Abstract 1115: β Adrenergic Phosphorylation of Contractile Proteins in Cultured Neonatal Rat Ventricular Myocytes
β agonists are known to induce inotropic and chronotopic responses in cultured neonatal rat ventricular myocytes (NRVM), in part, by phosphorylating several contractile and Ca2+ regulatory proteins. The objective of this study was to determine the phosphorylation states of troponin I (TnI) and myosin binding protein-C (MyBP-C) following treatment with isoproterenol (Iso), a β1 and β2 agonist. Iso is believed to stimulate contractility by activating protein kinase A, elevating cAMP levels and phosphorylating TnI and MyBP-C, among others, in the NRVM. Using antibodies that recognize the phosphorylated TnI and MyBP-C, we have demonstrated that Iso-induced phosphorylation of TnI increased ~ 70%, while phosphorylation of MyBP-C was depressed ~ 90% (P<0.05 for both proteins, N= 6 experiments). One dimensional isoelectric focusing (IEF) further revealed that phosphorylation of Ser23/24 was responsible for the change in the phosphorylation state of TnI. In contrast, the phosphorylation state of MyBP-C was not altered following Iso-exposure, and the protein remained as mono- or di-phosphorylated in both control or Iso-treated cultures. However, the level of total phosphorylation of this protein declined significantly with Iso treatment. Propranolol (Pro), a β1 and β2 antagonist, blocked the changes in TnI and MyBP-C phosphorylation by Iso. Since both of these contractile proteins have been implicated in regulating actomyosin cross-bridge formation and contractility in the adult heart, the results of this study suggest that TnI plays a more central role in modulating contractility in NRVM than does MyBP-C. These observations further imply that MyBP-C may have a more structural role in stabilizing thick filaments than influencing cross-bridge formation in developing hearts.