TARGETING OF PKA BY AKAP100 IN AGING AND FAILING HEARTS

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The severity of human heart failure parallels increased sympathetic drive, elevated circulating catecholamines and a diminished inotropic response to beta-adrenergic stimulation. A dominant view has been that the observed down-regulation of beta- receptors in failing hearts predicts altered downstream activity of the beta-adrenergic pathway. However recent evidence suggests that regulation distal to receptor activation of adenylyl cyclase may be equally or more important. The subcellular distribution of cAMP-dependent protein kinase (PKA) is regulated in many tissues by compartmentalization of PKA by A-kinase anchoring proteins (AKAPs). However, to date there is little information as to how AKAPs regulate PKA activity in the heart. We recently showed co-localization of RII, the regulatory subunit of PKA II, with the cardiac-specific A-kinase anchoring protein, AKAP100, at the junctional sarcoplasmic reticulum (jSR), nucleus and intercalated disc of cardiac muscle cells. We showed that AKAP binding of PKA is regulated by RII autophosphorylation and that RII autophosphorylation is decreased in failing human hearts. These findings implicate AKAP100 dependent PKA targeting in the regulation of substrate phosphorylation in the heart and provide evidence for altered AKAP targeting of PKA in failing hearts. Our goal is to investigate the role of AKAP100 in regulating PKA- dependent substrate phosphorylation in normal and failing hearts. In Specific Aim 1, we will investigate the distribution of AKAP100 and AKAP: RII binding in failing and non-failing human hearts, and will determine the effect on AKAP:RII interation of unloading the failing heart by implantation of a left ventricular assist device (LVAD). This Aim will be addressed by immunofluorescent confocal microscopy, immunogold EM, surface plasmon resonance and Western and Northern blotting. In Specific Aim 2 we will study the functional role of AKAP100 in the regulation of PKA-dependent substrate phosphorylation in cardiac muscle cells by inhibition of AKAP100: RII binding. This will be achieved by adenovirus expression of the inhibitory peptide Ht31. We will assess changes in substrate phosphorylation, cell function, myofibrillar Ca2+ sensitivity and subcellular distribution of RII by confocal microscopy. Specific Aim 3 will investigate molecular interactions between AKAP100, PKA and a PKA substrate, the ryanodine receptor (RyR). This will be carried out by fluorescence resonance energy transfer (FRET) of fusion proteins of RII, catalytic subunit of PKA (C), AKAP100, Ht31 and RyR, each linked to a variant of green fluorescent protein (GFP) and expressed in CHO cells. Overall these studies will allow us to determine the role of AKAP targeting of PKA in the heart and the implications of altered targeting of PKA in heart failure.