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Perturbed vitamin-A metabolism is associated with type 2 diabetes and mitochondrial dysfunction that are pathophysiologically linked to the development of diabetic cardiomyopathy (DCM). However, the mechanism, by which vitamin A might regulate mitochondrial energetics in DCM has previously not been explored. To test the hypothesis that vitamin-A deficiency accelerates the onset of cardiomyopathy in diet-induced obesity (DIO), we subjected mice with lecithin retinol acyltransferase (Lrat) germline deletion, which exhibit impaired vitamin-A stores, to vitamin A-deficient high-fat diet (HFD) feeding. Wild-type mice fed with a vitamin A-sufficient HFD served as controls. Cardiac structure, contractile function, and mitochondrial respiratory capacity were preserved despite vitamin-A deficiency following 20 wk of HFD feeding. Gene profiling by RNA sequencing revealed that vitamin A is required for the expression of genes involved in cardiac fatty acid oxidation, glycolysis, tricarboxylic acid cycle, and mitochondrial oxidative phosphorylation in DIO as expression of these genes was relatively preserved under vitamin A-sufficient HFD conditions. Together, these data identify a transcriptional program, by which vitamin A preserves cardiac energetic gene expression in DIO that might attenuate subsequent onset of mitochondrial and contractile dysfunction.

AimsPathological overload triggers maladaptive myocardial remodelling that leads to heart failure. Recent studies have shown that long non-coding RNAs (lncRNAs) contribute to the development of cardiac disease. This study investigates two recently discovered and secreted lncRNAs,Gadlor1andGadlor2(Gadlor 1/2).Methods and ResultsIn the heart,Gadlor 1/2are mainly expressed in endothelial cells and to a lesser extent in fibroblasts and cardiomyocytes.Gadlor1/2are upregulated in failing mouse hearts as well as in the myocardium and serum of heart failure patients. Interestingly,Gadlor1/2are secreted from endothelial cells (EC) within extracellular vesicles (EV) and are taken up by cardiomyocytes.Gadlor1/2 knock-out (Gadlor-KO) and overexpression during experimental cardiac pressure overload by transverse aortic constriction (TAC) was analysed.Gadlor-KO mice exhibited reduced cardiomyocyte hypertrophy, enhanced angiogenesis, diminished myocardial fibrosis, and improved cardiac function, but paradoxically suffered from sudden death during prolonged overload.Gadlor1/2overexpression, in turn, aggravated hypertrophy, fibrosis and cardiac dysfunction during TAC. Mechanistically,Gadlor1/2inhibited angiogenic gene expression in endothelial cells, while promoting the expression of pro-fibrotic genes in cardiac fibroblasts. We employed RNA antisense purification coupled with mass spectrometry (RAP-MS) and identified the calcium/calmodulin-dependent protein kinase type II (CaMKII) as interaction partner ofGadlor1/2in cardiomyocytes. By activating CaMKII,Gadlor1/2promoted hypertrophic gene-expression and excitation-contraction coupling in these cells. Accordingly,Gadlorablation entailed reduced hypertrophy, but also increased diastolic calcium levels in the cytosol and arrhythmic beating in cardiomyocytes.ConclusionGadlor1andGadlor2are novel lncRNAs that are mainly enriched in EC-derived EVs and are upregulated in mouse and human hearts during pathological overload.Gadlor1/2affect multiple cardiac cell types and inhibit angiogenesis, induce myocardial fibrosis and hypertrophy as well as alterations in calcium homeostasis in cardiomyocytes. We suggest that inhibition ofGadloreffects in cardiac non-myocytes could be a therapeutic strategy in heart failure.Translational PerspectiveHere, we characterized two secreted lncRNAsGadlor1/2, which are conserved in humans. We observed increasedGadlor1/2levels in human failing myocardium and a negative correlation betweenGADLOR2serum levels and left ventricular ejection fraction in heart failure patients.Gadlor1/2ablation in mice reduces cardiac hypertrophy and fibrosis and increases angiogenesis, but slows calcium re-uptake into the sarcoplasmic reticulum (SR) and might thereby trigger arrhythmia and sudden death when cardiac overload persists in the long term. Inhibition ofGadlor1/2effects in endothelial cells and fibroblasts, on the other hand, could be beneficial by increasing angiogenesis and reducing fibrosis in the heart.

Heart failure due to high blood pressure or ischemic injury remains a major problem for millions of patients worldwide. Despite enormous advances in deciphering the molecular mechanisms underlying heart failure progression, the cell-type specific adaptations and especially intercellular signaling remain poorly understood. Cardiac fibroblasts express high levels of cardiogenic transcription factors such as GATA-4 and GATA-6, but their role in fibroblasts during stress is not known. Here, we show that fibroblast GATA-4 and GATA-6 promote adaptive remodeling in pressure overload induced cardiac hypertrophy. Using a mouse model with specific single or double deletion of Gata4 and Gata6 in stress activated fibroblasts, we found a reduced myocardial capillarization in mice with Gata4/6 double deletion following pressure overload, while single deletion of Gata4 or Gata6 had no effect. Importantly, we confirmed the reduced angiogenic response using an in vitro co-culture system with Gata4/6 deleted cardiac fibroblasts and endothelial cells. A comprehensive RNA-sequencing analysis revealed an upregulation of anti-angiogenic genes upon Gata4/6 deletion in fibroblasts, and siRNA mediated downregulation of these genes restored endothelial cell growth. In conclusion, we identified a novel role for the cardiogenic transcription factors GATA-4 and GATA-6 in heart fibroblasts, where both proteins act in concert to promote myocardial capillarization and heart function by directing intercellular crosstalk.

Aims Chronic heart failure is becoming increasingly prevalent and is still associated with a high mortality rate. Myocardial hypertrophy and fibrosis drive cardiac remodelling and heart failure, but they are not sufficiently inhibited by current treatment strategies. Furthermore, despite increasing knowledge on cardiomyocyte intracellular signalling proteins inducing pathological hypertrophy, therapeutic approaches to target these molecules are currently unavailable. In this study, we aimed to establish and test a therapeutic tool to counteract the 22 kDa calcium and integrin binding protein (CIB) 1, which we have previously identified as nodal regulator of pathological cardiac hypertrophy and as activator of the maladaptive calcineurin/NFAT axis. Methods and results Among three different sequences, we selected a shRNA construct (shCIB1) to specifically down-regulate CIB1 by 50% upon adenoviral overexpression in neonatal rat cardiomyocytes (NRCM), and upon overexpression by an adeno-associated-virus (AAV) 9 vector in mouse hearts. Overexpression of shCIB1 in NRCM markedly reduced cellular growth, improved contractility of bioartificial cardiac tissue and reduced calcineurin/NFAT activation in response to hypertrophic stimulation. In mice, administration of AAV-shCIB1 strongly ameliorated eccentric cardiac hypertrophy and cardiac dysfunction during 2 weeks of pressure overload by transverse aortic constriction (TAC). Ultrastructural and molecular analyses revealed markedly reduced myocardial fibrosis, inhibition of hypertrophy associated gene expression and calcineurin/NFAT as well as ERK MAP kinase activation after TAC in AAV-shCIB1 vs. AAV-shControl treated mice. During long-term exposure to pressure overload for 10 weeks, AAV-shCIB1 treatment maintained its anti-hypertrophic and anti-fibrotic effects, but cardiac function was no longer improved vs. AAV-shControl treatment, most likely resulting from a reduction in myocardial angiogenesis upon downregulation of CIB1. Conclusions Inhibition of CIB1 by a shRNA-mediated gene therapy potently inhibits pathological cardiac hypertrophy and fibrosis during pressure overload. While cardiac function is initially improved by shCIB1, this cannot be kept up during persisting overload.

Rationale: Myocardial endothelial cells promote cardiomyocyte hypertrophy, possibly through the release of growth factors. The identity of these factors, however, remains largely unknown, and we hypothesized here that the secreted CTRP9 (C1q-tumor necrosis factor–related protein-9) might act as endothelial-derived protein to modulate heart remodeling in response to pressure overload. Objective: To examine the source of cardiac CTRP9 and its function during pressure overload. Methods and Results: CTRP9 was mainly derived from myocardial capillary endothelial cells. CTRP9 mRNA expression was enhanced in hypertrophic human hearts and in mouse hearts after transverse aortic constriction (TAC). CTRP9 protein was more abundant in the serum of patients with severe aortic stenosis and in murine hearts after TAC. Interestingly, heterozygous and especially homozygous knock-out C1qtnf9 (CTRP9) gene-deleted mice were protected from the development of cardiac hypertrophy, left ventricular dilatation, and dysfunction during TAC. CTRP9 overexpression, in turn, promoted hypertrophic cardiac remodeling and dysfunction after TAC in mice and induced hypertrophy in isolated adult cardiomyocytes. Mechanistically, CTRP9 knock-out mice showed strongly reduced levels of activated prohypertrophic ERK5 (extracellular signal-regulated kinase 5) during TAC compared with wild-type mice, while CTRP9 overexpression entailed increased ERK5 activation in response to pressure overload. Inhibition of ERK5 by a dominant negative MEK5 mutant or by the ERK5/MEK5 inhibitor BIX02189 blunted CTRP9 triggered hypertrophy in isolated adult cardiomyocytes in vitro and attenuated mouse cardiomyocyte hypertrophy and cardiac dysfunction in vivo, respectively. Downstream of ERK5, we identified the prohypertrophic transcription factor GATA4, which was directly activated through ERK5-dependent phosphorylation. Conclusions: The upregulation of CTRP9 during hypertrophic heart disease facilitates maladaptive cardiac remodeling and left ventricular dysfunction and might constitute a therapeutic target in the future.

Cachexia is associated with poor prognosis in chronic heart failure patients, but the underlying mechanisms of cachexia triggered disease progression remain poorly understood. Here, we investigate whether the dysregulation of myokine expression from wasting skeletal muscle exaggerates heart failure. RNA sequencing from wasting skeletal muscles of mice with heart failure reveals a reduced expression of Ostn, which encodes the secreted myokine Musclin, previously implicated in the enhancement of natriuretic peptide signaling. By generating skeletal muscle specific Ostn knock-out and overexpressing mice, we demonstrate that reduced skeletal muscle Musclin levels exaggerate, while its overexpression in muscle attenuates cardiac dysfunction and myocardial fibrosis during pressure overload. Mechanistically, Musclin enhances the abundance of C-type natriuretic peptide (CNP), thereby promoting cardiomyocyte contractility through protein kinase A and inhibiting fibroblast activation through protein kinase G signaling. Because we also find reduced OSTN expression in skeletal muscle of heart failure patients, augmentation of Musclin might serve as therapeutic strategy., Cachexia is associated with poor prognosis in heart failure. Here the authors show that mice and patients with cardiac cachexia display reduced skeletal muscle expression and circulating levels of Musclin. Musclin ablation in skeletal muscle worsens, while its muscle-specific overexpression ameliorates heart failure in mice.

Pathological cardiac overload induces myocardial protein synthesis and hypertrophy, which predisposes to heart failure. To inhibit hypertrophy therapeutically, the identification of negative regulators of cardiomyocyte protein synthesis is needed. Here, we identified the tumor suppressor protein TIP30 as novel inhibitor of cardiac hypertrophy and dysfunction. Reduced TIP30 levels in mice entailed exaggerated cardiac growth during experimental pressure overload, which was associated with cardiomyocyte cellular hypertrophy, increased myocardial protein synthesis, reduced capillary density, and left ventricular dysfunction. Pharmacological inhibition of protein synthesis improved these defects. Our results are relevant for human disease, since we found diminished cardiac TIP30 levels in samples from patients suffering from end‐stage heart failure or hypertrophic cardiomyopathy. Importantly, therapeutic overexpression of TIP30 in mouse hearts inhibited cardiac hypertrophy and improved left ventricular function during pressure overload and in cardiomyopathic mdx mice. Mechanistically, we identified a previously unknown anti‐hypertrophic mechanism, whereby TIP30 binds the eukaryotic elongation factor 1A (eEF1A) to prevent the interaction with its essential co‐factor eEF1B2 and translational elongation. Therefore, TIP30 could be a therapeutic target to counteract cardiac hypertrophy.

Capillary endothelial cells influence myocardial growth and function during pathological stress by releasing paracrine factors. We found that the transcription factor GATA2 is suppressed in cardiac endothelial cells by mechanical stimuli, and that GATA2 is downregulated in human failing hearts. To investigate the functional consequence of reduced endothelial GATA2 expression, we exposed endothelial cell specific, inducible GATA2 knock-out (G2-EC-KO) or wild-type (WT) mice to pressure overload through transverse aortic constriction (TAC). G2-EC-KO mice developed aggravated heart failure after TAC, but not enhanced fibrosis or capillary rarefaction. Investigation of stress signaling pathways revealed a prominent activation of p38 MAP kinases and Akt in cardiomyocytes after TAC only in WT mice, but not in G2-EC-KO mice, which in addition exerted increased calcineurin/NFAT activation. Transcriptional profiling revealed a strong upregulation of two distinct previously unknown long non-coding (lnc) RNAs in cardiac endothelial cells from G2-EC-KO mice, which we termed GADLOR1 and GADLOR2 and which were also induced in human failing hearts. Both RNAs were also induced in cultured cardiac endothelial cells in vitro after ablation of GATA2 and were found to be secreted within extracellular vesicles. Isolated cardiomyocytes incubated with extracellular vesicles from GATA2 depleted endothelial cells efficiently incorporated GADLOR1 and 2. Uptake of GADLORs by cardiomyocytes led to a profound reduction of p38 MAPK and Akt activation. Proteomic screening revealed that GADLOR1/2 bind the Ras-like protein TC21 in cardiomyocytes and block downstream signaling by preventing TC21 binding to its target PI3K. Application of both lncRNAs to the mouse myocardium by exosomal gene-transfer triggered aggravated cardiac dysfunction and disturbed stress signaling, while in turn inhibition of GADLOR1/2 by specific GapmeRs rescued stress signaling and cardiac dysfunction in G2-EC-KO mice during TAC. In summary, GATA2 protects the heart during pressure overload by suppressing the endothelial release of two long non-coding RNAs, which interfere with stress signaling in cardiomyocytes and ultimately induce heart failure.

Background: Here, we examined the currently unknown cardiac expression and function of Tip30, which has previously emerged as a tumor suppressor gene. Results: Myocardial TIP30 mRNA and protein were significantly upregulated in response to experimental transverse aortic constriction (TAC). TIP30 contributed to cardiac compensation, since a reduction of cardiac TIP30 in heterozygous Tip30 knock-out mice (HET) led to exaggerated hypertrophy and cardiac dysfunction compared to wild-type mice (WT) after TAC. In turn, TIP30 overexpression by an adenovirus in isolated neonatal cardiomyocytes or by an adeno-associated-virus (AAV9) in mouse hearts led to reduced hypertrophy after pro-hypertrophic stimulation in cells and reduced hypertrophy and improved cardiac function after TAC in mice. Interestingly, cardiac TIP30 levels were strongly diminished in mouse models of genetic cardiomyopathy (mdx mice) and in endstage human cardiomyopathy. Reduced cardiac TIP30 contributed to disease progression, since reconstitution of myocardial TIP30 via AAV9 in mdx mice prevented hypertrophy and improved cardiac function. A protein interaction screen and subsequent characterization showed that TIP30 interacts with the middle domain of the eukaryotic translation factor 1A1 (eEF1A1). As revealed by immunoprecipitation and in situ proximity ligation assay, the cellular interaction of eEF1A1 and its essential cofactor eEF1B was diminished by TIP30 overexpression, but enhanced in cardiomyocytes isolated from HET mice after pro-hypertrophic stimulation. Because eEF1A1 is a crucial mediator of translational elongation, we hypothesized that TIP30 regulates cardiac hypertrophy by interfering with protein synthesis. Indeed, overexpression of TIP30 inhibited cardiac protein synthesis during pro-hypertrophic stimulation, while reduced TIP30 levels in HET (vs. WT) mice triggered enhanced protein synthesis after TAC. Interestingly, administration of the eEF1A1 inhibitor narciclasine ablated the increased hypertrophy in HET mice after TAC. Conclusion: TIP30 inhibits protein synthesis by binding to eEF1A1 and inhibiting its interaction with eEF1B. It thereby reduces pathological hypertrophy and supports cardiac compensation during overload.

Background— In comparison with men, women have a better prognosis when experiencing aortic valve stenosis, hypertrophic cardiomyopathy, or heart failure. Recent data suggest that androgens like testosterone or the more potent dihydrotestosterone contribute to the development of cardiac hypertrophy and failure. Therefore, we analyzed whether antiandrogenic therapy with finasteride, which inhibits the generation of dihydrotestosterone by the enzyme 5-α-reductase, improves pathological ventricular remodeling and heart failure. Methods and Results— We found a strongly induced expression of all 3 isoforms of the 5-α-reductase (Srd5a1 to Srd5a3) in human and mouse hearts with pathological hypertrophy, which was associated with increased myocardial accumulation of dihydrotestosterone. Starting 1 week after the induction of pressure overload by transaortic constriction, mice were treated with finasteride for 2 weeks. Cardiac function, hypertrophy, dilation, and fibrosis were markedly improved in response to finasteride treatment in not only male, but also in female mice. In addition, finasteride also very effectively improved cardiac function and mortality after long-term pressure overload and prevented disease progression in cardiomyopathic mice with myocardial Gαq overexpression. Mechanistically, finasteride, by decreasing dihydrotestosterone, potently inhibited hypertrophy and Akt-dependent prohypertrophic signaling in isolated cardiac myocytes, whereas the introduction of constitutively active Akt blunted these effects of finasteride. Conclusions— Finasteride, which is currently used in patients to treat prostate disease, potently reverses pathological cardiac hypertrophy and dysfunction in mice and might be a therapeutic option for heart failure.