Anesthetic agents can provide organ protection in the setting of ischemia-reperfusion injury, which is clinically important in both the peri-operative and post-operative setting. The cellular and molecular mechanisms by which anesthetics preserve organ systems and promote cell survival from an ischemic challenge are not clearly established. It is believed that the cellular actions of anesthetics in the CNS are mediated via interactions with G-protein coupled receptors (GPCRs), particularly the GABAA receptors. However, because anesthetics readily pass through cell membranes, they may also directly interact with soluble intracellular proteins resulting in direct regulation and/or an allosteric modulation of molecular interactions with other signaling molecules derived from GPCR stimulation. Activation of protein kinase C epsilon (PKCe) has been shown to play a key role in mediating anesthesia-induced myocardial protection, however the cellular and molecular mechanisms of activation, whether by stimulation of GPCRs, or direct activation of the enzyme, have not been investigated and therefore represent a clinically important area of laboratory based research. Our primary goal is to identify the cellular signaling pathways by which the intravenous anesthetic, propofol, acts as a ligand to activate PKCe, and to delineate the molecular mechanism by which interaction and activation of the enzyme occurs. Our overarching hypothesis is that propofol activates the PKCe isoform indirectly via actions on GPCRs, and directly via a molecular interaction with the enzyme at or near the diacylglycerol/phorbol ester binding domain. To achieve our goal, we will utilize a gain or loss of function approach using isolated cardiomyocytes from wild type and PKCe null mice in combination with recombinant PKCe and synthesized PKCe regulatory sub-domains (C1A and C1B) to investigate cellular and molecular mechanisms of PKCe activation. This innovative approach encompasses the use of PKCs activator and inhibitor peptides, down regulation and re-expression of PKCe in cultured cells, recombinant PKCe and synthesized sub-domains combined with photoactivable diazirine propofol analogs to assess direct propofol-induced activation of PKCe. Endpoint measurements include intracellular Ca2+ concentration, contractility, myofilament Ca2+ sensitivity, protein phosphorylation, PKCe activity, translocation and autophosphorylation, and molecular binding studies. Our experimental approach is comprehensive, ranging from molecular interactions to functional assessments of cellular regulation. Cardiomyocytes and the PKCe isoform were chosen as the model system to investigate because they represent a direct extension of our studies from the previous funding period, and because anesthetics are believed to provide myocardial protection during ischemia-reperfusion injury via activation of PKCe.
Specific Aim 1 will determine the role of PKCe in mediating propofol-induced effects on intracellular Ca2+ concentration and myofilament Ca2+ sensitivity, the key regulators of myocardial contractility.
Specific Aim 2 will identify the cellular signaling pathways involved in propofol-induced activation of PKCe.
Specific Aim 3 will determine the molecular mechanism of interaction between propofol and PKCe. We believe these studies represent a logical extension of our previous work in cardiomyocytes and provide an innovative approach to better understand the cellular and molecular mechanisms of anesthetic action in the heart. ? ? ?
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