Decreased cardiac response to beta-adrenergic stimulation is linked to development of heart failure. During heart failure, down-regulation of the beta-adrenergic receptor takes place, however downstream alterations in the pathway, i.e. regulation of substrate phosphorylation by cAMP-dependent protein kinase (PKA), are also involved. The major hypothesis tested in this proposal is that regulation of interaction between Rll and A-kinase anchoring proteins (AKAPs) provides a novel mechanism of regulating the cardiac response to activation of the beta-adrenergic signaling pathway. PKA, primarily PKA Type II (PKA II), is targeted to its substrates by high affinity binding via the regulatory subunits, Rll, of PKA, to AKAPs. AKAPs, in turn bind to the PKA substrate. This facilitates PKA substrate phosphorylation by increasing the local concentration of PKA catalytic subunits, C, upon an elevation in cAMP. We showed by surface plasmon resonance (SPR) and by Rll overlay, that Rll phosphorylation by C on serine 96 (S96) increases AKAP affinity for Rll. Also, expression of mutant Rll. (RllS96D) which mimics phosphorylated Rll (RII-P), showed increased co-localization with AKAP15/18, and also with mAKAP, as compared with RllS96A, which mimics unphosphorylated Rll. These results are consistent with our SPR and Rll overlay data with Rll vs RII-P which also show increased binding of RII-P vs Rll to AKAPs. We also demonstrated that phosphorylation of Rll, and PKA substrates (Tnl, MBP-C and PLB), is decreased in failing human hearts. We predict that reduced Rll phosphorylation in failing hearts results in decreased AKAP affinity for PKA, decreased AKAP-targeted PKA activity and decreased phosphorylation of substrates to which AKAP:PKA is targeted, i.e. alpha-subunit of L-type Ca2+ channels and the Ryanodine Receptor of the SR (RyR). We recently demonstrated that expression of a competing Rll binding peptide, Ht31, by adenoviral (Ad) gene transfer into adult cardiac myocytes disrupts PKA: AKAP binding, decreasing PKA-dependent phosphorylation of myofibrillar PKA substrates (Tnl and MBP-C). We observed an increased contractile response to isoproterenol vs controls, but no significant effect on Ca2+ cycling. We hypothesize that regulation of binding of PKA to AKAPs (by altered Rll phosphorylation or disruption of RlI:AKAP interaction) is a novel mechanism to regulate cardiac function. We also hypothesize that altered Rll targeting contributes to decreased contractility in heart failure. We will address three Specific Aims: (1): to investigate the effect of altered Rll phosphorylation on targeting of PKA, and other signaling proteins, in order to test whether Rll phosphorylation increases Rll: AKAP interaction in cardiac cells: (2) to investigate the effect of altered PKA targeting on localized PKA activity, substrate phosphorylation, cardiac myocyte function and co-localization of PKA and AKAP15/18 and with mAKAP; (3) to determine whether altered PKA targeting via AKAPs regulates cardiac contractility by Ad gene transfer into failing rat hearts in vivo. These studies will provide new insights into the functional significance of AKAP:PKA targeting in the heart. In the long term, results of these studies may offer novel approaches to heart failure therapy.
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