SUMMARY Heart failure (HF) is driven by the interplay between master regulatory transcription factors and dynamic alterations in chromatin structure. While pathologic gene transactivation in this context is known to be associated with recruitment of histone acetyl-transferases and local chromatin hyperacetylation, the role of epigenetic reader proteins in cardiac biology is unknown. We therefore undertook a first study of acetyl-lysine reader proteins, or bromodomains, in HF. Using a chemical genetic approach, we establish a central role for BET-family bromodomain proteins in gene control during HF pathogenesis. BET inhibition potently suppresses cardiomyocyte hypertrophy in vitro and pathologic cardiac remodeling in vivo. Integrative transcriptional and epigenomic analyses reveal that BET proteins function mechanistically as pause-release factors critical to activation of canonical master regulators and effectors that are central to HF pathogenesis and relevant to the pathobiology of failing human hearts. This study implicates epigenetic readers in cardiac biology and identifies BET co-activator proteins as therapeutic targets in HF.

Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease 1, 2, but the molecular basis is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (e.g. short or long QT syndromes, heart failure) 3-5 or pattern (e.g. Brugada syndrome) 6 of myocardial repolarization. Here we provide the first molecular evidence that links circadian rhythms to vulnerability in ventricular arrhythmias in mice. Specifically, we show that cardiac ion channel expression and QT interval duration (an index of myocardial repolarization) exhibit endogenous circadian rhythmicity under the control of a novel clock-dependent oscillator, Krüppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of KChIP2, a critical subunit required for generating the transient outward potassium current (Ito). 7 Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. In sum, these findings identify circadian transcription of ion channels as a novel mechanism for cardiac arrhythmogenesis.

Summary A ketogenic diet (KD) recapitulates certain metabolic aspects of dietary restriction such as reliance on fatty acid metabolism and production of ketone bodies. We investigated whether KD might, like dietary restriction, affect longevity and healthspan in C57BL/6 male mice. We find that an isoprotein KD, fed on alternate weeks to prevent obesity (Cyclic KD), reduces mid-life mortality but does not affect maximum lifespan. A non-ketogenic high-fat diet (HF) fed similarly may have an intermediate effect on mortality. Cyclic KD improves memory performance in old age, while modestly improving composite healthspan measures. Gene expression analysis identifies down-regulation of insulin, TOR, and fatty acid synthesis pathways as mechanisms common to KD and HF. However, up-regulation of PPARα target genes is unique to KD, consistent across tissues, and preserved in old age. In all, we show that a non-obesogenic ketogenic diet improves survival, memory, and healthspan in aging mice.

In the postabsorptive state, certain tissues, including the brain, require glucose as the sole source of energy. After an overnight fast, hepatic glycogen stores are depleted, and gluconeogenesis becomes essential for preventing life-threatening hypoglycemia. Mice with a targeted deletion of KLF15, a member of the Krüppel-like family of transcription factors, display severe hypoglycemia after an overnight (18 hr) fast. We provide evidence that defective amino acid catabolism promotes the development of fasting hypoglycemia in KLF15-/- mice by limiting gluconeogenic substrate availability. KLF15-/- liver and skeletal muscle show markedly reduced mRNA expression of amino acid-degrading enzymes. Furthermore, the enzymatic activity of alanine aminotransferase (ALT), which converts the critical gluconeogenic amino acid alanine into pyruvate, is decreased (approximately 50%) in KLF15-/- hepatocytes. Consistent with this observation, intraperitoneal injection of pyruvate, but not alanine, rescues fasting hypoglycemia in KLF15-/- mice. We conclude that KLF15 plays an important role in the regulation of gluconeogenesis.

Cardiac hypertrophy is a common response to injury and hemodynamic stress and an important harbinger of heart failure and death. Herein, we identify the Kruppel-like factor 15 (KLF15) as an inhibitor of cardiac hypertrophy. Myocardial expression of KLF15 is reduced in rodent models of hypertrophy and in biopsy samples from patients with pressure-overload induced by chronic valvular aortic stenosis. Overexpression of KLF15 in neonatal rat ventricular cardiomyocytes inhibits cell size, protein synthesis and hypertrophic gene expression. KLF15-null mice are viable but, in response to pressure overload, develop an eccentric form of cardiac hypertrophy characterized by increased heart weight, exaggerated expression of hypertrophic genes, left ventricular cavity dilatation with increased myocyte size, and reduced left ventricular systolic function. Mechanistically, a combination of promoter analyses and gel-shift studies suggest that KLF15 can inhibit GATA4 and myocyte enhancer factor 2 function. These studies identify KLF15 as part of a heretofore unrecognized pathway regulating the cardiac response to hemodynamic stress.

Despite current standard of care, the average 5-year mortality after an initial diagnosis of heart failure (HF) is about 40%, reflecting an urgent need for new therapeutic approaches. Previous studies demonstrated that the epigenetic reader protein bromodomain-containing protein 4 (BRD4), an emerging therapeutic target in cancer, functions as a critical coactivator of pathologic gene transactivation during cardiomyocyte hypertrophy. However, the therapeutic relevance of these findings to human disease remained unknown. We demonstrate that treatment with the BET bromodomain inhibitor JQ1 has therapeutic effects during severe, preestablished HF from prolonged pressure overload, as well as after a massive anterior myocardial infarction in mice. Furthermore, JQ1 potently blocks agonist-induced hypertrophy in human induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs). Integrated transcriptomic analyses across animal models and human iPSC-CMs reveal that BET inhibition preferentially blocks transactivation of a common pathologic gene regulatory program that is robustly enriched for NFκB and TGF-β signaling networks, typified by innate inflammatory and profibrotic myocardial genes. As predicted by these specific transcriptional mechanisms, we found that JQ1 does not suppress physiological cardiac hypertrophy in a mouse swimming model. These findings establish that pharmacologically targeting innate inflammatory and profibrotic myocardial signaling networks at the level of chromatin is effective in animal models and human cardiomyocytes, providing the critical rationale for further development of BET inhibitors and other epigenomic medicines for HF.

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Background: Heart failure with preserved ejection fraction (HFpEF) constitutes half of all HF yet lacks effective therapy. Understanding its myocardial biology remains limited due to a paucity of heart tissue molecular analysis. Methods: We performed RNA sequencing on right ventricular septal endomyocardial biopsies prospectively obtained from patients with consensus criteria for HFpEF (n=41) and contrasted to RV-septal tissue from HF with reduced EF (HFrEF, n=30) and donor controls (CON, n=24). Principal component analysis (PCA) and hierarchical clustering tested for transcriptomic distinctiveness between groups and impact of co-morbidities, and differential gene expression with pathway enrichment contrasted HF groups to CON. Within HFpEF, non-negative matrix factorization (NMF) and weighted gene co-expression analysis (WGCNA) identified molecular subgroups and the resulting clusters were correlated with hemodynamic and clinical data. Results: HFpEF patients were more often women (59%), African American (68%), obese (median BMI 41), and hypertensive (98%), with clinical HF characterized by 65% NYHA III-IV, nearly all on a loop diuretic, and 70% with a HF hospitalization in the prior year. PCA separated HFpEF from HFrEF and CON with minimal overlap and this persisted after adjusting for primary co-morbidities: BMI, sex, age, diabetes, and renal function. Hierarchical clustering confirmed group separation. Nearly half the significantly altered genes in HFpEF versus CON (1882 up, 2593 down) changed in the same direction in HFrEF; however, 5745 genes were uniquely altered between HF groups. Compared to CON, uniquely upregulated genes in HFpEF were enriched in mitochondrial ATP synthesis/electron transport, pathways downregulated in HFrEF. HFpEF-specific down-regulated genes engaged endoplasmic reticulum stress, autophagy, and angiogenesis. BMI differences largely accounted for HFpEF upregulated genes whereas neither this nor broader co-morbidity adjustment altered pathways enriched in downregulated genes. NMF identified three HFpEF transcriptomic subgroups with distinctive pathways and clinical correlates, including a group closest to HFrEF with higher mortality, and a mostly female group with smaller hearts and pro-inflammatory signaling. These groupings remained after sex adjustment. WGCNA analysis yielded analogous gene-clusters and clinical groupings. Conclusions: HFpEF exhibits distinctive broad transcriptomic signatures and molecular subgroupings with particular clinical features and outcomes. The data reveal new signaling targets to consider for precision therapeutics.