The increase of left ventricular mass represents the structural

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1 17 -Estradiol Attenuates the Development of Pressure-Overload Hypertrophy Martin van Eickels, MD; Christian Grohé, MD; Jack P.M. Cleutjens, PhD; Ben J. Janssen, PhD; Hein J.J. Wellens, MD; Pieter A. Doevendans, MD Background Cardiac hypertrophy is an independent risk factor for cardiovascular morbidity and mortality in men and in women. Epidemiological studies indicate that estrogen replacement therapy is cardioprotective; the mechanisms involved in this process, however, are poorly understood. We therefore studied the effect of 17 -estradiol (E 2 ) on the development of pressure-overload hypertrophy. Methods and Results Ovariectomized mice receiving E 2 or placebo underwent transverse aortic constriction (TAC) or sham operation. TAC led to a significant increase in ventricular mass compared with sham operation. E 2 treatment reduced cardiac hypertrophy by 31% and 26% compared with placebo 4 and 8 weeks after TAC, whereas it had no effect on the degree of pressure overload, as determined by hemodynamic measurements. Furthermore, E 2 blocked the increased phosphorylation of p38-mitogen activated protein kinase (MAPK) observed in the placebo-treated animals with TAC. No differences were observed in the phosphorylation of extracellular signal regulated kinase (ERK) 1/2 and c-jun N-terminal kinase (JNK) 1/2 between the groups. E 2 had no effect on the expression of angiotensin-converting enzyme (ACE) or the angiotensin II type 1 receptor. Ventricular atrial natriuretic peptide (ANP) expression was detected only in the animals with TAC. Compared with placebo, E 2 treatment led to an increased expression of ANP in animals with pressure overload. Conclusions Here, we show that E 2 attenuates the hypertrophic response to pressure overload in mice. This observation demonstrates that hormone replacement therapy with E 2 has direct effects on the heart and may be beneficial in the treatment of postmenopausal women to reduce cardiac hypertrophy. (Circulation. 2001;104: ) Key Words: hormones hypertrophy myocardium sex The increase of left ventricular mass represents the structural mechanism of adaptation of the heart in response to pressure overload. The resulting left ventricular hypertrophy is an important negative predictor of cardiac morbidity and mortality and displays significant sex-based differences. 1,2 Estrogen is known to have multiple protective effects on the cardiovascular system. 3 The role of estrogen in the development of cardiac hypertrophy, however, is poorly understood. See p 1333 We have shown previously that cardiac myocytes and cardiac fibroblasts contain both known estrogen receptor isoforms, called and. 4 Via these receptors, estrogen can regulate the cardiac expression of endothelial and inducible NO synthase and connexin Estrogen also modulates the activity of the mitogen-activated protein kinase (MAPK) pathways in cardiac myocytes. 6 The MAPK signaling pathways consist of a sequence of successively acting kinases that ultimately result in the dual phosphorylation and activation of effector kinases such as p38-mapks, c-jun N-terminal kinases (JNKs), and extracellular signal-regulated kinases (ERKs), which subsequently phosphorylate a large array of targets, leading to altered gene expression patterns. 7 These signaling cascades play an important role in the initiation of cardiac hypertrophy and in the development of heart failure Furthermore, we have shown previously that estrogen downregulates the activity of the renin-angiotensin system in the vasculature of normotensive and of hypertensive rats. 11,12 The inhibition of the renin-angiotensin system has an antihypertrophic effect and is used widely in the therapy of patients with cardiac hypertrophy. 13 In addition, it is known that estrogen can increase the expression of atrial natriuretic peptide (ANP). 14,15 ANP has long been known to be a marker of cardiac hypertrophy. More recent studies, however, have suggested that ANP possesses antihypertrophic properties. 16,17 Taken together, these data led us to the hypothesis that estrogen may modulate the development of cardiac hypertrophy. We therefore studied the effects of 17 -estradiol (E 2 ), the predominant estrogen in premenopausal women, on the Received April 9, 2001; revision received June 4, 2001; accepted June 5, From the Cardiovascular Research Institute Maastricht, University of Maastricht (M.v.E., J.P.M.C., B.J.J.), and the Department of Cardiology, Academisch Ziekenhuis Maastricht (H.J.J.W., P.A.D.), Maastricht, Netherlands; and the Medizinische Universitäts-Poliklinik, Bonn, Germany (C.G.). Correspondence to Martin van Eickels, MD, Division of Cardiology, Department of Internal Medicine II, University of Bonn, Sigmund Freud Straße 25, Bonn, Germany. mvaneickels@lifespan.org or vaneickels@altavista.com 2001 American Heart Association, Inc. Circulation is available at

2 1420 Circulation September 18, 2001 Effects of TAC and E 2 Treatment on Body and Organ Weights, Blood Pressure, and Myocyte Size Sham TAC Placebo E 2 Placebo E 2 4 Weeks 8 Weeks 4 Weeks 8 Weeks 4 Weeks 8 Weeks 4 Weeks 8 Weeks BW, g VW, mg * * 174 6* 159 8* VW/TL, mg/mm * * * * Lung weight, mg * * Uterus weight, mg P sys,mmhg * 115 8* 118 3* 115 9* Myocyte CSA, m * 199 9* BW indicates body weight; VW, ventricular weight; TL, tibial length; P sys, prestenotic systolic pressure; and CSA, myocyte cross-sectional area. All values are mean SEM. Four weeks, n 7; 8 weeks, n 6. *P 0.05 for TAC vs sham, P 0.05 for E 2 vs placebo. development of pressure-overload hypertrophy and the activation of the pathways mentioned above. Methods Animals Ten-week-old female C57BL/6 mice were housed under standard conditions. Animals were anesthetized with ketamine (100 mg/kg body weight [BW] IP) and xylazine (10 mg/kg BW IP) for ovariectomy, pellet placement, and transverse aortic constriction (TAC). The study was approved by the animal ethics committee of the University of Maastricht. Estrogen Replacement One week after ovariectomy, a 60-day-release pellet containing 0.18 mg E 2 or placebo was implanted subcutaneously. E 2 serum levels were measured with a radioimmunoassay (DPC Biermann) in a subset of animals. To measure the magnitude of proliferation of cardiac cells, a subgroup of animals with pressure overload received bromodeoxyuridine (BrdU) (2.5 mg per 21-day-release pellet) for the last 14 days of the study period. All pellets were purchased from Innovative Research of America. Surgical Procedures and Hemodynamics One week after the pharmacological intervention, TAC was performed, as described previously. 18 Sham-operated animals underwent an identical operation without placement of the constricting suture. There was no difference in the mortality between the E 2 - and the placebo-treated animals. Four and 8 weeks (n 7 and n 6 per group) after TAC or the sham procedure, animals were anesthetized with pentobarbital (100 mg/kg BW IP). Pressure-transducing catheters were introduced into the right and the left carotid arteries, and systolic pressure before and after the stenosis was measured as described previously. 19 Tissue Preparation and Histology Hearts were arrested in diastole with CdCl 2 (0.1 mol/l IV). For morphometric analysis, hearts were fixed in 10% formalin and embedded in paraffin as described previously. 20 For protein extraction, hearts were excised, washed in ice-cold PBS, and frozen. All external fluid was completely removed before the organs were weighed. Transverse sections of the heart were stained with hematoxylin and eosin, picrosirius red, modified Azan, or anti-brdu antibodies. The analysis of the collagen content was performed with a computerized morphometry system as described previously. 20 Cross-sectional areas of 50 (sham) or 100 (TAC) myocytes in which the centrally located nucleus and a clear staining of the cell borders could be visualized were measured in the transversely cut papillary muscles of each animal. Immunoblot Analysis Total heart lysates (40 g/lane) were analyzed by standard immunoblotting procedures as described previously. 5,6,21 24 Equal loading was checked by stripping and reprobing the membrane with troponin C. The following primary antibodies were used: ACE (BMA Biomedicals AG); ANP (Phoenix Pharmaceuticals Inc); angiotensin type 1 receptor (AT 1 R), ERK1/2, JNK, MKP1, p38-mapk, 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 GmbH). 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. Statistical Analysis Data are shown as mean SEM. Means were compared by ANOVA, followed by Bonferroni s test for multiple comparisons. Differences were considered significant at P Results Estrogen replacement led to a reconstitution of physiological estrogen levels (sham pg/ml, TAC pg/ml, n 5 per group). All measured E 2 levels in animals receiving placebo were 5 pg/ml. Uterus weight was measured to demonstrate the effectiveness of ovariectomy and E 2 substitution in all animals (Table). TAC led to a significant increase in ventricular mass 4 and 8 weeks after the intervention. The degree of ventricular hypertrophy was significantly lower in E 2 -treated than placebo-treated animals with pressure overload (Figure 1A). Compared with placebo, E 2 treatment led to a significant reduction of the ventricular weight (VW), the VW/BW ratio, and the VW/tibial length ratio in animals with pressure overload (Figure 2 and Table). No differences were observed between the E 2 - and the placebo-treated animals that were sham-operated (Figure 2 and Table). The lung weight was increased in the animals with TAC; however, this increase reached statistical significance only in the placebo-treated animals (Table). There were no differences in BW between the groups (Table). Because E 2 can alter blood pressure, we tested whether the observed antihypertrophic effect of estrogen could be medi-

3 van Eickels et al Estrogen and Pressure-Overload Hypertrophy 1421 Figure 2. A, Ventricular weight (VW)/BW ratios from animals with (TAC) and without (sham) pressure overload that were treated with either E 2 or placebo (P) 4 and 8 weeks after intervention. B, Systolic pressure gradients across stenosis 4 and 8 weeks after intervention: 4 weeks, n 7 per group; 8 weeks, n 6 per group. *P 0.05 for TAC vs sham, P 0.05 for E 2 vs placebo. Figure 1. A, Transverse sections of hearts stained with hematoxylin-eosin from animals with (TAC) and without (sham) pressure overload that were treated with either E 2 or placebo 4 weeks after TAC (bars 1 mm). B, Picrosirius red stained anterior septal sections from animals with (TAC) and without (sham) pressure overload that were treated with either E 2 or placebo 4 weeks after TAC to demonstrate interstitial fibrosis (bars 100 m). ated by a change in the degree of pressure overload. In our model of TAC, E 2 had no influence on the degree of pressure overload as determined by the pressure gradient or the prestenotic pressure (Figure 2B). Histological analysis revealed that pressure overload led to progressive cardiac fibrosis (Figure 1B). No differences in collagen content (E % and placebo %) or proliferation of interstitial cells, measured by BrdU labeling (E % and placebo %), however, were observed in the animals with ventricular hypertrophy. TAC led to a significant increase of myocyte size, as determined by myocyte cross-sectional area. This increase was significantly attenuated by treatment with E 2 compared with placebo (Table). To further elucidate the mechanism involved in the observed antihypertrophic effect of estrogen, we studied pathways involved in the development and progression of cardiac hypertrophy, which also have been shown to be regulated by E 2. Immunoblot analysis revealed that E 2 blocked the increased phosphorylation of p38-mapk in ovariectomized animals with pressure-overload hypertrophy, whereas it exerted no effect in sham-operated animals (Figures 3A and 4A). No differences could be observed between the study groups with regard to the phosphorylation level of ERK1/2 and JNK (Figure 3A). In the sham-operated animals, the levels of AT 1 R expression were slightly lower in E 2 -treated animals; however, this difference did not reach statistical significance. In the hypertrophied hearts, no differences in the expression levels of AT 1 R or ACE were detected between the E 2 - and the placebo-treated animals (Figure 3B). Compared with placebo, E 2 treatment led to an increase in ANP expression in the hypertrophied ventricles. No significant levels of ANP were detectable in the ventricles of shamoperated animals (Figures 3C and 4B). Discussion Here, we show for the first time that hormone replacement therapy with physiological doses of E 2 can attenuate the development of pressure-overload hypertrophy. In contrast to a previous study in spontaneously hypertensive rats, the antihypertrophic effect observed in our study was not mediated by the blood pressure lowering effect of E We did not observe E 2 -associated differences in cardiac fibrosis and cell proliferation in the heart. In fact, myocyte cross-sectional area was attenuated by estrogen treatment compared with placebo. Therefore, our study supports the hypothesis that E 2 has direct effects on cardiac myocytes and the heart. To further elucidate the mechanisms involved, we studied the activation of MAPK 7 9,27 and the expression of ACE, AT 1 R, 13,15 and ANP, 14,17 all of which have been shown to

4 1422 Circulation September 18, 2001 Figure 3. Ventricular expression of (A) phospho-p38-mapk, p38-mapk, phospho-erk1/2, ERK1/2, phospho-jnk, and JNK; (B) AT 1 R and ACE; and (C) ANP in animals with (TAC) and without (sham) pressure overload that were treated with either E 2 or placebo (P), as determined by immunoblot analysis. play important roles in the development and progression of cardiac hypertrophy and are estrogen responsive. A previous study suggested that the activation of p38- MAPK is important for the maintenance of the hypertrophic response over a longer period of time. 27 Therefore, the inhibition of the p38-mapk phosphorylation by E 2 treatment may represent one of the mechanisms by which E 2 exerts its antihypertrophic effect in our model of pressure overload. ERK1/2 and JNK were not activated in all the animals with pressure overload, possibly because these pathways may be more important in the induction of the hypertrophic response and have returned to baseline after 4 weeks. Inhibition of the renin-angiotensin system plays an important role in the development of cardiac hypertrophy. On the basis of our previous findings that E 2 reduces the expression of the AT 1 R, 11,12 we hypothesized that a similar mechanism could be involved in the observed antihypertrophic effect of E 2. No such differences were observed, however, in the hypertrophied hearts. Recent studies have identified the antihypertrophic properties of ANP. 16,17 Furthermore, it has been shown previously that E 2 increases the expression of ANP. 14,28 In concordance with these results, E 2 led to an increase in ANP expression in the ventricles of animals with pressure overload compared with placebo. These findings suggest that the antihypertrophic effect of E 2 may be mediated by the increased expression of ANP. The development of cardiac hypertrophy is a complex process involving signal integration of multiple pathways. 7,29 Therefore, it may well be that the observed antihypertrophic effect of E 2 is mediated by the modulation of not one but several of these pathways. To further elucidate the mechanisms involved in E 2 s antihypertrophic effects, it will be necessary to identify the essential signaling molecules in- Figure 4. Densitometric analysis of phospho-p38-mapk/p38- MAPK (A) and ANP (B) immunoblots. n 6 per group. *P 0.05 for TAC vs sham, P 0.05 for E 2 vs placebo. volved in the development of cardiac hypertrophy, their time course of activation, and the cross talk between them. Overall, our results suggest that the lack of estrogen may be responsible for the increase in ventricular hypertrophy observed in postmenopausal women. Furthermore, these findings may constitute a novel approach in the treatment of postmenopausal women at risk of developing cardiac hypertrophy. Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (Gr729/8 1), BONFOR (O708), the Cardiovascular Biology Foundation in Delft, and the Bekales Foundation. Dr van Eickels received a postdoctoral fellowship from the German Academic Exchange Service (DAAD). We thank Richard H. Karas, MD, PhD, for his valuable comments, Peter Leenders and Jacques Debets for expert assistance with the animal surgery, and Hanne Bock for expert technical assistance with the immunoblots. References 1. Krumholz HM, Larson M, Levy D. Sex differences in cardiac adaptation to isolated systolic hypertension. Am J Cardiol. 1993;72: Liao Y, Cooper RS, Mensah GA, et al. Left ventricular hypertrophy has a greater impact on survival in women than in men. Circulation. 1995; 92: Mendelsohn ME, Karas RH. The protective effects of estrogen on the cardiovascular system. N Engl J Med. 1999;340: Grohé C, Kahlert S, Löbbert K, et al. Cardiac myocytes and fibroblasts contain functional estrogen receptors. FEBS Lett. 1997;416: Nüdling S, Kahlert S, Löbbert K, et al. 17 Beta-estradiol stimulates expression of endothelial and inducible NO synthase in rat myocardium in-vitro and in-vivo. Cardiovasc Res. 1999;43: Nüdling S, Kahlert S, Löbbert K, et al. Differential effects of 17betaestradiol on mitogen-activated protein kinase pathways in rat cardiomyocytes. FEBS Lett. 1999;454: Sugden PH, Clerk A. Stress-responsive mitogen-activated protein kinases (c-jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ Res. 1998;83: Bueno OF, De Windt LJ, Lim HW, et al. The dual-specificity phosphatase MKP-1 limits the cardiac hypertrophic response in vitro and in vivo. Circ Res. 2001;88:88 96.

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