Methods and results: Ischemia was induced in rats via a 45 min occlusion of the left anterior descending coronary artery followed by 1 h, 48 h, or 3 week reperfusion. EPI (10 mg/kg) was administered IV 15 min prior to reperfusion for the single dose group and again 12 h later for the double dose group. Controls received water. Experiments also utilized cultured neonatal rat ventricular myocytes (NRVM) and myoblasts. A single dose of EPI reduced infarct size by 27% at 48 h and 28% at 3 week. Double dose treatment further decreased infarct size by 80% at 48 h, and 52% by 3 weeks. The protective effect of EPI on mitochondrial function was evident after 1h of reperfusion when mitochondria demonstrated less respiratory inhibition, lower mitochondrial Ca2+ load, and a preserved pool of NADH that correlated with higher tissue ATP levels. Mechanistic studies in NRVM revealed that EPI acutely stimulated maximal rates of respiration, an effect that was blocked by inhibitors of the mitochondrial pyruvate carrier, nitric oxide synthase, or soluble guanylyl cyclase. In myoblasts, knockdown of components of the mitochondrial pyruvate carrier blocked EPI-induced respiratory stimulation.