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

Transgenic mice with cardiac specific overexpression 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 overexpression on contraction were examined in mouse ventricular myocytes, studied at 37°C, in the absence of adrenergic stimulation. In myocytes voltage-clamped with microelectrodes (18–26 M ; 2.7 M KCl) to minimize intracellular dialysis, contractions were significantly larger in ARK-1 cells than in wild-type myocytes. In contrast, when cells were dialyzed with patch pipette solution (1–3 M ; 0 mM NaCl, 70 mM KCl, 70 mM potassium aspartate, 4 mM MgATP, 1 mM MgCl2, 2.5 mM KH2PO4, 0.12 mM CaCl2, 0.5 mM EGTA, and 10 mM 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 M), 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 M), contractions were larger in ARK-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 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 up-regulation of ARK-1 on the cAMP-protein kinase A pathway. Cardiac contraction is initiated by a rise in intracellular free Ca , derived primarily from internal stores in the sarcoplasmic reticulum (SR; Bers, 2001). Ca release from the SR is triggered by excitation of the sarcolemma through a process called excitation-contraction (EC) coupling (Bers, 2001). In the healthy heart, Ca cycling between intraand extracellular compartments is tightly controlled. However, disruptions in Ca cycling can impair cardiac contractile function in diseases such as congestive heart failure (Marks et al., 2002; Pieske et al., 2002). Alterations in the expression, function, or regulation of various components of EC coupling are thought to contribute to contractile dysfunction in heart failure (Striessnig, 1999). The sympathetic nervous system is activated in heart failure to compensate for diminished contractile function via activation of cardiac -adrenergic receptors ( ARs) (Keys and Koch, 2004). Stimulation of cardiac ARs activates adenylyl cyclase, which increases intracellular cAMP levels and leads to phosphorylation of protein targets via protein kinase A (Wallukat, 2002). However, chronic adrenergic stimulation results in phosphorylation and desensitization of ARs, which further impairs contractile function in the failing heart (Hausdorff et al., 1990; Post