Spatiotemporal regulation of beta adrenoceptor signaling in cardiacmyocytes

描述（由申请人提供）：我们的长期目标是了解在生理和病理生理条件下心肌细胞中cAMP/PKA信号传导的时空调节机制及其在心脏治疗中的意义。在心力衰竭期间，发生B肾上腺素能受体（AR）的下调;然而，该途径的下游改变，即cAMP依赖性蛋白激酶A（PKA）对底物磷酸化的调节，可能先于下调。主要假设是bAR亚型相关的磷酸二酯酶（PDE）异构体控制cAMP/PKA信号的时空传播和PDE的抑制蛋白依赖性隔离提供了调节心脏对肾上腺素能刺激的反应的新机制。我们将使用两个功能测量的心肌细胞收缩和实时测量的cAMP/PKA活性下的bAR亚型的调节，以揭示潜在的功能调节的bAR信号从细胞表面的细胞内传播到心肌细胞的细胞内区室的机制。目标1.表征亚型特异性bAR/PDE 4D复合物对心肌细胞中时空cAMP/PKA信号传播的影响。我们将描述不同复合物中PDE 4Ds与bAR亚型的相关性，PDE 4Ds从复合物中的激动剂依赖性解离机制（通过PKA和/或GRK磷酸化），以及PDE 4Ds对不同细胞隔室中cAMP/PKA活性、底物磷酸化、钙信号传导和心肌细胞收缩的影响。目标2.表征心肌细胞中bAR刺激期间barrestin 1和2控制时空cAMP/PKA传播的机制。我们将研究PDE 4D亚型与心肌细胞中bAR亚型激活的arrestins和PKA的相关性，arrestin 2和3在控制不同细胞室中bAR亚型诱导的cAMP/PKA活性、底物磷酸化、钙信号传导和心肌细胞收缩中的不同作用。我们还将研究b1和b2 AR对cAMP信号时空传播的相互作用。目标3.目的研究心衰心肌细胞中bAR/PDE 4D复合物调控cAMP/PKA信号传导的时空变化。我们将研究从横主动脉缩窄（TAC）诱导的心脏肥大和心力衰竭大鼠分离的肌细胞中bAR/PDE 4D复合物的完整性，以及改变bAR与PDE 4D亚型的相关性是否有助于抑制时空cAMP信号传播和降低心脏收缩反应。我们将尝试通过腺病毒基因转移PDE 4D突变体到TAC治疗动物的心肌中来选择性地抑制特定的PDE 4D亚型，并检查体内心脏性能。

DESCRIPTION (provided by applicant): Our long term goal is to understand mechanisms that govern spatiotemporal regulation of cAMP/PKA signaling in cardiac myocytes under physiological and pathophysiological conditions, and their implication in cardiac therapy. During heart failure, down-regulation of the b adrenergic receptor (AR) takes place; however downstream alterations in the pathway, i.e. regulation of substrate phosphorylation by cAMP- dependent protein kinase A (PKA), may precede the down-regulation. The major hypothesis here is that spatiotemporal propagation of cAMP/PKA signaling controlled by bAR subtype-associated phosphodiesterase (PDE) isoforms and arrestin-dependent sequestration of PDEs provides novel mechanism on regulating cardiac response to adrenergic stimulation. We will use both functional measurement of myocyte contraction and real-time measurement of cAMP/PKA activities under bAR subtype regulation to uncover the mechanism underlying functional regulation of intracellular propagation of bAR signaling from the cell surface to intracellular compartments in cardiac myocytes. Aim 1. To characterize the effects of subtype-specific bAR/PDE4D complexes on spatiotemporal cAMP/PKA signaling propagation in cardiac myocytes. We will characterize the association of PDE4Ds with bAR subtypes in different complexes, mechanisms of agonist-dependent dissociation of PDE4Ds from the complexes (via phosphorylation by PKA and/or GRK), and the effects of PDE4Ds on cAMP/PKA activities in different cellular compartments, substrate phosphorylation, calcium signaling, and cardiac myocyte contraction. Aim 2. To characterize the mechanism(s) by which barrestin 1 and 2 control spatiotemporal cAMP/PKA propagation during bAR stimulation in cardiac myocytes. We will examine the association of PDE4D isoforms with bAR subtype-activated arrestins and PKA in cardiac myocytes, the differential roles of arrestin 2 and 3 in controlling bAR subtype-induced cAMP/PKA activities in different cellular compartments, substrate phosphorylation, calcium signaling, and cardiac myocyte contraction. We will also examine the interactive effects of b1 and b2AR on the spatiotemporal propagation of cAMP signaling. Aim 3. To investigate the alteration(s) of bAR/PDE4D complexes in controlling spatiotemporal cAMP/PKA signaling propagation in cardiac myocytes from failing hearts. We will examine the integrity of bAR/PDE4D complexes in myocytes isolated from transverse aortic constriction (TAC)-induced cardiac hypertrophy and heart failure rats, and whether altered bAR association with PDE4D isoforms contributes to depressed spatiotemporal cAMP signal propagation and decreased cardiac contractile responses. We will attempt to selectively inhibit a specific PDE4D isoform by adenovirus gene transfer of PDE4D mutants into myocardium of TAC-treated animals, and examine cardiac performance in vivo.