Protein kinase A-mediated phosphorylation contributes to enhanced contraction observed in mice that overexpress beta-adrenergic receptor kinase-1.
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.
Mueller
Erin E
EE
Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada.
Grandy
Scott A
SA
Howlett
Susan E
SE
D000262:Adenylyl Cyclases / Q000187:drug effects / Q000502:physiology; D000818:Animals; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000502:physiology; D002304:Cardiac Pacing, Artificial; D002469:Cell Separation; D048429:Cell Size; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D003627:Data Interpretation, Statistical; D003956:Dialysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D054769:G-Protein-Coupled Receptor Kinase 2; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects; D066298:In Vitro Techniques; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D051379:Mice; D017551:Microdialysis; D009200:Myocardial Contraction / Q000235:genetics* / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000187:drug effects; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology; D051552:beta-Adrenergic Receptor Kinases / Q000096:biosynthesis* / Q000235:genetics*
D020746:Calcium Channels, L-Type; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D013449:Sulfonamides; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C496568:GRK2 protein, mouse; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; D000262:Adenylyl Cyclases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide