Abstract 140: Role of miR-181c in Mitochondrial Matrix Calcium Accumulation During Ischemia/Reperfusion Injury in the Heart

Background: We have identified a microRNA, miR-181c, which can translocate into the mitochondria of cardiomyocytes, and regulate the mitochondrial gene mt-COX1. Recently, we have also demonstrated that miR-181c/d -/- mice are protected against ischemia/reperfusion (I/R) injury by attenuating oxidative stress in the heart. Previous data also suggest that overexpression of miR-181c in the heart can activate Ca 2+ entry into mitochondria. Here, we investigate the mechanism by which miR-181c regulates Ca 2+ influx into the mitochondrial matrix. Methods and Results: We found both Mitochondrial Calcium Uptake 1 (MICU1) and mitochondrial respiratory complex IV (COX IV) expression are markedly higher in the miR-181c/d -/- mouse heart. Immunoprecipitated with MICU1, and then immunoblot for different sub-units of COX IV confirmed a protein-protein interaction between MICU1 and COX IV. We have also found significantly less Pyruvate Dehydrogenase (PDH) activity in neonatal mouse ventricular myocytes (NMVMs) isolated from miR-181c/d -/- mouse compared to C57BL6 (WT), suggesting significantly lower mitochondrial Ca 2+ -concentrations in the miR-181c/d -/- group. Utilizing a coverslip induced I/R-model, we observe that siRNAs against MICU1 (si-MICU1) during the ischemic phase significantly increase Ca 2+ -entry into the mitochondria of the NMVMs. Lowering MICU1 also significantly increases Ca 2+ -entry into the mitochondria after 30 min of ischemia in miR-181c/d -/- NMVMs. Furthermore, 30 min ischemia followed by 30 min reperfusion in NMVM monolayers led to significantly less oscillatory instability in mitochondrial inner membrane potential (ΔΨ m ) in miR-181c/d -/- NMVMs compared with WT NMVMs. However, using si-MICU1 in the miR-181c/d -/- NMVM group attenuated mitochondrial protection against I/R-injury. Conclusions: MICU1 is directly associated with complex IV. Thus, miR-181c can regulate mitochondrial Ca 2+ -entry by targeting mt-COX1 during I/R injury.