Spatiotemporal regulation of beta adrenoceptor signaling in cardiacmyocytes

总结 我们的长期目标是了解cAMP/PKA的时空调控机制 在生理和病理生理条件下心肌细胞中的信号传导， 心脏治疗在心力衰竭期间，发生B肾上腺素能受体（AR）的下调; 然而，下游改变的途径，即调节底物磷酸化的cAMP- 依赖性蛋白激酶A（PKA）可能先于下调。这里的主要假设是， bAR亚型相关的cAMP/PKA信号转导的时空传播 磷酸二酯酶（PDE）同工型和PDE的抑制蛋白依赖性螯合提供了新的 调节心脏对肾上腺素能刺激反应的机制。我们将使用两个函数 在bAR下测量心肌细胞收缩和实时测量cAMP/PKA活性 亚型调节，以揭示细胞内增殖的功能调节机制， 心肌细胞中bAR信号从细胞表面传递到细胞内。目标1.到 表征亚型特异性bAR/PDE4D复合物对时空cAMP/PKA信号传导的影响 在心肌细胞中增殖。我们将描述PDE4D与bAR亚型的相关性， 不同的复合物，PDE4D从复合物的激动剂依赖性解离的机制（通过 磷酸化的PKA和/或GRK），以及PDE4Ds对cAMP/PKA活性的影响，在不同的细胞中， 细胞区室、底物磷酸化、钙信号传导和心肌细胞收缩。目的 2.表征barrestin 1和2控制时空cAMP/PKA的机制 在心肌细胞中bAR刺激期间的增殖。我们将研究PDE4D与 心肌细胞中bAR亚型激活的arrestins和PKA的亚型，arrestin的不同作用 2和3在控制bAR亚型诱导的cAMP/PKA活性的不同细胞区室中， 底物磷酸化、钙信号传导和心肌细胞收缩。我们亦会研究 b1和b2AR对cAMP信号传导的时空传播的相互作用。目标3.到 研究bAR/PDE 4D复合物在控制时空cAMP/PKA信号传导中的改变 在衰竭心脏的心肌细胞中的增殖。我们将检查bAR/PDE4D的完整性， 从横向主动脉缩窄（TAC）诱导的心脏肥大中分离的肌细胞中的复合物， 心力衰竭大鼠，以及与PDE4D亚型相关的bAR改变是否有助于抑郁症的发生。 时空cAMP信号传播和降低的心脏收缩反应。我们将尝试 通过将PDE4D突变体的腺病毒基因转移到 TAC处理的动物的心肌，并检查体内心脏性能。

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.