Abstract 13709: Cardioprotective Mechanisms of FTO-Regulated m 6 A in Heart Failure

Abstract

Introduction: Post-transcriptional modifications of mRNA play an important role in cardiac remodeling. We recently reported an aberrant increase in global N6-methyladenosine (m 6 A) in failing hearts associated with reduction in expression of m 6 A eraser, fat and obesity-associated protein (FTO). Overexpression of FTO increased m 6 A, and improved cardiomyocyte and cardiac function in both in vitro and in vivo models for myocardial infarction (MI). Here, we investigated the mRNA targets of FTO and mechanism of FTO-dependent cardiac protein expression in failing hearts. Hypothesis: We hypothesized that FTO mediates cardioprotection via demethylation of selective mRNA transcripts in the nucleus and cytoplasm of cardiomyocytes. Methods and Results: To identify subcellular localization of FTO-mRNA interaction, we examined FTO expression in human left ventricular cardiomyocytes under hypoxia in vitro and in mouse and human failing hearts in vivo . FTO was detected in both nucleus and cytoplasm of healthy cardiomyocytes and tissues, but not in hypoxic cardiomyocytes and MI mouse and human heart tissues. To reveal functional targets of FTO, we performed m 6 A RNA sequencing of nuclear and cytoplasmic RNA in cultured human cardiomyocytes. We identified distinct sets of RNA transcripts in nucleus and cytoplasm of cardiomyocytes involved in cardiac contraction, angiogenesis, cardiomyogenesis, hypermethylated by FTO. Methylated RNA immunoprecipitation experiments revealed that SERCA2a (involved in cardiomyocytes contraction) and FSTL1 (pro-angiogenic) mRNAs directly bind to FTO. Loss of FTO resulted in decreased nuclear export of SERCA2a and FSTL1 mRNAs. Our stable isotope labeling (SILAC) experiment confirmed that FTO regulates de-novo synthesis of SERCA2A and FSTL1 proteins. Conclusion: We conclude that FTO selectively targets the cardioprotective transcripts, regulates their nuclear export and protein expression. Our study will reveal FTO binding sites on cardiac transcripts and a novel mechanism of FTO-dependent protein expression in cardiomyocytes.