Background and Aim

It is widely known that epigenetic deregulation is associated with CVD pathophysiology. The epigenetic modification mainly N6-methyladenosine (m6A) is the most conserved internal modification in RNA, which functions as an additional layer of transcriptional and translational control. However, extensive research is required to understand the regulatory role of m6A methylation in the heart. Recent publication in our lab highlighted the deregulation of m6A methylation in cardiac hypertrophy and heart failure, which also imparts changes in protein translation independent of transcription. Further, the role of regulatory m6A in normal cardiac function were studied by cardiomyocyte specific knockout of m6A demethylase FTO (FTO-cKO) in rodents and our data showed that FTO depletion exacerbated cardiac phenotype by quickening heart failure progression with reduced hypertrophy. I also analysed the effect of regulatory m6A methylation in the normal and pressure overloaded FTO-cKO rodents using the widely available computational algorithms. Consequently, I investigated the functional mechanism of FTO demethylase in the regulation of caridac hypertrophy and homeostasis using iPSC-Cardiomyocytes (CMs).

Materials and Methodology

Firstly, the control and FTO-cKO group mice were subjected to sham and TAC (transverse aortic constriction) operation which is the model of cardiac hypertrophy and heart failure. Secondly, hearts were isolated from both the control and FTO-cKO mice which underwent sham and TAC; RNA extraction and m6A RNA immunoprecipitation (meRIP) were performed from the isolated mice heart tissue; and highthroughput m6A sequencing and analysis were performed in order the study the effect of regulatory m6A methylation. Additionally for the functional investigation, firstly, I analysed the effect of apoptosis and mTORC1 upon FTO ablation in both rodents and iPSC-CMs. Moreover, the interelation between FTO demethylase, mTORC1 and autophagy in the regulation of cardiac apoptosis were identified by 1. inhibiting mTORC1 pathway with specific inhibitors upon FTO ablation and  also by 2. inhibiting 'late autophagy' in FTO silenced iPSC-CMs.

Results

In consistent with our previous data, deregulation of m6A methylation observed in failing heart tissue compared to the healthy tissue. Moreover, GO term analysis showed that the hypomethylated transcripts in failing heart are mainly involved in the pathways of apoptosis and collagen formation, whereas the hypermethylated transcripts are part of RNA metabolism and cell cycle progression. Mechanistically, I showed that FTO ablation significantly increases both cardiac apoptosis and  mTORC1 signaling pathway in both rodents and iPSC-CMs. Furthermore, I also showed that inhibition of mTORC1 pathway attenuates cardiac apoptosis in FTO silenced iPSC-CMs. Moreover, I tested the autophagic flux upon FTO silecing by inhibiting late-autophagy and consequently, FTO ablation cause impaired autophagic flux and exacerbates cardiac apoptosis.

Clinical relevance