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.