Spatiotemporal regulation of beta adrenoceptor signaling in cardiacmyocytes

摘要 我们的长期目标是了解cAMP/PKA的时空调控机制 生理和病理生理条件下心肌细胞内的信号转导及其在心肌损伤中的意义 心脏治疗。在心力衰竭期间，b肾上腺素能受体(AR)发生下调； 然而，该通路的下游变化，即cAMP对底物磷酸化的调节- 依赖蛋白激酶A(PKA)可能先于下调。这里的主要假设是 BAR亚型相关调控的cAMP/PKA信号的时空传播 磷酸二酯酶(PDE)亚型和芳香素依赖的PDE封存提供了新的 调节心脏对肾上腺素能刺激反应的机制。我们将使用这两种功能 BAR下心肌细胞收缩及cAMP/PKA活性的实时测定 亚型调控揭示细胞内增殖的功能调控机制 心肌细胞中从细胞表面到细胞内室的BAR信号。目标1.目标 亚型特异性BAR/PDE4D复合体对cAMP/PKA信号转导的影响 在心肌细胞中繁殖。我们将描述PDE4D与BAR亚型之间的关联 不同的络合物，依赖激动剂的PDE4D从络合物中解离的机制(通过 PKA和/或GRK的磷酸化)，以及PDE4DS对不同组织中cAMP/PKA活性的影响 细胞间隔、底物磷酸化、钙信号和心肌细胞收缩。目标 2.阐明Barrestin 1和Barrestin 2调控时空cAMP/PKA的机制(S) BAR刺激在心肌细胞中的传播。我们将研究PDE4D的关联性 心肌细胞中含有bar亚型激活的阻滞素和PKA的异构体，芳香素的不同作用 2和3在控制bar亚型诱导的不同细胞间cAMP/PKA活性方面， 底物磷酸化、钙信号和心肌细胞收缩。我们还将研究 B1和b2AR在cAMP信号时空传播中的交互作用。目标3.至 BAR/PDE4D复合体时空调控cAMP/PKA信号的改变(S)研究 衰竭心脏在心肌细胞中的繁殖。我们将检查BAR/PDE4D的完整性 横断性主动脉缩窄(TAC)诱导的心肌肥厚和心肌细胞复合体 心力衰竭大鼠，以及与PDE4D亚型相关的BAR改变是否导致抑郁 CAMP信号的时空传播和心脏收缩反应的降低。我们将尝试 腺病毒转导PDE4D突变体选择性抑制特定的PDE4D亚型 TAC处理的动物的心肌组织，并检测体内的心功能。

Summary 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.