Abstract 301: An m6A Demethylase, FTO Mediates Post-transcriptional mRNA Modifications to Regulate Cardiac and Cardiomyocyte Function

Abstract

Background: Post-transcriptional modifications in the form of m6A (N6-methyladenosine) regulate mRNA fate and translation, miRNA biogenesis, lncRNA function and several cellular processes. However, m6A mechanisms in the mature post-mitotic tissues such as the mammalian heart remain unexplored. Here, we investigated the role of transcriptome-wide m6A in cardiac protein expression as well as lncRNA function. Methods: Using biochemical assays, we investigated the spatiotemporal gene expression patterns of m6A regulators in non-failing, failing (ischemic) human hearts, in mouse and pig MI models. In mouse heart, we mapped transcriptome-wide m6A by developing state-of-the-art MeRIP-seq coupled with novel bioinformatics analysis. To investigate the functional relevance of m6A to cardiac proteome, we used SILAC-LC-MS. By silencing (siRNA) m6A-reader proteins, we investigated the role of m6A in cardiomyocyte mRNA stability, decay and nuclear export. For in vivo myocardial gene delivery, we used AAV9 and adenovirus vectors. Finally, we performed immunohistology in cardiomyocytes and mouse heart tissues to study nuclei size, fibrosis and angiogenesis. Results: We discovered that the expression of m6A demethylase, FTO is decreased in ischemic myocardium and cardiomyocytes, thus in vivo FTO gene delivery resulted in attenuation of ischemia-induced increase in m6A and decrease in cardiac contractile function post-MI. FTO overexpression in mouse heart and human cardiomyocytes revealed selective demethylation (MeRIP-seq) of cardiac contractile transcripts resulting in induced contractile protein expression (SILAC-LC-MS) thus rescuing heart function post-MI. Mechanistically, we demonstrate that the cardioprotective mechanism of FTO is mediated by selective demethylation of cardiac contractile transcripts under ischemia, which prevents mRNA degradation as well as enhanced nuclear compaction. Finally, we demonstrate that FTO overexpression in mouse models of MI resulted in decreased fibrosis and enhanced angiogenesis. Conclusion: Our study provides the first description of a cardiac active m6A demethylase working at post-transcriptional level as a critical regulator of cardiac contractile function, fibrosis and angiogenesis.