Progress from this laboratory has indicated that endotoxin administration altered canine myocardial metabolism through a variety of different membrane-associated enzyme and receptor systems, and that the mechanisms responsible for the endotoxin-induced alteration can be categorized into two conceptual frameworks: one by an imbalance in the phosphorylation/dephosphorylation of membrane proteins and the other by changes in membrane lipid microenvironment in response to phospholipase A activation. During the course of our studies we have switched animal species from dog to rat due to the fact that dog heart is technically not suitable for the perfusion-rated phosphorylation- dephosphorylation studies. Since the pathological manifestation in sepsis is known to differ from that in endotoxemia and since polymicrobial septic model induced by cecal ligation and puncture (CLP) resembles more closely the clinical state of sepsis, the primary aim of this proposal is to extend our previous studies to CLP model (rat) in search for the understanding of the underlying mechanisms responsible for the myocardial dysfunction during sepsis. The specific objectives include: studies of intracellular redistribution of beta and alpha-adrenergic receptors and muscarinic cholinergic receptors between the sarcolemma and the light vesicles during early (9 hr post CLP) and late (18 hr post CLP) phases of sepsis, and investigation of the altered receptor phosphorylation/dephosphorylation as an underlying mechanism; investigations of the phosphorylation/dephosphorylation of Na+ pump and its relationship to the externalization and internalization of (Na+ plus K+)-ATPase in rat heart during sepsis; studies of the sepsis-induced alterations in the sarcolemmal Na+-Ca+ exchange, ATP-dependent Ca2+ transport, and dihydropyridine-sensitive Ca2+ channel, and investigation of altered sarcolemmal phosphorylation/dephosphorylation and of membrane lipid modification as underlying mechanisms; studies of the ATP-dependent Ca2+ uptake by cardiac sarcoplasmic reticulum and its regulation by the phosphorylation/dephosphorylation of phospholamban during various phases of sepsis; investigation of the Ca2+-induced Ca2+ release and ryanodine binding in junctional sarcoplasmic reticulum and their regulation by membrane lipid reorganization; studies of the sepsis- induced alterations in the phosphorylation/dephosphorylation of contractile proteins and their association with changes in myofibrillar ATPase activities; investigations of the dynamics of protein kinases A, C, and M and studies of membrane phospholipid composition, and their association with alterations in various enzyme/receptor systems during different phases of sepsis. Since protein phosphorylation/dephosphorylation and membrane lipid organization are regulated by various metabolic and pharmacologic agents, a therapeutic intervention will be attempted using liposomes as a membrane modifier and a drug carrier system.
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