Female gender and estrogen-replacement therapy in postmenopausal women are associated with improved heart failure survival, and physiological replacement of 17beta-estradiol (E2) reduces infarct size and cardiomyocyte apoptosis in animal models of myocardial infarction (MI). Here, we characterize the molecular mechanisms of E2 effects on cardiomyocyte survival in vivo and in vitro. Ovariectomized female mice were treated with placebo or physiological E2 replacement, followed by coronary artery ligation (placebo-MI or E2-MI) or sham operation (sham) and hearts were harvested 6, 24, and 72 hours later. After MI, E2 replacement significantly increased activation of the prosurvival kinase, Akt, and decreased cardiomyocyte apoptosis assessed by terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) staining and caspase 3 activation. In vitro, E2 at 1 or 10 nmol/L caused a rapid 2.7-fold increase in Akt phosphorylation and a decrease in apoptosis as measured by TUNEL staining, caspase 3 activation, and DNA laddering in cultured neonatal rat cardiomyocytes. The E2-mediated reduction in apoptosis was reversed by an estrogen receptor (ER) antagonist, ICI 182,780, and by phospho-inositide-3 kinase inhibitors, LY294002 and Wortmannin. Overexpression of a dominant negative-Akt construct also blocked E2-mediated reduction in cardiomyocyte apoptosis. These data show that E2 reduces cardiomyocyte apoptosis in vivo and in vitro by ER- and phospho-inositide-3 kinase-Akt-dependent pathways and support the relevance of these pathways in the observed estrogen-mediated reduction in myocardial injury.

Little is known about the interaction of 17β‐estradiol (E2) and the vasoactive endothelin system in the heart. Endothelin signaling is activated in a failing heart and may contribute to myocardial dysfunction and remodeling. Therefore, we investigated the regulation of proteins of the endothelin system (ppET‐1, ECE and ETA‐R and ETB‐R) in the hearts of female spontaneously hypertensive rats (SHR) with respect to E2. Relative expression levels of the respective cardiac mRNA obtained from sham‐operated, ovariectomized and ovariectomized E2‐substituted SHR were quantified by real‐time PCR. Ovariectomy led to a significant upregulation of the ETB‐R mRNA (2.6±0.8‐fold) in the left ventricular myocardium, which was not attendant with an alteration of ETA‐R, ECE and ppET‐1 mRNA expression. An upregulation of the relative expression level of ETB‐R protein due to ovariectomy was also demonstrated by radioligand binding assay. Upregulation of both ETB‐R mRNA and ETB‐R protein expression was completely inhibited by E2 replacement. To confirm these results in in vitro experiments, we quantified the mRNA of ET‐R subtypes from isolated cardiomyocytes in the presence and absence of E2 (10−8 M, 24 h). Our data showed a markedly downregulated level of ETB‐R mRNA in cardiomyocytes stimulated with E2. ETB‐R downregulation was not attendant with the alteration of ETA‐R, ECE and ppET‐1 mRNA expression. Taken together, these data demonstrate that estrogen regulates the expression of ETB‐R in rat ventricular myocardium in vivo and in vitro. These observations may help to understand gender‐based differences found in cardiovascular disease. British Journal of Pharmacology (2003) 140, 195–201. doi:10.1038/sj.bjp.0705409

Physiological effects of estrogen on myocardium are mediated by two intracellular estrogen receptors, ERalpha and ERbeta, that regulate transcription of target genes through binding to specific DNA target sequences. To define the role of ERbeta in the transcriptional activation of both endothelial (eNOS) and inducible nitric oxide synthase (iNOS) in cardiac myocytes, we used the complete ER-specific antagonist R,R-tetrahydrochrysene (R,R-THC). R,R-THC inhibited activation of iNOS/eNOS promoter-luciferase reporter constructs (iNOS/eNOS-Luc) in a dose-dependent fashion in COS7 cells selectively transfected with ERbeta, but failed to influence ERalpha-mediated increase of iNOS/ eNOS-Luc. In neonatal rat cardiomyocytes transfected with eNOS-Luc or iNOS-Luc, incubation with 17betaestradiol (E2, 10(-8) M) for 24 h stimulated expression of eNOS and iNOS. R,R-THC (10(-5) M) completely inhibited this effect. Furthermore, eNOS and iNOS protein expression in cardiac myocytes induced by E2 was completely blocked by R,R-THC as shown by immunoblot analysis. Taken together, these results show that ERbeta mediates transcriptional activation of eNOS and iNOS by E2.

Estrogen and insulin-like-growth factor 1 (IGF-1) are potent mitogenic stimuli that share important properties in the control of cellular proliferation. However, the coupling between the signaling cascades of estrogen receptors α and β and the IGF-1 receptor (IGF-1R) is poorly understood. Therefore, we selectively transfected estrogen receptor α or β in COS7 and HEK293 cells, which contain IGF-1R. In presence of estrogen receptor α but not β, 17β-estradiol (E2) rapidly induces phosphorylation of the IGF-1R and the extracellular signal-regulated kinases 1/2. Furthermore, upon stimulation with E2, estrogen receptor α but not β bound rapidly to the IGF-1R in COS7 as well as L6 cells, which express all investigated receptors endogenously. Control experiments in the IGF-1R-deficient fibroblast cell line R− showed that after stimulation with E2 only estrogen receptor α bound to the transfected IGF-1R. Overexpression of dominant negative mitogen-activated protein kinases kinase inhibited this effect. Finally, estrogen receptor α but not β is required to induce the activation of the estrogen receptor-responsive reporter ERE-LUC in IGF-1-stimulated cells. Taken together, these data demonstrate that ligand bound estrogen receptor α is required for rapid activation of the IGF-1R signaling cascade.

OBJECTIVESNO production has been attributed to play a major role in cardiac diseases such as cardiac hypertrophy and cardiac remodeling after myocardial infarction which display significant gender-based differences. Therefore we assessed the effect of 17 beta-estradiol (E2) on estrogen receptor (ER) alpha and beta and endothelial and inducible NO synthase in neonatal and adult rat cardiomyocytes.METHODSThe presence of ER alpha and ER beta was demonstrated by immunofluorescence and western blot analysis as well as the expression pattern of inducible NO synthase (iNOS) and endothelial NOS (eNOS) in isolated cardiomyocytes from neonatal and adult rats. Furthermore, regulation of myocardial iNOS and eNOS expression by estrogen was evaluated in the myocardium from ovariectomized or sham-operated adult Wistar-Kyoto rats.RESULTSIncubation with E2 led to translocalization of the ER into the nucleus and increased receptor protein expression. E2 stimulated expression of iNOS and eNOS in both neonatal and adult cardiac myocytes. Coincubation with the pure anti-estrogen ICI 182,780 inhibited upregulation of ER and NOS expression. In ovariectomized rats myocardial iNOS and eNOS protein levels were significantly lower compared to sham-operated female animals.CONCLUSIONTaken together, these results show that E2 stimulates the expression of iNOS/eNOS in neonatal and adult cardiomyocytes in-vivo and in-vitro. These novel findings provide a potential mechanism of how estrogen may modulate NOS expression and NO formation in the myocardium.

Cardiac myocytes contain functional estrogen receptors, however, the effect of estrogen on growth-related signaling pathways such as mitogen-activated protein kinases (MAPK) in the pathogenesis of cardiac disease is unclear. MAPKs are critically involved in regulatory signaling pathways which ultimately lead to cardiac hypertrophy. Here we show that 17beta-estradiol (E2) activates extracellular signal-regulated kinase (ERK1/2), c-Jun-NH2-terminal protein kinase (JNK) and p38 in rat cardiomyocytes in a distinctive pattern. As shown by immunoblot analysis and phosphorylation assays, E2 (10(-9) M) induced a rapid and transient activation of ERK1/2 and a rapid but sustained increase of JNK phosphorylation. In contrast, E2 had only a marginal effect on p38 activation. Furthermore, MAPK phosphatase expression was induced by E2 and E2-stimulated expression of endothelial and inducible NO synthase was inhibited by PD 98059, an inhibitor of the ERK pathway. These novel observations may help to explain the role of estrogen in gender-based differences found in cardiac disease.