Knockdown of METTL4 Reduces Mitochondrial DNA 6mA Modification and Alleviates Myocardial Ischemia-Reperfusion Injury

Abstract

Background: Myocardial ischemia-reperfusion injury (MIRI) is damage caused by restoring coronary blood supply after myocardial ischemia. Recombinant mouse methyltransferase-like protein 4 (METTL4), a N6-methyladenin (6mA) methylase-related protein, is associated with cardiovascular disease. However, the role and mechanism of METTL4 in MIRI have yet to be clarified. This study aims to explore whether METTL4 regulates MIRI by mitochondrial DNA 6mA modification. Methods: We first constructed a MIRI rats model and an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model from mouse cardiomyocytes (HL-1). The 6mA level of mitochondrial DNA, METTL4 expression, and mitochondrial DNA (mtDNA) copy number were confirmed using Dot blot, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) in the rat and cell models, respectively. Functionally, cell proliferation and apoptosis were assessed by cell counting kit-8 (CCK-8) and flow cytometry, respectively. In addition, the levels of adenosine triphosphate (ATP) and mitochondria-related genes were monitored by enzyme-linked immunosorbent assay (ELISA) and qRT-PCR. Results: Our results showed that mitochondrial DNA 6mA and METTL4 levels were higher in MIRI model rats and OGD/R HL-1 model cells (p < 0.001). Then, we showed that knockdown of METTL4 induces proliferation and attenuates apoptosis of OGD/R HL-1 cells (p < 0.01, p < 0.001). We also found that METTL4 silencing reduces mitochondrial DNA 6mA modification and increases mitochondrial DNA (mtDNA) copy in OGD/R HL-1 cells (p < 0.001). The results indicated that silencing METTL4 reduces ATP and increases the levels of mitochondria-related genes in OGD/R HL-1 cells (p < 0.01, p < 0.001). Conclusions: METTL4 silencing reduces mitochondrial DNA 6mA modification, increases mitochondrial DNA copy number, improves mitochondrial dysfunction, and inhibits apoptosis. Therefore, inhibition of METTL4 might be a therapeutic strategy for MIRI.