TARGETING OF PKA BY AKAP100 IN AGING AND FAILING HEARTS

人类心力衰竭的严重程度与交感神经驱动增加、循环儿茶酚胺升高和对β-肾上腺素能刺激的变力反应减弱平行。 一种主流观点认为，在衰竭心脏中观察到的β受体下调预示着β肾上腺素能途径的下游活性改变。 然而，最近的证据表明，腺苷酸环化酶受体激活远端的调节可能同样重要或更重要。 cAMP依赖性蛋白激酶（PKA）的亚细胞分布在许多组织中通过A-激酶锚定蛋白（AKAP）对PKA的区室化来调节。 然而，迄今为止，关于AKAP如何调节心脏中PKA活性的信息很少。 我们最近发现，PKA II的调节亚基RII与心脏特异性A激酶锚定蛋白AKAP 100在心肌细胞的连接肌浆网（jSR）、细胞核和闰盘处共定位。 我们发现PKA的AKAP结合受RII自身磷酸化的调节，并且RII自身磷酸化在衰竭的人类心脏中减少。这些发现暗示AKAP 100依赖性PKA靶向调节心脏中的底物磷酸化，并为衰竭心脏中PKA的AKAP靶向改变提供证据。我们的目标是研究AKAP 100在正常和衰竭心脏中调节PKA依赖性底物磷酸化的作用。在特定目标1中，我们将研究AKAP 100和AKAP：RII结合在衰竭和非衰竭人类心脏中的分布，并将确定通过植入左心室辅助装置（LVAD）卸载衰竭心脏对AKAP：RII相互作用的影响。 这一目标将通过免疫荧光共聚焦显微镜、免疫金EM、表面等离子体共振和Western和北方印迹来解决。 在具体目标2中，我们将研究AKAP 100在心肌细胞中通过抑制AKAP 100：RII结合来调节PKA依赖性底物磷酸化的功能作用。 这将通过腺病毒表达抑制肽Ht 31来实现。我们将通过共聚焦显微镜评估底物磷酸化、细胞功能、肌原纤维Ca 2+敏感性和RII亚细胞分布的变化。 具体目标3将研究AKAP 100，PKA和PKA底物，ryanodine受体（RyR）之间的分子相互作用。 这将通过RII、PKA（C）的催化亚基、AKAP 100、Ht 31和RyR的融合蛋白的荧光共振能量转移（FRET）来进行，每种融合蛋白与绿色荧光蛋白（GFP）的变体连接并在CHO细胞中表达。 总的来说，这些研究将使我们能够确定AKAP靶向PKA在心脏中的作用以及PKA靶向改变在心力衰竭中的意义。

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.