ryanodine and chelation of intracellular Ca²⁺ interrupted delayed afterdepolarizations (DADs) but did not alter EADs (Marban et al. 1986)). Alternatively, long APDs can cause an imbalance between Ca²⁺ influx and efflux, resulting in SR Ca²⁺ overload which promotes spontaneous SR Ca²⁺ release then activation of a forward-mode Na⁺–Ca²⁺ exchanger (NCX) current, I_NCX, which can depolarize the plateau potential to re-activate I_Ca,L (Volders et al. 2000)).

Dual optical mapping of APs and transients in the Langendorff rabbit model of drug-induced LQT2 revealed that adult females were more prone to EADs and TdP and that the arrhythmia phenotype was reversed in pre-pubertal hearts (Liu et al. 2005)). elevation preceded EAD upstrokes at the origins of EADs and when paced at 1.2 s cycle length, marked oscillations preceded the occurrence of EADs (Choi et al. 2002; Nemec et al. 2010)). In pre-pubertal male and adult female hearts with LQT2, EADs originated at the base and not the apex of the epicardium (Sims et al. 2008)). In freshly isolated ventricular myocytes, peak I_Ca,L density and Cav1.2α channel protein were 25–30% greater at the base than the apex of adult female and pre-pubertal male hearts (Sims et al. 2008)). Western blot analysis and voltage-clamp studies showed that the higher level of Cav1.2α at the base of the adult female heart was matched by a regional elevation of NCX and I_NCX (Chen et al. 2011)). Moreover, incubation of myocytes with oestrogen (1 nm) revealed a regional genomic upregulation of NCX mediated by oestrogen receptors, enhanced transcription and biosynthesis of NCX channel protein (Chen et al. 2011)).

Pham et al. (2002) reported a transmural dispersion of I_Ca,L in female but not in male rabbit hearts with I_Ca,L density being higher on the epicardium than the endocardium and no male–female differences on the endocardium. These findings are congruent to humans since cardiac contractility is greater in women than men (Merz et al. 1996)) and female myocytes may have greater I_Ca,L (Verkerk et al. 2005)). Our findings in rabbit hearts (Sims et al. 2008)) were consistent with previous studies (Pham et al. 2002)) and extended them by revealing marked apex–base differences of Cav1.2α and I_Ca,L densities in female but not in male rabbit hearts.

However, the mechanism(s) underlying sex differences in ion channel expression remain a matter of conjecture. Castration with and without hormone replacement in experimental animals suggested that oestrogen promotes EADs and TdP but such experiments are difficult to interpret. Sham surgeries alone cause long-lasting (>2 weeks, Tong L, Wagner R and Salama G, unpublished observations) shortening of action potential durations, castration has marked effects on all organs, bound and free serum levels of oestrogen are typically not measured (either before or after hormone replacement), nor is the serum concentration of sex hormone binding globulins (SHBG).

The present study investigates the mechanisms that upregulate I_Ca,L at the base of female hearts by investigating the effects of the predominant sex steroids 17β-oestradiol (E2) and progesterone on isolated cardiomyocytes.

Methods

Ethical approval

All protocols were first approved by the University of Pittsburgh Institutional Animal Care and Use Committee and were in accordance with the current Guide for the Care and Use of Laboratory Animals published by the National Institute of Health. In all the studies described in this article, the rabbits were first killed and the hearts were removed to isolate and culture adult ventricular myocytes for 0 to 3 days. The rabbits were obtained from an approved commercial vendor, Myrtle's Rabbitry, and were housed in the animal facilities of the University of Pittsburgh according to Federal Regulations of the USA. The authors have read and examined the rules and regulations in the UK as set by the Medical Research Council and found them to be in agreement and congruent with the policies of the University of Pittsburgh and The Journal of Physiology as stipulated in the reporting of ethical matters (Drummond, 2009)).

Cell isolation and incubation

Ventricular myocytes were isolated from adult (3 months old) male and female New Zealand white rabbits, as previously described (Sims et al. 2008)). Briefly, rabbits were anaesthetized with pentobarbital (50 mg kg⁻¹) and pretreated with heparin (200 U kg⁻¹). Hearts were excised and Langendorff-perfused with a Tyrode solution containing (in mmol l⁻¹): 140 NaCl, 5.4 KCl, 1.8 CaCl₂, 1 MgCl₂, 5.5 glucose and 10 Hepes, at pH 7.4 for 5 min, then Ca²⁺-free Tyrode solution for 10 min, after which collagenase type II (0.6 mg ml⁻¹) and 0.02% bovine serum albumin were added for a 20 min digestion at 35°C, followed by Tyrode solution containing 50 μmol l⁻¹ CaCl₂ and 0.02% BSA for 10 min. The hearts were removed and placed in a high K⁺, Kraft–Bruhe (KB) solution containing (in mmol l⁻¹): 110 potassium glutamate, 10 KH₂PO₄, 25 KCl, 2 MgSO₄, 20 taurine, 5 creatine, 0.5 EGTA, 5 Hepes and 20 glucose (pH 7.4). Sections of epicardium ∼1 mm in depth were excised from the apex (3–6 mm from the tip of the heart) and base (1–4 mm below the atrium) regions of the left ventricle then isolation proceeded separately for each region. The tissues were minced and myocytes were obtained by filtering through a 100 μm nylon mesh. Cells were allowed to settle, the supernatant was aspirated, and the pellets were washed twice and re-suspended in an incubation medium: DMEM (free of phenol red) with 5% fetal bovine serum (FBS) and 100 μg ml⁻¹ primocin (InvivoGen) for 2–72 h at 37°C. In some experiments, cells were incubated with dimethyl sulfoxide (DMSO at 1:10,000 dilution, control), 17-β-oestradiol (E₂) and/or other agents as described for each experiment.

Electrophysiology

I_Ca,L was measured with the whole-cell configuration of the patch-clamp technique with pipettes filled with (in mmol l⁻¹):130 CsCl, 20 tetraethylammonium chloride (TEA-Cl), 5 MgATP, 5 EGTA, 0.1 Tris-GTP and 5 Hepes (pH 7.2 with CsOH) (Sims et al. 2008)). The external solution contained (in mmol l⁻¹): 140 NaCl, 10 CsCl, 1 CaCl₂, 1 MgCl₂, 10 glucose and 10 Hepes (pH 7.4 with NaOH). Currents were measured with an EPC9/2 amplifier (Heka Instruments) at 36 ± 0.5°C. Cells were held at –80 mV and then –40 mV for 80 ms. Membrane currents were elicited by 200 ms steps from –30 to +60 mV in increments of 10 mV applied every 6 s. Recording were made ∼3–5 min after gaining whole-cell access and after I_Ca,L stabilized. Capacitance measurements were obtained from membrane test parameters. There were no significant differences in the capacitance of apex vs. base myocytes (Sims et al. 2008)) or when incubated with (75.3 ± 6.2 pF) or without (79.5 ± 6.7 pF) E2. Data are expressed as I_Ca,L density in pA pF⁻¹, abbreviated as I_Ca,L.

The voltage-dependent kinetics of I_Ca,L was analysed as previously described (Sims et al. 2008)). The Ca,L(g) conductance was calculated from: g = I/(V_m−V_rev), where I is the current at V_m and V_rev is the apparent reversal potential. The ratio of g/g_max (where g_max is the maximum conductance) was plotted against V_m. The relationship between g/g_max and V_m was fitted to a Boltzman function: g/g_max= 1/ (1 + expµ(V_0.5– V_m)/k½), where V_0.5 is the half-maximum activation voltage and k is the slope factor of the steady-state activation curve. To measure the voltage-dependent inactivation, the normalized current at the test potential (I/I_max) was plotted against V_m. The curve was fitted to a Boltzman function: I/I_max= 1/(1 + exp µ(V_0.5– V_m)/k), where V_0.5 is the half-maximum activation voltage and k is the slope factor of the steady-state inactivation curve. The activation kinetics of I_Ca,L was measured for each depolarizing step as the time from the onset of the voltage step to the peak current. To determine the time-course of inactivation, the decay phase of I_Ca,L was fitted to a bi-exponential function: I = A₁× exp(–t/τ_fast) + A₂× exp(–t/τ_slow) + A₀, where τ_fast and τ_slow are the time constants of the slow and fast exponential components; A₁ and A₂ are the amplitudes of the fast and slow exponential components; and A₀ indicates the amplitude of the sustained component.

Immunocytochemistry

Cav1.2 (α1C) protein labelling and imaging in cardiac myocytes was performed as described previously (Zhang et al. 2005)). Myocytes were incubated on laminin-coated coverslips for 1 day, fixed with 2% paraformaldehyde and permeabilized (0.1% Triton-X 100). Cav1.2 (α1C) monoclonal antibody (1:200, Alomone Labs: catalogue no. ACC-003) was incubated for 60 min then with goat anti-rabbit Alexa 488 (5 μg ml⁻¹, Molecular Probes). Immunofluorescence was analysed with an Olympus-1000 Fluoview confocal microscope and Metamorph (Version 7.1, Molecular Devices).

Single-cell mRNA levels by quantitative real-time RT-PCR

Cav1.2 α mRNA level in single cells was measured by reverse transcription polymerase chain reaction (RT-PCR) as previously described (Schultz et al. 2001; Parhar et al. 2003)). Briefly, the content of each myocyte was aspirated in a micropipette filled with 6 μl of RNase-free water and was then ejected into a PCR tube (200 μl) and stored in liquid nitrogen until use. The reverse transcriptase reaction was done according to manufacturer's instructions (Invitrogen catalogue no. 18064022). For the first round PCR (volume 20 μl), each mixture contained: 7 μl of RT product, 10 μl of 2× Redmix Plus (including Taq, dNTP and MgCl₂) and 0.5 μm of primer. The cycle condition was as follows: 94°C 5 min; 94°C 30 s, 58°C 30 s, 72°C 30 s, 35 cycles and 72°C 7 min. The reaction mixture (20 μl) for real-time PCR was as follows: 2× SYBR Green PCR master mix (Applied Biosystems) 10 μl, 0.25 μm primers and 6 μl of dsDNA from first round PCR product. The real-time PCR reaction was performed using the ABI PRISM 7000 Sequence Detection System under the conditions: 95°C for 10 min, followed by 75 cycles at 95°C for 15 s and 60°C for 1 min. The primers for rabbit Cav1.2 were: forward: 5′ CCT GTT TGG CAA CCA TGT CA 3′; and reverse: 5′ GTG GGC GCT GCG TAG TG 3′. (Armoundas et al. 2007)). GAPDH was used as internal reference to normalize the relative mRNA level.

Western blots

Tissues were dissected from the apex (3 mm from the tip) and base (3 mm below the left atrium) of the epicardium of the left ventricular free wall and stored in liquid nitrogen. Samples were pulverized in liquid nitrogen and homogenized on ice. Lysis buffer contained (in mm): 150 NaCl, 1 EDTA, 2.5 MgCl₂, 20 Hepes, 1% Triton-X 100, 0.5 dithiothreitol and 0.1% SDS. Protease inhibitors and phosphatase inhibitor cocktail (PhosSTOP, Roche) were added to the lysis buffer. The supernatant resulting from centrifugation (120,000 g for 5 min) was used to measure protein concentration with the BioRad assay (BioRad Laboratories, Hercules, CA, USA) and run on SDS-PAGE gels with 30 μg of protein per lane. ERα antibody (Thermo Scientific catalogue no. MA3-310) and ERβ (Santa Cruz catalogue no. sc-53494) antibody were used at 1:1000 dilutions.

Data analysis

Clampfit 9.0, SPSS13.0 and Sigmaplot11.0 were used for data analysis. All I_Ca,L were normalized for cell capacitance (i.e. pA pF⁻¹) to allow comparison between cells of various sizes. All data were expressed as mean ± SEM. Individual group statistical comparison were analysed by unpaired Student t test with Bonferroni correction, and multiple group comparisons were evaluated by 2-way ANOVA. A probability value of P < 0.05 was considered statistically significant. Each experimental group is described as n/N, where n is the number of cells and N is the number of hearts.

Results

Effect of oestrogen on I_Ca,L

Left ventricular myocytes isolated from the epicardium, base and apex, were incubated in DMSO (1/10,000, controls) or E2 (1 nm) and the current density-to-voltage (I–V) relationship was measured for I_Ca,L from freshly isolated myocytes (2–4 h) on days 0, 1, 2 and 3. As shown in Fig. 1A, overnight incubation with E2 significantly increased I_Ca,L in base myocytes at –10 to +20 mV (P < 0.05) compared to controls that were treated with DMSO (1/10,000). E2 incubation increased I_Ca,L without altering the shape of I–V plots. On days 2 and 3, E2 increased I_Ca,L further whereas in the absence of E2, I_Ca,L decreased (Fig. 1B and C)). As summarized in Fig. 1D, E2 treatment significantly increased peak I_Ca,L (0 mV) on day 1 (10.69 ± 0.67 pA pF⁻¹, controls, n/N = 17/4; 14.19 ± 0.92 pA pF⁻¹, E2, n/N = 13/4; P < 0.01). The higher I_Ca,L with E2 incubation remained statistically significant on day 2 (9.57 ± 0.53 pA pF⁻¹ control, n/N = 17/4 vs. 14.46 ± 1.11 pA pF⁻¹, E2, n/N = 15/4, P < 0.01) and day 3 (8.00 ± 0.62 pA pF⁻¹ control, n/N = 11/2 vs. 15.99 ± 1.25 pA pF⁻¹, E2, n/N = 8/2, P < 0.01). Moreover, I_Ca,L had a tendency to decrease in base myocytes incubated in E2-free medium and this decrease was statistically significant on day 3 compared to days 0, 1 and 2 (P < 0.05) for myocytes in the control groups. Note that on day 0 (2–4 h post-isolation), base myocytes had the same I_Ca,L± E2, because E2 did not acutely alter I_Ca,L.

Figure 1. Effect of E2 on I_Ca,L in female base and apex myocytes.

A–C, I–V plots for I_Ca,L measured in female base myocytes incubated in the absence (filled circles) or presence (open circles) of E2 (1 nm) for 1 (A), 2 (B) or 3 days (C), respectively; †P < 0.05, ‡P < 0.01 E2 vs. control. D, summary of mean peak I_Ca,L (at 0 mV) in female base myocytes incubated ± E2 (1 nm) in freshly isolated cells and after 1, 2 and 3 days. E, I–V plots of I_Ca,L in female apex myocytes incubated in the absence (filled squares) or presence (open squares) of E2 (1 nm) for 1 day. †P < 0.01 for E2 on day 2 and 3 vs. without E2 on days 0, 1, 2 or 3; ‡P < 0.01 E2 vs. control. F, summary of mean I_Ca,L± SEM at 0 mV in female apex myocytes incubated ± E2 (1 nm) for 1, 2 and 3 days. E2 had no significant effect on I_Ca,L at the apex.

Myocytes from the apex had stable I_Ca,L from 2–72 h and treatment with E2 (1 nm) did not alter I_Ca,L. I–V plots recorded on day 1 were virtually identical for control and E2-treated cells from the apex (Fig. 1E)). The summary histogram (Fig. 1F)) shows that peak I_Ca,L did not change ± E2 incubation for apex myocytes (8.35 ± 0.44 pA pF⁻¹ in control, n/N = 19/4 vs. 8.16 ± 0.44 pA pF⁻¹, E2, n/N = 17/4, P > 0.05).

We also tested the effect of E2 on I_Ca,L in female myocytes isolated from the base of the endocardium (Fig. 2A and B)) (n/N = 9/2 for each ± E2 group) and in male myocytes isolated from the apex and base of the epicardium (n/N = 9/2 for each ± E2 group) (Fig. 2C and D)). In both cases, E2 had no significant effect on the I–V relationship of I_Ca,L after 24 h incubation with E2 (Fig. 2)).

Figure 2. Effect of oestrogen on I_Ca,L density of female epicardial vs. endocardial base myocytes and male epicardial apex and base ventricular myocytes.

Myocytes were isolated from the epicardium and endocardium of the base of female hearts and from the epicardium at the base and apex of male rabbit hearts. The myocytes were incubated with or without E2 (1 nm) for day 1 then I_Ca,L density was analysed. A, I–V plots for I_Ca,L measured in female base epicardial and endocardial myocytes incubated in the absence (filled circles) or presence (open circles) of E2 (1 nm) for 1 day. B, summary of mean I_Ca,L± SEM at 0 mV in female base epicardial and endocardial myocytes incubated ± E2 (1 nm) for 1 day. C, I–V plots for I_Ca,L measured in male apex and base epicardial myocytes incubated in the absence (filled circles) or presence (open circles) of E2 (1 nm) for 1 day. D, summary of mean I_Ca,L± SEM at 0 mV in male epicardial myocytes isolated from the base and apex, incubated without (filled columns) and with (open columns) E2 (1 nm) for 1 day.

Thus, oestrogen was found to upregulate I_Ca,L in the epicardium at the base of female hearts and when myocytes are isolated, I_Ca,L declined in an E2-free environment (Fig. 1A–D)). In contrast, E2 did not alter the density of I_Ca,L in female apex myocytes (Fig. 1E and F)) and female base endocardial cells (Fig. 2A and B)). Similarly, male epicardial cells (Fig. 2C and D)) were impervious to E2.

Concentration-dependent effects of oestrogen and progesterone

Various oestrogen and progesterone concentrations were tested on female epicardial base myocytes. Figure 3A shows I–V plots for I_Ca,L measured after a 1 day incubation with different E2 concentrations (10, 100 or 1000 pm). E2 at 100 and 1000 pm increased the current density without altering the shape of I–V plots. A summary histogram shows that 10 pm E2 did not alter I_Ca,L (10.59 ± 0.84 pA pF⁻¹ control vs. 11.07 ± 0.77 pA pF⁻¹, n/N = 10/2 for each group, P > 0.05) whereas 100 and 1000 pm produced statistically significant increases in I_Ca,L (12.90 ± 0.46 pA pF⁻¹, 100 pm E2 and 14.16 ± 0.9 pA pF⁻¹, 1 nm E2; n/N = 10/2 for each group, P < 0.05 vs. control) (Fig. 3B)). Thus, E2 increased I_Ca,L in a concentration-dependent manner in the physiological range for female rabbits (0.1–1 nm) (Bahr et al. 1976)).

Figure 3. Effect of oestrogen and progesterone on I_Ca,L.

A, I–V plots of I_Ca,L recorded from female base cardiomyocytes incubated without (filled circles) or with 17-β-estradiol (E2 = 10 (open squares), 100 (filled squares) or 1000 (open circles) pm) for 1 day. B, summary of mean I_Ca,L (0 mV) in female base cardiomyocytes incubated without (control) or with E2 (10, 100 or 1000 pm) for 24 h, †P < 0.05, ‡P < 0.01 vs. control. C, I–V plots of I_Ca-L recorded from female base cardiomyocytes incubated without (open squares) or with progesterone (P4 = 0.1 (open circles), 1 (filled circles) or 10 (open squares) μm) for 1 day. Progesterone (1 and 10 μm) did not alter the I–V plots for I_Ca,L even after incubating female base cardiomyocytes for 1.5 and 2 days. D, summary of mean I_Ca,L (0 mV) in female base cardiomyocytes incubated without (control) or with progesterone (P4 = 100 nm, 1 μm or 10 μm) for 1 day.

Progesterone (P4) was similarly tested as a possible mechanism to modulate I_Ca,L. I–V plots measured from controls and myocytes incubated with P4 (0.1, 1 and 10 μm) for 1, 2 and 3 days did not change I_Ca,L despite concentrations of P4 well above physiological levels in female rabbits (6.5 nm) (Bahr et al. 1976)). Figure 3C and D illustrates the lack of progesterone effect after 1 day of incubation.

Role of oestrogen receptors

E2 can regulate cell function through classical or non-classical genomic pathways that are mediated by oestrogen receptors (ERs) or through non-genomic pathways that bypass ERs (Bjornstrom & Sjoberg, 2005; Levin, 2005)). The role of ERs on I_Ca,L regulation was tested in female base cardiomyocytes incubated with E2 (1 nm) ± the ER antagonist, Fulvestrant (ICI 182,780, ICI, 10 μm) for 1 day. I–V plots from ICI, E2 and E2 + ICI-treated myocytes showed that ICI suppressed the effect of E2 on I_Ca,L (Fig. 4A)). I–V plots from control (see Fig. 1A)) and ICI-treated myocytes were indistinguishable. Peak I_Ca,L density (0 mV) was not significantly different for control and E2 + ICI-treated myocytes which were lower than in E2-treated cells (10.44 ± 0.91 pA pF⁻¹, E2 + ICI vs. 14.25 ± 1.08 pA pF⁻¹, E2, n/N = 9/2, P < 0.05). Although E2 acted via ERs, Fulvestrant did not identify the ER subtype, ERα or ERβ, since it is not a selective antagonist (Robertson, 2001)). To identify the ER subtype, cardiomyocytes were incubated with a selective agonist for ERα (PPT, 5 nm) and/or ERβ (DPN; 5 nm). Figure 4C shows the I–V relationships for four groups of female base myocytes: controls, PPT, DPN and PPT + DPN-treated cells. PPT but not DPN significantly increased I_Ca,L in the voltage range of –20 to +20 mV (Fig. 4C)). Peak I_Ca-L were significantly higher in PPT compared to controls and DPN-treated myocytes (15.29 ± 0.74 pA pF⁻¹, n/N = 18/4 PPT vs. 11.35 ± 0.63 pA pF⁻¹ controls, n/N = 30/5, P < 0.01) and 12.65 ± 0.60 pA pF⁻¹, n/N = 18/4, P < 0.01). I_Ca,L also increased in myocytes treated with both PPT and DPN for 24 h (17.02 ± 0.77 pA pF⁻¹, n/N = 12/2, P < 0.01 vs. controls). No statistically significant differences were found for peak current between PPT and PPT + DPN-treated cells (P > 0.05), suggesting that ERα is the major ER subtype involved in I_Ca,L. upregulation.

Figure 4. Effect of ER antagonist and agonists on cardiac I_Ca,L.

A, superposition of I–V plots from female base myocytes, controls (open squares), with E2 (open circles) (1 nm) or E2 ± ER antagonist (filled squares) ICI 182,780 (ICI = 1 μm). †P < 0.05 E2 vs. ICI and ICI + E2. B, summary histograms of mean ± SEM of I_Ca,L (0 mV) in female base myocytes controls, treated with E2 or E2 + ICI, ‡ and †P < 0.05. C, superposition of I–V plots from female base myocytes, controls (open circles), incubated with ERα agonist (open squares) PPT (5 nm), ERβ agonist (filled circles) DPN (5 nM) or PPT + DPN (filled squares) for 1 day. D, summary of mean ± SEM of I_Ca,L (0 mV) at female base myocytes in each group. ‡P < 0.05 PPT or PPT + DPN vs. control, †P < 0.05 PPT vs. DPN, NS: PPT vs. PPT + DPN.

We tested the regional distribution of ERα and ERβ to test the possibility that the regional effects of E2 were caused by non-uniform expression levels of ERs. Figure 5A shows the protein distribution of ERα or ERβ from four female and four male rabbit hearts where epicardial tissues were taken from the apex and base areas of the left ventricles. When normalized with respect to β-actin, ERα or ERβ protein densities were not significantly different as a function of sex or location. A summary histogram of ERα and ERβ protein density confirmed the uniform distribution of ERs. Thus, the different responses of epicardial myocytes from the base and apex to E2 cannot be explained based on a non-uniform distribution of ERs.

Figure 5. Regional distributions of ERα and ERβ at the base and apex of male and female hearts.

Aa, Western blots for ERα (top lanes) and ERβ (middle lanes) are shown for 8 tissue samples, 4 samples from the apex (A1–4) and from the base (B1–4) of female hearts 1–4 and these are aligned with 8 corresponding samples of β-actin (bottom lanes). Ab, the same experiment as in Aa but with tissues from male hearts. B, summary histograms of the relative density of ERα and ERβ normalized with respect to β-actin. The density of each ER did not significantly vary between apex vs. base or between male vs. female hearts.

Effects of E2 on I_Ca,L activation and inactivation properties

E2 may regulate I_Ca,L by cell signalling mechanisms involving PKA or CAMKII-dependent phosphorylation or changes in ancillary peptides that interact with main channel proteins (α subunit), which tend to shift the I–V relationship and alter gating properties (Mikala et al. 1998; Blaich et al. 2010)). Although Figs 1–3 showed that E2 increased I_Ca,L without shifting the I–V relationship, the effect of E2 (1 nm) on the kinetics of I_Ca,L was measured in female base myocytes treated with or without E2 after 1 day (n/N = 9/2 for each group) (Fig. 6A)). As shown in Fig. 6B, there were no significant differences in the half-maximum activation voltage (–7.52 ± 1.30, E2 vs. –5.83 ± 2.40 mV, controls, P > 0.05) and the slope factor of the steady-state activation curve (4.34 ± 0.55, E2 vs. 4.44 ± 0.26, controls, P > 0.05). The half-maximum inactivation voltage was similar in E2-treated and control cells (–17.10 ± 0.40 mV, E2 vs. –16.19 ± 0.64 mV, controls, P > 0.05) and the slope factor of the steady-state inactivation curve between both groups (–4.00 ± 0.13, E2 vs. –4.07 ± 0.29, controls, P > 0.05).

Figure 6. Effect of E2 on voltage dependence and kinetics of I_Ca,L.

A, representative traces of I_Ca,L from female base myocytes incubated without (filled circle) or with 1 nm E2 (open circle) for 1 day. B, steady-state I_Ca,L activation and inactivation curves, female base myocytes incubated without (filled circles) or with E2 (open circles) for 1 day. C, activation kinetics of I_Ca,L expressed as the time to peak, from onset of voltage step to the peak of current amplitude are plotted vs. V_m (mV), female base myocytes incubated without (filled circles) or with E2 (open circles) for 1 day. D, voltage dependence of the time-courses of inactivation: τ_fast (circles) and τ_slow (triangles), female base myocytes incubated without (filled symbols) or with E2 (open symbols) for 1 day.

The time to peak measured at different membrane potentials did not significantly change by E2 treatment for 1 day (Fig. 6C, P > 0.05). Both the fast and slow components of I_Ca,L at different voltage steps were similar ± E2 (Fig. 6D)). At 0 mV, τ_fast was 12.40 ± 1.41 ms in control and 10.99 ± 1.97 ms in E2; τ_slow was 56.48 ± 6.23 ms in control and 52.44 ± 7.73 ms in E2 (P > 0.05). These data demonstrated that E2 increased I_Ca,L without altering gating properties.

E2 and Cav1.2α protein and mRNA levels

To test if E2 enhanced I_Ca,L by stimulating de novo expression of L-type Ca²⁺ channel protein, Cav1.2α was measured by immunocytochemistry on myocytes from the base and the apex treated with E2 or DMSO for 1 day. Figure 7A shows the antibody labelling of Cav1.2 (α1C) protein on an isolated cardiomyocyte. When myocytes from the apex were cultured for 1 day, the levels of Cav1.2 (α1C) were similar if incubated with (Fig. 7B)) or without E2 (Fig. 7A)). However, the intensity of Cav1.2 (α1C) was considerably higher in base myocytes treated with E2 (Fig. 7D)) compared to base myocytes incubated without E2 (Fig. 7C)). The statistical significance analysed by quantitative measurements of fluorescence intensity of confocal images is summarized in Fig. 7E. Cav1.2 (α1C) label was measured from apex myocytes (n/N = 27/3) and was set arbitrarity to 100% and had an SEM of 5.11%. When incubated with E2, apex myocytes (n/N = 21/3) had intensities of 102.45 ± 3.58%. Control myocytes from base (n = 16/3) had intensities of 110.77 ± 0.95% which increased to 127.59 ± 0.56% when incubated with E2 (P < 0.01, control base vs. E2 base). The relative increase of channel protein caused by incubation with E2 (Fig. 7D)) is consistent with the increase in peak I_Ca,L.

Figure 7. Effect of oestrogen on Cav1.2α levels.

Representative immunocytochemistry of Cav1.2α in apex female myocytes incubated 1 day without (A) and with (B) E2 (1 nm). C and D, as for A and B but with myocytes from the base. E, summary of relative Cav1.2α intensity from control apex (A), E2-treated apex (B), control base (C) and E2-treated base (D) myocytes. †P < 0.05 E2 base vs. control base. F, quantitative real-time RT-PCR showed that E2-treated myocytes had higher mRNA levels (E2: 1.297 ± 0.123, n = 6, 2 hearts) compared to control (Ctrl: 0.988 ± 0.085, n = 6, 2 hearts. †P < 0.04 compared to Ctrl).

Single-cell quantitative real-time RT-PCR was used to determine whether E2 increased the expression levels of Cav1.2 (α1C) by a genomic mechanism which would be associated with an increase in mRNA levels. Female base myocytes were incubated with or without E2 (1 nm) for 1 day (n/N = 6/2 for each group) followed by quantitative real-time RT-PCR analysis. As shown in Fig. 7F, mRNA levels were 30% more elevated in myocytes treated with E2 compared to controls (P < 0.04).

Inhibition of transcription or translation suppresses I_Ca,L upregulation

The E2 upregulation of Cav1.2 expression (Fig. 7)) may occur via an increase of mRNA and de novo protein biosynthesis or enhanced translation but stable transcription. Female base cardiomyocytes were incubated with E2 (1 nm) and either actinomycin D (AmD, 4 μm) or cycloheximide (CHX, 70 μm) for 24 h to respectively block transcription or translation, as previously described (Benitah & Vassort, 1999)). I–V relationships for E2-treated myocytes, E2 + AmD and E2 + CHX are superimposed in Fig. 8A and B. Peak I_Ca,L are summarized in Fig. 8C. Overnight incubation with E2 increased I_Ca,L in base myocytes (–14.80 ± 1.12 pA pF⁻¹, E2 vs. –11.18 ± 0.77 pA pF⁻¹, controls, n/N = 7/2 for both groups,P < 0.05). E2 treatment resulted in a 32.6% increase of peak I_Ca,L. AmD or CHX alone had no effect on basal I_Ca,L (without E2), in agreement with findings by Benitah & Vassort (1999). However, AmD or CHX blocked the upregulation of I_Ca,L by E2 (–10.77 ± 0.89 pA pF⁻¹, AmD + E2, –10.86 ± 0.82 pA pF⁻¹, CHX + E2, n/N = 9/2). Thus, the increase in cardiac I_Ca,L after incubation with E2 involves a genomic upregulation.

Figure 8. Inhibition of transcription or translation during E2-induced up-regulation of I_Ca,L.

A, superposition of I–V plots from female base myocytes incubated with DMSO (control, filled circles) (dilution 1:10,000), E2(open circles) (1 nm), actinomycin D (filled squares) (AmD, 4 μm) or AmD + E2 (open squares) for 1 day. †P < 0.05. B, superposition of I–V plots from female base myocytes incubated with DMSO (control, filled circles) (dilution 1:10,000), E2 (open circles; 1 nm), cycloheximide (filled squares; CHX, 70 μm) or CHX + E2 (open squares) for 1 day. †P < 0.05. C, summary histograms of mean ± SEM of I_Ca-L (0 mV) in female base myocytes in each group for 1 day. †P < 0.05 E2 vs. control

Discussion

The main findings are that oestrogen but not progesterone upregulates I_Ca,L in cardiomyocytes isolated from the base but not the apex, which accounts for the regional and sex heterogeneities of I_Ca,L that were reported in intact female rabbit hearts. E2, at concentrations as low as 0.1 nm, upregulated I_Ca,L at the base of the female epicardium by a genomic mechanism mediated by ERα but not ERβ. E2 did not change I_Ca,L kinetics or gating properties and increased the mRNA and protein levels of Cav1.2α. Consistent with these findings, the upregulation of I_Ca,L by E2 was blocked by the inhibition of transcription or translation. E2 (1 nm) elicited similar increases of channel protein and of peak I_Ca,L without altering activation or inactivation kinetics of I_Ca,L, which supported the interpretation that the increase in current was due to an increase in the copy number of functional channels. Put together, these data provide compelling evidence that E2 upregulates L-type Ca²⁺ channels by a regional genomic mechanism.

Effects of E2 on I_Ca,L and TdP risk are closely related

Animal studies showed that OVX reduces the risk of dofetilide (I_Kr blocker)-induced EAD (Pham et al. 2001)), and E2 replacement in OVX rabbits increased the incidence of drug-induced EAD (Drici et al. 1996)). A similar finding was reported in papillary muscles of OVX rabbits (Hara et al. 1998)). Optical mapping of APs and from Langendorff rabbit hearts with LQT2 revealed that aberrations of handling at the base of the epicardium was associated with higher I_Ca,L density, which played a major role in the induction of EADs and TdP (Choi et al. 2002; Liu et al. 2005; Sims et al. 2008; Nemec et al. 2010)). E2 and I_Ca,L levels were known to be important determinants of arrhythmia risk in LQT2; the current study shows that E2 upregulates I_Ca,L by a regional genomic mechanism mediated by ERα. In addition, we recently showed that E2 upregulates the Na⁺–Ca²⁺ exchange (NCX1) current, I_NCX, by increasing mRNA levels and protein expression of NCX1 by a regional genomic mechanism similar to that shown here for Cav1.2α (Chen et al. 2011)). Moreover, we showed that the higher levels of I_Ca,L and I_NCX at the base of female hearts account for sex differences in LQT2-related arrhythmias (Liu et al. 2005; Sims et al. 2008; Chen et al. 2011)) and since both currents are upregulated by E2, E2 underlies sex-related arrhythmogenic effects. The greater susceptibility of women to ventricular arrhythmias has been attributed to a ‘reduced repolarization reserve’ mostly due to a greater suppression of I_Kr compared to men (Roden, 2006)). However, I_Kr blockade alone may not be sufficient to trigger EADs. However, when combined with a higher I_Ca,L, female hearts have a greater likelihood of intracellular Ca²⁺ overload, spontaneous Ca²⁺ release from the sarcoplasmic reticulum and EADs caused by the reactivation of I_Ca,L during the AP plateau due a -mediated enhanced depolarizing Na⁺–Ca²⁺ exchange current. Consistent with this interpretation is that APD prolongation is not a good predictor of TdP vulnerability. For instance, male pre-pubertal rabbit hearts were shown to be more susceptible to EADs and TdP in drug-induced LQT2, despite having shorter APDs than pre-pubertal females (Liu et al. 2005)). Moreover, pre-pubertal male base myocytes had greater I_Ca,L density than females implicating overload as a major determinant of EADs and TdP rather than APDs (Sims et al. 2008)).

Genomic regulation in myocyte cultures

The genomic regulation of cardiac ion channels was studied by incubating adult primary cardiomyocyte cultures with E2 which increased Cav1.2α levels and I_Ca,L whereas progesterone did not. Issues regarding the stability of cardiomyocytes in culture have been addressed in several studies of cell signalling and regulation of ion channel expression. For instance, rabbit cardiac I_Ca,L were shown to remain stable for 4 days even though contractility decreased (Mitcheson et al. 1997, 1998)). Cardiac ATP, CrP and LDH levels have been found to remain stable in long-term culture (Decker et al. 1990)) and mouse cardiomyocytes had normal signalling responses for 3 days (Sambrano et al. 2002)). Here, the effects of E2 were tested for up to 3 days post-isolation but most of the E2-dependent changes occurred in 1 day, the time needed to obtain significant genomic upregulation of I_Ca,L. Our cultured cardiomyocytes retained their differentiated state for 3 days based on whole-cell membrane capacitance (Chen et al. 2011)), organization of T-tubules and stability of I–V plots. Moreover, I_Ca,L measured from the apex remained stable over 3 days post-isolation and for each heart, data from the apex served as internal control from changes in I_Ca,L measured at the base. It should be noted that the stability of I_Ca,L density has been traditionally used as a measure of phenotype stability for cultured adult myocytes, with varying results. The current data provide compelling evidence that I_Ca,L density alone is not sufficient to measure phenotype change of adult myocytes and close attention must be paid to the age, sex and location within the heart of cardiac myocytes.

Regional genomic regulation of Cav1.2α

It is unlikely that E2 acts by non-classical, transcription-independent effects of ER or by causing the indirect phosphorylation of Cav1.2α, because the elevation of peak I_Ca,L by E2 required ∼1 day of incubation in E2. Acute effects of E2 and progesterone have been reported to decrease I_Ca,L. E2 at 300 nm–10 μm decreased I_Ca,L by 15–20% acutely in human atrial, guinea pig and rat ventricular myocytes (Meyer et al. 1998)) and P4 (40 nm) suppressed the cAMP-dependent activation of I_Ca,L in guinea pig myocytes (Nakamura et al. 2007)). However, the steroid concentrations used in many studies tend to be significantly higher than the maximum physiological concentrations of these steroids and most studies ignored the binding of steroids to sex hormone binding globulins (SHBG) such that the concentrations of ‘free’ steroids are ∼1% of total steroid levels. In rabbits, the bio-available concentrations of E2 are considerably lower than the often stated ‘total physiological’ concentration (E2, ∼0.3 nm; Bahr et al. 1976)) because of high-affinity binding to SHBG (Araujo et al. 2008)). In our pilot studies using rabbit ventricular myocytes from the base of the epicardium, E2 and P4 at up to 10 μm failed to impart an acute effect on I_Ca,L.

We presented compelling evidence that E2 regulates I_Ca,L through a genomic mechanism that involves stimulation of transcription: (1) E2 acted in ∼1 day, too slowly to suggest a direct interaction or a cell-signalling mechanism; (2) E2 could act by ER-dependent or -independent mechanisms (Bjornstrom & Sjoberg, 2005; Levin, 2005)); however, the inhibition of E2 regulation of I_Ca,L by fulvestrant, an antagonist of ERα and ERβ, demonstrates that the mode of action of E2 occurs via one of its nuclear receptors. ERα and ERβ have been shown to yield different and sometimes opposing effects on cell function (Matthews & Gustafsson, 2003)), but our results show that an ERα agonist mimics the effects of E2 and ERβ has no significant effect on I_Ca,L. The latter implicates the ERα subtype as in the receptor mediating E2-dependent upregulation of Cav1.2α.

In silico analysis of the promoter region of human CACNA1C using BIOBASE Knowledge Library detected eight high-probability oestrogen receptor ERα binding sites within the first 500 nucleotide promoter region. The analysis suggests that ERα (but not ERβ) regulates the transcription of the gene that encodes for Cav1.2α in human hearts. The analogous search for ER binding sites was run on the rabbit CACNA1C promoter sequence (500 nucleotides before the start codon), obtained from the UCSC Genome Bioinformatics data base (at http://genome.ucsc.edu). The rabbit promoter sequence was transported to BIOBASE: EXPLAIN to search for ERα and ERβ binding sites and as for the human CACNA1C sequence, eight ERα (and no ERβ) response elements were detected at analogous positions within the rabbit promoter sites as for the human promoter sites. Besides the promoter region of CACNA1C, the search was carried out in the large intron found in both the human and rabbit CACNA1C. In both introns, 49 high-quality ERα binding sites were identified from an in silico analysis; these are putative sites of interaction of ERα and CACNA1C.

Evidence of direct interactions between CACNA1C and ERα was reported through chip-on-chip analysis of the human CACNA1C, which is available from the BIOBASE Knowledge Library. The latter analysis detected the same eight high-quality ERα binding sites in the promoter region and none were found in the intron in human MCF-7 cells (Welboren et al. 2009)). Chip-on-chip analysis provides compelling evidence of ERα genomic regulation of the human gene but such a study has not been carried out in the rabbit. The analysis of the rabbit sequence suggests that there are putative ERα binding sites but does not provide proof that ERα binding does occur and results in functional oestrogen response elements. Nevertheless, the prevalence of high-affinity ERα binding sites in analogous regions of the human and rabbit CACNA1C gene and chip-on-chip analysis that identified these ERα as biologically active supports the notion of a direct genomic regulation of Cav1.2α by ERα. Further studies will be needed in the rabbit to identify possible regulatory sites in CACNA1C introns and bioactive sites will have to be demonstrated by GENE SEEK ChIP assays performed in rabbit heart.

Both ERs can mitigate the response to vascular injury (Kim & Levin, 2006)) but in mice, only ERα appears to protect the heart from ischaemic injury (Wang et al. 2006)). In the ERα knockout mouse, I_Ca,L and Cav1.2 were upregulated (Johnson et al. 1997)), which is opposite and contrary to the positive transcriptional response of these genes to E2 in rabbits. Several factors could account for the different results: (a) E2 regulation of I_Ca,L and Cav1.2 appears to be restricted to specific regions of the heart, which was not investigated in the mouse, (b) species differences in regulation of this current are likely since the mouse and rabbit action potential differ significantly from endocrine and electrophysiological points of view, and (c) distinct transcriptional elements that control the expression of Cav1.2α may be differentially activated in mice and rabbits. Among the conserved cardiac transcriptional elements Nkx2.5, NFAT and CREB are found in the promoter region of the rat CACNA1C gene (Liu et al. 2000; Dai et al. 2002)). It is possible that E2ER suppression of Nkx2.5 and NFAT activation, which has been observed in other cell types (Qin et al. 2008; Marni et al. 2009)), may be responsible for reduced expression of Cav1.2α in E2-treated cells. In contrast, activation of CREB acts in the opposite direction by increasing Cav1.2α expression (Mayr & Montminy, 2001)). Ongoing studies in our group show that CREB activation is 2- to 3-fold greater at the base than the apex of female hearts and of male hearts. It is interesting to speculate that the regional CREB activation plus E2–ERα complex may be important to upregulate Cav1.2α expression. Further studies will be needed to determine the mechanisms underlying the regional transcriptional regulation of Cav1.2α or why E2 can upregulate I_Ca,L and I_NCX in myocytes from the base but not the apex of the epicardium.

Nevertheless, the upregulation of L-type Ca²⁺ channels and of Na⁺–Ca²⁺ exchange protein (Chen et al. 2011)) by E2 contribute to sex differences in Ca²⁺ handling which is accentuated during repolarization delays in LQT2 and underlie the propensity to EADs and arrhythmia risk (Sims et al. 2008)). Their role may have been missed because of a lack of investigation of apex–base regional heterogeneities of ion channel expression. These sex differences of I_Ca,L in rabbits may apply to human hearts where a trend towards higher I_Ca,L was reported in women than in men (Verkerk et al. 2005)). Our findings bring new insights to the actions of sex steroids, their role in the cardiac ion channel remodelling and implicate ERs as novel targets in the treatment of ventricular arrhythmia.

Glossary

Abbreviations

AmD

actinomycin D

APD

action potential duration

Ca,L

L-type calcium channel

CHX

cycloheximide

DPN

2,3-bis(4-hydroxyphenyl)-propionitrile

EADs

early afterdepolarization

E2

oestrogen

ER

oestrogen receptor

I_Ca,L

L-type Ca²⁺ current density

I_NCX

Na⁺–Ca²⁺ exchanger current

LQT

long QT

NCX

Na⁺–Ca²⁺ exchanger

n/N

number of myocytes/number of hearts

OVX

ovariectomy

PPT

4,4′,4″-(4-propyl-µ1H½-pyrazole -1,3,5-triyl)trisphenol

RT-PCR

reverse transcription polymerase chain reaction

SHBG

sex hormone binding globulins

SR

sarcoplasmic reticulum

TdP

Torsade de pointes

Author contributions

All authors approved the final version of the manuscript. XY and GC contributed equally to the collection, analysis and interpretation of data and to drafting the article, RP made significant contributions to the collection of Western blots and immuno-histochemistry, DD helped us elucidate the genomic regulation of L type calcium channels, FZ, thesis advisor of XY, provided partial financial support for XY and GS conceived and designed the study, contributed to the interpretation of data, drafted and revised the article for important intellectual content and provided the financial support for the study.