Elke Dworatzek

Female sex and estrogen receptor-β attenuate cardiac remodeling and apoptosis in pressure overload

We investigated sex differences and the role of estrogen receptor-beta (ERbeta) on myocardial hypertrophy in a mouse model of pressure overload. We performed transverse aortic constriction (TAC) or sham surgery in male and female wild-type (WT) and ERbeta knockout (ERbeta(-/-)) mice. All mice were characterized by echocardiography and hemodynamic measurements and were killed 9 wk after surgery. Left ventricular (LV) samples were analyzed by microarray profiling, real-time RT-PCR, and histology. After 9 wk, WT males showed more hypertrophy and heart failure signs than WT females. Notably, WT females developed a concentric form of hypertrophy, while males developed eccentric hypertrophy. ERbeta deletion augmented the TAC-induced increase in cardiomyocyte diameter in both sexes. Gene expression profiling revealed that WT male hearts had a stronger induction of matrix-related genes and a stronger repression of mitochondrial genes than WT female hearts. ERbeta(-/-) mice exhibited a different transcriptional response. ERbeta(-/-)/TAC mice of both sexes exhibited induction of proapoptotic genes with a stronger expression in ERbeta(-/-) males. Cardiac fibrosis was more pronounced in male WT/TAC than in female mice. This difference was abolished in ERbeta(-/-) mice. The number of apoptotic nuclei was increased in both sexes of ERbeta(-/-)/TAC mice, most prominent in males. Female sex offers protection against ventricular chamber dilation in the TAC model. Both female sex and ERbeta attenuate the development of fibrosis and apoptosis, thus slowing the progression to heart failure.

Regression of Myocardial Hypertrophy After Aortic Valve Replacement Faster in Women

Background— In patients with aortic stenosis, pressure overload induces cardiac hypertrophy and fibrosis. Female sex and estrogens influence cardiac remodeling and fibrosis in animal models and in men. Sex differences and their molecular mechanisms in hypertrophy regression after aortic valve replacement have not yet been studied. Methods and Results— We prospectively obtained preoperative and early postoperative echocardiography in 92 patients, 53 women and 39 men, undergoing aortic valve replacement for isolated aortic stenosis. We analyzed in a subgroup of 10 patients matrix gene expression in left ventricular (LV) biopsies. In addition, we determined the effect of 17&bgr;-estradiol on collagen synthesis in isolated rat cardiac fibroblasts. Preoperatively, women and men had similar ejection fraction. Similar percentages of women and men had increased LV diameters (37% and 38%). Women more frequently exhibited LV hypertrophy than men (women: 86%; men: 56%; P<0.01). Postoperatively, increased LV diameters persisted in 34% of men but only in 12% of women (P<0.023). LV hypertrophy reversed more frequently in women than in men, leading to a similar prevalence of LV hypertrophy after surgery (women: 45%; men: 36%). In surgical biopsies, men had significantly higher collagen I and III and matrix metalloproteinase 2 gene expression than women. In isolated rat cardiac fibroblasts, 17&bgr;-estradiol significantly increased collagen I and III gene expressions in male cells but decreased it in female cells. Conclusion— Women adapt to pressure overload differently from men. Less fibrosis before surgery may enable faster regression after surgery.

17β-Estradiol Inhibits Matrix Metalloproteinase-2 Transcription via MAP Kinase in Fibroblasts

Aims Female sex and sex hormones contribute to cardiac remodelling. 17β-estradiol (E2) is involved in the modulation of extracellular matrix composition and function. Here, we analysed the effect of E2 on matrix metalloproteinase (MMP)-2 gene expression and studied the underlying molecular mechanisms in rat cardiac fibroblasts and in a human fibroblast cell line. Methods and results In adult rat cardiac fibroblasts, E2 significantly decreased MMP-2 gene expression in an estrogen receptor (ER)-dependent manner. Transient transfection experiments of human MMP-2 (hMMP-2) promoter deletion constructs in a human fibroblast cell line revealed a regulatory region between −324 and −260 bp that is involved in E2/ERα-mediated repression of hMMP-2 gene transcription. Electrophoretic mobility shift assays (EMSA) and supershift analysis demonstrated the binding of transcription factor Elk-1 within this promoter region. Elk-1 was phosphorylated by E2 via the mitogen-activated protein kinase (MAPK) signalling pathway as shown by western blotting. Treatment of cells with the MAPK inhibitor PD98059 blocked the E2-dependent repression of hMMP-2 promoter activity as well as the endogenous MMP-2 mRNA levels in both human fibroblast cells and rat cardiac fibroblasts. Conclusion E2 inhibits MMP-2 expression via the ER and the MAPK pathway in rat cardiac fibroblasts and in a human fibroblast cell line. These mechanisms may contribute to sex-specific differences in fibrotic processes that are observed in human heart and other diseases.

Sex- and estrogen-dependent regulation of a miRNA network in the healthy and hypertrophied heart

BACKGROUND In pressure overload, profibrotic gene expression and cardiac fibrosis are more pronounced in males than in females. Sex-specific and estrogen-dependent regulation of microRNAs (miRNAs), such as miR-21, may be a potential mechanism leading to sex differences in fibrosis. OBJECTIVES To analyze the influence of sex, estrogen, and estrogen receptor beta (ERβ) on the expression of miR-21 and to identify additional miRNAs potentially involved in sex-specific pressure overload-induced cardiac remodeling. METHODS The sex-specific regulation of fibrosis-related miRNAs was analyzed in male and female wild type and ERβ-deficient mice after transverse aortic constriction (TAC), in rat fibroblasts, and in a cardiomyocyte-like cell line. RESULTS We report the sex-specific expression of functionally-related miR-21, -24, -27a, -27b, 106a, -106b and the regulation of their expression by estrogen in a sex-specific manner. These effects were abolished in ERβ-deficient mice. We demonstrate the presence of common functional target sites for these miRNAs on three repressors of the mitogen-activated protein kinase signaling pathway, i.e. Rasa1, Rasa2 and Spry1, which may all lead to cardiac fibrosis. As expected, transfection with miRNA mimics targeting these repressors induced ERK1/2 phosphorylation. CONCLUSIONS Estrogen regulates a network of miRNAs in a sex-specific manner via ERβ. Our data suggest that the sex-specific expression of these miRNAs may be related to sex differences in fibrosis after pressure overload.

Sex-dependent regulation of fibrosis and inflammation in human left ventricular remodelling under pressure overload.

Women with aortic stenosis develop a more concentric form of LV hypertrophy than men. However, the molecular factors underlying sex differences in LV remodelling are incompletely understood. We took an unbiased approach to identify sex‐specific patterns in gene expression and pathway regulation, and confirmed the most prominent findings in human hearts.

Sex differences in exercise-induced physiological myocardial hypertrophy are modulated by oestrogen receptor beta

AIMS Oestrogen receptor alpha (ERα) and beta (ERβ) are involved in the regulation of pathological myocardial hypertrophy (MH). We hypothesize that both ER are also involved in physiological MH. Therefore, we investigated the role of ER in exercise-induced physiological MH in loss-of-function models and studied potential mechanisms of action. METHODS AND RESULTS We performed 1 and 8 weeks of voluntary cage wheel running (VCR) with male and female C57BL/6J wild-type (WT), ERα- and ERβ-deleted mice. In line with other studies, female WT mice ran more than males (P ≤ 0.001). After 8 weeks of VCR, both sexes showed an increase in left ventricular mass (females: P ≤ 0.01 and males: P ≤ 0.05) with more pronounced MH in females (P < 0.05). As previously shown, female ERα-deleted mice run less than female WT mice (P ≤ 0.001). ERβ-deleted mice showed similar running performance as WT mice (females vs. male: P ≤ 0.001), but did not develop MH. Only female WT mice showed an increase in phosphorylation of serine/threonine kinase (AKT), ERK1/2, p38-mitogen-activated protein kinase (MAPK), and ribosomal protein s6, as well as an increase in the expression of key regulators of mitochondrial function and mitochondrial respiratory chain proteins (complexes I, III, and V) after VCR. However, ERβ deletion abolished all observed sex differences. Mitochondrial remodelling occurred in female WT-VCR mice, but not in female ERβ-deleted mice. CONCLUSION The sex-specific response of the heart to exercise is modulated by ERβ. The greater increase in physiological MH in females is mediated by induction of AKT signalling, MAPK pathways, protein synthesis, and mitochondrial adaptation via ERβ.

Cardiomyocyte-specific Estrogen Receptor Alpha Increases Angiogenesis, Lymphangiogenesis and Reduces Fibrosis in the Female Mouse Heart Post-Myocardial Infarction.

Experimental studies showed that 17β-estradiol (E2) and activated Estrogen Receptors (ER) protect the heart from ischemic injury. However, the underlying molecular mechanisms are not well understood. To investigate the role of ER-alpha (ERα) in cardiomyocytes in the setting of myocardial ischemia, we generated transgenic mice with cardiomyocyte-specific overexpression of ERα (ERα-OE) and subjected them to Myocardial Infarction (MI). At the basal level, female and male ERα-OE mice showed increased Left Ventricular (LV) mass, LV volume and cardiomyocyte length. Two weeks after MI, LV volume was significantly increased and LV wall thickness decreased in female and male WT-mice and male ERα-OE, but not in female ERα-OE mice. ERα-OE enhanced expression of angiogenesis and lymphangiogenesis markers (Vegf, Lyve-1), and neovascularization in the peri-infarct area in both sexes. However, attenuated level of fibrosis and higher phosphorylation of JNK signaling pathway could be detected only in female ERα-OE after MI. In conclusion, our study indicates that ERα protects female mouse cardiomyocytes from the sequelae of ischemia through induction of neovascularization in a paracrine fashion and impaired fibrosis, which together may contribute to the attenuation of cardiac remodelling.

Nuclear Factor-κB Regulates Estrogen Receptor-α Transcription in the Human Heart

Estrogen receptor (ER)-mediated effects have been associated with the modulation of myocardial hypertrophy in animal models and in humans, but the regulation of ER expression in the human heart has not yet been analyzed. In various cell lines and tissues, multiple human estrogen receptor α (hERα) mRNA isoforms are transcribed from distinct promoters and differ in their 5′-untranslated regions. Using PCR-based strategies, we show that in the human heart the ERα mRNA is transcribed from multiple promoters, namely, A, B, C, and F, of which the F-promoter is most frequently used variant. Transient transfection reporter assays in a human cardiac myocyte cell line (AC16) with F-promoter deletion constructs demonstrated a negative regulatory region within this promoter. Site-directed mutagenesis and electrophoretic mobility shift assays indicated that NF-κB binds to this region. An inhibition of NF-κB activity by parthenolide significantly increased the transcriptional activity of the F-promoter. Increasing NF-κB expression by tumor necrosis factor-α reduced the expression of ERα, indicating that the NF-κB pathway inhibits expression of ERα in human cardiomyocytes. Finally, 17β-estradiol induced the transcriptional activity of hERα promoters A, B, C, and F. In conclusion, inflammatory stimuli suppress hERα expression via activation and subsequent binding of NF-κB to the ERα F-promoter, and 17β-estradiol/hERα may antagonize the inhibitory effect of NF-κB. This suggests interplay between estrogen/estrogen receptors and the pro-hypertrophic and inflammatory responses to NF-κB.

Sex and Sex Hormone–Dependent Cardiovascular Stress Responses

Different stimuli confer mechanical and hormonal stress to the heart. Pressure overload (PO) affects the myocardium, kidney, and peripheral fat. The latter may be considered simultaneously as a novel end organ and as a mediator in cardiovascular damage and, by its interplay with the heart, contributes to the development of pathological myocardial hypertrophy (MH). In contrast to pathological MH, in physiological MH the heart serves as an end organ for recurrent pressure spikes without the development of pathology. Complex stressors such as mineralocorticoid excess and salt challenge induce complex pathological cardiac and renal phenotypes. Understanding the underlying sex-specific mechanisms mediating these processes may help to design new therapeutic strategies. The major goal of this review is to analyze sex differences in physiological and pathological forms of cardiovascular stress and its effects on different organs, including the heart, kidney, and peripheral fat. Exercise-induced physiological MH differs markedly from pathological MH resulting from hypertension, valve disease, or other challenges. Physiological MH is reversible, associated with improved cardiac function and lack of fibrosis.1 At the level of cardiac metabolism, MH during the course of physical training is accompanied by enhanced fatty acid oxidation and reduced glucose use. ### Clinical Impact and Relevance in Humans Studies in humans comparing exercise-mediated MH in both sexes are limited. Left ventricular mass (LVM) is significantly increased in male endurance athletes by 36±14% compared with untrained control subjects.2 In young adult elite athletes, both sexes exhibited similar increases in LVM and LVM indices when compared with their sex-matched sedentary controls.3 However, limitations of these studies in humans have to be taken into account: despite a comparable training duration (hours/week and years at elite level) in female and male athletes, no detailed information about training intensity was provided.3 Putative sex differences in training intensity might have influenced exercise-induced cardiac remodeling. A lower …

17β-Estradiol-induced interaction of ERα with NPPA regulates gene expression in cardiomyocytes

AIMS 17β-Oestradiol (E2) and its receptors (ERα and ERβ) are important regulators of physiological and pathological processes in the cardiovascular system. ER act in concert with other regulatory factors mediating oestrogenic effects. However, the underlying mechanisms modulating ER transcriptional activity are not fully elucidated. To gain better understanding of E2-induced ERα action in the human heart, we aimed to identify and functionally analyse interaction partners of ERα. METHODS AND RESULTS Using yeast two-hybrid assays with a human heart cDNA library, we identified atrial natriuretic peptide precursor A (NPPA), a well-known cardiac hypertrophy marker, as a novel ERα interaction partner interacting in an E2-dependent manner. Mutation analyses and immunofluorescence data indicated that the LXXLL motif within NPPA is necessary for its E2-induced interaction with ERα, its action as a co-repressor of ERα, and its translocation into the nucleus of human and rat cardiomyocytes. Expression analysis and chromatin immunoprecipitation assays in a human left ventricular cardiomyocyte cell line, AC16, showed that NPPA interacts with E2/ERα, suppressing the transcriptional activity of ERα on E2-target genes, such as NPPA, connexin43, αactinin-2, nuclear factor of activated T-cells, and collagens I and III. CONCLUSION We characterize for the first time an E2-regulated interaction of NPPA with ERα in cardiomyocytes, that may be crucial in physiological and/or pathological cardiac processes, thereby representing a potential therapeutic target.