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

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Abstract
Ionic currents and unloaded cell shortening were recorded from guinea‐pig ventricular myocytes with single electrode voltage clamp techniques and video edge detection at 37 °C. Patch pipettes (1–3 MΩ) were used to provide intracellular dialysis with pipette solutions. Na+ currents were blocked with 200 μm lidocaine. Contractions initiated by the voltage‐sensitive release mechanism (VSRM) and Ca2+‐induced Ca2+ release (CICR) in response to L‐type Ca2+ current (ICa,L) were separated with voltage clamp protocols. 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 ICa,L‐induced contractions were demonstrable in 100 % of cells. Addition of 10 or 50 μm 8‐Br‐cAMP to the pipette increased the percentage of cells exhibiting VSRM contractions to 68 and 93 %, respectively. With 50 μm 8‐Br‐cAMP, contractions initiated by the VSRM and ICa,L were not significantly different in amplitude. 8‐Br‐cAMP‐supported VSRM contractions had characteristics of the VSRM shown previously in undialysed myocytes. Cd2+ (100 μm) blocked ICa,L and ICa,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. Without 8‐Br‐cAMP in the pipette, contraction‐voltage relations determined with steps from a post‐conditioning potential (Vpc) of either −40 or −65 mV were bell shaped, with a threshold near −35 mV. With 50 μm 8‐Br‐cAMP in the pipette, contraction‐voltage relations from a Vpc 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 Vpc was −40 mV. H‐89, which inhibits cAMP‐dependent protein kinase A (PKA), significantly reduced the amplitudes of VSRM contractions by approximately 84 % with 50 μm 8‐Br‐cAMP in the pipette. H‐89 also significantly reduced the amplitudes of peak ICa,L and ICa,L contractions, although to a lesser extent. 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.