H-89 decreases the gain of excitation-contraction coupling and attenuates calcium sparks in the absence of beta-adrenergic stimulation.
This study used the selective protein kinase A (PKA) inhibitor H-89 (N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) to determine the role of basal PKA activity in modulating cardiac excitation-contraction coupling in the absence of β-adrenergic stimulation. Basal intracellular cyclic AMP (cAMP) levels measured in isolated murine ventricular myocytes with an enzyme immunoassay were increased upon adenylyl cyclase activation (forskolin; 1 and 10 μM) or phosphodiesterase inhibition (3-isobutyl-1-methylxanthine, IBMX; 300 μM). Forskolin and IBMX also caused concentration-dependent increases in peak Ca(2+) transients (fura-2) and cell shortening (edge-detector) measured simultaneously in field-stimulated myocytes (37 °C). Similar effects were seen upon application of dibutyryl cAMP. In voltage-clamped myocytes, H-89 (2 μM) decreased basal Ca(2+) transients, contractions and underlying Ca(2+) currents. H-89 also decreased diastolic Ca(2+) and the gain of excitation-contraction coupling (Ca(2+) release/Ca(2+) current), especially at negative membrane potentials. This was independent of alterations in sarcoplasmic reticulum (SR) Ca(2+) loading, as SR stores were unchanged by PKA inhibition. H-89 also decreased the frequency, amplitude and width of spontaneous Ca(2+) sparks measured in quiescent myocytes (loaded with fluo-4), but increased time-to-peak. Thus, H-89 suppressed SR Ca(2+) release by decreasing Ca(2+) current and by reducing the gain of excitation-contraction coupling, in part by decreasing the size of individual Ca(2+) release units. These data suggest that basal PKA activity enhances SR Ca(2+) release in the absence of ß-adrenergic stimulation. This may depress contractile function in models such as aging, where the cAMP/PKA pathway is altered due to low basal cAMP levels.
Departments of Pharmacology, 1459 Oxford Street, PO Box 15000 Dalhousie University Halifax, Nova Scotia, Canada B3H 4R2. Randi.Parks@dal.ca
Randi J
RJ
Parks
Susan E
SE
Howlett
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001481:Basal Metabolism / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D056966:Excitation Contraction Coupling / Q000187:drug effects*; D005260:Female; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects; D007546:Isoquinolines / Q000494:pharmacology*; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology*; D013997:Time Factors
D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D011943:Receptors, Adrenergic, beta; D013449:Sulfonamides; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010727:Phosphoric Diester Hydrolases; D000262:Adenylyl Cyclases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide