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生理和病理生理条件下心肌细胞cAMP/PKA信号的时空调控机制，及其在心脏治疗中的意义。心力衰竭时，b肾上腺素能受体（AR）下调；然而，该通路的下游改变，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。本文的主要假设是，由bAR亚型相关磷酸二酯酶（PDE）亚型控制的cAMP/PKA信号的时空传播和PDE的抑制蛋白依赖的隔离为调节心脏对肾上腺素能刺激的反应提供了新的机制。我们将使用心肌细胞收缩的功能测量和实时测量bAR亚型调控下cAMP/PKA活性来揭示心肌细胞中bAR信号从细胞表面到细胞内区室的细胞内传播的功能调节机制。目的1。表征亚型特异性bAR/PDE4D复合物对心肌细胞cAMP/PKA信号时空传播的影响。我们将描述PDE4Ds与不同复合物中bAR亚型的关联，激动剂依赖的PDE4Ds从复合物中解离的机制（通过PKA和/或GRK的磷酸化），以及PDE4Ds对不同细胞室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。研究bAR刺激心肌细胞时barrestin 1和barrestin 2调控cAMP/PKA时空增殖的机制。我们将研究PDE4D亚型与心肌细胞中bAR亚型激活的阻滞蛋白和PKA的关系，阻滞蛋白2和3在控制bAR亚型诱导的不同细胞室cAMP/PKA活性、底物磷酸化、钙信号传导和心肌细胞收缩中的差异作用。我们还将研究b1和b2AR对cAMP信号时空传播的相互作用。目标3。探讨bAR/PDE4D复合物在控制衰竭心肌细胞cAMP/PKA信号时空传播中的作用。我们将检测横断主动脉收缩（TAC）诱导的心脏肥厚和心力衰竭大鼠分离的肌细胞中bAR/PDE4D复合物的完整性，以及bAR与PDE4D同工异构体关联的改变是否会抑制cAMP信号的时空传播和心脏收缩反应的降低。我们将尝试通过腺病毒将PDE4D突变体基因转移到tac处理动物的心肌中，选择性地抑制特定的PDE4D异构体，并在体内检测心脏性能。

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.