Protein Kinase A-Mediated Phosphorylation Contributes to Enhanced Contraction Observed in Mice That Overexpress β-Adrenergic Receptor Kinase-1

Abstract

Transgenic mice with cardiac specific overexpression of beta-adrenergic receptor kinase-1 (betaARK-1) exhibit reduced contractility in the presence of adrenergic stimulation. However, whether contractility is altered in the absence of exogenous agonist is not clear. Effects of betaARK-1 overexpression on contraction were examined in mouse ventricular myocytes, studied at 37 degrees C, in the absence of adrenergic stimulation. In myocytes voltage-clamped with microelectrodes (18-26 MOmega; 2.7 M KCl) to minimize intracellular dialysis, contractions were significantly larger in betaARK-1 cells than in wild-type myocytes. In contrast, when cells were dialyzed with patch pipette solution (1-3 MOmega; 0 mM NaCl, 70 mM KCl, 70 mM potassium aspartate, 4 mM MgATP, 1 mM MgCl(2), 2.5 mM KH(2)PO(4), 0.12 mM CaCl(2), 0.5 mM EGTA, and 10 mM HEPES), the extent of cell shortening was similar in wild-type and betaARK-1 myocytes. Furthermore, when cells were dialyzed with solutions that contained phosphodiesterase-sensitive sodium-cAMP (50 microM), the extent of cell shortening was similar in wild-type and betaARK-1 myocytes. However, when patch solutions were supplemented with phosphodiesterase-resistant 8-bromo-cAMP (50 muM), contractions were larger in betaARK-1 than wild-type cells. This difference was eliminated by the protein kinase A inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89). Interestingly, Ca(2+) current amplitudes and inactivation rates were similar in betaARK-1 and wild-type cells in all experiments. These results suggest components of the adenylyl cyclase-protein kinase A pathway are sensitized by chronically increased betaARK-1 activity, which may augment contractile function in the absence of exogenous agonist. Thus, changes in contractile function in myocytes from failing hearts may reflect, in part, effects of chronic up-regulation of betaARK-1 on the cAMP-protein kinase A pathway.