Guillaume van Eys, Christian Grohй and Pieter A. Doevendans Fawzi A. Babiker, Daniel Lips

13 Pages·2006·0.95 MB·English

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
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(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
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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
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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.
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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
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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
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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
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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.

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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.

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