Cooperative binding of ETS2 and NFAT link Erk1/2 and calcineurin signaling in the pathogenesis of cardiac hypertrophy

BACKGROUND: Cardiac hypertrophy is an independent risk factor for heart failure, a leading cause of morbidity and mortality globally. The calcineurin/NFAT (nuclear factor of activated T cells) pathway and the MAPK/Erk (extracellular signal-regulated kinase) pathway contribute to the pathogenesis of cardiac hypertrophy as an inter-dependent network of signaling cascades. However, how these pathways interact remains unclear, and specifically few direct targets responsible for the pro-hypertrophic role of NFAT have been described. METHODS: By engineering a cardiomyocyte-specific ETS2 (a member of E26 transformationspecific sequence (ETS)-domain family) knockout mice, we investigated the role of ETS2 in cardiac hypertrophy. Primary cardiomyocytes were also used to evaluate ETS2 function in cell growth. RESULTS: ETS2 is phosphorylated and activated by Erk1/2 upon hypertrophic stimulation in both mouse (n = 3) and human heart samples (n = 8-19). Conditional deletion of ETS2 in mouse cardiomyocytes protects against pressure overload-induced cardiac hypertrophy (n = 6-11). Furthermore, silencing of ETS2 in the hearts of calcineurin transgenic mice significantly attenuates hypertrophic growth and contractile dysfunction (n = 8). As a transcription factor, ETS2 is capable of binding to the promoters of hypertrophic marker genes, such as ANP, BNP and Rcan1.4 (n = 4). Additionally, we report that ETS2 forms a complex with NFAT to stimulate transcriptional activity through increased NFAT binding to the promoters of at least two hypertrophy-stimulated genes, Rcan1.4 and miR-223 (n = 4-6). Suppression of miR-223 in cardiomyocytes inhibits calcineurin-mediated cardiac hypertrophy (n = 6), revealing miR-223 as a novel pro-hypertrophic target of the calcineurin-NFAT and Erk1/2-ETS2 pathways. CONCLUSIONS: In aggregate, our findings point to a critical role for ETS2 in calcineurin-NFAT pathway-driven cardiac hypertrophy and unveil a previously unknown molecular connection between the Erk1/2 activation of ETS2 and expression of NFAT/ETS2 target genes

Xbp1s-FoxO1 axis governs lipid accumulation and contractile performance in heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure and one for which no efficacious therapies exist. Obesity and lipid mishandling greatly contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are largely unknown. Here, we report that cardiomyocyte steatosis in HFpEF is coupled with increases in the activity of the transcription factor FoxO1 (Forkhead box protein O1). FoxO1 depletion, as well as over-expression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) in cardiomyocytes each ameliorates the HFpEF phenotype in mice and reduces myocardial lipid accumulation. Mechanistically, forced expression of Xbp1s in cardiomyocytes triggers ubiquitination and proteasomal degradation of FoxO1 which occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-box-containing protein 1) a novel and direct transcriptional target of Xbp1s. Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of cardiometabolic HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes

Remodeling of substrate consumption in the murine sTAC model of heart failure

BACKGROUND: Energy metabolism and substrate selection are key aspects of correct myocardial mechanical function. Myocardial preference for oxidizable substrates changes in both hypertrophy and in overt failure. Previous work has shown that glucose oxidation is upregulated in overpressure hypertrophy, but its fate in overt failure is less clear. Anaplerotic flux of pyruvate into the tricarboxylic acid cycle (TCA) has been posited as a secondary fate of glycolysis, aside from pyruvate oxidation or lactate production. METHODS AND RESULTS: A model of heart failure that emulates both valvular and hypertensive heart disease, the severe transaortic constriction (sTAC) mouse, was assayed for changes in substrate preference using metabolomic and carbon-13 flux measurements. Quantitative measures of O(2) consumption in the Langendorff perfused mouse heart were paired with (13)C isotopomer analysis to assess TCA cycle turnover. Since the heart accommodates oxidation of all physiological energy sources, the utilization of carbohydrates, fatty acids, and ketones were measured simultaneously using a triple-tracer NMR method. The fractional contribution of glucose to acetyl-CoA production was upregulated in heart failure, while other sources were not significantly different. A model that includes both pyruvate carboxylation and anaplerosis through succinyl-CoA produced superior fits to the data compared to a model using only pyruvate carboxylation. In the sTAC heart, anaplerosis through succinyl-CoA is elevated, while pyruvate carboxylation was not. Metabolomic data showed depleted TCA cycle intermediate pool sizes versus the control, in agreement with previous results. CONCLUSION: In the sTAC heart failure model, the glucose contribution to acetyl-CoA production was significantly higher, with compensatory changes in fatty acid and ketone oxidation not reaching a significant level. Anaplerosis through succinyl-CoA is also upregulated, and is likely used to preserve TCA cycle intermediate pool sizes. The triple tracer method used here is new, and can be used to assess sources of acetyl-CoA production in any oxidative tissue

Cytosolic DNA sensing promotes macrophage transformation and governs myocardial ischemic injury

BACKGROUND: Myocardium irreversibly injured by ischemic stress must be efficiently repaired to maintain tissue integrity and contractile performance. Macrophages play critical roles in this process. These cells transform across a spectrum of phenotypes to accomplish diverse functions ranging from mediating the initial inflammatory responses that clear damaged tissue to subsequent reparative functions that help rebuild replacement tissue. Although macrophage transformation is crucial to myocardial repair, events governing this transformation are poorly understood. METHODS: Here, we set out to determine whether innate immune responses triggered by cytoplasmic DNA play a role. RESULTS: We report that ischemic myocardial injury, along with the resulting release of nucleic acids, activates the recently described cyclic GMP-AMP synthase-stimulator of interferon genes pathway. Animals lacking cyclic GMP-AMP synthase display significantly improved early survival after myocardial infarction and diminished pathological remodeling, including ventricular rupture, enhanced angiogenesis, and preserved ventricular contractile function. Furthermore, cyclic GMP-AMP synthase loss of function abolishes the induction of key inflammatory programs such as inducible nitric oxide synthase and promotes the transformation of macrophages to a reparative phenotype, which results in enhanced repair and improved hemodynamic performance. CONCLUSIONS: These results reveal, for the first time, that the cytosolic DNA receptor cyclic GMP-AMP synthase functions during cardiac ischemia as a pattern recognition receptor in the sterile immune response. Furthermore, we report that this pathway governs macrophage transformation, thereby regulating postinjury cardiac repair. Because modulators of this pathway are currently in clinical use, our findings raise the prospect of new treatment options to combat ischemic heart disease and its progression to heart failure

Polycystin-1 assembles with Kv channels to govern cardiomyocyte repolarization and contractility

BACKGROUND: Polycystin-1 (PC1) is a transmembrane protein originally identified in autosomal dominant polycystic kidney disease where it regulates the calcium-permeant cation channel polycystin-2. Autosomal dominant polycystic kidney disease patients develop renal failure, hypertension, left ventricular hypertrophy, and diastolic dysfunction, among other cardiovascular disorders. These individuals harbor PC1 loss-of-function mutations in their cardiomyocytes, but the functional consequences are unknown. PC1 is ubiquitously expressed, and its experimental ablation in cardiomyocyte-specific knockout mice reduces contractile function. Here, we set out to determine the pathophysiological role of PC1 in cardiomyocytes. METHODS: Wild-type and cardiomyocyte-specific PC1 knockout mice were analyzed by echocardiography. Excitation-contraction coupling was assessed in isolated cardiomyocytes and human embryonic stem cell-derived cardiomyocytes, and functional consequences were explored in heterologous expression systems. Protein-protein interactions were analyzed biochemically and by means of ab initio calculations. RESULTS: PC1 ablation reduced action potential duration in cardiomyocytes, decreased Ca(2+) transients, and myocyte contractility. PC1-deficient cardiomyocytes manifested a reduction in sarcoendoplasmic reticulum Ca(2+) stores attributable to a reduced action potential duration and sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA) activity. An increase in outward K(+) currents decreased action potential duration in cardiomyocytes lacking PC1. Overexpression of full-length PC1 in HEK293 cells significantly reduced the current density of heterologously expressed Kv4.3, Kv1.5 and Kv2.1 potassium channels. PC1 C terminus inhibited Kv4.3 currents to the same degree as full-length PC1. Additionally, PC1 coimmunoprecipitated with Kv4.3, and a modeled PC1 C-terminal structure suggested the existence of 2 docking sites for PC1 within the N terminus of Kv4.3, supporting a physical interaction. Finally, a naturally occurring human mutant PC1(R4228X) manifested no suppressive effects on Kv4.3 channel activity. CONCLUSIONS: Our findings uncover a role for PC1 in regulating multiple Kv channels, governing membrane repolarization and alterations in SERCA activity that reduce cardiomyocyte contractility

NAD(+) repletion reverses heart failure with preserved ejection fraction

RATIONALE: Heart failure with preserved ejection fraction (HFpEF) is a mortal clinical syndrome without effective therapies. We recently demonstrated in mice that a combination of metabolic and hypertensive stress recapitulates key features of human HFpEF. OBJECTIVE: Using this novel preclinical HFpEF model, we set out to define and manipulate metabolic dysregulations occurring in HFpEF myocardium. METHODS AND RESULTS: We observed impairment in mitochondrial fatty acid oxidation associated with hyperacetylation of key enzymes in the pathway. Down-regulation of sirtuin 3 and deficiency of NAD(+) secondary to an impaired NAD(+) salvage pathway contribute to this mitochondrial protein hyperacetylation. Impaired expression of genes involved in NAD(+) biosynthesis was confirmed in cardiac tissue from HFpEF patients. Supplementing HFpEF mice with nicotinamide riboside or a direct activator of NAD(+) biosynthesis led to improvement in mitochondrial function and amelioration of the HFpEF phenotype. CONCLUSIONS: Collectively, these studies demonstrate that HFpEF is associated with myocardial mitochondrial dysfunction and unveil NAD(+) repletion as a promising therapeutic approach in the syndrome

Activation of autophagic flux blunts cardiac ischemia/reperfusion injury

RATIONALE: Reperfusion injury accounts for up to half of myocardial infarct size, and meaningful clinical therapies targeting it do not exist. We have reported previously that autophagy is reduced during reperfusion and that HDAC (histone deacetylase) inhibition enhances cardiomyocyte autophagy and blunts ischemia/reperfusion (I/R) injury when administered at the time of reperfusion. However, whether inducing autophagy per se, as opposed to other effects triggered by HDAC inhibition, is sufficient to protect against reperfusion injury is not clear. OBJECTIVE: We set out to test whether augmentation of autophagy using a specific autophagy-inducing peptide, TB (Tat-Beclin), protects the myocardium through reduction of reactive oxygen species (ROS) during reperfusion injury. METHODS AND RESULTS: Eight- to 12-week-old, WT (wild type) C57BL6 mice and tamoxifen-inducible cardiomyocyte-specific ATG7 KO (ATG7 knockout) mice (to test the dependency on autophagy) were randomized into 2 groups: exposed to a control TS (Tat-scrambled) peptide or a TB peptide. Each group was subjected to I/R surgery (45-minute coronary ligation, 24-hour reperfusion). Infarct size, systolic function, autophagic flux, and ROS were assayed. Cultured neonatal rat ventricular myocytes were exposed to TB during simulated I/R injury. ATG7 knockdown by small interfering RNA in neonatal rat ventricular myocytes was used to evaluate the role of autophagy. TB treatment at reperfusion reduced infarct size by 20% (absolute reduction; 50% relative reduction) and improved contractile function. Improvement correlated with increased autophagic flux in the border zone with less oxidative stress. ATG7 KO mice did not manifest TB-promoted cardioprotection during I/R. In neonatal rat ventricular myocytes subjected to I/R, TB reduced cell death by 41% and reduced I/R-induced ROS generation. Conversely, ATG7 knockdown in neonatal rat ventricular myocytes abolished these beneficial effects of TB on cell death and ROS reduction. CONCLUSIONS: Induction of autophagy at the time of reperfusion is sufficient to mitigate myocardial reperfusion injury by reducing ROS and cell death. Maintenance of appropriate autophagic flux may emerge as a viable clinical therapy to reduce reperfusion injury in acute myocardial infarction

Epigenetic reader BRD4 (Bromodomain-Containing Protein 4) governs nucleus-encoded mitochondrial transcriptome to regulate cardiac function

BACKGROUND: BET (Bromodomain and Extra-Terminal) epigenetic reader proteins, in particular BRD4, have emerged as potential therapeutic targets in a number of pathological conditions, including cancer and cardiovascular disease. Small molecule BET protein inhibitors, such as JQ1, have demonstrated efficacy in reversing cardiac hypertrophy and heart failure in preclinical models. Yet, genetic studies elucidating the biology of BET proteins in the heart have not been conducted to validate pharmacological findings and unveil potential pharmacological side effects. METHODS: By engineering a cardiomyocyte-specific BRD4 (bromodomain-containing protein 4) knockout mouse, we investigated the role of BRD4 in cardiac pathophysiology. We performed functional, transcriptomic, and mitochondrial analysis to evaluate BRD4 function in developing and mature hearts. RESULTS: Unlike pharmacological inhibition, loss of BRD4 protein triggered progressive declines in myocardial function, culminating in dilated cardiomyopathy. Transcriptome analysis of BRD4 knockout mouse heart tissue identified early and specific disruption of genes essential to mitochondrial energy production and homeostasis. Functional analysis of isolated mitochondria from these hearts confirmed that BRD4 ablation triggered significant changes in mitochondrial electron transport chain protein expression and activity. Computational analysis identified candidate transcription factors participating in the BRD4-regulated transcriptome. In particular, ESRRα (estrogen-related receptor alpha), a key nuclear receptor in metabolic gene regulation, was enriched in promoters of BRD4-regulated mitochondrial genes. CONCLUSIONS: In aggregate, we describe a previously unrecognized role for BRD4 in regulating cardiomyocyte mitochondrial homeostasis, observing that its function is indispensable to the maintenance of normal cardiac function