Role of SUMOylation and deSUMOylation of mitochondrial fission proteins in myocardial ischaemia-reperfusion injury

Background Restoration of blood supply to the heart after a prolonged ischaemic episode causes myocardial ischaemia-reperfusion (I/R) injury. Paradoxically, short periods of ischaemia and reperfusion, known as preconditioning, has a potent cardioprotective effect. One key aspect of I/R injury is cytotoxic mitochondrial fission that allows cytochrome c release and promotes apoptotic cell death. Dynamin-related protein 1 (Drp1) is a large GTPase that is recruited from the cytosol to the mitochondrial outer membrane to drive fission. Drp1 function is regulated by posttranslational modifications, including conjugation to small ubiquitin-like modifier (SUMO)−2/3. SUMO-2/3-ylation decreases Drp1 partitioning to the mitochondrial membrane, which reduces stress-induced fission and apoptosis. We have shown previously that, in neurons, Drp1 SUMO-2/3ylation is controlled by the deSUMOylating enzyme SENP3, levels of which are reduced during ischaemia but restored upon reperfusion. Objective Here we investigated alterations in SUMOylation and Drp1 partitioning in heart during ischaemia, I/R and preconditioning. Methods: Isolated hearts from male Wistar rats were perfused using Langendorff apparatus with Krebs Henseleit solution. Hearts were randomly divided into 4 groups (with n=5–6 per group): control (50 min perfusion); ischaemia (20 min perfusion+30 min ischaemia); preconditioning (3 short cycles of I/R, 2 and 3 mins, respectively, followed by 30 min of ischaemia); I/R (30 min of ischaemia followed by 2 hour of reperfusion). All samples were immediately subjected to subcellular fractionation, then frozen on dry ice and used for Western blot analysis.