Abstract
Objective: Cardiac fibrosis is one of the most warranted cardiac impairments which ultimately leads to heart failure. N6-methyladenosine (m6A) is an abundant and conserved chemical modification in eukaryotic mRNA and is associated with mRNA metabolism. Recently, we showed that m6A mRNA methylation regulates the angiogenic potential of the endothelial cells following ischemic injury. However, the role of m6A mRNA methylation in cardiac fibrosis following ischemic injury is still elusive and needs improvement in treatment strategy. Methods: First we analyzed the available data set for the fibrosis-regulating targets in Sham and MI hearts. Further, we studied methylation patterns in vivo by inhibiting methylation in MI mice using STM2457. Then we assessed cardiac function using echocardiography and harvested tissue samples for further biochemical analysis. We also performed in vitro experiment to investigate the expression pattern of the screened gene (Fibronectin) in hypoxic cardiac fibroblast as an ischemic model. We also performed an Actinomycin D assay to check the role of methylation on FN mRNA stability. Fibrosis and other phenotypic statuses were studied using several molecular techniques including ICC/IHC. Finally, siMETTL3 was used to study rescue experiments. Results: We have shown upregulated FN expression post-MI, and also several methylation sites on FN mRNA in-silico. Histological and biochemical analysis showed increased fibrosis and m6A mRNA methylation in mice post-MI. Hypoxic stress in fibroblast induces mRNA methylation, which leads to FN mRNA stabilization and deposition of FN at the scar site by activated fibroblast. In contrast, METTL3 inhibition using STM 2457 significantly improved cardiac function. METTL3 inhibition significantly reduced FN mRNA methylation and FN expression, and ultimately reduced cardiac fibrosis. Conclusion: We observed that increased FN mRNA (m6A) methylation enhanced cardiac fibrosis following MI. Inhibition of mRNA methylation using STM2457 in in-vivo, and siRNA in-vitro reduced hypoxia-induced FN deposition and fibrosis, which suggests STM2457 could be a potential therapeutic molecule either alone or in combination with current regimens to curb methylation-induced cardiac fibrosis.
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