Estrogen Receptor b Protects the Murine Heart Against Left Ventricular Hypertrophy Fawzi A. Babiker, Daniel Lips, Rainer Meyer, Els Delvaux, Pieter Zandberg, Ben Janssen, Guillaume van Eys, Christian Grohé and Pieter A. Doevendans Arterioscler Thromb Vasc Biol. 2006;26:1524-1530; originally published online April 20, 2006; doi: 10.1161/01.ATV.0000223344.11128.23 Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2006 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://atvb.ahajournals.org/content/26/7/1524 Data Supplement (unedited) at: http://atvb.ahajournals.org/content/suppl/2006/04/24/01.ATV.0000223344.11128.23.DC1.html Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published inArteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in thePermissions and Rights Question and Answerdocument. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online at: http://atvb.ahajournals.org//subscriptions/ Downloaded from http://atvb.ahajournals.org/ by guest on February 20, 2013 (cid:1) Estrogen Receptor Protects the Murine Heart Against Left Ventricular Hypertrophy Fawzi A. Babiker, Daniel Lips, Rainer Meyer, Els Delvaux, Pieter Zandberg, Ben Janssen, Guillaume van Eys, Christian Grohe´, Pieter A. Doevendans Background—Leftventricularhypertrophy(LVH)displayssignificantgender-baseddifferences.17(cid:1)-estradiol(E2)plays animportantroleinthisprocessbecauseitcanattenuatepressureoverloadhypertrophyvia2distinctestrogenreceptors (ERs): ER(cid:2)and ER(cid:1). However, which ER is critically involved in the modulation of LVH is poorly understood. We therefore used ER(cid:2)-deficient (ER(cid:2)(cid:1)/(cid:1)) and ER(cid:1)-deficient (ER(cid:1)(cid:1)/(cid:1)) mice to analyze the respective ER-mediated effects. Methods and Results—Respective ER-deficient female mice were ovariectomized and were given E2 or placebo subcutaneouslyusing60-dayreleasepellets.After2weeks,theyunderwenttransverseaorticconstriction(TAC)orsham operation.InER(cid:2)(cid:1)/(cid:1)animals,TACledtoasignificantincreaseinventricularmasscomparedwithshamoperation.E2 treatment reduced TAC induced cardiac hypertrophy significantly in wild-type (WT) and ER(cid:2)(cid:1)/(cid:1) mice but not in ER(cid:1)(cid:1)/(cid:1) mice. Biochemical analysis showed that E2 blocked the increased phosphorylation of p38–mitogen-activated protein kinase observed in TAC-treated ER(cid:2)(cid:1)/(cid:1) mice. Moreover, E2 led to an increase of ventricular atrial natriuretic factor expression in WT and ER(cid:2)(cid:1)/(cid:1) mice. Conclusions—ThesefindingsdemonstratethatE2,throughER(cid:1)-mediatedmechanisms,protectsthemurineheartagainst LVH. (Arterioscler Thromb Vasc Biol. 2006;26:1524-1530.) Key Words: hypertrophy (cid:1) hormones (cid:1) myocardium (cid:1) gender Theincreaseofleftventricularmassrepresentsastructural hormone. However, the underlying mechanisms of E2 pro- mechanism of compensation of the heart in response to tectionofthemyocardiumarenotfullyunderstood.Myocytes pressure overload. The resulting left ventricular hypertrophy and fibroblasts contain functional estrogen receptors (ERs) (LVH) is an important, independent negative predictor of ER(cid:2)andER(cid:1).Viathesereceptors,E2modulatestheactivity cardiac morbidity and mortality.1 LVH displays significant ofthemitogen-activatedproteinkinase(MAPK)pathwaysin gender-based differences. Premenopausal women have a cardiacmyocytes.6TheMAPKsignalingpathwaysconsistof lower prevalence of LVH than men.2 The Coronary Artery a sequence of successively acting kinases that ultimately Risk Development In young Adults (CARDIA) study dem- result in the dual phosphorylation and activation of effector onstrated a higher prevalence of LVH in men, even after kinases such as p38-MAPKs, c-Jun N-terminal kinases correction for a large number of risk factors. It further (JNKs), and extracellular signal-regulated kinases (ERKs), demonstratedthatthedifferenceinleftventricularsizebegins which subsequently phosphorylate a large array of targets, early in life (ie, before menopause), suggesting that intrinsic leading to altered gene expression patterns.7 These signaling factorsareinvolvedintheinductionofLVH.2Sexhormones cascades play an important role in the initiation of cardiac such as estrogen have been attributed to play an important hypertrophy and in the development of heart failure.7–10 E2 roleinthepathogenesisofcardiovasculardisease.Therecent caninhibitp38-MAPKphosphorylationandthusp38-MAPK clinical trials with respect to the therapeutic role of 17(cid:1)- activation.11Furthermore,itisknownthatE2canincreasethe estradiol (E2) vascular disease are controversial.3 However, expression of the atrial natriuretic factor (ANF), which thepotentialofE2asatherapeuticoptioninthemodulation recently has been shown to possess antihypertrophic ef- of cardiac disease remains poorly understood. It has been fects.5,11–13SignificantincreasesinANFmRNAaredetected demonstratedthatestrogensareabletoattenuatehypertrophic inthemouseventriclethatischallengedbyaorticbanding.11 responses.4,5 E2 appears to act as a cardioprotective steroid However, little is known about the respective role of the OriginalreceivedJune15,2005;finalversionacceptedApril11,2006. From the Department of Cardiology (F.A.B., D.L., E.D.), Cardiovascular Research Institute Maastricht, University Hospital Maastricht, the Netherlands; Physiologisches Institut II (R.M.), Bonn Germany; Department of Pharmacology (P.Z.), Organon NV, the Netherlands; Department of Pharmacology(B.J.),CardiovascularResearchInstituteMaastricht,MaastrichtUniversity,theNetherlands;DepartmentofMolecularGenetics(G.v.E.), Cardiovascular Research Institute Maastricht, Maastricht University, the Netherlands; Medizinische Universita¨tspoliklinik (C.G.), Bonn, Germany; InteruniversityCardiologyInstituteoftheNetherlands(P.A.D.)andDepartmentofCardiology(P.A.D.),HeartLungCenterUtrecht,theNetherlands. CorrespondencetoDrC.Grohe´,MedizinischeUniversita¨ts-Poliklinik,Wilhelmstr.35-37,Universita¨tsklinikumBonn,53111Bonn,Germany.E-mail [email protected] ©2006AmericanHeartAssociation,Inc. ArteriosclerThrombVascBiol.isavailableathttp://www.atvbaha.org DOI:10.1161/01.ATV.0000223344.11128.23 Downloaded from http://atvb.ahajou1rn5a2l4s.org/ by guest on February 20, 2013 Babiker et al ER(cid:1)Protects the Murine Heart 1525 distinct ERs. We recently reported the effects of E2 on the collagencontentwasperformedwithacomputerizedmorphometry development of pressure-overload hypertrophy and the acti- systemasdescribedpreviously.21 vationofsignalingpathwaysofMAPKs.11Furthermore,new Immunoblot Analysis studies suggest that ER(cid:1)plays an important role in cardiac Total heart lysates (40 (cid:3)g per lane) were analyzed by standard disease.14 Here, we further define the role of ERs in this immunoblotting procedures as described previously.22 For details, process. For this goal, we used ER(cid:2)-deficient (ER(cid:2)(cid:1)/(cid:1)) and seetheonlinesupplement. ER(cid:1)-deficient (ER(cid:1)(cid:1)/(cid:1)) mice. We found that cardioprotec- tiveeffectsofE2onLVHaremediatedbyER(cid:1)andnotER(cid:2). Real-Time Polymerase Chain Reaction Analysis These effects are paralleled by an increase in the expression Detailsofthereal-timeRT-PCRhavebeendescribedpreviously.23 The primer sequences used for real time PCR are: ANF 5(cid:4) primer of ANF and a decrease in the phosphorylation of p38. (5(cid:4)-CCTGTGTACAGTGCGGTGTC),ANF3(cid:4)primer(5(cid:4)-TCC TCC AGG TGG TCT AGC A), cyclophillin 5(cid:4) primer (5(cid:4)-CAA Materials and Methods ATGCTGGACCAAACACAA),cyclophillin3(cid:4)primer(5(cid:4)-TTC ACC TTC CCA AAG ACC ACA T). The CT measurement is Animals defined at the fractional cycle number at which the amount of ER(cid:2)(cid:1)/(cid:1) transgenic mice were generated using C57BL/6 as back- amplified target reaches a fixed threshold above background Sybr ground as described previously.15 These mice, which have been Green fluorescence. The amount of target in the cDNA sample extensively studied, do not express ER(cid:2)protein in any tissue.15–18 relativetocyclophilinewascalculated. ER(cid:1)(cid:1)/(cid:1) mice were generated and provided by Organon (Oss, the Netherlands). For details, see the online supplement, available at Statistical Analysis http://atvb.ahajournals.org. Mice showing germline transmission were again crossed with C57BL/6 mice (F2(cid:2) F3 generation). Dataareshownasmean(cid:5)SEM.MeanswerecomparedbyANOVA, followed by Bonferroni test for multiple comparisons. Differences Subsequently, mice from the same litters were used for breeding. wereconsideredsignificantatP(cid:6)0.05.24 Wild-type(WT)littermatesfromtherespectivegenotypeswereused inthestudy.Onlyfemalemiceof(cid:3)10weeksagewereincorporated Results intothisstudy. Inourstudy,wedividedthecardiacanalysisoftheanimalsin Experimental Procedures a total of 16 different groups, as shown in the Table. The All animals (300 WT and knockout mice) were housed under different groups underwent either sham or TAC surgical standardconditions.Animalswereanesthetizedwithketamine(100 proceduresandwereovariectomizedafterE2substitution.A mg/kg body weight [BW] IP) and xylazine (10 mg/kg BW IP) for complete phenotypic analysis of both cardiac and endocrine ovariectomy, pellet placement, and transverse aortic constriction parameters was performed to study the receptor-mediated (TAC).Thestudywasapprovedbytheanimalethicscommitteeof theUniversityofMaastricht. effects in all animal groups studied (Table). E2 replacement ledtoareconstitutionofphysiologicalE2levels(122pg/mL Estrogen Replacement in E2-treated versus (cid:6)5 pg/mL in placebo-treated). Uterus Two weeks after ovariectomy, a 60-day-release pellet containing weight(UW)wasmeasuredtodemonstratetheeffectiveness 0.18 mg E2 or placebo was implanted subcutaneously. All pellets of ovariectomy and E2 substitution in all animals. In all were purchased from Innovative Research of America. E2 serum groups (8 conditions with TAC or sham and placebo or E2 levelsweremeasuredwitharadioimmunoassay(DPCBiermann)in asubsetofanimals. treatment for ER(cid:2)(cid:1)/(cid:1) as well as ER(cid:1)(cid:1)/(cid:1)), the UW/BW and UW/tibia length (TL) ratios showed a significant difference Surgical Procedures and Hemodynamics between placebo and E2-treated mice (Table). In E2-treated Ovariectomy was performed by a standard bilateral abdominal WT and ER(cid:1)(cid:1)/(cid:1) mice, the UW/BW ratios are significantly approach.Theuteruswasleftremainingtostudytheresponsiveness higher than that of E2-treated ER(cid:2)(cid:1)/(cid:1) mice (Table). To- to hormone replacement therapy. Afterward, placebo or E2- gether,wewereabletodemonstratethatovariectomyleadsto containingpelletswereimplantedintheuppernecksubcutaneously. Two weeks after the pharmacological intervention, TAC was per- uterus atrophy on the basis of E2 withdrawal, and E2 formed, as described previously.11 Sham-operated animals under- replacement restored UW. There were no significant differ- went an identical operation without placement of the constricting ences in BW between the groups and no significant changes suture. Assessment of left ventricular function was performed as in lung weight (Table). describedpreviously.19,20Conductanceandpressureinputwasdig- In all animal groups, TAC led to a significant increase in itizedwithaConduct-PCdataacquisitionsystem(CDLeycomBV). Average values for mean arterial pressure, heart rate, systolic and ventricularmass4weeksaftertheintervention.E2treatment diastolic LV pressure, and left ventricular end-diastolic pressure led to a significant reduction of the increase of ventricular were determined. The mortality in all treatment groups during the weight(VW)andtheVW/TLratioinWTandER(cid:2)(cid:1)/(cid:1)mice surgerydidnotdiffersignificantlybetweengroups.Inparticular,no (Figure 1). No differences were observed between sham- increasedmortalitywasfoundintheestrogentreatmentgroupsand theER(cid:1)animalgroups. operated mice (Figure 1; data not shown). Also in ER(cid:1)(cid:1)/(cid:1) mice and their WT littermates, TAC led to significant Tissue Preparation and Histology increaseinventricularmass4weeksaftertheintervention.In Hearts were arrested in diastole with CdCl (0.1 mol/L IV). For WT mice, TAC the degree of ventricular hypertrophy were 2 morphometric analysis, hearts were fixed in 10% formalin and significantly lower in E2-treated compared with placebo- embeddedinparaffinasdescribedpreviously.21Forproteinextrac- treated mice. Interestingly, E2 treatment in ER(cid:1)(cid:1)/(cid:1) mice tion,heartswereexcisedandwashedinice-coldPBS.Allexternal resultedinahigherlevelofhypertrophycomparedwithWT fluidwascompletelyremovedbeforetheorganswereweighedand frozen.Transversesectionsoftheheartwerestainedwithhematox- mice. Similar results were found when we used VW/BW ylin and eosin, sirius red, or modified Azan. The analysis of the (please see the online supplement; data not shown). No Downloaded from http://atvb.ahajournals.org/ by guest on February 20, 2013 1526 Arterioscler Thromb Vasc Biol. July 2006 EffectsofTACandE2TreatmentonBWandOrganWeight Sham TAC Placebo E2 Placebo E2 WT(n(cid:7)) 7 7 10 10 BW,g 24.00(cid:5)0.61 24.25(cid:5)1.50 23.55(cid:5)1.14 23.25(cid:5)0.67 VW,mg 114.56(cid:5)4 119.88(cid:5)12 161.91(cid:5)26* 131.75(cid:5)13*† VW/BW,mg/g 4.77(cid:5)0.19 4.94(cid:5)0.35 6.89(cid:5)1.16* 5.68(cid:5)0.75*† VW/TL,mg/mm 6.24(cid:5)0.24 6.93(cid:5)0.61 9.21(cid:5)1.59* 7.63(cid:5)0.74*† UW/BW,mg/g 0.63(cid:5)0.09 14.38(cid:5)1.82† 1.39(cid:5)0.21 11.89(cid:5)2.1† Lungweight,g 0.18(cid:5)0.06 0.18(cid:5)0.02 0.17(cid:5)0.01 0.17(cid:5)0.04 ER(cid:2)(cid:1)/(cid:1)(n(cid:7)) 7 7 10 10 BW,g 24.17(cid:5)1.56 26(cid:5)2.78 22.71(cid:5)2 24.08(cid:5)0.78 VW,mg 112.83(cid:5)10 119.20(cid:5)7 164.57(cid:5)29* 135.92(cid:5)16*† VW/BW,mg/g 4.68(cid:5)0.46 4.61(cid:5)0.20 7.34(cid:5)1.70* 5.65(cid:5)0.72*† VW/TL,mg/mm 6.55(cid:5)0.66 6.96(cid:5)0.42 9.93(cid:5)0.61* 7.99(cid:5)0.98*† UW/BW,mg/g 0.73(cid:5)0.15 3.26(cid:5)0.19‡ 0.71(cid:5)0.13 3.42(cid:5)0.35†‡ Lungweight,g 0.16(cid:5)0.02 0.15(cid:5)0.01 0.20(cid:5)0.09 0.16(cid:5)0.02 WT(n(cid:7)) 7 7 10 10 BW,g 23.71(cid:5)2.33 24.00(cid:5)1 22.66(cid:5)1.88 25.25(cid:5)1.56 VW,mg 119.14(cid:5)16 114.17(cid:5)7 170.71(cid:5)29* 131.50(cid:5)12*† VW/BW,mg/g 5.01(cid:5)0.28 4.75(cid:5)0.23 7.66(cid:5)1.95* 5.29(cid:5)0.81*† VW/TL,mg/mm 6.81(cid:5)0.84 6.75(cid:5)0.42 9.79(cid:5)1.77* 7.45(cid:5)0.89*† UW/BW,mg/g 1.33(cid:5)0.22 12.39(cid:5)1.22† 1.05(cid:5)0.07 14.85(cid:5)1.96† Lungweight,g 0.17(cid:5)0.02 0.14(cid:5)0.02 0.18(cid:5)0.03 0.16(cid:5)0.00 ER(cid:1)(cid:1)/(cid:1)(n(cid:7)) 7 7 10 10 BW,g 24.30(cid:5)1.90 25.86(cid:5)1.27 26.14(cid:5)1.06 24.71(cid:5)0.69 VW,mg 111.30(cid:5)6 122.29(cid:5)6 157.43(cid:5)13* 169.29(cid:5)32* VW/BW,mg/g 4.60(cid:5)0.28 4.73(cid:5)0.21 6.03(cid:5)0.53* 6.86(cid:5)1.25* VW/TL,mg/mm 6.42(cid:5)0.33 7.14(cid:5)0.39 8.78(cid:5)0.79* 9.76(cid:5)1.79* UW/BW,mg/g 1.16(cid:5)0.16 13.28(cid:5)3.19† 0.93(cid:5)0.11 15.77(cid:5)1.77† Lungweight,g 0.16(cid:5)0.02 0.16(cid:5)00.01 0.18(cid:5)0.01 0.16(cid:5)0.01 BWindicatesbodyweight;VW,ventricularweight;TL,tibiallength;UW,uterusweight.Allvalues areMean(cid:5)SEM.*P(cid:6)0.05forTACvssham,†P(cid:6)0.05forE2vsplacebo,‡P(cid:6)0.05forKOvsWT. significant differences were observed among the sham- Hemodynamic analysis showed that developed pressure, as operated mice (Figure 1). Weight analyses are in line with an indication of the quantity of afterload, was significantly morphometric analyses (please see the online supplement). increased in TAC ER(cid:2)(cid:1)/(cid:1) and ER(cid:1)(cid:1)/(cid:1) instrumented mice The most surprising finding was the lack of inhibition of comparedwithshammice(pleaseseetheonlinesupplement). hypertrophy in the ER(cid:1)(cid:1)/(cid:1) mice. Therefore, we performed E2 treatment had no influence on the degree of pressure invasive hemodynamic measurements to check whether the overloadcomparedwithplacebo-treatedmice(pleaseseethe blunted response had an effect on left ventricular function. online supplement). There was no significant difference Figure1.PhenotypicanalysisofVW/TL ratiosinallanimalmodels(A,ER(cid:2)(cid:1)/(cid:1);B, ER(cid:1)(cid:1)/(cid:1)).VW/TLratiosfromanimalswith TACorSHAMthatweretreatedwithE2or placebofor4weeksafterintervention.The degreeofventricularhypertrophywassignifi- cantlylowerinE2-treatedthanplacebo- treatedWTandER(cid:2)(cid:1)/(cid:1)micewithpressure overload.E2treatmentledtoasignificant reductioninthedegreeofhypertrophyinWT butnotinER(cid:1)(cid:1)/(cid:1)mice.Allvaluesare mean(cid:5)SEM;n(cid:7)for7shamand10forTAC pergroup.*P(cid:6)0.05forTACvssham; (cid:2)P(cid:6)0.05forE2vsplacebo. Downloaded from http://atvb.ahajournals.org/ by guest on February 20, 2013 Babiker et al ER(cid:1)Protects the Murine Heart 1527 Figure2.CardiacfunctionmeasuredinER(cid:1)(cid:1)/(cid:1) andWTmice.Representativeanalysisofcardiac output(CO;A),dP/dt (B),anddP/dt (C).D max min showsleftventricularinvivopressure–volume loopsinshamplacebo-treatedWTmice(ER(cid:1)(cid:1)/(cid:1) littermates;blackloops),TACplacebo-treated ER(cid:1)(cid:1)/(cid:1)mice(blueloops),andTACE2-treated ER(cid:1)(cid:1)/(cid:1)(redloops).RVUindicatesrelativevolume units.Pressuregradientsofallgroupsareshown. Allvaluesaremean(cid:5)SEM;n(cid:7)7forshamand10 forTACpergroup. between ER(cid:2)(cid:1)/(cid:1) and ER(cid:1)(cid:1)/(cid:1) mice. Cardiac performance re- WT and ER(cid:2)(cid:1)/(cid:1) mice with pressure overload hypertrophy, mainedatnormalWTlevelbecausenosignificantdifferencesin whereas it exerted no effect in sham-operated animals and cardiac output and heart rate were detected between groups ER(cid:1)(cid:1)/(cid:1) mice (Figure 3). No differences could be observed (pleaseseetheonlinesupplement).EvenintheER(cid:1)(cid:1)/(cid:1)mice,no between the study groups with regard to the phosphorylation deteriorationofcardiacfunctionwasobservedafter4weeksof levelofERK1/2andJNK(datanotshown). pressureoverload.Hypertrophicgrowthisthereforenotneces- Furthermore,TACledtoasignificantincreaseinANFexpres- sarytomaintaincardiacperformanceinthesituationofpressure sion in the hypertrophied ventricles of WT and ER(cid:2)(cid:1)/(cid:1) mice 4 overload.Thisisnotinaccordancewiththegeneralconsensus weeks after intervention compared with placebo-treated and that hypertrophy is an obligatory compensating mechanism to ER(cid:1)(cid:1)/(cid:1) mice (P(cid:6)(cid:5)0.05); however, in ER(cid:1)(cid:1)/(cid:1) mice, no such withstand augmented hemodynamic stress. In this study, ven- effect was found (Figure 3). No significant difference was seen tricular contraction and relaxation did not alter between geno- betweenplacebo-treatedanimalsinbothgroups.Also,E2-treated typesnorsubstitutiontherapiesafterTAC.Maximalderivative WTmiceshowedanonsignificantdifferencebetweenE2-treated of left ventricular pressure (dP/dt ) increased and minimal WTofER(cid:2)(cid:1)/(cid:1)andER(cid:1)(cid:1)/(cid:1)animals(Figure3). max derivative (dP/dt ) decreased, although no significant differ- min ences were found between groups (Figure 2). The pressure Discussion volume loops showed the absence of significant differences in ER-mediatedeffectsincardiovasculardiseaserequireabetter cardiac performance between placebo- and E2-treated ER(cid:1)(cid:1)/(cid:1) understandingbecauseofthecontroversialfindingsofprevi- (Figure 2), except for the TAC-induced systolic pressure rises. ousclinicalstudiesofhormonereplacementtherapy.25These DatainER(cid:2)(cid:1)/(cid:1)micearecomparable(datanotshown).E2had prospective studies focused on primary and secondary pre- noinfluenceonthedegreeofpressureoverloadasdeterminedby vention of ischemic heart disease. Despite these recent the pressure gradient or the prestenotic pressure (data not observations,littleisknownabouttheroleofERsincardiac shown). disease such as cardiac hypertrophy. A better understanding Toanalyzethemechanismsinvolvedintheobservedantihy- of the function of specific ERs in different tissues is impor- pertrophic effect of E2, we investigated critical target genes tant in the development and selection of new agents that involved in development and progression of cardiac hypertro- couldbeusedfortreatment.Currently,thebiologicalrolesof phy. In previous reports, these genes have been shown to be these2differentERsubtypesarenotclear.Itmayberelated regulatedbyE2.4ImmunoblotanalysisrevealedthatE2blocked to the selective actions of E2 in various target tissues. Also, theincreasedphosphorylationofp38-MAPKinovariectomized it is known that different E2 compounds have different Downloaded from http://atvb.ahajournals.org/ by guest on February 20, 2013 1528 Arterioscler Thromb Vasc Biol. July 2006 Figure3.Ventricularexpressionand phosphorylationofp38(shownasrepre- sentativeWesternblotandmeandensi- tometricalanalysis)andANF(real-time PCR).p38expressionandphosphoryla- tionwereevaluatedinWTorER(cid:2)(cid:1)/(cid:1)ani- malswithTACandtreatedwithE2or placebo(P)4weeksafterintervention(A andB).B,Phosphorylationofp38in ER(cid:2)(cid:1)/(cid:1)aspercentageoftotalp38.C andD,p38expressionandphosphoryla- tioninWTorER(cid:1)(cid:1)/(cid:1)animalswithTAC andtreatedwithE2orplacebo4weeks afterTACwereevaluated.D,Phosphory- lationofp38inER(cid:1)(cid:1)/(cid:1)aspercentageof totalp38.Atotalofn(cid:7)7animalsinthe shamgroupandatotalofn(cid:7)10animals intheTACgroupwereevaluated.E,ANF expressioninTACWTandER(cid:2)(cid:1)/(cid:1)ani- malstreatedwithE2orplacebo.F,ANF expressioninTACWTandER(cid:1)(cid:1)/(cid:1)ani- malstreatedwithE2orplacebo.Allval- uesaremean(cid:5)SEM.*P(cid:6)0.05forpla- cebovsE2;†P(cid:6)0.05forER(cid:2)(cid:1)/(cid:1)vs ER(cid:1)(cid:1)/(cid:1).Thevaluesarecorrectedby subtractingcyclophilinvaluesandpre- sentedasrelativevaluestosham animals. relative binding affinities for ER(cid:2) versus ER(cid:1).26,27 For tendency toward decreased hypertrophy in the absence of E2. instance, recent studies suggest that ER(cid:1) may inhibit the Together,ourstudysupportsthehypothesisthatE2hasdirect, stimulatory effects of ER(cid:2)on cellular proliferation.28 In the modulating effects on cardiac myocytes and the heart. Similar case of E2 signaling, cellular selectivity for one or the other results were also obtained by Skavdahl et al31 in a model of ERappearstoberegulatedbythecellularexpressionpattern hypertrophythatevaluatedgender-baseddifferencesandadded of the ERs and interacting coactivator and corepressor pro- the important observation that gender determines the hypertro- teins.29TheER(cid:2)appearstobemoreinvolvedinregulationof phicphenotype.Furthermore,Pelzeretal32demonstratedthatin uterinegrowththantheER(cid:1),ascanbededucedfromboththe ER(cid:1)(cid:1)/(cid:1) animals of a different genetic background than the uterinewetweightandtheuterinedryweightinER(cid:2)(cid:1)/(cid:1)mice animalsusedinourstudy,anincreaseofmortalitywasshown. (Table). This is in line with previous studies regarding the ThefindingsofPelzeretalunderlinetheimportanceofER(cid:1)for importanceofER(cid:2)fortheuterineresponse.15,30Ontheother the cardiovascular system, in particular for cardiac dimensions hand, ER(cid:1), as we proved in this study, mediates the attenu- andfunction.AlthoughER(cid:1)appearstobeofmajorimportance ationofpressureoverloadhypertrophybyE2.Becauseofthe intheER-dependentresponsesstudiedinthisinvestigation,ER(cid:1) overallnumberrequiredtostudyalltreatmentgroupstoreach also plays a role in other physiological contexts such as the statistical significance, we focused on the well-established development of cardiac arrhythmias after myocardial infarc- time point of 4 weeks after TAC. tion.14 There, ER(cid:1)was shown to play an important role in WhereasinER(cid:2)(cid:1)/(cid:1)mice,VWissignificantlyreduced(com- ventricular repolarization after myocardial infarction and the parable to WT) after addition of E2, no such effect is seen in regulationofthepotassiumchannelexpression.Furthermore,it ER(cid:1)(cid:1)/(cid:1).Moreover,inER(cid:1)(cid:1)/(cid:1)mice,therewasanonsignificant was demonstrated, using the same animal model, that ER(cid:1)is tendency toward hypertrophy when E2 was present and a necessary for normal morphology in several regions of the Downloaded from http://atvb.ahajournals.org/ by guest on February 20, 2013 Babiker et al ER(cid:1)Protects the Murine Heart 1529 centralnervoussystem.33Studiesontheseanimalsalsoindicate WynandPonfoundationtoP.A.D.,andbytheDeutscheForschungs- thatER(cid:1)hasanantiproliferativeeffectintheimmatureuterus gemeinschaft and BONFOR to C.G. and R.M. Additional support fromtheRESCARfoundationwasgreatlyappreciated. andintheprostate,atleastpartiallybybalancingtheprolifera- tive activity of ER(cid:2).34 To further elucidate the mechanisms Disclosures involved in the antihypertrophic effects of E2, it will be None. necessarytoidentifytheadditionalsignalingmoleculesinvolved intheseprotectiveeffects,theirtimecourseofactivation,andthe References cross-talkbetweenthem. 1. LiaoY,CooperRS,MensahGA,McGeeDL.Leftventricularhypertro- Inapreviousstudy,weshowedthatnodifferencesoccurinthe phy has a greater impact on survival in women than in men. Circu- expressionlevelsofERK1/2,JNK,angiotensinIItype1receptor,or lation.1995;92:805–810. angiotensin-converting enzyme.11 To further elucidate possible 2. GardinJM,WagenknechtLE,Anton-CulverH,FlackJ,GiddingS,Kurosaki T,WongND,ManolioTA.Relationshipofcardiovascularriskfactorsto mechanisms involved, we studied the activation of MAPK and echocardiographicleftventricularmassinhealthyyoungblackandwhite ANF. 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Nuclear hormone receptors: 1991;325:756–762. ligand-activatedregulatorsoftranscriptionanddiversecellresponses.Chem 38. GradyD,RubinSM,PetittiDB,FoxCS,BlackD,EttingerB,ErnsterVL, Biol.1996;3:529–536. Cummings SR. Hormone therapy to prevent disease and prolong life in 28. BarkhemT,NilssonS,GustafssonJA.Molecularmechanisms,physiological postmenopausalwomen.AnnInternMed.1992;117:1016–1037. consequencesandpharmacologicalimplicationsofestrogenreceptoraction. 39. MendelsohnME,KarasRH.ThetimehascometostoplettingtheHERStale AmJPharmacogenomics.2004;4:19–28. wagthedogma.Circulation.2001;104:2256–2259. Downloaded from http://atvb.ahajournals.org/ by guest on February 20, 2013 On Line supplemental data ATVB/2005/099283R1 Material and Methods Animals The mouse ERβ gene was isolated from an 129 Sv genomic BAC library(Genome Systems) by screening with a full length human ERβ cDNA probe. Two BAC clones were obtained and restriction enzyme mapping was performed using a panel of restriction enzymes and degenerated probes deduced from exon 1-4. Two fragments were identified: a 2.7 kb HindIII- EcoRV fragment located 5' of exon 1 and a 1.6 kb HindIII fragment located 3' of exon 2. Both fragments were cloned into the pKO gene targeting vector (Lexicon Genetics, Woodlands, USA) containing the pgk-neo gene for positive selection and the CMV-Tk gene for negative selection. ES cells were transfected with NotI linearized targeting vector using a BioRad Gene Pulser(230V). Targeted ES cells were identified by nested PCR analysis. PCR conditions used were 100 ng each of primer 5'-GGAGTGGCAGACAAGGGCA-3' and primer 5'- GATTCGCAGCGCATCGCC-3', 10 ng genomic DNA, DNA Taq polymerase(1U, Gibco BRL), a dATP, dCTP, dGTP, dTTP mixture (10 mM), Taq buffer (Gibco BRL) for 20 cycles Nested PCR was performed using the nested primers 5'-ACCCAACACCCTCTCTGGCC-3 and 5'-GGGCTCTATGGCTTCTGAGG-3' for 24 cycles. PCR products were analyzed on an 1% agarose gel and visualized with ethidium bromide. Positive clones obtained were analyzed by Southern blot analysis. Targeted clones were expanded and injected into blastocysts from C57Bl/6 mothers (Genome Systems), and where returned to pseudopregnant C57Bl/6 hosts to complete their development. Germ line transmission of the mutant allele was tested by PCR analysis of genomic tail DNA. Downloaded from http://atvb.ahajournals.org/ by guest on February 20, 2013 Immunoblot Analysis Equal loading was checked by stripping and reprobing the membrane with troponin C. The following primary antibodies were used: p38-mitogen-activated protein kinase (p38- MAPK), ERK1/2, JNK, phospho-JNK (Thr183/Tyr185) and troponin C (Santa Cruz Biotechnology Inc) and phospho-ERK1/2 (Thr202/Tyr204) and phospho-p-38 MAPK (Thr180/Tyr182) (New England Biolabs). Detection was performed with the enhanced chemiluminescence technique after incubation with a suitable secondary antibody coupled to horseradish peroxidase (ECL; Amersham Pharmacia Biotech). A computerized image acquisition system (Alpha Innotech Corp) was used for densitometric analysis. Figure I. Transverse sections of hearts stained with hematoxylin-eosin from WT and ERα-/ - animals with TAC that where treated with either E2 or placebo 4 weeks after intervention (A). Transverse sections of hearts stained with hematoxylin-eosin from WT and ERβ-/ animals - with TAC that where treated with either E2 or placebo 4 weeks after intervention (B). Left ventricular mass analysis for WT and ERα-/ (C). Left ventricular mass analysis for WT and - ERβ-/ (D). Ventricular (LV) external circumference analysis for WT and ERα-/ (E). - - Ventricular external circumference analysis for WT and ERβ-/ (F): All values are mean ± - SEM, n = 7 for sham and 10 for TAC per group. *Indicates P<0.05 for placebo vs. E2 treatments. Downloaded from http://atvb.ahajournals.org/ by guest on February 20, 2013