<p>Myocardial ischemia is the common etiology of heart failure (HF). However, the precise molecular mechanisms that govern the ischemic myocardium into HF remain poorly defined. Selective accumulation of succinate is a hallmark of ischemia. Succinate is a predominant regulator of protein succinylation modification. The present study unravels the novel role of succinate in ischemia-induced HF via succinylation. A clinical cohort study that was performed on 1554 Chinese patients with coronary artery disease (CAD) indicated that serum succinate levels were positively correlated with the increase of HF biomarker, decrease of left ventricular ejection fraction (LVEF), incidence of HF, and risk of death. In cardiomyocytes deprived of oxygen and glucose (OGD) and in mice subjected to ligation of the left anterior descending coronary artery (LAD), succinate levels and global protein succinylation were elevated. Succinylation proteomic analysis identified the α-subunit of mitochondrial ATP synthase (ATP5A1) as an important succinylated protein, and K531 was identified as the functional succinylation site. Ablation of succinylation by the ATP5A1-K531R mutant ameliorated OGD-induced cardiomyocyte death, mitochondrial dysfunction and energy metabolic dysfunction. K531R delivered by cardiac-specific adeno-associated virus (AAV9) achieved short-term cardioprotective effects against ischemic injury, and exerted prolonged protective effects against HF development. Sirtuin 5 was confirmed as a desuccinylase of ATP5A1, whereas carnitine palmitoyltransferase 1A (CPT1A) was recognized as a trans-succinylase. Mechanistically, succinylation of ATP5A1-K531 impeded the assembly of ATP synthase and impaired its activity. Elevated succinate potentially serves as a risk predictor of HF. Targeting desuccinylation of ATP5A1-K531 might be a promising therapeutic strategy.</p>