Spatiotemporal regulation of beta adrenoceptor signaling in cardiacmyocytes

心肌细胞β肾上腺素受体信号传导的时空调节

• 批准号: 8040850
• 负责人: YANG K XIANG
• 金额: $ 38.1万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8040850
• 关键词: A kinase anchoring protein; Adenoviruses; Adrenergic Agents; Adrenergic Receptor; Agonist; Animal Model; Animals; Arrestins; Binding; Biological Models; Biosensor; Calcium Signaling; Cardiac; Cardiac Myocytes; Cell membrane; Cell surface; Code; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Depressed mood; Diffusion; Disease; Dissociation; Down-Regulation; Fluorescence Resonance Energy Transfer; GRK; Gene Transfer; Genes; Goals; Health; Heart; Heart Hypertrophy; Heart failure; Measurement; Mus; Muscle Cells; Myocardial; Myocardium; PDE4D3; Pathway interactions; Patients; Performance; Phosphorylation; Physiological; Protein Isoforms; Proteins; Rattus; Regulation; Role; Second Messenger Systems; Series; Signal Pathway; Signal Transduction; Staging; Stress; System; Therapeutic; Time; United States; adrenergic; arrestin 2; base; constriction; in vivo; mutant; novel; phosphoric diester hydrolase; receptor; receptor coupling; response; second messenger; spatiotemporal; therapeutic target

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. PUBLIC HEALTH RELEVANCE: Heart failure is the major health issue contributing one of the deadliest diseases in the United States. In this proposed study, we are asking a critical question: how beta adrenergic signaling, the major signaling system that regulates cardiac function during stresses, is altered at the early stage of heart failure? These studies will provide novel information on using other proteins as therapeutic targets in the beta adrenergic signaling pathway in treating heart failure.

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 信号传导时空调节的机制及其在心脏治疗中的意义。心力衰竭期间，b 肾上腺素受体 (AR) 会发生下调；然而，该通路的下游变化，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。这里的主要假设是，bAR 亚型相关的磷酸二酯酶 (PDE) 亚型控制的 cAMP/PKA 信号的时空传播和 PDE 的抑制蛋白依赖性隔离提供了调节心脏对肾上腺素能刺激反应的新机制。我们将使用心肌细胞收缩的功能测量和 bAR 亚型调节下 cAMP/PKA 活性的实时测量来揭示心肌细胞中 bAR 信号从细胞表面到细胞内区室的细胞内传播功能调节的机制。目标 1. 表征亚型特异性 bAR/PDE4D 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 PDE4D 与 bAR 亚型的关联、PDE4D 从复合物中激动剂依赖性解离的机制（通过 PKA 和/或 GRK 磷酸化），以及 PDE4D 对不同细胞区室中 cAMP/PKA 活性的影响、底物磷酸化、钙信号传导和心肌细胞收缩。目标 2. 表征 barrestin 1 和 2 在 bAR 刺激心肌细胞期间控制时空 cAMP/PKA 传播的机制。我们将研究心肌细胞中 PDE4D 亚型与 bAR 亚型激活的抑制蛋白和 PKA 的关联、抑制蛋白 2 和 3 在控制不同细胞区室中 bAR 亚型诱导的 cAMP/PKA 活性中的不同作用、底物磷酸化、钙信号传导以及心肌细胞收缩。我们还将检查 b1 和 b2AR 对 cAMP 信号传导时空传播的相互作用。目标 3. 研究 bAR/PDE4D 复合物在控制衰竭心脏心肌细胞中时空 cAMP/PKA 信号传播中的变化。我们将检查从横主动脉缩窄 (TAC) 诱导的心脏肥大和心力衰竭大鼠中分离出的肌细胞中 bAR/PDE4D 复合物的完整性，以及改变 bAR 与 PDE4D 亚型的关联是否会导致时空 cAMP 信号传播抑制和心脏收缩反应降低。我们将尝试通过将 PDE4D 突变体的腺病毒基因转移到 TAC 处理的动物的心肌中来选择性抑制特定的 PDE4D 亚型，并检查体内心脏性能。 公共卫生相关性：心力衰竭是导致美国最致命的疾病之一的主要健康问题。在这项拟议的研究中，我们提出了一个关键问题：β肾上腺素能信号传导（在压力下调节心脏功能的主要信号传导系统）在心力衰竭的早期阶段是如何改变的？这些研究将为使用其他蛋白质作为β肾上腺素能信号通路中的治疗靶点来治疗心力衰竭提供新的信息。