Received for publication May 16, 2006.
Revised August 31, 2006.
Accepted for publication August 31, 2006.

Protein kinase A-mediated phosphorylation contributes to enhanced contraction observed in mice that overexpress adrenergic receptor kinase-1

Abstract

Transgenic mice with cardiac specific over-expression of adrenergic receptor kinase-1 (ARK-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 ARK-1 over-expression on contraction were examined in mouse ventricular myocytes, studied at 37^oC, in the absence of adrenergic stimulation. In myocytes voltage-clamped with microeletrodes (18-26M, 2.7 M KCl) to minimize intracellular dialysis, contractions were significantly larger in ARK-1 cells than wild-type myocytes. In contrast, when cells were dialyzed with patch pipette solution (1-3M; 0 mM NaCl, 70 KCl, 70 K-aspartate,4 MgATP, 1 MgCl₂, 2.5 KH₂PO₄, 0.12 CaCl₂, 0.5 EGTA, 10 HEPES), the extent of cell shortening was similar in wild-type and ARK-1 myocytes. Furthermore, when cells were dialyzed with solutions that contained phosphodiesterase-sensitive sodium-cAMP (50 µM), the extent of cell shortening was similar in wild-type and ARK-1 myocytes. However, when patch solutions were supplemented with phosphodiesterase-resistant 8-bromo-cAMP (50 µM), contractions were larger in ARK-1 cells than wild-type cells. This difference was eliminated by the protein kinase-A inhibitor, H-89. Interestingly, Ca²⁺ current amplitudes and inactivation rates were similar in ARK-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 ARK-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 upregulation of ARK-1 on the cAMP-protein kinase-A pathway.

Key words: Contraction, H89, cAMP, calcium current, phosphodiesterase, protein kinase A