ENPP2 Protects Mouse Myocardium from Ischemia-Reperfusion-Induced Ferroptosis Injury Via the SIRT1/PGC-1α/NRF1...

Ferroptosis is a critical contributor to ischemia-reperfusion (I/R) injury and subsequent organ failure. While ENPP2 has been implicated in regulating ferroptosis in cardiomyocytes, its specific role in myocardial I/R injury remains unclear. This study aims to elucidate the function of ENPP2-mediated ferroptosis in myocardial ischemia-reperfusion injury (MI/RI), to provide novel insights into potential therapeutic strategies. A mouse model of MI/RI was established and subjected to interventions with ENPP2 overexpression and/or SIRT1 knockdown. In vitro, cardiomyocytes were treated with palmitate and subjected to hypoxia/reoxygenation (H/R) to simulate I/R injury. These cells received treatments with ENPP2 overexpression (oe-ENPP2), SIRT1 overexpression (oe-SIRT1), PGC-1α silencing (si-PGC-1α), and/or SIRT1 knockdown (sh-SIRT1). Additionally, Erastin-induced ferroptosis in cardiomyocytes was used to assess the protective effects of oe-ENPP2. Ferroptosis was assessed through the lipid peroxidation (MDA, 4-HNE), iron and Fe²⁺ assays, GPx4 and SLC7A11 expression, and transmission electron microscope. Overexpression of ENPP2 significantly alleviated myocardial infarction in MI/RI mice, as indicated by the upregulation of GPx4 and SLC7A11 protein levels. In cardiomyocytes subjected to hypoxia/reoxygenation (H/R) or erastin-induced ferroptosis, oe-ENPP2 reduced apoptosis rates, preserved Fe²⁺ content, and restored GPx4 and SLC7A11 expression. Silencing PGC-1α blocked the protective effect of oe-ENPP2 against H/R-induced ferroptosis in HL-1 cells. Additionally, SIRT1 overexpression inhibited PGC-1α acetylation, whereas SIRT1 knockdown similarly reversed the anti-ferroptotic effects of oe-ENPP2 in H/R-treated HL-1 cells. SIRT1 silencing blocked the protective effects of oe-ENPP2 against myocardial infarction and fibrosis in MI/RI mice via the PGC-1α/NRF1 pathway. ENPP2 overexpression protects the mouse myocardium from I/R-induced ferroptosis injury via the SIRT1/PGC-1α/NRF1 pathway. These findings suggest a novel gene therapy strategy for mitigating myocardial I/R injury.