MicroRNA as an emerging cell-instructive material to regulate cardiomyocyte cell cycle and mitochondrial biogenesis

摘要

Introduction: Cardiac remodeling and heart failure post myocardial Infarction (Ml) is mainly due to inadequate regenerative capacity of mammalian heart, which is attributed to the limited proliferative potential of adult cardiomyocytes (ACMs). We identified miR-1825 as a master regulator, which induces ACM proliferation through recruiting other proliferation inducing miRs, including miR-199a. Moreover, the generation of reactive oxygen species (ROS) by mitochondrial oxidative phosptiorylation is known to induce DNA Damage Response (DDR) pathway, resulting in cell cycle arrest and apoptosis.Here we hypothesize that miR-1825 promotes proliferation of ACMs by inhibiting DNA damage and cell cycle inhibitors. Material and Methods: ACMs were isolated from 12 week old male rats and were transfected with cel-miR-67 (control), miR-1825, miR-199a, siRNA against Rb1 and Meis2 both individually and in combination. To label proliferating ACMs, EdU (5 μM) was added for 5 days and cells were harvested for staining, protein, and RNA analysis. Immuno-staining was performed for EdU, p-H3 (mitotic marker), and Troponin-Ⅰ (cardiac marker), Dihydro-rhodamine 123 (marker for mitochondrial ROS production), 8-OxoG (DNA damage marker) and VDAC (mitochondrial number). Results: Similar to our previous reports, miR-1825 induced 2310.4% and miR-199a induced 14.8±0.5% proliferation of ACMs, as shown by EdU incorporation. In addition, we identified that siRNA mediated silencing of Rb1 and Meis2 promoted 8.7±2.8% and 3.8±1.4% increases in proliferation, whereas simultaneous inhibition of both Rb1 and Meis2 induced 23±5.0% proliferation indicating a synergistic effect. p-H3+ ACMs following individual and combined silencing of Rb1 and Meis2 was observed to be 2.6%, 2.2%, and 7.6%, respectively. Western blotting confirmed silencing of Rb1 (0.14±0.02 fold) in miR-1825 transfected ACMs. In-silico analysis revealed a direct binding site for miR-199a on the 3'UTRs of both Rb1 and Meis2. Luciferase assay following miR-199a transfection, showed a 0.2910.06 fold and 0.0910.07 fold reduction of chemiluminescent activity with Rb1-3'UTR and Meis2-3'UTR, respectively confirming direct binding site. We also identified 25% less mitochondrial ROS production in ACMs transfected with miR-1825 and a 25% reduction in 8-OxoG levels. VDAC immuno-staining showed over 85% decrease in mitochondrial number in miR-1825 transfected group 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 upregulated. Our RNA sequencing data and in-silico analysis reveal miR-1825 regulating mitochondrial biogenesis indirectly through upregulating miR-199a expression and by subsequent down-regulation of miR-199a target SIRTUIN1I. SIRTUIN1 is a known inhibitor of PGC-1α and mitochondrial biogenesis. Discussion: Recent studies identified the role of oxidative stress and hypoxia on CM proliferation. In lower organisms, like zebrafish as well as in neonatal mammals, CMs proliferative window is maintained. However, increases in oxygen concentration after birth cause inhibition of cell cycle through DNA damage response'3]. Here we show that miR-1825 mediated increases in proliferation of naturally quiescent ACMs is through inhibition of mitochondrial biogenesis and reduced oxidative stress. Conclusion: We identified that the increase in ACM proliferation observed with miR-1825 is through miR-199a mediated inhibition of mitochondrial biogenesis and ROS production. This is a novel mechanism and has translational potential for inducing proliferation of ACMs post myocardial infarction. Moreover, miR-1825 and miR-199a mediated synergistic silencing of cell cycle inhibitors Rb1 and Meis2 promote robust proliferation of ACMs. This is a novel approach of using microRNA as cell-instructive material to regenerate the ischemic myocardium by promoting ACM proliferation.