Mitochondrial succinate transport is required for cardiac ischemia/reperfusion injury

Background: Succinate accumulates significantly during myocardial ischemia, and its rapid oxidation upon reperfusion is a critical driver of ischemia/reperfusion (I/R) injury. The transport of succinate across the mitochondrial inner membrane, particularly by the dicarboxylate carrier (DIC; SLC25A10), is hypothesized to play a crucial role in mediating these pathological succinate dynamics. However, tools to test this hypothesis by modulating mitochondrial succinate transport in biological systems are lacking. Methods: C57BL/6J mice, isolated Wistar Rat heart mitochondria, bovine heart mitochondrial membranes, C2C12 mouse myoblasts and primary adult cardiomyocytes were used as in vitro and in vivo models. Butylmalonate prodrugs were synthesized and tested. Isolated mitochondria were used to assess succinate- dependent respiration and reactive oxygen species (ROS) production. Cells were treated with succinate dehydrogenase (SDH) inhibitors or exposed to anoxia and butylmalonate esters. Mouse hearts were subjected to in vivo left anterior descending coronary artery ligation. Succinate and butylmalonate levels were measured by targeted liquid chromatography-tandem mass spectrometry, and infarct size by TTC (2,3,5-triphenyl-2H-tetrazolium chloride) staining. Results: Knockdown of DIC, but not of the oxoglutarate carrier OGC, in C2C12 cells prevented succinate accumulation by SDH inhibition and anoxia. The only extant DIC inhibitor butylmalonate, is limited by poor cell permeability. We synthesized diacetoxymethyl butylmalonate (DAB), which efficiently delivers butylmalonate intramitochondrially in isolated heart mitochondria and cells. DAB inhibited succinate- dependent respiration and ROS production. DAB prevented succinate accumulation in cells treated with SDH inhibitors. DAB delivered butylmalonate to cardiac mitochondria when administered to mice in vivo and reduced infarct size by perturbing mitochondrial succinate transport. Conclusions: The DIC is a key node in the cellular distribution of succinate, controlling its transport between mitochondria and the cytosol. These findings highlight the potential of DIC as a promising therapeutic target for conditions where succinate elevation contributes to pathogenesis, such as cardiac I/R injury.