AKAP-mediated targeting of protein kinase A regulates contractility in cardiac myocytes

Compartmentalization of cAMP-dependent protein kinase A (PKA) by A-kinase a nchoring proteins (AKAPs) targets PKA to distinct subcellular locations in many cell types. However, the question of whether AKAP-mediated PKA anchori ng in the heart regulates cardiac contractile function has not been address ed. We disrupted AKAP-mediated PKA anchoring in cardiac myocytes by introdu cing, via adenovirus-mediated gene transfer, Ht31, a peptide that binds the PKA regulatory subunit type II (RII) with high affinity. This peptide comp etes with endogenous AKAPs for RII binding. Ht31P (a proline-substituted de rivative), which does not bind RII, was used as a negative control. We then investigated the effects of Ht31 expression on RII distribution, Ca2+ cycl ing, cell shortening, and PKA-dependent substrate phosphorylation. By confo cal microscopy, we showed redistribution of RII from the perinuclear region and from periodic transverse striations in Ht31P-expressing cells to a dif fuse cytosolic localization in Ht31-expressing cells. In the presence of 10 nmol/L isoproterenol, Ht31-expressing myocytes displayed an increased rate and amplitude of cell shortening and relaxation compared with control cell s (uninfected and Ht31P-expressing myocytes); with isoproterenol stimulatio n we observed decreased time to 90% decline in Ca2+ but no Significant diff erence between Ht31-expressing and control cells in the rate of Ca2+ cyclin g or amplitude of the Ca2+ transient. The increase in PKA-dependent phospho rylation of troponin I and myosin binding protein C on isoproterenol stimul ation was significantly reduced in Ht31-expressing cells compared with cont rols. Our results demonstrate that, in response to beta -adrenergic stimula tion, cardiomyocyte function and substrate phosphorylation by PKA is regula ted by targeting of PKA. by AKAPs.