Abstract 12497: Regulation of Mitochondrial Biogenesis and Oxidative Phosphorylation by miR-1825 Promotes Proliferation and Survival of Quiescent Adult Cardiomyocytes

Abstract

Background: Generation of reactive oxygen species (ROS) by mitochondrial oxidative phosphorylation is known to induce DNA Damage Response (DDR) pathway resulting in cell cycle arrest and apoptosis in a number of cell types including Adult Cardiomyocytes (ACMs). In our recent studies, we have identified miR-1825 to be the most robust ACM proliferation inducing miR. Here we hypothesize that miR-1825 promotes proliferation by regulating mitochondrial biogenesis and ROS production. Methods and Results: ACMs isolated from 10 week old male rats were transfected with hsa-miR-1825 and cel-miR-67 (control). EdU was added (5μM) each day for 6 days and cells were used for immuno-staining, RNA and protein analysis. In addition to EdU, TnI, and DAPI staining, Dihydro-rhodamine 123 was used as a marker for mitochondrial ROS production, VDAC immuno-staining was used to detect mitochondrial number. We identified 25% less mitochondrial ROS production in ACMs transfected with miR-1825. Moreover, when ACMs were administered H2O2 (10μM), along with miR-1825 a significant decrease in apoptosis was detected (TUNEL+ ACM 0.53±0.10 fold less than control). In addition to reduction of mitochondrial ROS, miR-1825 transfected ACMs also showed a significant reduction in total ROS. VDAC immuno-staining showed over 85% decrease in mitochondrial number in miR-1825 transfected ACMs compared to control. Our whole transcriptome micro-array analysis of ACMs transfected with miR-1825 identified down regulation of genes involved in oxidative phosphorylation and TCA cycle; while cell-cycle genes like Cyclin-D and Cyclin-B were up regulated. Our RNA sequencing data and in-silico analysis reveal miR-1825 regulates mitochondrial biogenesis indirectly through up-regulating miR-199a expression and by subsequent down regulation of miR-199a target SIRTUIN1 which is a known inhibitor of PGC-1α and mitochondrial biogenesis. Conclusion: We identified that the increase in proliferation of ACMs observed with miR-1825 is through miR-199a mediated inhibition of PGC-1α expression which inhibits mitochondrial biogenesis and ROS production. This is a novel mechanism and has translational potential for inducing proliferation of ACM post myocardial infarction.