Abstract 3463: Important Role of Basic Fibroblast Growth Factor Signaling and p27kip1 in Adult Cardiomyocyte Cell Cycle Regulation
OBJECTIVE: Mammalian cardiomyocytes lose the ability to proliferate during terminal differentiation. We previously reported that co-expression of cyclin D1, tagged with a nuclear localization signal (D1NLS), and its partner, cyclin-dependent kinase 4 (CDK4) induces proliferation of rat neonatal cardiomyocytes. We also showed that down-regulation of the CDK inhibitor p27Kip1 by small interfering (si)RNA, or Skp2, a negative regulator of p27Kip1, significantly enhances the effect of D1NLS and CDK4 on neonatal cardiomyocyte proliferation. In this study, we analyzed the regulation of the cell cycle in adult rat cardiomyocytes in culture.
METHODS AND RESULTS: To test whether adult rat cardiomyocytes expressing D1NLS, CDK4, p27Kip1 siRNA, and Skp2 can activate the cell cycle, immunohistochemical analysis was performed using antibodies to Ki67 antigen and cardiomyocyte-specific marker tropomyosin. Genes were delivered by adenovirus vectors. The proportion of cardiomyocytes positive for Ki67 was approximately 80% 48 hours after infection with D1NLS/CDK4 in the presence of serum (10% fetal bovine serum). However, under serum-free conditions, Ki67-positive cells were hardly detected among D1NLS/CDK4 infected cardiomyocytes (1.7±1.5%). Co-expression of p27Kip1 siRNA partially mimicked the presence of serum (50.4±10.5%), whereas skp2 had no effect. Basic fibroblast growth factor (bFGF, 1ng/mL) treatment with D1NLS/CDK4 infection also increased Ki67-positive cells (54.7±10.8%) at the same level as p27Kip1 siRNA, however hepatocyte growth factor, insulin-like growth factor and platelet-derived growth factor had no effect. Furthermore, in the presence of bFGF, Skp2 increased the effect of D1NLS and CDK4 on Ki67 expression at similar level to p27Kip1 siRNA (63.9±2.1%).
CONCLUSIONS: Knockdown of p27Kip1 and FGF signaling are important for D1NLS/CDK4 expressing adult cardiomyocyte cell cycle activation. Further Skp2 does not mimic p27Kip1 siRNA in the absence of bFGF, suggesting that FGF signaling is essential for p27Kip1 degradation by Skp2. These differ from the case in neonatal cells and provide new insights in understanding the molecular mechanism by which mammalian cardiomyocytes cease to proliferate during terminal differentiation.