Beta-Adrenergic Receptor Function in Atrial Myocytes

心房肌细胞中的β-肾上腺素能受体功能

• 批准号: 7077777
• 负责人: STEPHEN Lloyd LIPSIUS
• 金额: $ 36.25万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-15 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7077777
• 关键词: atrium; beta adrenergic receptor; calcium channel; calcium flux; cardiac myocytes; cats; fibrosis; fluorescent dye /probe; focal adhesion kinase; immunocytochemistry; integrins; lamins; paxillin; phospholamban; phosphorylation; radiotracer; receptor coupling; voltage /patch clamp; western blottings

DESCRIPTION (provided by applicant): My long range goal is to elucidate the mechanisms by which beta-adrenergic receptor (beta-AR) stimulation regulates Ca2+ homeostasis and how the extracellular matrix-integrin-cytoskeletal complex remodels a-AR function in atrial muscle. Atrial muscle contains 3 functionally different beta-AR signaling pathways that couple to various G-proteins: Pathway A: Beta1-AR/Gs, Pathway B: Beta2-AR/Gs and Pathway C: Beta2-AR/Gs/Gi. We hypothesize that Pathways A and B act via global mechanisms while Pathway C acts via local mechanisms to regulate Ca2+ homeostasis as defined by stimulation of L-type Ca2+ current, intracellular Ca2+ content and release, contraction, and phosphorylation of phospholamban. Pathway C acts via IP3-mediated NO signaling to exert local control of Ca2+ homeostasis. Alternatively, Pathway C may act via cytosolic Ca2+-dependent phospholipase A2 signaling to locally regulate Ca2+ homeostasis. Atrial disease is commonly associated with atrial fibrosis, i.e. structural remodeling. Structural remodeling is associated with increases in extracellular matrix (ECM) proteins, and changes in integrin receptor signaling. Our preliminary findings indicate that the ECM protein laminin acts via beta1-integrin receptors and the actin-based cytoskeleton to selectively alter each Beta-AR signaling pathway. These ECM-integrin-mediated changes in beta-AR function are striking similar to the remodeling of Beta-AR function exhibited by the failing heart. We therefore hypothesize that stimulation of Beta1- integrin receptors by laminin initiates a cascade of signaling events that lead to the local recruitment and autophosphorylation of focal adhesion kinase (FAK) which in turn transduces a signal via Src-mediated tyrosine phosphorylation of paxillin to produce local alterations in the myocyte cytoskeleton that directly remodels Beta-AR regulation of Ca2+ homeostasis. We propose to use whole cell patch clamp recordings, epi- and confocal fluorescence measurements of [Ca2+]i and NOi, radioligand receptor binding, immunoblots, immuno-histochemistry, and viral transfection methods to determine the mechanisms underlying Beta-AR regulation of Ca2+ homeostasis and how stimulation of Beta-integrin receptors by laminin remodels these Beta-AR regulatory mechanisms. These experiments should provide a comprehensive understanding of the mechanisms by which beta-ARs regulate Ca2+ homeostasis in atrial myocytes, as well as important insight into how ECM-integrin-cytoskeletal signaling contributes to remodeling of beta-AR function in the diseased heart.

描述(申请人提供)：我的长期目标是阐明β-肾上腺素能受体(β-AR)刺激调节钙稳态的机制，以及细胞外基质-整合素-细胞骨架复合体如何重塑心房肌的α-AR功能。心房肌含有3条功能不同的β-AR信号通路，分别与不同的G蛋白偶联：通路A：β1-AR/Gs、通路B：β2-AR/Gs和通路C：β2-AR/Gs/Gi。我们假设途径A和B通过全局机制发挥作用，而途径C通过局部机制调节钙稳态，其定义是刺激L型钙电流、细胞内钙含量和磷蛋白的释放、收缩和磷酸化。途径C通过IP3介导的NO信号作用于局部钙稳态。或者，途径C可能通过胞内钙依赖的磷脂酶A2信号作用于局部调节钙稳态。心房疾病通常与心房纤维化有关，即结构重构。结构重塑与细胞外基质(ECM)蛋白的增加和整合素受体信号的改变有关。我们的初步发现表明，ECM蛋白层粘连蛋白通过β1整合素受体和基于肌动蛋白的细胞骨架选择性地改变每一条Beta-AR信号通路。这些ECM-整合素介导的β-AR功能改变与衰竭心脏表现出的β-AR功能重塑惊人地相似。因此，我们假设层粘连蛋白对β1整合素受体的刺激启动了一系列信号事件，导致粘着斑激酶(FAK)的局部募集和自动磷酸化，FAK进而通过Src介导的帕西林酪氨酸磷酸化转导信号，从而在心肌细胞细胞骨架中产生局部变化，直接改变β-AR对钙稳态的调节。我们建议使用全细胞膜片钳记录、[Ca~(2+)]i和NOI的表观和共聚焦荧光测量、放射性配基受体结合、免疫印迹、免疫组织化学和病毒转染法来确定β-AR调节钙稳态的机制，以及层粘连蛋白刺激β-整合素受体如何重塑这些β-AR调节机制。这些实验有助于全面了解β-AR调节心房肌细胞内钙稳态的机制，并对ECM-整合素-细胞骨架信号如何参与病变心脏β-AR功能的重塑提供重要的见解。