Nebulette knockout mice have normal cardiac function, but show Z-line widening and up-regulation of cardiac stress markers

Aims: Nebulette is a 109 kDa modular protein localized in the sarcomeric Z-line of the heart. In vitro studies have suggested a role of nebulette in stabilizing the thin filament, and missense mutations in the nebulette gene were recently shown to be causative for dilated cardiomyopathy and endocardial fibroelastosis in human and mice. However, the role of nebulette in vivo has remained elusive. To provide insights into the function of nebulette in vivo, we generated and studied nebulette-deficient (nebl-/-) mice. Methods and results: Nebl-/- mice were generated by replacement of exon 1 by Cre under the control of the endogenous nebulette promoter, allowing for lineage analysis using the ROSA26 Cre reporter strain. This revealed specific expression of nebulette in the heart, consistent with in situ hybridization results. Nebl-/- mice exhibited normal cardiac function both under basal conditions and in response to transaortic constriction as assessed by echocardiography and haemodynamic analyses. Furthermore, histological, IF, and western blot analysis showed no cardiac abnormalities in nebl-/- mice up to 8 months of age. In contrast, transmission electron microscopy showed Z-line widening starting from 5 months of age, suggesting that nebulette is important for the integrity of the Z-line. Furthermore, up-regulation of cardiac stress responsive genes suggests the presence of chronic cardiac stress in nebl-/- mice. Conclusion: Nebulette is dispensable for normal cardiac function, although Z-line widening and up-regulation of cardiac stress markers were found in nebl-/- heart. These results suggest that the nebulette disease causing mutations have dominant gain-of-function effects

Growth hormone-releasing hormone attenuates cardiac hypertrophy and improves heart function in pressure overload-induced heart failure

It has been shown that growth hormone-releasing hormone (GHRH) reduces cardiomyocyte (CM) apoptosis, prevents ischemia/reperfusion injury, and improves cardiac function in ischemic rat hearts. However, it is still not known whether GHRH would be beneficial for life-threatening pathological conditions, like cardiac hypertrophy and heart failure (HF). Thus, we tested the myocardial therapeutic potential of GHRH stimulation in vitro and in vivo, using GHRH or its agonistic analog MR-409. We show that in vitro, GHRH(1-44)NH2attenuates phenylephrine-induced hypertrophy in H9c2 cardiac cells, adult rat ventricular myocytes, and human induced pluripotent stem cell-derived CMs, decreasing expression of hypertrophic genes and regulating hypertrophic pathways. Underlying mechanisms included blockade of Gq signaling and its downstream components phospholipase Cβ, protein kinase Ce, calcineurin, and phospholamban. The receptor-dependent effects of GHRH also involved activation of Gαsand cAMP/PKA, and inhibition of increase in exchange protein directly activated by cAMP1 (Epac1). In vivo, MR-409 mitigated cardiac hypertrophy in mice subjected to transverse aortic constriction and improved cardiac function. Moreover, CMs isolated from transverse aortic constriction mice treated with MR-409 showed improved contractility and reversal of sarcolemmal structure. Overall, these results identify GHRH as an antihypertrophic regulator, underlying its therapeutic potential for HF, and suggest possible beneficial use of its analogs for treatment of pathological cardiac hypertrophy

Dietary essential amino acids for the treatment of heart failure with reduced ejection fraction

Aims: Heart failure with reduced ejection fraction (HFrEF) is a leading cause of mortality worldwide, requiring novel therapeutic and lifestyle interventions. Metabolic alterations and energy production deficit are hallmarks and thereby promising therapeutic targets for this complex clinical syndrome. We aim to study the molecular mechanisms and effects on cardiac function in rodents with HFrEF of a designer diet in which free essential amino acids - in specifically designed percentages - substituted for protein. Methods and results: Wild-type mice were subjected to transverse aortic constriction (TAC) to induce left ventricle (LV) pressure overload or sham surgery. Whole body glucose homeostasis was studied with glucose tolerance test, while myocardial dysfunction and fibrosis were measured with echocardiogram and histological analysis. Mitochondrial bioenergetics and morphology were investigated with oxygen consumption rate measurement and electron microscopy evaluation. Circulating and cardiac non-targeted metabolite profiles were analyzed by ultrahigh performance liquid chromatography-tandem mass spectroscopy, while RNA sequencing was used to identify signalling pathways mainly affected. The amino acid-substituted diet shows remarkable preventive and therapeutic effects. This dietary approach corrects the whole-body glucose metabolism and restores the unbalanced metabolic substrate usage - by improving mitochondrial fuel oxidation - in the failing heart. In particular, biochemical, molecular, and genetic approaches suggest that renormalization of branched-chain amino acid oxidation in cardiac tissue, which is suppressed in HFrEF, plays a relevant role. Beyond the changes of systemic metabolism, cell-autonomous processes may explain at least in part the diet's cardioprotective impact. Conclusion: Collectively, these results suggest that manipulation of dietary amino acids, and especially essential amino acids, is a potential adjuvant therapeutic strategy to treat systolic dysfunction and HFrEF in humans

Enhancers regulate anaerobic glycolysis in old cardiomyocytes

Heart failure is a typical age-associated pathology with high rates of mortality and morbidity. Age-related cardiac malfunctioning is multifactorial and cardiac remodelling contributes to this. A major event in aging-associated-cardiac remodelling is a metabolic shift in cardiomyocytes so that glycolysis increases at the expense of mitochondrial oxidative phosphorylation. This metabolism modification causes an \u201cenergy deficiency\u201d that contributes in the elderly to an impairment of cardiac contractility. Gene expression analysis in cardiac aging models shows a general down-regulation of genes involved in energy metabolism, including those associated with mitochondrial function (e.g., fatty-acid metabolism) and turnover. This finding thus supports the notion that cardiac aging-related metabolic remodelling is caused by changes in the expression of metabolic genes. Despite this, the molecular mechanisms causing these changes are not, as yet, fully unravelled. Enhancers play a key role in defining the gene expression program of heart development and cardiac hypertrophy. Therefore, alteration of the activity of enhancers could contribute in defining the gene expression changes responsible for metabolic remodelling in old cardiomyocytes. To test this hypothesis, we investigated the activity state of enhancers in relation to gene expression and metabolic changes occurring during cardiac aging. To this end, we integrated metabolic data with RNA-seq and ChIP-seq data for H3K27ac and H3K27me3 (two histone markers that define active and repressed enhancers, respectively) obtained from ventricular cardiomyocytes purified from the heart of mice at different ages (8 weeks, and 6 and 18 months old, corresponding to young, adult and old-age mice). Results show that a large fraction of enhancers undergo a change in activity, an event associated with variation in transcription of neighbouring genes: a set of enhancers switched from a state of little or no activity to an active state, such as those associated with genes involved in glycolysis, hypertrophy and dilated cardiomyopathy; in contrast, enhancers that switched from an active state to an inactive state were involved in cytoskeleton organization. By comparing the enhancer dataset with metabolic profiles, we found that the activation of enhancers neighbouring glycolytic genes was associated with an increase of metabolites of anaerobic glycolysis. Gene ontology analysis showed that the dataset of modulated enhancers overlapped partially with datasets identified for heart development, cardiomyocyte differentiation and cardiac hypertrophy, indicating the involvement during aging of enhancers implicated in heart development and disease. Interestingly, some of these enhancers were listed in Vista, a dataset of human and mouse enhancers validated in vivo. Together, these results demonstrate that enhancers are involved in promoting the gene expression changes \u2013 in particular, activation of anaerobic glycolysis \u2013 occurring in the cardiomyocyte during aging. These results suggest the possibility of modifying cardiac function during aging by modulating the activity of enhancers through epigenetic drugs

DNA hydroxymethylation controls cardiomyocyte gene expression in development and hypertrophy

Methylation at 5-cytosine (5-mC) is a fundamental epigenetic DNA modification associated recently with cardiac disease. In contrast, the role of 5-hydroxymethylcytosine (5-hmC)-5-mC's oxidation product-in cardiac biology and disease is unknown. Here we assess the hydroxymethylome in embryonic, neonatal, adult and hypertrophic mouse cardiomyocytes, showing that dynamic modulation of hydroxymethylated DNA is associated with specific transcriptional networks during heart development and failure. DNA hydroxymethylation marks the body of highly expressed genes as well as distal regulatory regions with enhanced activity. Moreover, pathological hypertrophy is characterized by a shift towards a neonatal 5-hmC distribution pattern. We also show that the ten-eleven translocation 2 (TET2) enzyme regulates the expression of key cardiac genes, such as Myh7, through 5-hmC deposition on the gene body and at enhancers. Thus, we provide a genome-wide analysis of 5-hmC in the cardiomyocyte and suggest a role for this epigenetic modification in heart development and disease

Histone methyltransferase G9a is required for cardiomyocyte homeostasis and hypertrophy

BACKGROUND: Correct gene expression programming of the cardiomyocyte underlies the normal functioning of the heart. Alterations to this can lead to the loss of cardiac homeostasis, triggering heart dysfunction. Although the role of some histone methyltransferases in establishing the transcriptional program of postnatal cardiomyocytes during heart development has been shown, the function of this class of epigenetic enzymes is largely unexplored in the adult heart. In this study, we investigated the role of G9a/Ehmt2, a histone methyltransferase that defines a repressive epigenetic signature, in defining the transcriptional program for cardiomyocyte homeostasis and cardiac hypertrophy. METHODS: We investigated the function of G9a in normal and stressed cardiomyocytes with the use of a conditional, cardiacspecific G9a knockout mouse, a specific G9a inhibitor, and highthroughput approaches for the study of the epigenome (chromatin immunoprecipitation sequencing) and transcriptome (RNA sequencing); traditional methods were used to assess cardiac function and cardiovascular disease. RESULTS: We found that G9a is required for cardiomyocyte homeostasis in the adult heart by mediating the repression of key genes regulating cardiomyocyte function via dimethylation of H3 lysine 9 and interaction with enhancer of zeste homolog 2, the catalytic subunit of polycomb repressive complex 2, and MEF2C-dependent gene expression by forming a complex with this transcription factor. The G9a-MEF2C complex was found to be required also for the maintenance of heterochromatin needed for the silencing of developmental genes in the adult heart. Moreover, G9a promoted cardiac hypertrophy by repressing antihypertrophic genes. CONCLUSIONS: Taken together, our findings demonstrate that G9a orchestrates critical epigenetic changes in cardiomyocytes in physiological and pathological conditions, thereby providing novel therapeutic avenues for cardiac pathologies associated with dysregulation of these mechanisms

The Histone Methyl-transferase G9a Defines the Epigenetic Landscape Underlying of the Homeostasis of Heart and the Cardiac Hypertrophy

Cardiac hypertrophy is an initially adaptive response of the myocardium to increased work overload that can progress to heart failure (HF)1. At the molecular level, it is associated with a specific gene expression program2. The role of histone methylation in regulating this program is poorly understood. Our group has shown that an epigenetic signature defined by methylation and acetylation of histone H3 regulates the gene expression changes accompanying cardiac hypertrophy3. However, the molecular pathways that define this signature are not elucidated yet. To try to answer this question, we will investigate the function of G9a - a histone methyl-transferase able to mono- and di-methylate histone H3 at Lys9, and to a lesser extent at Lys27- in cardiac hypertrophy and cardiomyocyte homeostasis. Here, we show that the histone methyltransferase G9a/Ehmt2 has a key role in maintaining cardiomyocyte homeostasis, repressing key genes of heart function through the dimethylation of lysine 9 of histone H3 and interaction with enhancer of zeste homolog 2, the catalytic subunit of polycomb repressive complex 2. We also found that G9a represses the transcriptional activity of MEF2C on genes encoding contractility and calcium signalling proteins, and that the G9a\u2013MEF2C complex is required for the maintenance of heterochromatin regions responsible for the silencing of key genes of heart developmental. Moreover, G9a is up-regulated in initial stages of cardiac hypertrophy and it promotes cardiac hypertrophy by repressing anti-hypertrophic genes. These findings support a dual role for G9a in the heart: in the normal heart, G9a acts in an anti-hypertrophic manner, whereas in the stressed heart it promotes cardiac hypertrophy4