Role of cAMP-dependent protein kinase A in activation of a voltage-sensitive release mechanism for cardiac contraction in guinea-pig myocytes

1. Ionic currents and unloaded cell shortening mere recorded from guinea-pig ventricular myocytes with single electrode voltage clamp techniques and video edge detection at 37 degrees C. Patch pipettes (1-3 M Omega) were used to provide intracellular dialysis with pipette solutions. 2. Na+ currents were blocked with 200 mu M lidocaine. Contractions initiated by the voltage-sensitive release mechanism (VSRM) and Ca2+-induced Ca2+ release (CICR) in response to L-type Ca2+ current (I-Ca,I-L) were separated with voltage clamp protocols. 3. Without 8-bromo cyclic adenosine 3',5'-monophosphate (8-Br-cAMP) in the pipette, small VSRM-induced contractions occurred transiently in only 13% of myocytes. In contrast, large I-Ca,I-L-induced contractions were demonstrable in 100% of cells. 4. Addition of 10 or 50 mu M 8-Br-cAMP to the pipette increased the percentage of cells exhibiting VSRM contractions to 68 and 93%, respectively. With 50 mu M 8-Br-cAMP, contractions initiated by the VSRM and I-Ca,I-L were not significantly different in amplitude. 5. 8-Br-cAMP-supported VSRM contractions had characteristics of the VSRM shown previously in undialysed myocytes. Cd2+ (100 mu M) blocked I-Ca,I-L and I-Ca,I-L contractions but not VSRM contractions. 8-Br-cAMP-supported contractions exhibited steady-state inactivation with parameters characteristic of the VSRM, as well as sigmoidal contraction-voltage relations. 6. Without 8-Br-cAMP in the pipette, contraction-voltage relations determined with steps from a post-conditioning potential (V-pc) of either -40 or -65 mV were bell shaped, with a threshold near -35 mV. With 50 mu M 8-Br-cAMP in the pipette, contraction-voltage relations from a V-pc of -65 mV were sigmoidal and the threshold shifted to near -55 mV. Contraction-voltage relations remained bell shaped in the presence of 8-Br-cAMP when the V-pc was -40 mV. 7. H-89, which inhibits cAMP-dependent protein kinase A (PKA), significantly reduced the amplitudes of VSRM contractions by approximately 84% with 50 mu M 8-Br-cAMP in the pipette. H-89 also significantly reduced the amplitudes of peak I-Ca,I-L and I-Ca,I-L contractions, although to a lesser extent. 8. We conclude that intracellular dialysis with patch pipettes disrupts the adenylyl cyclase-PKA phosphorylation cascade, and that the VSRM requires intracellular phosphorylation to be available for activation. Intracellular dialysis with solutions that do not maintain phosphorylation levels inhibits a major mechanism in cardiac excitation-contraction coupling.