DNA methylation-regulated APOLD1 protects cardiomyocytes from hypoxia-induced ferroptosis by activating Nrf2 pathway in vitro.

Cardiomyocyte ferroptosis inhibition can alleviate myocardial injury during myocardial infarction (MI). Apolipoprotein L domain containing 1 (APOLD1) is abnormally expressed in the MI model. This study planned to elucidate the mechanism by which APOLD1 modulated ferroptosis in hypoxia-induced cardiomyocyte injury. The levels of myocardial injury markers, such as creatine kinase isoenzyme myocardial band, cardiac troponin T, and lactate dehydrogenase, were measured using ELISA kits. Cell death rate was determined utilizing flow cytometry with the Calcein-AM/PI kit. Intracellular Fe2+ level was examined using ELISA kits. Methylation-Specific PCR analysis was utilized to evaluate the CpG methylation status within the APOLD1 promoter. Chromatin immunoprecipitation assay was employed to confirm the interactions between APOLD1 and DNA methyltransferases (DNMTs). In this study, we found that the overexpressed APOLD1 alleviated cardiomyocyte damage and ferroptosis. Furthermore, APOLD1 activated the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase (HO)-1 pathway, which was involved in the APOLD1-mediated protection, by enhancing its nuclear translocation. Hypoxia inhibited APOLD1 expression via the promotion of DNMTs-mediated CpG methylation. The methylation inhibitor, 5-Azacytidine, ameliorated hypoxia-induced cardiomyocyte ferroptosis by restoring APOLD1 expression. Therefore, our data clarified that DNA methylation-modulated APOLD1 could alleviate ferroptosis by activating the Nrf2/HO-1 signaling in hypoxia-induced cardiomyocytes.