Abstract 3462: Knock-Down of Cyclin-Dependent Kinase Inhibitors by RNAi Induces Cardiomyocyte Re-Entry in the Cell Cycle
Background. Mammalian cardiomyocytes (CM) have limited proliferation potential, and acutely injured mammalian hearts do not regenerate adequately. Indeed, mouse CM stop proliferating rapidly after birth and become terminally differentiated, at least in part, by the up-regulation of the Cyclin-Dependent Kinases Inhibitors (CKI) p21Waf1/Cip1/Sdi1 (p21) and p27Kip1 (p27). In this report, it was investigated whether the simultaneous p21 and p27 knock-down by RNAi induced CM proliferation.
Methods and results. Newborn mice CM in culture were transfected with small interfering RNAs (siRNA) targeting both p21 and p27, yielding a >70% decrease in protein expression compared to scrambled-siRNA. After 2 days, CKI knock-down CM, identified by alpha-sarcomeric actin staining, were almost twice control (1.9±0.1; p<0.006). To corroborate these data indicating CM proliferation, it was measured whether markers of cell cycle progression increased accordingly. It was found that CKI knock-down increased >5 fold the rate of cells undergoing DNA synthesis after transfection, as assessed by BrdU incorporation (p<0.0001). Mitotic figures were detectable only in CKI-siRNA transfected cells and AuroraB-positive mid body structures, a feature of cytokinesis, were present only upon CKI knock-down (6.5%±2.4). CM proliferation correlated with the re-expression of cyclin A and with the phosphorylation pRb-family proteins. Interestingly, the unscheduled entrance of differentiated CM in the cell cycle did not increase apoptosis significantly. We then tested whether CKI-knock-down induced cell cycle re-entry in isolated adult CM. We found that CKI knock-down increased almost 4 fold the rate of adult rat CM undergoing DNA synthesis (p<0.0002). Moreover, we injected CKI-siRNA lipofection mix in the left ventricle of adult mice. Preliminary evidence indicates that areas of decreased p21/p27 expression were present in the proximity of the injection site. In these areas, BrdU+ CM were found, while scrambled-siRNA injected hearts were BrdU negative.
Conclusions. The down modulation of p21 and p27 protein expression induces neonatal CM proliferation and cell cycle re-entry in adult CM. Our findings could have application in cardiac regenerative therapy.