Abstract 17229: Synergistic Inhibition of Retinoblastoma1 and Meis2 Promotes Robust Proliferation of Adult Cardiomyocytes
Background: Cardiac remodeling and heart failure post myocardial Infarction (MI) is mainly due to inadequate regenerative capacity of mammalian heart, which is attributed to limited proliferative potential of adult cardiomyocytes (ACMs). In our recent study, we identified miR-1825 as a master regulator, which induces ACM proliferation through recruiting other proliferation inducing miRs, including miR-199a. In this study, we hypothesize that miR-1825 promotes proliferation through miR-199a mediated inhibition of cell cycle inhibitors Retinoblastoma1 (Rb1) and Meis2.
Methods and Results: ACMs were isolated from 8-12 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 (siRb1, siMeis2, siRb1+siMeis2). To label proliferating ACMs, EdU (5 μM) was added each day for 5 days and cells were harvested/fixed for staining, protein, and RNA analysis. Immuno-staining was performed for EdU, pH3 (mitotic marker), and Troponin-I. Similar to our previous reports, miR-1825 induced 23±0.4% and miR-199a induced 14.8±0.5% proliferation, as shown by EdU+ ACMs. In addition, we identified siRNA mediated silencing of Rb1 and Meis2 promoted 8.7±2.8% and 3.8±1.4% increase in proliferation, whereas simultaneous inhibition of both Rb1 and Meis2 induced robust (23±5.0%) proliferation of ACMs. Mitosis, as identified by 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 Meis1/2 and Rb1 (0.14±0.02 fold) in miR-1825 transfected ACMs. Computational prediction and luciferase assay revealed a direct binding site for miR-199a on the 3’UTRs of both Rb1 and Meis2. Following miR-199a transfection, we found a 0.29±0.06 fold and 0.09±0.07 fold reduction of chemiluminescent activity with Rb1-3’UTR and Meis2-3’UTR, respectively. In addition, binding sites for miR-199a on both Rb1-3’UTR and Meis2-3’UTR are conserved across species as indicated by in-silico analysis.
Conclusion: miR-1825 and miR-199a mediated simultaneous silencing of cell cycle inhibitors Rb1 and Meis2 promotes robust proliferation of ACMs. This can be used to regenerate ischemic myocardium.
Author Disclosures: R. Pandey: None. J. Fritz: None. R.P. Ahmed: None.
- © 2015 by American Heart Association, Inc.