This is a continuation of five years of work examining alterations in cellular, molecular, and physiologic mechanisms of coronary microvascular regulation induced by cardioplegia, extracorporal artificial surface pump systems, warm ischemia/reperfusion, and associated pathologic processes. The Principal Investigator proposes to examine A) The changes in endothelial and vascular smooth muscle relaxation and vascular smooth muscle contractile mechanisms following hyperkalemic cardioplegia and cardiopulmonary bypass; B) mechanisms of altered arteriolar and venular permeability relative to complement activation, nitric oxide activity, and vascular endothelial growth factor; C) the influence of cardiopulmonary bypass and cardioplegia induced reductions in coronary and peripheral intrinsic vascular smooth muscle tone as this relates to protein kinase C activity, myosin light chain phosphorylation and inducible nitric oxide synthase activity; D) the influence of artificial surface/extracorporel circulation and cardioplegia on gene expression and protein synthesis of VEGF and basic fibroblast growth factor and their respective receptors; and E) the modulating influence of cardioplegia and ischemic cardiac preconditioning on altered coronary blood flow and vascular autoregulation during myocardial ischemia. Multiple sophisticated techniques in several models of ischemia reperfusion, cardiopulmonary bypass and cardioplegia will be utilized in vitro. The arteriolar and venular microvascular changes will be examined with video microscopy or intact isolated coronary microvessels (again, both arteriolar and venular) in order to examine altered microvascular vasodilator and microvascular smooth muscle contractile response to agonist and pressure stimulation. Confocal microscopy will be used to examine complement deposition on vascular tissue and PKC translocation. Fluorescent calcium indicators and 2D electrophoresis will be used to delineate intracellular calcium transients and myosin light chain phosphorylation altered expression of inducible NOS and angiogenic growth factors will be examined with immunohistochemistry and molecular techniques. In vivo measurements of myocardial perfusion and function will be correlated with in vitro findings.
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