Investigating the Role of Mitochondrial Fission in Cardiac Myocyte Hypoxia/Re-oxygenation-Induced Cell Death

Currently there is no effective pharmacological intervention to attenuate myocardialischemia reperfusion (IR) injury. Understanding the mechanisms that govern IR injury would benefit myocardial infarction, coronary bypass and organ transplantpatients. Recent studies suggest that cardiac mitochondria undergo excessive fragmentation during IR, a process that is called fission. Increased mitochondrial fission may lead to increased cell death during IR. Mitochondrial fission has been shown to be mediated by association of dynamin related protein-1(Drp1), a GTPasethat translocates from the cytosol to interact with outer mitochondrial membrane proteins, fission protein 1 and mitochondrial fission factor, to induce fission. We hypothesize that attenuating mitochondrial fission during IR may prove to be an effective strategy to mitigate IR injury. To test this hypothesis, we used a pharmacological inhibitor of Drp-1 GTPase activity, Mdivi-1(MW=353 g/mol). We subjected murine cardiac myocytes (HL-1 cells) to 12 hours of hypoxia in hypoxic buffer (pH 6.8, no glucose or pyruvate) and then 1 hour of re-oxygenation in normoxic media (pH 7.4), in the presence/absence of Mdivi-1 (5-25μM). We predicted that HL-1 cells subjected to simulated IR (SIR) would exhibit increased cell death in relation to their normoxic control counterparts, and that Mdivi-1 treated cells would have attenuated SIR-induced cell death. Cell death was determined by trypan blue (0.3%) staining. Preliminary data shows that control SIRcells (n=4) exhibited 25% cell death, by contrast Mdivi-1 treated cells only showed 7% (5μM, n=4) and 11% (25μM n=4) cell death. Normoxic untreated (n=3) and Mdivi-1 treated cells (5 and 25 μM; n=3) showed only 2% cell death in all study groups. The data suggests that inhibiting Drp-1 may mitigate cardiac cell death during SIR. Future studies will determine the role of Drp-1 in the regulation of mitochondrial dynamics during SIR