Changes in E2F complexes containing retinoblastoma protein family members and increased cyclin-dependent kinase inhibitor activities during terminal differentiation of cardiomyocytes.

Cardiomyocyte terminal differentiation was examined by studying the interaction of retinoblastoma protein (pRb) family members with E2F during the developmental transition from 17-day fetal to 2-day neonatal. Additionally, the expression pattern of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors responsible for modulating the phosphorylation of pRb were studied. p107, pRb, and p130 are regulators of cellular proliferation, differentiation, and cell cycle exit and entry, respectively. The active, underphosphorylated form of these proteins targets the E2F family of transcriptional factors that play a critical role in the control of genes associated with DNA synthesis. Electromobility shift analyses demonstrated E2F complexed with p107 in proliferating fetal cardiomyocytes, whereas in 2-day neonatal cells, E2F was principally associated with p130 and a low level of pRb. At the 2-day neonatal stage, decreased protein levels were observed for cyclins D2, D3, and E, and CDK2 and CDK4. No changes were observed in the mRNA levels of the D-cyclins in neonatal cells; however, the transcripts for cyclins A and E and CDK4 were diminished. In skeletal myoblasts, differentiation is associated with induction of p21, a CDK inhibitor, by a MyoD-dependent pathway. Although heart cells lack MyoD, CDK assays demonstrated that the activity of CDKs 2, 4, and 6 were downregulated in 2-day neonatal cells, and CDC2 was increased. RT-PCR indicated that p21 mRNA was induced 1.4-, 2.0-, and 3.1-fold in the 2-day neonatal, 7-day neonatal, and adult stages, respectively, compared to the 17-day fetal stage. At the protein level, p21 also increased at the 2-day neonatal stage. Kinase inhibitory immunodepletion assays showed that CDK inhibitory activity was markedly increased in the 2-day neonate. Although mRNA levels of the p27 CDK inhibitor were unchanged, its protein level and inhibitory effect on CDK2 and CDK4 were increased. Thus, cardiomyocytes retain the capacity to proliferate until the early neonatal period when a series of changes occur, including a switch in pRb partners, a decrease in CDK levels and induction of CDK inhibitory activity, which is associated with terminal differentiation. Copyright 1998 Academic Press Limited.