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.
