Beta-Adrenergic Receptor Function in Atrial Myocytes

描述（由申请人提供）：我的长期目标是阐明β -肾上腺素能受体（β - ar）刺激调节Ca2+稳态的机制，以及细胞外基质-整合素-细胞骨架复合物如何重塑心房肌a-AR功能。心房肌包含3条功能不同的β - ar信号通路，可与多种g蛋白偶联：途径A: Beta1-AR/Gs，途径B: Beta2-AR/Gs和途径C: Beta2-AR/Gs/Gi。我们假设途径A和B通过全局机制起作用，而途径C通过局部机制调节Ca2+稳态，通过刺激l型Ca2+电流、细胞内Ca2+含量和释放、收缩和磷蛋白磷酸化来定义。途径C通过ip3介导的NO信号发挥局部控制Ca2+稳态的作用。另外，途径C可能通过胞质内Ca2+依赖性磷脂酶A2信号传导来局部调节Ca2+稳态。心房疾病通常与心房纤维化有关，即结构重塑。结构重塑与细胞外基质（ECM）蛋白的增加和整合素受体信号传导的变化有关。我们的初步研究结果表明，ECM蛋白层粘连蛋白通过β -整合素受体和基于肌动蛋白的细胞骨架选择性地改变每个β - ar信号通路。这些ecm -整合素介导的β - ar功能变化与衰竭心脏所表现出的β - ar功能重塑惊人相似。因此，我们假设层粘连蛋白刺激Beta1-整合素受体引发一系列信号事件，导致局灶黏着激酶（FAK）的局部募集和自磷酸化，进而通过src介导的paxillin酪氨酸磷酸化转导信号，从而在肌细胞骨架中产生局部改变，直接重塑β - ar对Ca2+稳态的调节。我们建议使用全细胞膜片钳记录、[Ca2+]i和NOi的外焦和共聚焦荧光测量、放射配体受体结合、免疫印迹、免疫组织化学和病毒转染方法来确定β - ar调节Ca2+稳态的机制，以及层粘胶蛋白刺激β -整合素受体如何重塑这些β - ar调节机制。这些实验应该全面了解β - ar调节心房肌细胞Ca2+稳态的机制，以及ecm -整合素-细胞骨架信号传导如何促进患病心脏β - ar功能重塑的重要见解。

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.