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Am J Physiol Heart Circ Physiol 294: H2421-H2427, 2008. First published April 25, 2008; doi:10.1152/ajpheart.01225.2007
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Sex Steroids and Gender in Cardiovascular-Renal Physiology and Pathophysiology

Genomic deletion of estrogen receptors ER{alpha} and ERβ does not alter estrogen-mediated inhibition of Ca2+ influx and contraction in murine cardiomyocytes

Nina D. Ullrich,1 Andree Krust,2 Peter Collins,1 and Kenneth T. MacLeod1

1Imperial College London, Cardiac Medicine, National Heart and Lung Institute, London, United Kingdom; and 2Université Louis Pasteur, Institut de Génétique et Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Illkirch, Communauté Urbaine de Strasbourg, France

Submitted 22 October 2007 ; accepted in final form 21 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Estrogens modify contraction of vascular smooth muscle and cardiomyocytes, but suggestions that they confer protective effects on the cardiovascular system remain controversial. The negative inotropic effects of estrogens are a consequence of L-type Ca2+ channel inhibition, but the underlying mechanisms remain elusive. We tested the hypothesis that membrane-associated estrogen receptors (ER)-{alpha} and -β are involved. We measured the effect of estrogens on Ca2+ current (ICaL) in isolated ventricular cardiomyocytes of wild-type (WT), ER{alpha} knockout (ER{alpha}KO), and ERβKO mice using the whole cell patch-clamp technique at 37°C. No differences in current densities or inactivation profiles of ICaL were found under control conditions in WT, ER{alpha}KO, and ERβKO cardiomyocytes, suggesting that absence of either ER has no effect on functional properties of ICaL. In all groups, application of raloxifene (2 µM) or 17{alpha}- or 17β-estradiol (50 µM) reduced ICaL (P < 0.001). Raloxifene decreased ICaL by 44 ± 9% (mean ± SE) in WT (n = 5), 34 ± 5% in ER{alpha}KO (n = 5), and 30 ± 5% in ERβKO mice (n = 8). 17{alpha}-Estradiol reduced ICaL by 41 ± 10% in WT (n = 4), 34 ± 12% in ER{alpha}KO (n = 7), and 38 ± 8% in ERβKO mice (n = 7). 17β-Estradiol inhibited ICaL by 31 ± 4% in WT (n = 4), 28 ± 6% in ER{alpha}KO (n = 3), and 42 ± 3% in ERβKO mice (n = 5). Decreases in cell shortening occurred in parallel with these findings. Our results suggest that inhibition of ICaL and the decrease in contraction by estrogens do not depend on ER{alpha} or ERβ.

L-type calcium channel; excitation-contraction coupling; cardiac electrophysiology

PREVIOUS STUDIES ON ISOLATED cardiac cells showed that application of different estrogens and related compounds such as phytoestrogens provoked a rapid decrease in myocyte contraction via inhibition of the cardiac L-type Ca2+ channel (Cav1.2) that is involved in the initiation of the excitation-contraction coupling mechanism (6, 10, 14). Similar effects were observed with the related, but nonsteroidal, selective estrogen receptor modulators (SERMs), suggesting that an estrogen receptor(ER)-mediated intracellular signaling process inhibits the L-type Ca2+ current (ICaL) and decreases cell contraction (13). However, the direct involvement of any ER in this cardiac response could not be demonstrated.

ERs exist in the hearts of both humans and rodents. Generally, they are nuclear receptors that act as transcription factors to initiate a genomic response following estrogen binding. However, the cardiac (and vascular) effects of estrogen and related ER modulators described above are rapid (several seconds to minutes), and so it is unlikely that they are mediated by a modulation of transcription. Indeed, rapid responses (of endothelial cells) to estrogen occur in the presence of transcriptional inhibitors (2).

ERs are also linked with the plasma membrane, where they are involved in alternative signaling pathways that induce rapid, so-called nongenomic, responses (11, 12, 19, 24). These rapid responses to estrogens have been most convincingly described in endothelial cells, where they activate membrane-associated ER{alpha}, which subsequently binds to phosphatidylinositol-3 kinase (PI3K), which activates the PI3K/Akt pathway, leading to phosphorylation of the endothelial nitric oxide synthase (eNOS) and, consequently, to an increase in NO (8, 25). Similar nongenomic ER signaling can also be triggered by the SERM raloxifene (23).

We tested the hypothesis that ERs play a role in the estrogen-induced negative inotropic effect using cardiomyocytes isolated from the hearts of mice that lacked the gene for either ER{alpha} or ERβ. We compared the effects of estrogens on ICaL recorded in these cells with that measured in wild-type cells. We found no difference in the inhibitory effects of estrogens on any genotype, suggesting that their effects are not mediated via the ER but more directly on the L-type Ca2+ channel itself.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transgenic mice and isolation of cardiac ventricular myocytes. Mice of the C57Bl6 strain that were heterozygous for genomic deletion of estrogen receptor types {alpha} and β (ER{alpha} and ERβ) were kindly provided by Pierre Chambon (Strasbourg, France). Inbreeding and appropriate backbreeding of the heterozygotes yielded homozygote knockout ER{alpha} and ERβ mice. These were genotyped by PCR analysis of tail-tip biopsies to confirm the genomic knockout of either ER{alpha} or ERβ (4, 16). Wild-type littermates were used as controls. Left ventricular myocytes were isolated from the adult mice with the use of a Langendorff apparatus and enzymatic digestion according to previously described methods adapted for the mouse heart (27). For a small series of confirmatory experiments we used cardiomyocytes isolated from guinea pig hearts. The method used to isolate these cells was described previously (9). After isolation, myocytes were stored in Dulbecco's modified Eagle's medium solution at room temperature for up 8 h. All experiments conformed strictly to and were approved by the Institutional Animal Care and Use Committee at Imperial College London and are in accordance with the Declaration of Helsinki for the Care and Use of Laboratory Animals.

Genotyping by PCR. Mice were genotyped 6–8 wk after birth using genomic DNA from 1-mm tail-tip biopsies. Tissue samples were homogenized in standard TNES lysis buffer (10 mM Tris, 400 mM NaCl, 100 mM EDTA, and 0.6% SDS) containing 3% proteinase K (10 mg/ml) at 55°C overnight. Genomic DNA was isolated from whole cell extracts by ethanol precipitation and further used for PCR as described previously (4). Specific PCR primers for ER{alpha} were as follows: first primer pair, P1 (5'-TTGCCCGATAACAATAAC AT-3') and P2 (5'-ATTGTCTCTTTCTGACAC-3'); second primer pair, P3 (5'-GGCATTACCATTCTCCTGGGAGTCT-3') and P4 (5'-TCGCTTTCCTGAAGACCTTTCATAT-3'). For ERβ, three primers were used in one reaction: P4 (5'-TGAAGAGGAAGCTTGGCGGG-3'), P5 (5'-CACAGGACCAGACACCGTA-3'), and P6 (5'-TCATAGCCTGAAGAACGAGA-3'). The PCR program was performed as follows: 1 cycle at 94°C for 5 min, 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, and then a final extension cycle of 72°C for 5 min and storage at 4°C. PCR products were analyzed using 2% agarose gel electrophoresis. Wild-type animals revealed bands of 387 (P1/P2) and 815 bp (P3/P4) for ER{alpha} and a band of 155 bp (P4/P5/P6) for ERβ; homozygous knockout animals revealed no band for P1/P2, a band of 255 bp for P3/P4 for ER{alpha}, and a band of 200 bp (P4/P5/P6) for ERβ. Heterozygotes showed both bands for each primer combination.

Voltage-clamp studies. Membrane currents were measured at 37°C using whole cell procedures with an Axopatch amplifier controlled by pCLAMP 8 software (Axon Instruments, Foster City, CA). Capacity current and series resistance compensation were carried out using analog techniques. Cells were voltage-clamped using low resistance (3–6 M{Omega}) borosilicate glass micropipettes. Starting from a holding potential of –80 mV, the voltage protocol consisted of an initial 60-ms step to –40 mV to inactivate the sodium and any T-type Ca2+ current, followed by 200-ms test steps ranging from –60 to +60 mV in 5-mV increments. After the step family, another 100-ms test step to 0 mV was added to monitor changes in the voltage-dependent inactivation profile.

Solutions. The pipette solution contained (in mM) 140 CsCl, 10 tetraethylammonium (TEA)-Cl, 4 MgATP, 5 EGTA, 5 HEPES, 1 CaCl2, and 1 MgCl2 at pH 7.2, adjusted with CsOH. The external solution contained 110 NaCl, 5.4 CsCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 30 TEA-Cl, and 10 glucose at pH 7.4, adjusted with NaOH. Remaining background Cl– currents were subtracted from the Ca2+ current traces after application of 100 µM CdCl2. Raloxifene hydrochloride (LY 139481), a gift from Eli Lilly (Indianapolis, IN), 17{alpha}- and 17β-estradiol (Sigma-Aldrich), and the ER antagonist ICI 182,780 (Tocris) were dissolved in DMSO to form stock solutions.

Cell shortening. Cell shortening was measured as described previously (10). Cells were field-stimulated at a frequency of 0.5 Hz, a pulse length of 2 ms, and a voltage of 10–30 V by using two platinum electrodes placed in parallel on either side of the chamber. Cell shortening was monitored with a video edge detection system and was also recorded using pCLAMP 8 (Axon Instruments). Once steady-state contractions were achieved, raloxifene, 17{alpha}-estradiol, or 17β-estradiol was added to the superfusate, and the new steady-state contractions were recorded.

Data analysis. Electrophysiological and cell shortening data were analyzed using Clampfit (pCLAMP 8). Significance was tested using the Student's t-test or one-way ANOVA followed by Dunnett's multiple comparison post test. A value of P < 0.05 was considered significant. Pooled data are given as means ± SE, with n representing the number of cells tested. Cells were isolated from 10 wild-type (WT), 12 ER{alpha} knockout (ER{alpha}KO), and 11 ERβKO mice.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Comparison of ICaL in WT, ER{alpha}KO, and ERβKO cardiomyocytes. For control purposes, cardiac ICaL of all genotypes were evaluated. Analyses of the current-voltage relationship and the inactivation profile are summarized in Fig. 1. Currents were activated using the voltage step protocol displayed in Fig. 1A, inset. After subtraction of Cd2+-resistant background currents, peak Ca2+ currents (Ipeak) at each voltage step were extracted, normalized to the maximal current values (Imax) at 0 mV, and plotted against the clamp potential. The pattern of voltage-dependent current activation of the cardiac L-type Ca2+ channel remained unchanged in ER{alpha}KO and ERβKO compared with WT mice (Fig. 1A). In all genotypes, the activated current peaked near 0 mV and reversed at similar voltages of +58 ± 2 mV (WT, n = 12), +57 ± 2 mV (ER{alpha}KO, n = 15), and +56 ± 2 mV (ERβKO, n = 14). Figure 1B shows original sample traces of ICaL activated by a voltage step from –40 to 0 mV in WT, ER{alpha}KO, and ERβKO cardiomyocytes. At 0 mV there were no significant differences in the current densities of all three cell types (P = 0.2183, Fig. 1C). The inactivation profile of ICaL was studied with a voltage test step to 0 mV following prepulses to different potentials (see voltage protocol inset in Fig. 1D). Channel availability plotted as a function of prepotential resulted in the characteristic sigmoid curve for each cell type, and these were fitted with the Boltzmann equation (Fig. 1D). Comparison of the mid-voltage point (V0) and the slope factor (k) revealed no significant difference between the three fits, showing that genomic deletion of ER{alpha} or ERβ has no influence on the inactivation behavior of ICaL in cardiac cells (kWT = 5.2 ± 0.7, kER{alpha}KO = 5.8 ± 1.0, and kERβKO = 5.5 ± 0.6). V0 values for the three curves were –23.5 ± 0.7 mV (WT, n = 13), –20.6 ± 1.1 mV (ER{alpha}KO, n = 12), and –23.5 ± 0.7 mV (ERβKO, n = 20). These results suggest that there is no difference in the properties of ICaL in WT and ER{alpha}/βKO cardiomyocytes.


Figure 1
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  		Fig. 1. Current properties of L-type Ca2+ current (ICaL) in wild-type (WT), estrogen receptor-{alpha} knockout (ER{alpha}KO), and estrogen receptor-β knockout (ERβKO) cardiac cells. A: normalized current (I/Imax)-voltage (V) relationship of ICaL in ER{alpha}KO and ERβKO cells. Inset: corresponding voltage protocol. B: original sample traces of ICaL (bottom) during the activating voltage step from –40 to 0 mV (top). C: current densities at 0 mV. D: inactivation curve of ICaL. Fopen, channel availability. Data points were fitted with the Boltzmann equation: y = A2 + (A1 – A2)/{1 + exp[(V – V0)/k]}, where A1, A2, V0, and k are the initial value, final value, mid-point voltage, and slope factor, respectively. Inset: voltage protocol with the presteps (Epre) ranging from –45 to +5 mV, followed by the 100-ms test-step (Etest) to 0 mV.

 
Effect of 17β-estradiol on ICaL. It has been shown that 17β-estradiol induces a rapid inhibition of ICaL in cardiomyocytes of different species (1, 10, 18, 20, 26). We tested the effect of 50 µM 17β-estradiol on ICaL of WT, ER{alpha}KO, and ERβKO cardiac cells, and the experiments are summarized in Fig. 2. Application of the steroid resulted in an immediate decrease in ICaL, reaching new steady-state levels after ~2 min of superfusion. Figure 2A shows original current traces of each cell type in response to an activating voltage step from –40 to 0 mV under control conditions and at the end of 17β-estradiol application. Paired analysis was performed on currents before and at the end of steroid application and was normalized to Imax at 0 mV in control solution. The means of the single data points representing Ipeak at each test potential before and at the end of 17β-estradiol application are shown in the current-voltage relationships in Fig. 2B for WT, ER{alpha}KO, and ERβKO cardiomyocytes. In all cell types, ICaL was significantly reduced by 17β-estradiol (Student's t-test: P < 0.001 for each group; Fig. 2F), but when different genotypes are compared, there is no difference in the effect on ICaL (Fig. 2, D and F). In the presence of 17β-estradiol, ICaL decreased by 31 ± 4% in WT (n = 4), 28 ± 6% in ER{alpha}KO (n = 3), and 42 ± 3% in ERβKO cardiomyocytes (n = 5). These results suggest that the decrease of ICaL induced by 17β-estradiol is unaffected by the absence of ER{alpha} or ERβ. The inactivation profiles of ICaL during application of 17β-estradiol shifted to more negative potentials in WT [V0 (control) = –23.3 ± 0.8 mV, V0 (17β-estradiol) = –29.4 ± 0.9 mV, P = 0.0004] and ER{alpha}KO cardiomyocytes [V0 (control) = –20.6 ± 1.1 mV, V0 (17β-estradiol) = –25.3 ± 1.8 mV, P = 0.0379], but no difference was found in ERβKO cardiomyocytes [V0 (control) = –23.5 ± 0.7 mV, V0 (17β-estradiol) = –25.1 ± 1.0 mV, P = 0.2209]. In all three groups, the slope factor did not change in the presence of 17β-estradiol (PWT = 0.5650, PER{alpha}KO = 0.7184, and PERβKO = 0.8359). A one-way ANOVA also revealed no significant differences when comparing the inactivation curves of all ICaL in control (P = 0.0554) or during drug application (P = 0.0629, Fig. 2E).


Figure 2
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  		Fig. 2. Comparison of the effect of 17β-estradiol on ICaL in WT, ER{alpha}KO, and ERβKO cardiomyocytes. A: original current traces of ICaL in response to an activating voltage step from –40 to 0 mV in control solution (solid line) and at steady-state in 17β-estradiol (dotted line) for all 3 genotypes. B: summary of current-voltage relationships of ICaL before (Ctrl) and at the end of steroid application (Estr) for each cell type. Data points show paired analysis of peak Ca2+ currents (Ipeak) at different potentials in the respective solutions. C: inactivation curves for ICaL in control solution and 17β-estradiol. D: normalized ICaL current-voltage traces for each cell type. E: inactivation curves in 17β-estradiol for each cell type. F: mean data for the 17β-estradiol-mediated effect on ICaL at 0 mV. C, control. ***P < 0.001, t-test between C and βE2. P = 0.0851, ANOVA between WT, ER{alpha}KO, and ERβKO.

 
Effect of 17β-estradiol on cell shortening. The inhibitory effect of 17β-estradiol on Ca2+ influx via the L-type Ca2+ channel was confirmed by a decrease in the contractility of cardiomyocytes from each genotype. 17β-Estradiol significantly decreased cell contraction by 22 ± 5% in WT (n = 15), 26 ± 4% in ER{alpha}KO (n = 17), and 19 ± 3% in ERβKO cardiomyocytes (n = 19). The percentage reduction in contraction of cells isolated from the three genotypes was not different, suggesting that this effect is also not related to ER expression.

Effect of 17{alpha}-estradiol on ICaL. We considered that 17β-estradiol might have a direct effect on the L-type Ca2+ channel and therefore tested the ability of its stereoisomer, 17{alpha}-estradiol, which is hormonally inactive, to inhibit ICaL. Application of 17{alpha}-estradiol (50 µM) resulted in a strong inhibition of ICaL in all WT, ER{alpha}KO, and ERβKO cardiomyocytes. The potency of this effect was comparable to the results with 17β-estradiol. Figure 3 A shows original current traces in response to an activating voltage step from –40 to 0 mV for all genotypes. The solid line represents ICaL in control solution, and the dotted line shows ICaL at steady-state inhibition by 17{alpha}-estradiol. The current-voltage relationships of normalized currents in control conditions and in the presence of 17{alpha}-estradiol are illustrated in Fig. 3B for all cell types. 17{alpha}-Estradiol significantly reduced ICaL (P < 0.001) in each tissue by 41 ± 8% (WT, n = 4), 47 ± 12% (ER{alpha}KO, n = 7), and 49 ± 8% (ERβKO, n = 7). Again, the degree of current inhibition was not different in ER{alpha}KO and ERβKO compared with WT cardiomyocytes, as shown in Fig. 3, D and F. The V0 point on the inactivation curve of ICaL for ERβKO was shifted (Fig. 3C) by 17{alpha}-estradiol from –23.5 ± 0.8 to –27.2 ± 0.8 mV (P = 0.0112), but not for WT (P = 0.5389) or ER{alpha}KO (P = 0.8234), with no difference in the slope factors of any group. However, unlike the results obtained with 17β-estradiol, comparison of the effect of 17{alpha}-estradiol of the different genotypes showed significant differences of V0 (1-way ANOVA: P = 0.0126, Fig. 4E), but no difference was found when comparing V0 in control conditions (compare with Fig. 1D).


Figure 3
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  		Fig. 3. 17{alpha}-Estradiol inhibits ICaL in WT, ER{alpha}KO, and ERβKO cardiomyocytes. A: sample traces of ICaL in control solution (solid line) and 17{alpha}-estradiol (dotted line) for all 3 genotypes. B: current-voltage relationship of ICaL in control solution and 17{alpha}-estradiol. C: Boltzmann fits of the inactivation curves for WT, ER{alpha}KO, and ERβKO cardiomyocytes in control solution and 17{alpha}-estradiol. D: effect of 17{alpha}-estradiol on the current-voltage relationship of ICaL in all cells. E: comparison of V0 of Fopen from WT, ER{alpha}KO, and ERβKO cardiomyocytes in the presence of 17{alpha}-estradiol. F: mean data for the effect of 17{alpha}-estradiol on ICaL at 0 mV in all genotypes. ***P < 0.001, t-test between C and {alpha}E2. P = 0.8972, ANOVA between WT, ER{alpha}KO, and ERβKO.

 

Figure 4
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  		Fig. 4. Raloxifene decreases ICaL in WT, ER{alpha}KO, and ERβKO cardiomyocytes. A: sample traces of ICaL before (solid line) and at steady-state level during application of raloxifene (dotted line) in all 3 genotypes. B: current-voltage relationship of WT ICaL in control solution and raloxifene (Ral). C: current-voltage relationships for the effect of raloxifene on ICaL in ER{alpha}KO and ERβKO. D: mean data for the effects of raloxifene on ICaL at 0 mV in all genotypes. E: Boltzmann fits of the inactivation curves for WT, ER{alpha}KO, and ERβKO cardiomyocytes in raloxifene.

 
Effect of raloxifene on ICaL. SERMs all have high affinity for ERs, but whether they act as an agonist or antagonist depends on the type of compound as well as the tissue on which it is acting. SERMs can act via pathways that are distinct from the classic route of estrogen action. We have shown that the SERM raloxifene has rapid effects similar to those of estrogen on Ca2+ influx and contraction in single cardiomyocytes, but at lower, more pharmacophysiological concentrations (13), and we wanted to test whether these effects depended on the presence of ERs. In WT, 2 µM raloxifene significantly reduced ICaL (Fig. 4A) but did not affect the voltage dependence of current activation (Fig. 4B). Application of 2 µM raloxifene resulted in the same degree of inhibition of ICaL in ER{alpha}KO and ERβKO as in WT cardiomyocytes. Figure 4A shows single traces of ICaL before and at the end of raloxifene superfusion during a step from –40 to 0 mV in all genotypes. The full current-voltage relationships for ER{alpha}KO and ERβKO cardiomyocytes are summarized in Fig. 4C. Raloxifene decreased ICaL by 49 ± 10% in WT (n = 5), 34 ± 5% in ER{alpha}KO (n = 5), and 41 ± 8% in ERβKO cardiomyocytes (n = 8). The decrease was not different among the three groups (P = 0.4772). There was no difference in the V0 (P = 0.2916) or slope factor (P = 0.1862) in all genotypes (Fig. 4E). Thus, like 17{alpha}/β-estradiol, the effects of raloxifene on ICaL in cardiomyocytes appear to be independent of ER{alpha} or ERβ.

Effect of raloxifene on ICaL in the presence of ICI 182,780. After knockout of one ER, it is possible that the other might play a role in mediating a response. One way to rule out this possibility was to investigate the effect of SERM inhibition of ICaL in the presence of the specific ER antagonist ICI 182,780. Myocytes were incubated with 10 µM ICI 182,780 at room temperature for 1 h before being placed in the superfusion chamber or were acutely exposed to the same concentration of compound. ICaL was measured in the continuing presence of 10 µM ICI 182,780 before and after the addition of 2 µM raloxifene. We found that in cells isolated from both WT and ERβKO mouse hearts, application of ICI 182,780 did not alter the effects of raloxifene. Figure 5A shows that in the presence of ICI 182,780 and raloxifene, ICaL was inhibited by 63%, whereas in the same cell, raloxifene alone inhibited ICaL by 54%. We confirmed that this type of response was not unique to the mouse by undertaking similar experiments in the guinea pig. Figure 5B shows the current-voltage relationships for ICaL under control conditions, in the presence of 10 µM ICI 182,780 alone, and after subsequent addition of 2 µM raloxifene. Despite inhibition of both ERs with ICI 182,780, raloxifene had a marked inhibitory effect on ICaL.


Figure 5
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  		Fig. 5. A: raloxifene decreases ICaL in ERβKO cardiomyocytes in the presence of ICI 182,780. Sample traces of ICaL elicited by the voltage protocol (top) under control conditions (solid line), in the presence of 10 µM ICI 182,780 (ICI; dashed line), and in the presence of 10 µM ICI 182,780 and 2 µM raloxifene (ICI+Ral; dotted line). The percentage inhibition of ICaL is shown in the histograms immediately below the traces. B: effect of 10 µM ICI 182,780 (open circles) and 10 µM ICI 182,780 plus 2 µM raloxifene (shaded circles) on the current-voltage relationship of ICaL in guinea pig cardiomyocytes. Control, filled circles; n = 10.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Role of ERs in the estrogen-mediated effect of ICaL. In this study, we investigated the role of ER{alpha} and ERβ in the mechanism underlying estrogen-mediated inhibition of the cardiac L-type Ca2+ channel and reduction in myocyte contractility. In WT cardiac cells, estrogen induces a concentration-dependent decrease of ICaL (1, 10, 18, 20, 26), and as a consequence of the Ca2+-induced Ca2+ release mechanism that operates in heart, this effect results in a reduction of the Ca2+ transient and contractility (10).

Our current understanding is that cardiac muscle expresses both of the classic ERs, ER{alpha} and ERβ (9, 17, 18). Although ERs are best known for their function as nuclear receptors and their involvement in genomic regulation of transcriptional processes, they are also associated with the plasma membrane, often in various isoforms resulting from alternative splicing, where they may be involved with more direct and rapid intracellular signaling mechanisms. Since the inhibitory actions of estrogens on Ca2+ influx and myocyte contractility have been observed to occur within seconds, it is possible that plasma membrane ERs are responsible for mediating this effect (11, 12, 19, 24).

To investigate the hypothesis that ERs are involved in the rapid, nongenomic effects of estrogen, we compared the effect of estrogens on cardiomyocytes isolated from the left ventricles of WT mice with ER-deficient left ventricular cells isolated from ER{alpha}KO or ERβKO animals. The deletion of either ER gene was confirmed by PCR of tail-tip biopsies as detailed previously (4).

Selective knockout of the genes for either ER did not alter the basic function of ICaL. The results of our control experiments showed that the density and current-voltage relationship of the L-type Ca2+ channel, as well as the contractility of isolated cardiomyocytes, were similar in cells isolated from the respective genotype.

Application of 17β-estradiol had the same inhibitory effect on ICaL and on contraction in ER-deficient as in WT cells. The stereoisomer 17{alpha}-estradiol and the SERM raloxifene had similar effects on ICaL. These results suggest that the effect of estrogen on cardiac cells is not mediated via ERs. Although our experiments demonstrate that neither ER{alpha} nor ERβ seems to be necessary for the estrogen-induced decrease in ICaL and further coupled contractility, it is possible to argue that upon knockout of one ER, the other may compensate for any arising deficiency. The only way to rule out this possibility would be to investigate ER double-knockout animals. However, there are significant problems with breeding such animals, given that all ER{alpha}KO females are sterile and ERβKO females are either sterile or exhibit variable degrees of fertility (4).

ICI 182,780 is a potent steroidal antiestrogen that can bind to both ER subtypes. Specific inhibition of both ERs using this antagonist did not alter the effect of estrogens or soy-derived isoflavones on ICaL in cardiac cells (data not shown and Ref. 14). Although these observations also imply that estrogens do not act via ERs, ICI 182,780 has been demonstrated to have effects on various ion channels that may complicate the interpretation of results in the present experiments. For example, ICI 182,780 has depolarizing actions and modulates Ca2+-activated K+ channel activity in cultured endothelial cells and smooth muscle cells (3, 15, 31). We were therefore cautious of using ICI 182,780 to provide unequivocal evidence of ER blockade. In a series of experiments using this compound, we found supportive evidence of SERM action on ICaL, whereas ERs were inhibited.

Although our results strongly indicate that the inhibitory effect of estrogens and raloxifene are not mediated via the ER, there are other mechanisms that may be activated. In brain tissue, a plasma membrane-associated estrogen binding protein, ER-X, has been identified. It shares some homology with the COOH terminus of ER{alpha} but seems to be derived from a different gene (29). Although ER-X binds both 17{alpha}- and 17β-estradiol, its preferred ligand is the former, which elicits rapid and sustained activation of the MAPK isoforms ERK1 and ERK2 (28). However, ER-X or a similar isoform has not yet been identified in heart. There is also evidence for different types of surface membrane-located estrogen binding sites, which activate phospholipase C and adenylyl cyclase pathways via the G protein-coupled receptors in the brain (21) and modulate insulin secretion in pancreatic β-cells via cGMP protein kinase (22), but again, there is no evidence of such binding sites in the heart. We suspect that there may be more direct interaction of estrogens with ion channels. Recently, the existence of an estrogen binding site within the β-subunit of the voltage-gated K+ channel hSlo was demonstrated (30), and our own results suggest that estrogens affect Ca2+ channel inactivation.

Effect of estrogens on ICaL. In WT and KO cardiomyocytes, the activation kinetics of ICaL did not differ in the presence of estrogen or raloxifene compared with those measured in control conditions. In contrast, inactivation curves showed, under certain conditions, significant leftward shifts (P < 0.05) in V0. Cells from WT and ER{alpha}KO mice showed significant changes in the inactivation curve during administration of 17β-estradiol, whereas the V0 of the inactivation curve recorded from ERβKO cells was significantly shifted to more negative potentials during application of 17{alpha}-estradiol. A hyperpolarizing shift of voltage-dependent inactivation would decrease the fraction of ion channels that can be activated from a given membrane potential. The reasons for stereoisomers having differing effects remain to be resolved. Genetic deletion of an estrogen receptor per se does not affect the inactivation mechanism(s) of the L-type Ca2+ channel, but it does alter the effect of estrogen or raloxifene on channel inactivation while leaving activation and conducting properties unchanged.

Nakajima et al. (20) also found a concentration-dependent shift of the inactivation curve to more negative potentials in the presence of 17β-estradiol. The synthetic estrogen diethylstilbestrol had similar effects, but other steroid hormones, such as testosterone and progesterone, did not. In earlier work (14), we showed that some soy-derived isoflavones (genistein and equol) significantly inhibited the peak L-type Ca2+ current, but we did not investigate Ca2+ channel inactivation. Therefore, despite sharing some similar structural features, there are differences in the action of these hormones and isoflavones.

Other ion channels also appear to respond to the enantiomers of estrogen differently. Whereas 17β-estradiol rapidly decreased TEA- and 4-aminopyridine-sensitive K+ currents, 17{alpha}-estradiol had no significant effect (5). Fatehi et al. (5) suggested three possibilities for 17β-estradiol action: the hormone may directly change the conformation of the pore close to the external mouth of the channel, physically occlude the channel in its open state, and/or modulate channel function by estrogen-induced phosphorylation of tyrosine residues.

The effect of estrogen on ion channels does not always result in channel inhibition. In hippocampal neurons, 17β-estradiol induced activation of the neuronal Ca2+ channel (32), which increased intracellular Ca2+ concentration ([Ca2+]i). This rise in [Ca2+]i activates Src and ERK signaling pathways that are involved in estrogen-induced neurite outgrowth and neuroprotective effects (32). Thus the final effect of a direct action of estrogens on ion channels may depend on the type of channel and may possibly involve different channel subunits and signaling factors.

The high concentration of estrogen used in the experiments can be legitimately questioned, since circulating levels are much lower. Two points ought to be considered. It has been shown that estrogen can be locally synthesized in the presence of the cytochrome CYP450 aromatase, which turns precursor steroids into estrogen by aromatization (7). Grohe et al. (7) found expression of CYP450 aromatase and sufficient local biosynthesis of estrogen in cardiomyocytes such that local estrogen levels might rise to concentrations well above normal circulating levels.

Second, we tested the effect of raloxifene on ICaL at much lower concentrations and obtained the same results. We chose to use a concentration of 2 µM raloxifene for these experiments, because this corresponded with the EC50 of raloxifene on cell contraction obtained from earlier work (13), in which we demonstrated significant effects at therapeutic (nanomolar) concentrations.

In summary, our findings suggest that ER{alpha} and ERβ are not involved in the estrogen-mediated inhibition of cardiac ventricular myocyte contraction and may point to a more direct interaction between estrogen and the ion channel protein that initiates contraction, the L-type Ca2+ channel. It is likely that this action will not be confined to ion channels in the heart but may be a more ubiquitous mechanism that underlies some of the effects of estrogens on the cardiovascular system.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
We are grateful to the British Heart Foundation for financial support.


   ACKNOWLEDGMENTS
 
We are very grateful to Mathew Edenbrow and Husam Ria for expert technical assistance with this work.


   FOOTNOTES
 
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
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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