oestrogen upregulates l-type ca2+ channels via oestrogen-receptor- by a regional genomic mechanism...

J Physiol 590.3 (2012) pp 493–507 493

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Oestrogen upregulates L-type Ca2+ channels viaoestrogen-receptor-α by a regional genomic mechanism infemale rabbit hearts

Xiaoyan Yang1,2, Guojun Chen1, Rita Papp1, Donald B. DeFranco3, Fandian Zeng2 and Guy Salama1

1Cardiovascular Institute and 3Department of Pharmacology and Chemical Biology, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261,USA2Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, China

Non-technical summary Women during their child-bearing years have longer QT intervals intheir electrocardiograms than men and are more susceptible to lethal arrhythmias elicited bydrugs that delay repolarization. Current theories posit that women have a reduced ‘repolarizationreserve’ due to reduced potassium currents resulting in longer QT and greater repolarizationdelays. We proposed an alternative mechanism of higher calcium currents in women which wouldlikewise prolong QT intervals, delay repolarization while increasing the force of contractions andintracellular calcium load. Here, we show that physiological concentrations of oestrogen increasethe calcium current only in cells from the base of the heart, by increasing messenger RNA andproteins levels that encode for the calcium current. Moreover, oestrogen acts by interacting withoestrogen receptors (ER)α but not ERβ which may explain why hormone replacement therapyincreases the risk of arrhythmia and offers a possible protective solution of using an oestrogenmimetic that selectively binds to ERβ.

Abstract In type-2 long QT (LQT2), adult women and adolescent boys have a higher risk oflethal arrhythmias, called Torsades de pointes (TdP), compared to the opposite sex. In rabbithearts, similar sex- and age-dependent TdP risks were attributed to higher expression levels ofL-type Ca2+ channels and Na+–Ca2+ exchanger, at the base of the female epicardium. Here, theeffects of oestrogen and progesterone are investigated to elucidate the mechanisms whereby ICa,L

density is upregulated in adult female rabbit hearts. ICa,L density was measured by the whole-cellpatch-clamp technique on days 0–3 in cardiomyocytes isolated from the base and apex of adultfemale epicardium. Peak ICa,L was 28% higher at the base than apex (P < 0.01) and decreasedgradually (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 ICa,L (∼2-fold) in female base but notendo-, apex or male myocytes. Progesterone (0.1–10 μM) had no effect at base myocytes. Anagonist of the α- (PPT, 5 nM) but not the β- (DPN, 5 nM) subtype oestrogen receptor (ERα/ERβ)upregulated ICa,L like E2. Western blots detected similar levels of ERα and ERβ in male andfemale hearts at the base and apex. E2 increased Cav1.2α (immunocytochemistry) and mRNA(RT-PCR) levels but did not change ICa,L kinetics. ICa,L upregulation by E2 was suppressed bythe 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 ICa,L by a regionalgenomic mechanism involving ERα which is a known determinant of sex differences in TdP riskin LQT2.

X. Yang and G. Chen contributed equally to this work.

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society DOI: 10.1113/jphysiol.2011.219501

494 X. Yang and others J Physiol 590.3

(Resubmitted 25 August 2011; accepted after revision 28 November 2011; first published online 28 November 2011)Corresponding author G. Salama: University of Pittsburgh, Department of Medicine, Cardiovascular Institute, 3550Terrace Street, Suite S 628 Scaife Hall, Pittsburgh, PA 15261, USA. Email: [email protected]

Abbreviations AmD, actinomycin D; APD, action potential duration; Ca,L, L-type calcium channel; CHX, cyclo-heximide; DPN, 2,3-bis(4-hydroxyphenyl)-propionitrile; EADs, early afterdepolarization; E2, oestrogen; ER, oestrogenreceptor; ICa,L, L-type Ca2+ current density; INCX, 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-[1H]-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.

Introduction

The congenital form of long QT type 2 (LQT2) is causedby mutations of the K+ channel protein HERG that resultin a loss of function of the rapid component of delayedrectifying K+ current, IKr, a prolongation of the actionpotential duration (APD) and QT interval (Morita et al.2008). Although the incidence of all forms of congenitalLQT is rare (<1/5000), drug-induced LQT2 remains aserious public health problem because a wide range ofcardiac and non-cardiac drugs suppress IKr, prolong APDsand promote early afterdepolarizations (EADs) that leadto Torsade de pointes (TdP) (Splawski et al. 2000; Vincent,2000; Drici & Clement, 2001; Levine et al. 2008; Moritaet al. 2008). Women are known to be at higher risk tocongenital 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 (Goldenberget al. 2008). Rabbits exhibit the same sex differencesin arrhythmia risk with adult females (>8 weeks) beingmore prone to TdP and the arrhythmia phenotype beingreversed in young rabbits (Liu et al. 2005) (<42 days),before the surge of steroids (de Turckheim et al. 1983). Infemales, ovariectomy (OVX) reduced dofetilide-inducedAPD 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 E2promotes TdP in female hearts.

There is general agreement that TdP is initiated byEADs that are caused by the re-activation of L-typeCa2+ channels. However, controversies persist regardingthe mechanisms that re-activate the L-type Ca2+ current(ICa,L) during long APs and whether or not an elevationof intracellular Ca2+ (Ca2+

i ) precedes and initiates EADs.Some studies found that the re-activation of ICa,L occurredspontaneously, independent of Ca2+ release from thesarcoplasmic reticulum (SR) because in ferret hearts,ryanodine and chelation of intracellular Ca2+ inter-rupted delayed afterdepolarizations (DADs) but did notalter EADs (Marban et al. 1986). Alternatively, longAPDs can cause an imbalance between Ca2+ influx andefflux, resulting in SR Ca2+ overload which promotes

spontaneous SR Ca2+ release then activation of aforward-mode Na+–Ca2+ exchanger (NCX) current, INCX,which can depolarize the plateau potential to re-activateICa,L (Volders et al. 2000).

Dual optical mapping of APs and Ca2+i transients in

the Langendorff rabbit model of drug-induced LQT2revealed that adult females were more prone to EADsand TdP and that the arrhythmia phenotype was reversedin pre-pubertal hearts (Liu et al. 2005). Ca2+

i elevationpreceded EAD upstrokes at the origins of EADs andwhen paced at 1.2 s cycle length, marked Ca2+

i oscillationspreceded the occurrence of EADs (Choi et al. 2002;Nemec et al. 2010). In pre-pubertal male and adultfemale hearts with LQT2, EADs originated at the baseand not the apex of the epicardium (Sims et al. 2008). Infreshly isolated ventricular myocytes, peak ICa,L densityand Cav1.2α channel protein were 25–30% greater atthe base than the apex of adult female and pre-pubertalmale hearts (Sims et al. 2008). Western blot analysis andvoltage-clamp studies showed that the higher level ofCav1.2α at the base of the adult female heart was matchedby a regional elevation of NCX and INCX (Chen et al.2011). Moreover, incubation of myocytes with oestrogen(1 nM) revealed a regional genomic upregulation of NCXmediated by oestrogen receptors, enhanced transcriptionand biosynthesis of NCX channel protein (Chen et al.2011).

Pham et al. (2002) reported a transmural dispersion ofICa,L in female but not in male rabbit hearts with ICa,L

density being higher on the epicardium than the end-ocardium and no male–female differences on the end-ocardium. These findings are congruent to humans sincecardiac contractility is greater in women than men (Merzet al. 1996) and female myocytes may have greater ICa,L

(Verkerk et al. 2005). Our findings in rabbit hearts (Simset al. 2008) were consistent with previous studies (Phamet al. 2002) and extended them by revealing markedapex–base differences of Cav1.2α and ICa,L densities infemale but not in male rabbit hearts.

However, the mechanism(s) underlying sex differencesin ion channel expression remain a matter of conjecture.Castration with and without hormone replacement inexperimental animals suggested that oestrogen promotes

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

J Physiol 590.3 Regional genomic upregulation of ICa,L by oestrogen via ERα 495

EADs and TdP but such experiments are difficultto interpret. Sham surgeries alone cause long-lasting(>2 weeks, Tong L, Wagner R and Salama G, unpublishedobservations) shortening of action potential durations,castration has marked effects on all organs, bound andfree serum levels of oestrogen are typically not measured(either before or after hormone replacement), nor is theserum concentration of sex hormone binding globulins(SHBG).

The present study investigates the mechanisms thatupregulate ICa,L at the base of female hearts by investigatingthe 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 ofPittsburgh Institutional Animal Care and Use Committeeand were in accordance with the current Guide for the Careand Use of Laboratory Animals published by the NationalInstitute of Health. In all the studies described in thisarticle, the rabbits were first killed and the hearts wereremoved to isolate and culture adult ventricular myocytesfor 0 to 3 days. The rabbits were obtained from an approvedcommercial vendor, Myrtle’s Rabbitry, and were housedin the animal facilities of the University of Pittsburghaccording to Federal Regulations of the USA. The authorshave read and examined the rules and regulations in theUK as set by the Medical Research Council and found themto be in agreement and congruent with the policies of theUniversity of Pittsburgh and The Journal of Physiology asstipulated in the reporting of ethical matters (Drummond,2009).

Cell isolation and incubation

Ventricular myocytes were isolated from adult (3 monthsold) male and female New Zealand white rabbits, as pre-viously described (Sims et al. 2008). Briefly, rabbits wereanaesthetized with pentobarbital (50 mg kg−1) and pre-treated with heparin (200 U kg−1). Hearts were excised andLangendorff-perfused with a Tyrode solution containing(in mmol l−1): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 5.5glucose and 10 Hepes, at pH 7.4 for 5 min, then Ca2+-freeTyrode solution for 10 min, after which collagenase typeII (0.6 mg ml−1) and 0.02% bovine serum albumin wereadded for a 20 min digestion at 35◦C, followed by Tyrodesolution containing 50 μmol l−1 CaCl2 and 0.02% BSAfor 10 min. The hearts were removed and placed in a highK+, 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 theheart) and base (1–4 mm below the atrium) regions ofthe left ventricle then isolation proceeded separately foreach region. The tissues were minced and myocytes wereobtained by filtering through a 100 μm nylon mesh. Cellswere allowed to settle, the supernatant was aspirated, andthe pellets were washed twice and re-suspended in anincubation medium: DMEM (free of phenol red) with5% 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 at1:10,000 dilution, control), 17-β-oestradiol (E2) and/orother agents as described for each experiment.

Electrophysiology

ICa,L was measured with the whole-cell configuration ofthe patch-clamp technique with pipettes filled with (inmmol 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 externalsolution contained (in mmol l−1): 140 NaCl, 10 CsCl,1 CaCl2, 1 MgCl2, 10 glucose and 10 Hepes (pH 7.4with NaOH). Currents were measured with an EPC9/2amplifier (Heka Instruments) at 36 ± 0.5◦C. Cells wereheld at –80 mV and then –40 mV for 80 ms. Membranecurrents were elicited by 200 ms steps from –30 to +60 mVin increments of 10 mV applied every 6 s. Recording weremade ∼3–5 min after gaining whole-cell access and afterICa,L stabilized. Capacitance measurements were obtainedfrom membrane test parameters. There were no significantdifferences 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 ICa,L

density in pA pF−1, abbreviated as ICa,L.The voltage-dependent kinetics of ICa,L was analysed

as previously described (Sims et al. 2008). The Ca,L(g)conductance was calculated from: g = I/(V m−V rev), whereI is the current at V m and V rev is the apparentreversal potential. The ratio of g/gmax (where gmax isthe maximum conductance) was plotted against V m.The relationship between g/gmax and V m was fitted to aBoltzman function: g/gmax = 1/ (1 + exp[(V 0.5 – V m)/k]),where V 0.5 is the half-maximum activation voltage andk is the slope factor of the steady-state activationcurve. To measure the voltage-dependent inactivation,the normalized current at the test potential (I/Imax) wasplotted against V m. The curve was fitted to a Boltzmanfunction: I/Imax = 1/(1 + exp [(V 0.5 – V m)/k), where V 0.5

is the half-maximum activation voltage and k is the slopefactor of the steady-state inactivation curve. The activationkinetics of ICa,L was measured for each depolarizing stepas the time from the onset of the voltage step to the peakcurrent. To determine the time-course of inactivation, the



decay phase of ICa,L was fitted to a bi-exponential function:I = A1 × exp(–t/τfast) + A2 × exp(–t/τslow) + A0, whereτfast and τslow are the time constants of the slow and fastexponential components; A1 and A2 are the amplitudesof the fast and slow exponential components; and A0

indicates the amplitude of the sustained component.

Immunocytochemistry

Cav1.2 (α1C) protein labelling and imaging in cardiacmyocytes was performed as described previously (Zhanget al. 2005). Myocytes were incubated on laminin-coatedcoverslips for 1 day, fixed with 2% paraformaldehydeand permeabilized (0.1% Triton-X 100). Cav1.2 (α1C)monoclonal antibody (1:200, Alomone Labs: catalogueno. ACC-003) was incubated for 60 min then withgoat anti-rabbit Alexa 488 (5 μg ml−1, Molecular Probes).Immunofluorescence was analysed with an Olympus-1000Fluoview confocal microscope and Metamorph (Version7.1, Molecular Devices).

Single-cell mRNA levels by quantitative real-timeRT-PCR

Cav1.2 α mRNA level in single cells was measured byreverse 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 ina micropipette filled with 6 μl of RNase-free water and wasthen ejected into a PCR tube (200 μl) and stored in liquidnitrogen until use. The reverse transcriptase reaction wasdone according to manufacturer’s instructions (Invitrogencatalogue no. 18064022). For the first round PCR (volume20 μl), each mixture contained: 7 μl of RT product, 10 μlof 2× Redmix Plus (including Taq, dNTP and MgCl2) and0.5 μM of primer. The cycle condition was as follows: 94◦C5 min; 94◦C 30 s, 58◦C 30 s, 72◦C 30 s, 35 cycles and 72◦C7 min. The reaction mixture (20 μl) for real-time PCRwas as follows: 2× SYBR Green PCR master mix (AppliedBiosystems) 10 μl, 0.25 μM primers and 6 μl of dsDNAfrom first round PCR product. The real-time PCR reactionwas performed using the ABI PRISM 7000 SequenceDetection 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 GTTTGG CAA CCA TGT CA 3′; and reverse: 5′ GTG GGCGCT GCG TAG TG 3′. (Armoundas et al. 2007). GAPDHwas used as internal reference to normalize the relativemRNA 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 liquidnitrogen. Samples were pulverized in liquid nitrogen andhomogenized on ice. Lysis buffer contained (in mM): 150NaCl, 1 EDTA, 2.5 MgCl2, 20 Hepes, 1% Triton-X 100,0.5 dithiothreitol and 0.1% SDS. Protease inhibitors andphosphatase inhibitor cocktail (PhosSTOP, Roche) wereadded to the lysis buffer. The supernatant resulting fromcentrifugation (120,000 g for 5 min) was used to measureprotein concentration with the BioRad assay (BioRadLaboratories, Hercules, CA, USA) and run on SDS-PAGEgels with 30 μg of protein per lane. ERα antibody (ThermoScientific catalogue no. MA3-310) and ERβ (Santa Cruzcatalogue no. sc-53494) antibody were used at 1:1000dilutions.

Data analysis

Clampfit 9.0, SPSS13.0 and Sigmaplot11.0 were used fordata analysis. All ICa,L were normalized for cell capacitance(i.e. pA pF−1) to allow comparison between cells of varioussizes. All data were expressed as mean ± SEM. Individualgroup statistical comparison were analysed by unpairedStudent t test with Bonferroni correction, and multiplegroup comparisons were evaluated by 2-way ANOVA. Aprobability value of P < 0.05 was considered statisticallysignificant. Each experimental group is described as n/N ,where n is the number of cells and N is the number ofhearts.

Results

Effect of oestrogen on ICa,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 ICa,L from freshly iso-lated myocytes (2–4 h) on days 0, 1, 2 and 3. As shownin Fig. 1A, overnight incubation with E2 significantlyincreased ICa,L in base myocytes at –10 to +20 mV(P < 0.05) compared to controls that were treated withDMSO (1/10,000). E2 incubation increased ICa,L withoutaltering the shape of I–V plots. On days 2 and 3, E2increased ICa,L further whereas in the absence of E2, ICa,L

decreased (Fig. 1B and C). As summarized in Fig. 1D,E2 treatment significantly increased peak ICa,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). Thehigher ICa,L with E2 incubation remained statisticallysignificant 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, ICa,L had a tendency to decrease



in base myocytes incubated in E2-free medium and thisdecrease was statistically significant on day 3 comparedto days 0, 1 and 2 (P < 0.05) for myocytes in the controlgroups. Note that on day 0 (2–4 h post-isolation), basemyocytes had the same ICa,L ± E2, because E2 did notacutely alter ICa,L.

Myocytes from the apex had stable ICa,L from 2–72 hand treatment with E2 (1 nM) did not alter ICa,L. I–Vplots 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 peakICa,L did not change ± E2 incubation for apex myo-cytes (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 ICa,L in female myo-cytes isolated from the base of the endocardium (Fig. 2Aand B) (n/N = 9/2 for each ± E2 group) and in male myo-cytes isolated from the apex and base of the epicardium

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Figure 1. Effect of E2 on ICa,L in female base and apex myocytesA–C, I–V plots for ICa,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 ICa,L (at 0 mV) in female base myocytes incubated ± E2 (1 nM) in freshly isolated cells andafter 1, 2 and 3 days. E, I–V plots of ICa,L in female apex myocytes incubated in the absence (filled squares) orpresence (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, 2or 3; ‡P < 0.01 E2 vs. control. F, summary of mean ICa,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 ICa,L at the apex.



(n/N = 9/2 for each ± E2 group) (Fig. 2C and D). In bothcases, E2 had no significant effect on the I–V relationshipof ICa,L after 24 h incubation with E2 (Fig. 2).

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

Concentration-dependent effects of oestrogen andprogesterone

Various oestrogen and progesterone concentrations weretested on female epicardial base myocytes. Figure 3A showsI–V plots for ICa,L measured after a 1 day incubation with

different E2 concentrations (10, 100 or 1000 pM). E2 at 100and 1000 pM increased the current density without alteringthe shape of I–V plots. A summary histogram shows that10 pM E2 did not alter ICa,L (10.59 ± 0.84 pA pF−1 controlvs. 11.07 ± 0.77 pA pF−1, n/N = 10/2 for each group,P > 0.05) whereas 100 and 1000 pM produced statisticallysignificant increases in ICa,L (12.90 ± 0.46 pA pF−1,100 pM E2 and 14.16 ± 0.9 pA pF−1, 1 nM E2; n/N = 10/2for each group, P < 0.05 vs. control) (Fig. 3B). Thus, E2increased ICa,L in a concentration-dependent manner inthe physiological range for female rabbits (0.1–1 nM) (Bahret al. 1976).

Progesterone (P4) was similarly tested as a possiblemechanism to modulate ICa,L. I–V plots measured fromcontrols and myocytes incubated with P4 (0.1, 1 and10 μM) for 1, 2 and 3 days did not change ICa,L despiteconcentrations of P4 well above physiological levels infemale rabbits (6.5 nM) (Bahr et al. 1976). Figure 3C and

A B

C D

Figure 2. Effect of oestrogen on ICa,L density of female epicardial vs. endocardial base myocytes andmale epicardial apex and base ventricular myocytesMyocytes were isolated from the epicardium and endocardium of the base of female hearts and from theepicardium at the base and apex of male rabbit hearts. The myocytes were incubated with or without E2 (1 nM)for day 1 then ICa,L density was analysed. A, I–V plots for ICa,L measured in female base epicardial and endocardialmyocytes incubated in the absence (filled circles) or presence (open circles) of E2 (1 nM) for 1 day. B, summary ofmean ICa,L ± SEM at 0 mV in female base epicardial and endocardial myocytes incubated ± E2 (1 nM) for 1 day.C, I–V plots for ICa,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 ICa,L ± SEM at 0 mV in male epicardialmyocytes isolated from the base and apex, incubated without (filled columns) and with (open columns) E2 (1 nM)for 1 day.



D illustrates the lack of progesterone effect after 1 day ofincubation.

Role of oestrogen receptors

E2 can regulate cell function through classical ornon-classical genomic pathways that are mediated byoestrogen receptors (ERs) or through non-genomicpathways that bypass ERs (Bjornstrom & Sjoberg, 2005;Levin, 2005). The role of ERs on ICa,L regulation was testedin 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-treatedmyocytes showed that ICI suppressed the effect of E2 onICa,L (Fig. 4A). I–V plots from control (see Fig. 1A) andICI-treated myocytes were indistinguishable. Peak ICa,L

density (0 mV) was not significantly different for controland 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). AlthoughE2 acted via ERs, Fulvestrant did not identify the ER sub-type, ERα or ERβ, since it is not a selective antagonist(Robertson, 2001). To identify the ER subtype, cardio-myocytes were incubated with a selective agonist for ERα(PPT, 5 nM) and/or ERβ (DPN; 5 nM). Figure 4C shows theI–V relationships for four groups of female base myocytes:controls, PPT, DPN and PPT+ DPN-treated cells. PPT butnot DPN significantly increased ICa,L in the voltage rangeof –20 to +20 mV (Fig. 4C). Peak ICa−L were significantlyhigher in PPT compared to controls and DPN-treatedmyocytes (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) and12.65 ± 0.60 pA pF−1, n/N = 18/4, P < 0.01). ICa,L alsoincreased in myocytes treated with both PPT and DPNfor 24 h (17.02 ± 0.77 pA pF−1, n/N = 12/2, P < 0.01 vs.controls). No statistically significant differences werefound for peak current between PPT and PPT +

Figure 3. Effect of oestrogen and progesterone on ICa,LA, I–V plots of ICa,L recorded from female base cardiomyocytes incubated without (filled circles) or with17-β-estradiol (E2 = 10 (open squares), 100 (filled squares) or 1000 (open circles) pM) for 1 day. B, summary ofmean ICa,L (0 mV) in female base cardiomyocytes incubated without (control) or with E2 (10, 100 or 1000 pM) for24 h, †P < 0.05, ‡P < 0.01 vs. control. C, I–V plots of ICa−L recorded from female base cardiomyocytes incubatedwithout (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 ICa,L even after incubating female basecardiomyocytes for 1.5 and 2 days. D, summary of mean ICa,L (0 mV) in female base cardiomyocytes incubatedwithout (control) or with progesterone (P4 = 100 nM, 1 μM or 10 μM) for 1 day.



DPN-treated cells (P > 0.05), suggesting that ERα is themajor ER subtype involved in ICa,L. upregulation.

We tested the regional distribution of ERα and ERβto test the possibility that the regional effects of E2 werecaused by non-uniform expression levels of ERs. Figure 5Ashows the protein distribution of ERα or ERβ from fourfemale and four male rabbit hearts where epicardial tissueswere taken from the apex and base areas of the leftventricles. When normalized with respect to β-actin, ERαor ERβ protein densities were not significantly differentas a function of sex or location. A summary histogramof ERα and ERβ protein density confirmed the uniformdistribution of ERs. Thus, the different responses ofepicardial myocytes from the base and apex to E2 cannotbe explained based on a non-uniform distribution ofERs.

Effects of E2 on ICa,L activation and inactivationproperties

E2 may regulate ICa,L by cell signalling mechanismsinvolving PKA or CAMKII-dependent phosphorylation

or changes in ancillary peptides that interact with mainchannel proteins (α subunit), which tend to shift the I–Vrelationship and alter gating properties (Mikala et al. 1998;Blaich et al. 2010). Although Figs 1–3 showed that E2increased ICa,L without shifting the I–V relationship, theeffect of E2 (1 nM) on the kinetics of ICa,L was measured infemale 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-maximumactivation voltage (–7.52 ± 1.30, E2 vs. –5.83 ± 2.40 mV,controls, P > 0.05) and the slope factor of the steady-stateactivation curve (4.34 ± 0.55, E2 vs. 4.44 ± 0.26, controls,P > 0.05). The half-maximum inactivation voltage wassimilar in E2-treated and control cells (–17.10 ± 0.40 mV,E2 vs. –16.19 ± 0.64 mV, controls, P > 0.05) and theslope factor of the steady-state inactivation curve betweenboth groups (–4.00 ± 0.13, E2 vs. –4.07 ± 0.29, controls,P > 0.05).

The time to peak measured at different membranepotentials did not significantly change by E2 treatmentfor 1 day (Fig. 6C, P > 0.05). Both the fast and slowcomponents of ICa,L at different voltage steps were

Figure 4. Effect of ER antagonist and agonists on cardiac ICa,LA, 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, summaryhistograms of mean ± SEM of ICa,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 ICa,L (0 mV) at female base myocytes in each group. ‡P < 0.05 PPT or PPT + DPNvs. control, †P < 0.05 PPT vs. DPN, NS: PPT vs. PPT + DPN.



Figure 5. Regional distributions of ERα and ERβ atthe base and apex of male and female heartsAa, Western blots for ERα (top lanes) and ERβ (middlelanes) are shown for 8 tissue samples, 4 samples from theapex (A1–4) and from the base (B1–4) of female hearts1–4 and these are aligned with 8 corresponding samplesof β-actin (bottom lanes). Ab, the same experiment as inAa but with tissues from male hearts. B, summaryhistograms of the relative density of ERα and ERβ

normalized with respect to β-actin. The density of eachER did not significantly vary between apex vs. base orbetween male vs. female hearts.

similar ± E2 (Fig. 6D). At 0 mV, τfast was 12.40 ± 1.41 msin control and 10.99 ± 1.97 ms in E2; τslow was56.48 ± 6.23 ms in control and 52.44 ± 7.73 ms in E2(P > 0.05). These data demonstrated that E2 increasedICa,L without altering gating properties.

E2 and Cav1.2α protein and mRNA levels

To test if E2 enhanced ICa,L by stimulating de novoexpression of L-type Ca2+ channel protein, Cav1.2α wasmeasured by immunocytochemistry on myocytes fromthe base and the apex treated with E2 or DMSO for1 day. Figure 7A shows the antibody labelling of Cav1.2(α1C) protein on an isolated cardiomyocyte. When myo-

cytes from the apex were cultured for 1 day, the levels ofCav1.2 (α1C) were similar if incubated with (Fig. 7B) orwithout E2 (Fig. 7A). However, the intensity of Cav1.2(α1C) was considerably higher in base myocytes treatedwith E2 (Fig. 7D) compared to base myocytes incubatedwithout E2 (Fig. 7C). The statistical significance analysedby quantitative measurements of fluorescence intensity ofconfocal images is summarized in Fig. 7E. Cav1.2 (α1C)label was measured from apex myocytes (n/N = 27/3) andwas 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 frombase (n = 16/3) had intensities of 110.77 ± 0.95% whichincreased to 127.59 ± 0.56% when incubated with E2

Figure 6. Effect of E2 on voltage dependenceand kinetics of ICa,LA, representative traces of ICa,L from female basemyocytes incubated without (filled circle) or with 1 nM

E2 (open circle) for 1 day. B, steady-state ICa,L

activation and inactivation curves, female basemyocytes incubated without (filled circles) or with E2(open circles) for 1 day. C, activation kinetics of ICa,L

expressed as the time to peak, from onset of voltagestep to the peak of current amplitude are plotted vs.Vm (mV), female base myocytes incubated without(filled circles) or with E2 (open circles) for 1 day. D,voltage dependence of the time-courses ofinactivation: τ fast (circles) and τ slow (triangles), femalebase myocytes incubated without (filled symbols) orwith E2 (open symbols) for 1 day.



(P < 0.01, control base vs. E2 base). The relative increaseof channel protein caused by incubation with E2 (Fig. 7D)is consistent with the increase in peak ICa,L.

Single-cell quantitative real-time RT-PCR was used todetermine whether E2 increased the expression levels ofCav1.2 (α1C) by a genomic mechanism which wouldbe associated with an increase in mRNA levels. Femalebase myocytes were incubated with or without E2(1 nM) for 1 day (n/N = 6/2 for each group) followedby quantitative real-time RT-PCR analysis. As shownin Fig. 7F , mRNA levels were 30% more elevatedin myocytes treated with E2 compared to controls(P < 0.04).

Inhibition of transcription or translation suppressesICa,L upregulation

The E2 upregulation of Cav1.2 expression (Fig. 7)may occur via an increase of mRNA and de novoprotein biosynthesis or enhanced translation but stabletranscription. Female base cardiomyocytes were incubatedwith E2 (1 nM) and either actinomycin D (AmD, 4 μM)or cycloheximide (CHX, 70 μM) for 24 h to respectivelyblock transcription or translation, as previously described(Benitah & Vassort, 1999). I–V relationships forE2-treated myocytes, E2 + AmD and E2 + CHXare superimposed in Fig. 8A and B. Peak ICa,L are

Figure 7. Effect of oestrogen on Cav1.2α levelsRepresentative 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 intensityfrom control apex (A), E2-treated apex (B), control base (C) and E2-treated base (D) myocytes. †P < 0.05 E2 basevs. 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 comparedto Ctrl).



summarized in Fig. 8C. Overnight incubation with E2increased ICa,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 forboth groups,P < 0.05). E2 treatment resulted in a 32.6%increase of peak ICa,L. AmD or CHX alone had no effecton basal ICa,L (without E2), in agreement with findings byBenitah & Vassort (1999). However, AmD or CHX blockedthe upregulation of ICa,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 ICa,L afterincubation with E2 involves a genomic upregulation.

Discussion

The main findings are that oestrogen but not progesteroneupregulates ICa,L in cardiomyocytes isolated from the basebut not the apex, which accounts for the regional andsex heterogeneities of ICa,L that were reported in intactfemale rabbit hearts. E2, at concentrations as low as 0.1 nM,upregulated ICa,L at the base of the female epicardium by agenomic mechanism mediated by ERα but not ERβ. E2 didnot change ICa,L kinetics or gating properties and increasedthe mRNA and protein levels of Cav1.2α. Consistentwith these findings, the upregulation of ICa,L by E2 wasblocked by the inhibition of transcription or translation.E2 (1 nM) elicited similar increases of channel protein and

of peak ICa,L without altering activation or inactivationkinetics of ICa,L, which supported the interpretation thatthe increase in current was due to an increase in the copynumber of functional channels. Put together, these dataprovide compelling evidence that E2 upregulates L-typeCa2+ channels by a regional genomic mechanism.

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

Animal studies showed that OVX reduces the riskof dofetilide (IKr blocker)-induced EAD (Pham et al.2001), and E2 replacement in OVX rabbits increasedthe incidence of drug-induced EAD (Drici et al. 1996).A similar finding was reported in papillary muscles ofOVX rabbits (Hara et al. 1998). Optical mapping of APsand Ca2+

i from Langendorff rabbit hearts with LQT2revealed that aberrations of Ca2+

i handling at the baseof the epicardium was associated with higher ICa,L density,which played a major role in the induction of EADs andTdP (Choi et al. 2002; Liu et al. 2005; Sims et al. 2008;Nemec et al. 2010). E2 and ICa,L levels were known tobe important determinants of arrhythmia risk in LQT2;the current study shows that E2 upregulates ICa,L bya regional genomic mechanism mediated by ERα. Inaddition, we recently showed that E2 upregulates theNa+–Ca2+ exchange (NCX1) current, INCX, by increasing

Figure 8. Inhibition of transcription ortranslation during E2-inducedup-regulation of ICa,LA, superposition of I–V plots from femalebase 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 femalebase 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 ofICa−L (0 mV) in female base myocytes ineach group for 1 day. †P < 0.05 E2 vs.control



mRNA levels and protein expression of NCX1 by aregional genomic mechanism similar to that shown herefor Cav1.2α (Chen et al. 2011). Moreover, we showedthat the higher levels of ICa,L and INCX at the base offemale hearts account for sex differences in LQT2-relatedarrhythmias (Liu et al. 2005; Sims et al. 2008; Chenet al. 2011) and since both currents are upregulated byE2, E2 underlies sex-related arrhythmogenic effects. Thegreater susceptibility of women to ventricular arrhythmiashas been attributed to a ‘reduced repolarization reserve’mostly due to a greater suppression of IKr comparedto men (Roden, 2006). However, IKr blockade alonemay not be sufficient to trigger EADs. However, whencombined with a higher ICa,L, female hearts have a greaterlikelihood of intracellular Ca2+ overload, spontaneousCa2+ release from the sarcoplasmic reticulum and EADscaused by the reactivation of ICa,L during the AP plateaudue a Ca2+

i -mediated enhanced depolarizing Na+–Ca2+

exchange current. Consistent with this interpretation isthat APD prolongation is not a good predictor of TdPvulnerability. For instance, male pre-pubertal rabbit heartswere shown to be more susceptible to EADs and TdPin drug-induced LQT2, despite having shorter APDsthan pre-pubertal females (Liu et al. 2005). Moreover,pre-pubertal male base myocytes had greater ICa,L densitythan females implicating Ca2+

i overload as a majordeterminant of EADs and TdP rather than APDs (Simset al. 2008).

Genomic regulation in myocyte cultures

The genomic regulation of cardiac ion channels wasstudied by incubating adult primary cardiomyocytecultures with E2 which increased Cav1.2α levels andICa,L whereas progesterone did not. Issues regarding thestability of cardiomyocytes in culture have been addressedin several studies of cell signalling and regulation of ionchannel expression. For instance, rabbit cardiac ICa,L wereshown to remain stable for 4 days even though contra-ctility decreased (Mitcheson et al. 1997, 1998). CardiacATP, CrP and LDH levels have been found to remainstable in long-term culture (Decker et al. 1990) andmouse cardiomyocytes had normal signalling responsesfor 3 days (Sambrano et al. 2002). Here, the effects ofE2 were tested for up to 3 days post-isolation but mostof the E2-dependent changes occurred in 1 day, the timeneeded to obtain significant genomic upregulation of ICa,L.Our cultured cardiomyocytes retained their differentiatedstate for 3 days based on whole-cell membrane capacitance(Chen et al. 2011), organization of T-tubules and stabilityof I–V plots. Moreover, ICa,L measured from the apexremained stable over 3 days post-isolation and for eachheart, data from the apex served as internal control fromchanges in ICa,L measured at the base. It should be noted

that the stability of ICa,L density has been traditionallyused as a measure of phenotype stability for culturedadult myocytes, with varying results. The current dataprovide compelling evidence that ICa,L density alone isnot sufficient to measure phenotype change of adult myo-cytes and close attention must be paid to the age, sex andlocation 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 causingthe indirect phosphorylation of Cav1.2α, becausethe elevation of peak ICa,L by E2 required ∼1 day ofincubation in E2. Acute effects of E2 and progesteronehave been reported to decrease ICa,L. E2 at 300 nM–10 μM

decreased ICa,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-dependentactivation of ICa,L in guinea pig myocytes (Nakamuraet al. 2007). However, the steroid concentrations usedin many studies tend to be significantly higher than themaximum physiological concentrations of these steroidsand most studies ignored the binding of steroids tosex hormone binding globulins (SHBG) such that theconcentrations of ‘free’ steroids are ∼1% of total steroidlevels. In rabbits, the bio-available concentrations ofE2 are considerably lower than the often stated ‘totalphysiological’ concentration (E2, ∼0.3 nM; Bahr et al.1976) because of high-affinity binding to SHBG (Araujoet al. 2008). In our pilot studies using rabbit ventricularmyocytes from the base of the epicardium, E2 and P4 atup to 10 μM failed to impart an acute effect on ICa,L.

We presented compelling evidence that E2 regulatesICa,L through a genomic mechanism that involvesstimulation of transcription: (1) E2 acted in ∼1 day, tooslowly to suggest a direct interaction or a cell-signallingmechanism; (2) E2 could act by ER-dependent or-independent mechanisms (Bjornstrom & Sjoberg, 2005;Levin, 2005); however, the inhibition of E2 regulationof ICa,L by fulvestrant, an antagonist of ERα and ERβ,demonstrates that the mode of action of E2 occurs via oneof its nuclear receptors. ERα and ERβ have been shownto yield different and sometimes opposing effects on cellfunction (Matthews & Gustafsson, 2003), but our resultsshow that an ERα agonist mimics the effects of E2 and ERβhas no significant effect on ICa,L. The latter implicates theERα subtype as in the receptor mediating E2-dependentupregulation of Cav1.2α.

In silico analysis of the promoter region of humanCACNA1C using BIOBASE Knowledge Library detectedeight high-probability oestrogen receptor ERα bindingsites within the first 500 nucleotide promoter region.The analysis suggests that ERα (but not ERβ) regulates



the transcription of the gene that encodes for Cav1.2αin human hearts. The analogous search for ER bindingsites was run on the rabbit CACNA1C promoter sequence(500 nucleotides before the start codon), obtainedfrom the UCSC Genome Bioinformatics data base (athttp://genome.ucsc.edu). The rabbit promoter sequencewas transported to BIOBASE: EXPLAIN to search for ERαand ERβ binding sites and as for the human CACNA1Csequence, eight ERα (and no ERβ) response elements weredetected at analogous positions within the rabbit promotersites as for the human promoter sites. Besides the promoterregion of CACNA1C, the search was carried out in the largeintron found in both the human and rabbit CACNA1C.In both introns, 49 high-quality ERα binding sites wereidentified from an in silico analysis; these are putative sitesof interaction of ERα and CACNA1C.

Evidence of direct interactions between CACNA1C andERα was reported through chip-on-chip analysis of thehuman CACNA1C, which is available from the BIOBASEKnowledge Library. The latter analysis detected the sameeight high-quality ERα binding sites in the promoterregion and none were found in the intron in humanMCF-7 cells (Welboren et al. 2009). Chip-on-chip analysisprovides compelling evidence of ERα genomic regulationof the human gene but such a study has not been carriedout in the rabbit. The analysis of the rabbit sequencesuggests that there are putative ERα binding sites butdoes not provide proof that ERα binding does occurand results in functional oestrogen response elements.Nevertheless, the prevalence of high-affinity ERα bindingsites in analogous regions of the human and rabbitCACNA1C gene and chip-on-chip analysis that identifiedthese ERα as biologically active supports the notion ofa direct genomic regulation of Cav1.2α by ERα. Furtherstudies will be needed in the rabbit to identify possibleregulatory sites in CACNA1C introns and bioactive siteswill have to be demonstrated by GENE SEEK ChIP assaysperformed in rabbit heart.

Both ERs can mitigate the response to vascular injury(Kim & Levin, 2006) but in mice, only ERα appearsto protect the heart from ischaemic injury (Wang et al.2006). In the ERα knockout mouse, ICa,L and Cav1.2 wereupregulated (Johnson et al. 1997), which is opposite andcontrary to the positive transcriptional response of thesegenes to E2 in rabbits. Several factors could account forthe different results: (a) E2 regulation of ICa,L and Cav1.2appears to be restricted to specific regions of the heart,which was not investigated in the mouse, (b) speciesdifferences in regulation of this current are likely sincethe mouse and rabbit action potential differ significantlyfrom endocrine and electrophysiological points of view,and (c) distinct transcriptional elements that control theexpression of Cav1.2α may be differentially activated inmice and rabbits. Among the conserved cardiac trans-criptional 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 suppressionof Nkx2.5 and NFAT activation, which has been observedin other cell types (Qin et al. 2008; Marni et al. 2009),may be responsible for reduced expression of Cav1.2α inE2-treated cells. In contrast, activation of CREB acts inthe opposite direction by increasing Cav1.2α expression(Mayr & Montminy, 2001). Ongoing studies in our groupshow that CREB activation is 2- to 3-fold greater at the basethan the apex of female hearts and of male hearts. It is inter-esting to speculate that the regional CREB activation plusE2–ERα complex may be important to upregulate Cav1.2αexpression. Further studies will be needed to determinethe mechanisms underlying the regional transcriptionalregulation of Cav1.2α or why E2 can upregulate ICa,L andINCX in myocytes from the base but not the apex of theepicardium.

Nevertheless, the upregulation of L-type Ca2+ channelsand of Na+–Ca2+ exchange protein (Chen et al. 2011) byE2 contribute to sex differences in Ca2+ handling whichis accentuated during repolarization delays in LQT2 andunderlie the propensity to EADs and arrhythmia risk (Simset al. 2008). Their role may have been missed because of alack of investigation of apex–base regional heterogeneitiesof ion channel expression. These sex differences of ICa,L

in rabbits may apply to human hearts where a trendtowards higher ICa,L was reported in women than in men(Verkerk et al. 2005). Our findings bring new insights tothe actions of sex steroids, their role in the cardiac ionchannel remodelling and implicate ERs as novel targets inthe treatment of ventricular arrhythmia.

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Author contributions

All authors approved the final version of the manuscript. XYand GC contributed equally to the collection, analysis andinterpretation of data and to drafting the article, RP madesignificant contributions to the collection of Western blots andimmuno-histochemistry, DD helped us elucidate the genomicregulation of L type calcium channels, FZ, thesis advisor of XY,provided partial financial support for XY and GS conceived anddesigned the study, contributed to the interpretation of data,drafted and revised the article for important intellectual contentand provided the financial support for the study.

Acknowledgements

This work was supported by NIH-NHLBI grants HL57929 andHL70722 (to G.S.) and by China NSFC grant: 81000080 (to X.Y.).The authors have no conflicts to declare.


oestrogen upregulates l-type ca2+ channels via oestrogen-receptor- by a regional genomic mechanism...

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