alpha-Adrenergic stimulation induces phosphorylation of retinoblastoma protein in neonatal rat ventricular myocytes.

Mammalian cardiac myocytes become postmitotic shortly after birth, and the subsequent myocardial growth in adaptation to increasing workloads becomes primarily dependent on hypertrophy of existing myocytes. Although hypertrophic growth of cardiac myocytes has been extensively studied by using both in vitro and in vivo models, the molecular mechanism controlling the switch from hyperplastic to hypertrophic growth of cardiac myocytes is largely unknown. Since the majority of terminally differentiated cardiac myocytes are growth-arrested in G1/G0 phase, it has been hypothesized that the retinoblastoma protein (Rb) or its related pocket proteins which block G1/S transition becomes constitutively active during myocardial terminal differentiation. To test this hypothesis, we studied the regulation of Rb activity by alpha-adrenergic stimulation in neonatal rat ventricular myocytes which are mostly postmitotic in culture. Our results demonstrate that Rb is predominantly in the active hypo-phosphorylated state in control neonatal ventricular myocytes. alpha-Adrenergic stimulation activates G1/S transition in foetal but not neonatal rate ventricular myocytes. Although alpha-adrenergic stimulation does not activate G1/S transition in neonatal myocytes, it induces hyperphosphorylation of Rb to the same extent as in proliferating skeletal-muscle myoblasts or foetal ventricles. Hyper- but not hypo-phosphorylated Rb in stimulated neonatal myocytes or proliferating skeletal-muscle myoblasts fails to bind to the transcription factor, E2F, suggesting that hyper-phosphorylated Rb is inactive. Therefore F1/S transition could also be blocked at steps in addition to Rb inactivation during terminal differentiation and these blockades are refractory to alpha-adrenergic stimulation.