MDH1 K298 succinylation stabilizes redox homeostasis to protect against cardiac ferroptosis in ischemia–reperfusion injury

INTRODUCTION: Lysine succinylation is an emerging post-translational modification critically involved in cardiovascular pathophysiology. Malate dehydrogenase 1 (MDH1), a core enzyme of the malate-aspartate shuttle that maintains cardiomyocyte redox homeostasis, is implicated in myocardial injury, yet the regulatory role and specific mechanism of MDH1 succinylation in myocardial ischemia/reperfusion (I/R) injury remain incompletely understood. OBJECTIVE: This study aims to elucidate the functional role and underlying molecular mechanism of site-specific lysine succinylation of MDH1 in myocardial I/R injury. METHODS: Global succinylome profiling was performed on cardiac tissue from murine I/R models. Target succinylation was validated by immunoprecipitation and Western blot. AAV9 vectors encoding wild-type MDH1 (MDH1-WT) or succinylation-deficient mutant (MDH1-K298R) were constructed for cardiac-specific delivery. Molecular docking and co-immunoprecipitation identified the upstream succinyltransferase, and virtual screening identified Ethyl rosmarinate (ER) as a Carnitine palmitoyltransferase 1A (CPT1A)-stabilizing compound. RESULTS: MDH1 K298 succinylation was significantly decreased in I/R-injured hearts compared with sham-operated control hearts. In vivo cardiac overexpression of MDH1-WT suppressed ferroptosis and ameliorated myocardial I/R injury, whereas MDH1-K298R failed to confer such protection, indicating that the cardioprotective effect of MDH1 is dependent on K298 succinylation. Mechanistically, K298 succinylation enhanced MDH1 protein stability by inhibiting its ubiquitin-proteasomal degradation, thereby preserving redox homeostasis required for glutathione peroxidase 4 (GPX4) activity. Importantly, CPT1A was identified as the succinyltransferase responsible for MDH1 K298 succinylation. ER alleviated myocardial I/R injury by preventing Carnitine palmitoyltransferase 1A (CPT1A) degradation and consequently enhancing MDH1 K298 succinylation. CONCLUSION: Our findings uncover a critical cardioprotective role of the CPT1A-MDH1 succinylation axis via suppressing ferroptosis during myocardial I/R injury, and identify ER as a CPT1A-stabilizing compound with promising therapeutic potential for ischemic heart disease.