Abstract 326: FTO-mediated mRNA Demethylation Regulates Cardiac Contractile Protein Expression and Function

Abstract

Background: Exciting new discoveries in RNA biology underscore the importance of post-transcriptional chemical modifications to mRNAs (epitranscriptome) in regulating RNA stability, nuclear export, cellular compartmentalization, splicing, translation and degradation. The most abundant and functionally relevant modification in RNA, N6-methyladenosine (m6A) is reversibly demethylated by one of the m6A demethylases, fat mass and obesity-associated protein (FTO) whose function in the mammalian heart remains incompletely understood. Materials and Methods: We used clinical human samples, preclinical pig and mouse models and primary cardiomyocytes to study m6A and FTO in the heart and in cardiomyocytes. We modulated FTO expression using AAV9 (in vivo), adenovirus (in vivo and in vitro) and siRNAs (in vitro). We investigated m6A-induced changes to contractile protein expression using m6A RNA immunoprecipitation sequencing (MeRIP-seq) and stable isotope labeling of amino acids in cell culture (SILAC). Results: We discovered in human heart failure that reduced FTO expression is associated with aberrant increase in m6A mRNA methylation, which is conserved in swine and mouse models of myocardial ischemia (MI). AAV9-mediated FTO gene delivery in mouse MI attenuated m6A increase and improved cardiac function with enhanced contractility, angiogenesis and reduced fibrosis. At the molecular level, FTO-mediated mRNA demethylation serves to increase contractile protein expression in mouse hearts as well as in isolated primary cardiomyocytes. By comparing human and mouse transcriptome-wide m6A maps with SILAC proteomic profiling from cardiomyocytes, we identified FTO-mediated m6A demethylation is transcript-specific and leads to altered protein expression of several key contractile, angiogenic and regenerative proteins. Conclusion: Using new RNA-based investigations, we uncovered a novel regulatory layer beyond the genome working at the level of epitranscriptome governing cardiac function. Our findings on the dynamic nature of the cardiac m6A-epitranscriptome will lead to deeper understanding of the mechanism of cardiac remodeling on one hand and innovative therapeutic interventions on the other.