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Women and men, female and male animals and cells are biologically different, and acknowledgement of this fact is critical to advancing medicine. However, incorporating concepts of sex-specific analysis in basic research is largely neglected, introducing bias into translational findings, clinical concepts and drug development. Research funding agencies recently approached these issues but implementation of policy changes in the scientific community is still limited, probably due to deficits in concepts, knowledge and proper methodology. This expert review is based on the EUGenMed project (www.eugenmed.eu) developing a roadmap for implementing sex and gender in biomedical and health research. For sake of clarity and conciseness, examples are mainly taken from the cardiovascular field that may serve as a paradigm for others, since a significant amount of knowledge how sex and oestrogen determine the manifestation of many cardiovascular diseases (CVD) has been accumulated. As main concepts for implementation of sex in basic research, the study of primary cell and animals of both sexes, the study of the influence of genetic vs. hormonal factors and the analysis of sex chromosomes and sex specific statistics in genome wide association studies (GWAS) are discussed. The review also discusses methodological issues, and analyses strength, weaknesses, opportunities and threats in implementing sex-sensitive aspects into basic research., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.)

Aims: Dilated cardiomyopathy (DCM) is an important cause of heart failure with a strong familial component. We performed an exome-wide array-based association study (EWAS) to assess the contribution of missense variants to sporadic DCM., Methods and Results: 116,855 single nucleotide variants (SNVs) were analyzed in 2796 DCM patients and 6877 control subjects from 6 populations of European ancestry. We confirmed two previously identified associations with SNVs in BAG3 and ZBTB17 and discovered six novel DCM-associated loci (Q-value<0.01). The lead-SNVs at novel loci are common and located in TTN, SLC39A8, MLIP, FLNC, ALPK3 and FHOD3. In silico fine mapping identified HSPB7 as the most likely candidate at the ZBTB17 locus. Rare variant analysis (MAF<0.01) demonstrated significant association for TTN variants only (P = 0.0085). All candidate genes but one (SLC39A8) exhibit preferential expression in striated muscle tissues and mutations in TTN, BAG3, FLNC and FHOD3 are known to cause familial cardiomyopathy. We also investigated a panel of 48 known cardiomyopathy genes. Collectively, rare (n = 228, P = 0.0033) or common (n = 36, P = 0.019) variants with elevated in silico severity scores were associated with DCM, indicating that the spectrum of genes contributing to sporadic DCM extends beyond those identified here., Conclusion: We identified eight loci independently associated with sporadic DCM. The functions of the best candidate genes at these loci suggest that proteostasis regulation might play a role in DCM pathophysiology.

Frequently, pharmacomechanisms are not fully elucidated. Therefore, drug use is linked to an elevated interindividual diversity of effects, whether therapeutic or adverse, and the role of biological sex has as yet unrecognized and underestimated consequences. A pharmacogenomic approach could contribute towards the development of an adapted therapy for each male and female patient, considering also other fundamental features, such as age and ethnicity. This would represent a crucial step towards precision medicine and could be translated into clinical routine. In the present review, we consider recent results from pharmacogenomics and the role of sex in studies that are relevant to cardiovascular therapy. We focus on genome-wide analyses, because they have obvious advantages compared with targeted single-candidate gene studies. For instance, genome-wide approaches do not necessarily depend on prior knowledge of precise molecular mechanisms of drug action. Such studies can lead to findings that can be classified into three categories: first, effects occurring in the pharmacokinetic properties of the drug, e.g. through metabolic and transporter differences; second, a pharmacodynamic or drug target-related effect; and last diverse adverse effects. We conclude that the interaction of sex with genetic determinants of drug response has barely been tested in large, unbiased, pharmacogenomic studies. We put forward the theory that, to contribute towards the realization of precision medicine, it will be necessary to incorporate sex into pharmacogenomics., (© 2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Major differences between men and women exist in epidemiology, manifestation, pathophysiology, treatment, and outcome of cardiovascular diseases (CVD), such as coronary artery disease, pressure overload, hypertension, cardiomyopathy, and heart failure. Corresponding sex differences have been studied in a number of animal models, and mechanistic investigations have been undertaken to analyze the observed sex differences. We summarize the biological mechanisms of sex differences in CVD focusing on three main areas, i.e., genetic mechanisms, epigenetic mechanisms, as well as sex hormones and their receptors. We discuss relevant subtypes of sex hormone receptors, as well as genomic and nongenomic, activational and organizational effects of sex hormones. We describe the interaction of sex hormones with intracellular signaling relevant for cardiovascular cells and the cardiovascular system. Sex, sex hormones, and their receptors may affect a number of cellular processes by their synergistic action on multiple targets. We discuss in detail sex differences in organelle function and in biological processes. We conclude that there is a need for a more detailed understanding of sex differences and their underlying mechanisms, which holds the potential to design new drugs that target sex-specific cardiovascular mechanisms and affect phenotypes. The comparison of both sexes may lead to the identification of protective or maladaptive mechanisms in one sex that could serve as a novel therapeutic target in one sex or in both., (Copyright © 2017 the American Physiological Society.)

Background: We recently demonstrated that the heart of late pregnant (LP) rodents is more prone to ischemia/reperfusion (I/R) injury compared to non-pregnant rodents. Lipids, particularly polyunsaturated fatty acids, have received special attention in the field of cardiovascular research. Here, we explored whether Intralipid (ITLD) protects the heart against I/R injury in LP rodents and investigated the mechanisms underlying this protection., Methods and Results: In-vivo female LP rat hearts or ex-vivo isolated Langendorff-perfused LP mouse hearts were subjected to ischemia followed by reperfusion with PBS or ITLD (one bolus of 5mg/kg of 20% in in-vivo and 1% in ex-vivo). Myocardial infarct size, mitochondrial calcium retention capacity, genome-wide expression profiling, pharmacological inhibition and co-immunoprecipitation were performed. One bolus of ITLD at reperfusion significantly reduced the in-vivo myocardial infarct size in LP rats (23.3±2% vs. 55.5±3.4% in CTRL, p<0.01). Postischemic administration of ITLD also protected the LP hearts against I/R injury ex-vivo. ITLD significantly increased the threshold for the opening of the mitochondrial permeability transition pore in response to calcium overload (nmol-calcium/mg-mitochondrial protein: 290±17 vs. 167±10 in CTRL, p<0.01) and significantly increased phosphorylation of STAT3 (1.8±0.08 vs. 1±0.16 in CTRL, p<0.05) and GSK-3β (2.63±0.55 vs. 1±0.0.34 in CTRL, p<0.05). The ITLD-induced cardioprotection was fully abolished by Stattic, a specific inhibitor of STAT3. Transcriptome analysis revealed caveolin 2 (Cav2) was significantly upregulated by ITLD in hearts of LP rats under I/R injury. Co-immunoprecipitation experiments showed that Cav2 interacts with STAT3., Conclusions: ITLD protects the heart in late pregnancy against I/R injury by inhibiting the mPTP opening through Cav2/STAT3/GSK-3β pathway., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Chronic increased workload of the human heart causes ventricular hypertrophy, re-expression of the atrial essential myosin light chain (hALC-1), and improved contractile function. Although hALC-1 is an important positive inotropic regulator of the human heart, little is known about its regulation. Therefore, we investigated the role of the sex hormone 17β-estradiol (E2) on hALC-1 gene expression, the underlying molecular mechanisms, and the impact of this regulatory process on cardiac contractile function. We showed that E2 attenuated hALC-1 expression in human atrial tissues of both sexes and in human ventricular AC16 cells. E2 induced the nuclear translocation of estrogen receptor alpha (ERα) and hALC-1 in AC16 cells, where they cooperatively regulate the transcriptional activity of hALC-1 gene promoter. E2-activated ERα required the estrogen response element (ERE) motif within the hALC-1 gene promoter to reduce its transcriptional activity (vehicle: 15.55 ± 4.80 vs. E2: 6.51 ± 3.69; ~2 fold). This inhibitory effect was potentiated in the presence of hALC-1 (vehicle: 11.13 ± 3.66 vs. E2: 2.18 ± 1.10; ~5 fold), and thus, hALC-1 acts as a co-repressor of ERα-mediated transcription. Yeast two-hybrid screening of a human heart cDNA library revealed that ERα interacts physically with hALC-1 in the presence of E2. This interaction was confirmed by Co-Immunoprecipitation and immunofluorescence in human atrium. As a further novel effect, we showed that chronic E2-treatment of adult mouse cardiomyocytes overexpressing hALC-1 resulted in reduced cell-shortening amplitude and twitching kinetics of these cells independent of Ca 2+ activation levels. Together, our data showed that the expression of hALC-1 gene is, at least partly, regulated by E2/ERα, while hALC-1 acts as a co-repressor. The inotropic effect of hALC-1 overexpression in cardiomyocytes can be significantly repressed by E2.

Background: Estrogen improves cardiac recovery after ischemia/reperfusion (I/R) by yet incompletely understood mechanisms. Mitochondria play a crucial role in I/R injury through cytochrome c-dependent apoptosis activation. We tested the hypothesis that 17β-estradiol (E2) as well as a specific ERβ agonist improve cardiac recovery through estrogen receptor (ER)β-mediated mechanisms by reducing mitochondria-induced apoptosis and preserving mitochondrial integrity., Methods: We randomized ovariectomized C57BL/6N mice 24h before I/R to pre-treatment with E2 or a specific ERβ agonist (ERβA). Isolated hearts were perfused for 20min prior to 30min global ischemia followed by 40min reperfusion., Results: Compared with controls, ERβA and E2 treated groups showed a significant improvement in cardiac recovery, i.e. an increase in left ventricular developed pressure, dP/dtmax and dP/dtmin. ERβA and E2 pre-treatment led to a significant reduction in apoptosis with decreased cytochrome c release from the mitochondria and increased mitochondrial levels of anti-apoptotic Bcl2 and ACAA2. Protein levels of mitochondrial translocase inner membrane (TIM23) and mitochondrial complex I of respiratory chain were increased by ERβA and E2 pre-treatment. Furthermore, we found a significant increase of myosin light chain 2 (MLC2) phosphorylation together with ERK1/2 activation in E2, but not in ERβA treated groups., Conclusions: Activation of ERβ is essential for the improvement of cardiac recovery after I/R through the inhibition of apoptosis and preservation of mitochondrial integrity and can be a achieved by a specific ERβ agonist. Furthermore, E2 modulates MLC2 activation after I/R independent of ERβ.

Impairment of skeletal muscle function has been associated with changes in ovarian hormones, especially estradiol. To elucidate mechanisms of estradiol on skeletal muscle strength, the hormone's effects on phosphorylation of the myosin regulatory light chain (pRLC) and muscle contractility were investigated, hypothesizing an estradiol-specific beneficial impact. In a skeletal muscle cell line, C2C12, pRLC was increased by 17β-estradiol (E2) in a concentration-dependent manner. In skeletal muscles of C57BL/6 mice that were E2 deficient via ovariectomy (OVX), pRLC was lower than that from ovary-intact, sham-operated mice (Sham). The reduced pRLC in OVX muscle was reversed by in vivo E2 treatment. Posttetanic potentiation (PTP) of muscle from OVX mice was low compared with that from Sham mice, and this decrement was reversed by acute E2 treatment, demonstrating physiological consequence. Western blot of those muscles revealed that low PTP corresponded with low pRLC and higher PTP with greater pRLC. We aimed to elucidate signaling pathways affecting E2-mediated pRLC using a kinase inhibitor library and C2C12 cells as well as a specific myosin light chain kinase inhibitor in muscles. PI3K/Akt, MAPK, and CamKII were identified as candidate kinases sensitive to E2 in terms of phosphorylating RLC. Applying siRNA strategy in C2C12 cells, pRLC triggered by E2 was found to be mediated by estrogen receptor-β and the G protein-coupled estrogen receptor. Together, these results provide evidence that E2 modulates myosin pRLC in skeletal muscle and is one mechanism by which this hormone can affect muscle contractility in females., (Copyright © 2016 the American Physiological Society.)

Biological sex significantly affects the presentation, outcome of treatment and progression of disease. However, the role of sex has yet underestimated consequences for physiology and pathology. We put forward that a better understanding of the effects of sex in pathophysiology and the underlying mechanisms is necessary. This may facilitate the identification of targets that respond to specific therapies, thereby contributing towards a more appropriate and individualised medical care for both men and women.

Purpose: Aging has severe implications for tissue damage and is a major risk factor for disease. However, the effects of aging on cardiac extracellular matrix (ECM) components in individuals free of cardiovascular disease are incompletely understood. We aimed at the characterization of the effects of aging on major ECM proteins in the heart of men and women., Experimental Design: Left ventricular (LV) samples of nondiseased human hearts technically unusable for transplantation obtained from general organ donors (n = 31; age 17-68 years; 48% women) were used for protein isolation. We separated the group into 17-40 years (n = 7 men and 7 women) and 50-68 years (n = 9 men and 8 women)., Results: Analysis of ECM proteins demonstrated an age-dependent sex-specific regulation of collagen type I and III (interaction p < 0.05), type VI (interaction p = 0.01), tissue inhibitor of metalloproteinase 3 (interaction p < 0.05), SMAD2 (interaction p < 0.05), and SMAD3 (interaction p = 0.001). Overall, the levels of these proteins in younger individuals were lower in women than men, while in older individuals they were higher in women than men., Conclusions and Clinical Relevance: This age-mediated myocardial ECM remodeling might play a key role in the limited ability of the aging heart to adapt adequately to altered work load and to respond to tissue damage. Therapeutic agents that target ECM homeostasis represent promising prevention strategies., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In pressure overload (PO), sex differences in humans and rodents have been well documented and estrogen receptor (ER) β is considered cardioprotective. However, the underlying mechanisms are poorly understood. Our aim was to investigate sex- and ERβ-specific effects in protein abundance in PO employing a 2-dimensional gel electrophoresis/mass spectrometry-based proteomics approach. We hypothesized major sex differences and ERβ-specific alterations consistent with cardioprotection in females. Two-month old male and female wild-type (WT) and ERβ knockout (BERKO) mice were subjected to transverse aortic constriction (TAC) for 9 weeks (n = 4/group). In WT mice, hypertrophy was significantly more pronounced in males than females, while this sex difference was abolished in BERKO mice. We found 82 protein spots modulated between TAC and sham in WT males, 31 in WT females, 114 in BERKO males, and 87 in BERKO females (P ≤ 0.05). Our analysis revealed in WT and BERKO females an altered pattern of various proteins involved in structure and suggests a link between female sex and cytoskeletal integrity. In males, a set of proteins was identified that associate with mitochondrial bioenergetics and energy supply. We confirmed protein regulation by immunoblotting analysis. In conclusion, the proteomic response of the heart to PO is significantly modulated by ERβ and sex. We put forward that the observed differences may identify sex-specific targets for the treatment of heart failure, contributing toward more personalized medical care.

Aims: 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., Methods and Results: Echocardiography was performed in 104 patients (53.8% women) with aortic stenosis before aortic valve replacement. LV mass, LV end-diastolic diameter, and relative wall thickness were included in a factor analysis to generate an index classifying LV remodelling as adaptive or maladaptive. Maladaptive remodelling was present in 64.6% of male and in 32.7% of female patients (P < 0.01). Genome-wide expression profiling of LV samples was performed in a representative subgroup of 19 patients (52.6% women) compared with samples from healthy controls (n = 18). Transcriptome characterization revealed that fibrosis-related genes/pathways were induced in male overloaded ventricles, while extracellular matrix-related and inflammatory genes/pathways were repressed in female overloaded ventricles (adjusted P < 0.05). We confirmed gene regulation by quantitative real-time reverse transcription-polymerase chain reaction and immunoblotting analysis, and we further demonstrate the relevance of our findings by histological documentation of higher fibrosis in men than in women., Conclusion: We conclude that in pressure overload distinct molecular processes are regulated between men and women. Maladaptive LV remodelling occurs more frequently in men and is associated with greater activation of profibrotic and inflammatory markers. Collectively, sex-specific regulation of these processes may contribute to sex differences in the progression to heart failure., (© 2014 The Authors. European Journal of Heart Failure © 2014 European Society of Cardiology.)

Objectives: The purpose of this study was to test whether adaptive or maladaptive remodeling is associated with survival in women and men after aortic valve replacement (AVR)., Background: Women with isolated aortic valve stenosis (AS) develop more concentric left ventricular hypertrophy (LVH) than men in similar disease states. We recently reported less up-regulation of profibrotic genes at AVR and faster LVH regression post-operatively in women than in men, suggesting that there are sex differences in the adaptation to pressure overload and its regression., Methods: The study cohort included 128 patients (age 70.0 ± 9.6 years, 49% women) undergoing AVR for AS. Echocardiography was obtained before and 4.0 ± 1.6 years after surgery. Factor analysis was used to classify LVH as adaptive (combining smaller left ventricular [LV] mass/diameters and greater relative wall thicknesses) or maladaptive. Myocardial tissue samples from the LV septum were obtained during AVR to analyze cardiac fibrosis and associated key molecular regulators., Results: Before AVR, LVH was classified as adaptive in 62% of women and 45% of men (p < 0.050). Four years after AVR, adaptive LVH was observed in 75% of women and 49% of men (p < 0.031). At surgery, more cardiac fibrosis was present in men compared with women (p < 0.05). Higher levels of transforming growth factor beta 1 (p < 0.01), SMAD2 phosphorylation (p < 0.001), and periostin expression (p < 0.05) were found in men than in women. Women with maladaptive LVH had worse survival than women with adaptive LVH (p < 0.050), whereas the pattern of LVH did not affect survival in men (p < 0.307)., Conclusions: Women more frequently exhibit adaptive LV remodeling with less fibrosis than men. Maladaptive LVH is associated with worse survival in women. Thus, sex should be considered as a strong modulating factor when management of patients with AS is discussed., (Copyright © 2014 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Estrogen regulates several biological processes in health and disease. Specifically, estrogen exerts antihypertrophic effects in the diseased heart. However, its role in the healthy heart remains elusive. Our initial aim was to identify the effects of 17β-estradiol (E2) on cardiac morphology and global gene expression in the healthy mouse heart. Two-month-old C57BL/6J mice were ovariectomized and treated with E2 or vehicle for 3 months. We report that E2 induced physiological hypertrophic growth in the healthy C57BL/6J mouse heart characterized by an increase in nuclear β-catenin. Hypothesizing that β-catenin mediates these effects of E2, we employed a model of cardiac β-catenin deletion. Our surprising finding is that E2 had the opposite effects in wild-type littermates, which were actually on the C57BL/6N background. Notably, E2 exerted no significant effect in hearts of mice with depleted β-catenin. We further demonstrate an E2-dependent increase in glycogen synthase kinase 3β (GSK3β) phosphorylation and endosomal markers in C57BL/6J but not C57BL/6N mice. Together, these findings indicate an E2-driven inhibition of GSK3β and consequent activation of β-catenin in C57BL/6J mice, whereas the opposite occurs in C57BL/6N mice. In conclusion, E2 exerts divergent effects on postnatal cardiac growth in mice with distinct genetic backgrounds modulating members of the GSK3β/β-catenin cascade.

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., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

The modulation of cardiac growth by estrogen in healthy mice is not completely understood. The aim was to investigate the effects of estrogen on cardiac growth in healthy mice lacking either estrogen receptor (ER) α or β. Wild-type (WT), ERα knockout (ERKO) and ERβ knockout (BERKO) 2-month-old mice were ovariectomized and randomly assigned to groups receiving an estradiol (E2)-containing or soy-free (control, CON) diet (n=5-7/group). After three months of E2 administration, WT and BERKO mice had significantly lower body weight, higher relative uterus and heart weight than CON mice, while there was no major E2 effect in ERKO mice. Furthermore, there was a higher concentration of E2-responsive genes Igf1 and Myocd in WT and BERKO but not in ERKO mice. Together, these findings indicate that the estrogenic regulation of cardiac growth in healthy mice is primarily mediated through ERα and not ERβ., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

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., (© 2013.)

The isoflavone genistein is used as a pharmacological compound and as a food supplement. The duration and the level of exposure of humans to genistein are considerable. However, the magnitude of genistein-supplemented dietary interventions necessary to induce any changes in the heart has not been studied so far. The aim of this study was to investigate the dose-dependent effects of dietary genistein in the disease- and stress-free mouse heart. Female C57BL/6J mice at the age of 2 months were ovariectomized and randomly assigned to feed on diets with seven different genistein doses (0.01, 0.03, 0.1, 0.3, 1, 3 and 10 g genistein/kg food) for 3 months. Mice with intact ovaries or ovariectomized fed on soy-free diets were used as controls. Ovariectomy led to an increase in body weight, while the two highest genistein doses prevented this increase. Absolute uterus weight was decreased in the ovariectomized group and all genistein groups except for the 10 g/kg food group compared with the intact ovaries/soy-free group. Considering cardiac mass, although the 3 and 10 g/kg food groups had significantly lower absolute heart weight than all other groups, heart-to-body-weight ratios did not differ between these two groups and the intact ovaries/soy-free group, while all remaining groups had smaller ratios. Next, we observed dose-dependent effects of genistein on cardiac gene expression. The present findings indicate that exposure of female mice to the soy isoflavone genistein influences body weight and cardiac mass and gene expression in a dose-dependent manner. Human exposure to dietary genistein supplements may influence cardiac function.

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.

Background: Availability of oxygen and nutrients in the coronary circulation is a crucial determinant of cardiac performance. Nutrient composition of coronary blood may significantly vary in specific physiological and pathological conditions, for example, administration of special diets, long-term starvation, physical exercise or diabetes. Quantitative analysis of cardiac metabolism from a systems biology perspective may help to a better understanding of the relationship between nutrient supply and efficiency of metabolic processes required for an adequate cardiac output., Results: Here we present CardioNet, the first large-scale reconstruction of the metabolic network of the human cardiomyocyte comprising 1793 metabolic reactions, including 560 transport processes in six compartments. We use flux-balance analysis to demonstrate the capability of the network to accomplish a set of 368 metabolic functions required for maintaining the structural and functional integrity of the cell. Taking the maintenance of ATP, biosynthesis of ceramide, cardiolipin and further important phospholipids as examples, we analyse how a changed supply of glucose, lactate, fatty acids and ketone bodies may influence the efficiency of these essential processes., Conclusions: CardioNet is a functionally validated metabolic network of the human cardiomyocyte that enables theorectical studies of cellular metabolic processes crucial for the accomplishment of an adequate cardiac output.

Objectives: This study investigated the effects of 17β-estradiol (E2) on gene regulation in human cardiac tissues. We hypothesized that a candidate E2 effect is cardiomyocyte (CM)- and sex-specific, conserved between humans and mice, and that E2 impairs contractile function in male CMs only., Background: Both men and women produce E2 locally from androgenic precursors. E2 regulates cardiovascular function, but specific mechanisms, protective or harmful, are not fully understood., Methods: We performed genome-wide expression profiling of E2-treated cardiac tissues from men and women, and studied gene expression and function in CMs from hearts of male and female E2-treated mice., Results: We found 36 E2-dependent genes regulated in a sex-specific manner. Of these, after E2 exposure, the myosin regulatory light chain interacting protein (MYLIP) gene was induced in tissues of men only. Focusing on Mylip and employing isolated mouse CMs, we confirmed our hypotheses that the E2 effect is CM- and sex-specific and conserved between humans and mice. The E2-treatment led to impaired contractile function in male CMs only, which was characterized by increased Mylip mRNA and protein levels, and decreased myosin regulatory light chain (Mrlc) protein. Our report is the first to our knowledge to show that cardiac Mrlc is an in vivo substrate for Mylip, leading to augmented Mrlc ubiquitination. Of relevance, we found that MYLIP expression levels rise with increasing age in hearts of men., Conclusions: E2 directly influences cardiac gene regulation, and E2 actions may be different between the sexes. Since E2 levels rise in older and/or obese men, pharmacological targeting of MYLIP in men with elevated E2 levels could possibly decrease their risk for the development or progression of cardiovascular disease., (Copyright © 2012 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Epidemiological data reveal that the overall risk for heart disease is lower for premenopausal women compared to age-matched men. However, the beneficial effect for the female sex is lost upon menopause. Thus, it has been suggested that estrogens convey the protective effect for the female sex against heart disease. Numerous natural plant products, i.e., phytoestrogens (PE), interfere with or alter the development or function of the endocrine system. Although PEs have been studied intensively with regard to the effects on the reproductive organs, such as the uterus or mammary gland, surprisingly little data are available about the effects of PEs on the heart. Here, we conducted a long-term study with ovariectomized mice to examine putative estrogenic effects of the PEs genistein (GEN), resveratrol (RES), and equol (EQ), using estradiol (E2) as a reference compound on heart size, morphology, and cardiac gene expression. We report for the first time significant changes in these parameters by GEN and E2. Changes in the size of cardiomyocytes were observed by GEN and E2. In line with these observations, cardiac expression of insulin-like growth factor 1 ( IGF1) was significantly induced by both GEN and E2. Thus, we speculate that endocrine active compounds, like the isoflavone GEN, which is used as a food additive or as a drug for the treatment of menopausal symptoms, may directly affect heart function., (© Georg Thieme Verlag KG Stuttgart · New York.)

Cardiac hypertrophy, the adaptive response of the heart to overload, is a major risk factor for heart failure and sudden death. Estrogen (E2) and estrogen receptor beta (ERbeta) offer protection against hypertrophy and in the transition to heart failure. However, the underlying pathways remain incompletely defined. We employed a publicly available microarray dataset of female wild-type (WT) and ERbeta knockout (BERKO) mice subjected to pressure overload-induced hypertrophy to perform a systematic investigation of the mechanisms involved in the protection conferred by the E2/ERbeta axis. We show that considerably more genes were modulated in response to pressure overload in BERKO mice than in WT mice. The majority of the identified candidates in BERKO mice were induced, while those in WT mice were repressed. Pathway analysis revealed a similar pattern. This study is the first to demonstrate that the lack of ERbeta led to a significant increase of inflammatory pathways. Mitochondrial bioenergetics- and oxidative stress-related pathways were also modulated. In conclusion, ERbeta acquires the role of gatekeeper of the genomic response of the heart to pressure overload-induced hypertrophy. This may offer the molecular explanation for its cardioprotective role. We consider the present study to be a useful resource and that it will contribute to downstream functional analysis and to the characterization of pathways with previously unknown role in hypertrophy.

Background: Hemodynamic load regulates myocardial function and gene expression. We tested the hypothesis that afterload and preload, despite similar average load, result in different phenotypes., Methods and Results: Afterload and preload were compared in mice with transverse aortic constriction (TAC) and aortocaval shunt (shunt). Compared with sham mice, 6 hours after surgery, systolic wall stress (afterload) was increased in TAC mice (+40%; P<0.05), diastolic wall stress (preload) was increased in shunt (+277%; P<0.05) and TAC mice (+74%; P<0.05), and mean total wall stress was similarly increased in TAC (69%) and shunt mice (67%) (P=NS, TAC versus shunt; each P<0.05 versus sham). At 1 week, left ventricular weight/tibia length was significantly increased by 22% in TAC and 29% in shunt mice (P=NS, TAC versus shunt). After 24 hours and 1 week, calcium/calmodulin-dependent protein kinase II signaling was increased in TAC. This resulted in altered calcium cycling, including increased L-type calcium current, calcium transients, fractional sarcoplasmic reticulum calcium release, and calcium spark frequency. In shunt mice, Akt phosphorylation was increased. TAC was associated with inflammation, fibrosis, and cardiomyocyte apoptosis. The latter was significantly reduced in calcium/calmodulin-dependent protein kinase IIdelta-knockout TAC mice. A total of 157 mRNAs and 13 microRNAs were differentially regulated in TAC versus shunt mice. After 8 weeks, fractional shortening was lower and mortality was higher in TAC versus shunt mice., Conclusions: Afterload results in maladaptive fibrotic hypertrophy with calcium/calmodulin-dependent protein kinase II-dependent altered calcium cycling and apoptosis. Preload is associated with Akt activation without fibrosis, little apoptosis, better function, and lower mortality. This indicates that different loads result in distinct phenotype differences that may require specific pharmacological interventions.

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.