Figure 7: AAV9-miR-294 enhances cardiomyocyte cell cycle in vivo after myocardial injury.

A) Histological analysis of heart sections reveals increased Ki67+/actin+ cells 8 weeks after MI in AAV9-miR-294 group compared to control. Ki67 (green), Actin (red) and nuclei (blue), scale bar = 40μm. Insets represent higher magnification at 20μm. Quantification in (B) (n=5). C) Increased p-histone 3/Actin+ small myocytes in AAV9-miR-294 hearts compared to controls. Inset shows small pH3 myocytes in the border zone area (scale bar = 20μm). p-histone H3 (green), actin (red), nuclei (blue). Quantification in (D) (n=5). E) Analysis of cytokinesis marker auroraB shows increased aurB+/actin+ cells in AAV9-miR-294 hearts compared to controls (n=5) (Scale bar = 40μm). Inset shows a AurB+ myocyte in the border zone area (scale bar = 20μm). AuroraB (green), actin (red) and nuclei (blue). Quantification in (F). G-H) EdU detection of the hearts at 8 weeks showed high number of EdU+ cells in infarct, border zone and remote zone in AAV9-miR-294 group compared to control with significantly higher number of EdU+ myocytes respectively (I) (n=5). EdU (green), actin (red), nuclei (blue), scale bar = 40μm. J) Schematic representation of single myocyte isolation from both AAV9-GFP and AAV9-miR-294 hearts followed by EdU detection and seahorse assays on the isolated myocytes 8 weeks after MI (n=3). K) AAV9-miR-294 hearts showed significantly higher number of EdU labeled adult myocytes compared to control hearts. EdU (green), nuclei (blue) bright field (BF). Inset shows an EdU+ isolated adult myocyte from an AAV9-miR-294 heart. Quantification in (L) (n=3). M) Distribution of EdU+ myocytes in the AAV9-miR-294 hearts showed higher % of mononucleated and tri-nucleated EdU+ myocytes. AAV9-Ctrl vs. AAV9-miR-294 *p < 0.05, **p < 0.01, ***p < 0.001, data was assessed using unpaired student’s t test