Anesthesia and Cardiomyocyte Signal Transduction

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The overall goal of this application is to identify fundamental cellular mechanisms by which the intravenous anesthetic, propofol, alters cardiac function. Our objectives are: 1) to investigate the actions of propofol on cellular mechanisms involved in steady state regulation of myocardial contractility and 2) to explore the actions of propofol on catecholamine activation of the heart. The rationale for the study is that induction of anesthesia with propofol is frequently associated with cardiovascular depression in patients with and without cardiac disease. The overarching hypothesis is that propofol alters cellular mechanisms involved in the regulation of intracellular free Ca2+ concentration ([Ca2+]i) and/or myofilament Ca2+ sensitivity at the level of the cardiomyocyte via an activation of distinct PKC isoforms. Using freshly dispersed adult rat ventricular myocytes, we will investigate the extent to which PKC activation is involved in propofol-induced changes in the cellular and subcellular mechanisms that regulate ion channel conductances and [Ca2+]i, sarcoplasmic reticulum (SR) Ca2+ handling and myofilament Ca2+ sensitivity. We present compelling preliminary evidence that propofol alters multiple cellular mechanisms involved in the regulation of [Ca2+]i and/or myofilament Ca2+ sensitivity via activation of PKC. Specific Aim 1 will investigate the effects of propofol on cellular mechanisms that regulate myofilament Ca2+ sensitivity (myofibrillar protein phosphorylation and intracellular pH). Specific Aim 2 will identify actions of propofol on cellular mechanisms that regulate [Ca2+]i (K+ and Ca2+ Currents, SR Ca2+ handling). Specific Aim 3 will identify site(s) in the signal transduction pathway and the cellular mechanism by which propofol alters the inotropic response to beta1 adrenoreceptor activation of cardiomyocytes. Specific Aim 4 will identify site(s) in the signal transduction pathway and the cellular mechanism by which propofol alters the inotropic response to alpha1a adrenoreceptor activation of cardiomyocytes. A variety of experimental preparations and techniques are utilized, including: 1) isolated SR vesicles to directly measure "real time" Ca2+ uptake; 2) purified myofibrils to measure contractile protein phosphorylation and myofibrillar actomyosin ATPase; 3) Western blot analysis and confocal microscopy to monitor translocation of PKC isoforms to distinct intracellular sites; 4) field-stimulated cardiomyocytes to simultaneously measure [Ca2+]i or pHi and shortening; 5) measurement of cAMP and IP3 accumulation; 6) electrophysiological measurements of Ca2+ and K+ channel activity; 7) Rat-1 fibroblasts selectively transfected with the alpha1a or beta1 adrenergic receptor for assessment of ligand binding; 8) inhibitor peptides selective for distinct PKC isoforms to identify isoform-specific changes in cellular mechanisms and function. These studies will yield novel information about cellular mechanisms of propofol-induced changes in myocardial regulation.