The long term goal of the proposed work is to investigate cellular mechanisms which contribute to metabolic derangements in skeletal muscle during septic injury. This study is designed to test the hypothesis that alterations in cellular calcium occurring in skeletal music during the course of septic injury impair the regulation of muscle's protein degradation and thus contribute to unabated net degradation of skeletal muscle proteins. Experiments will be carried out in two rat models of sepsis, viz, the intra abdominal abscess (IAA) and cecal-ligation-puncture (CLP) models. In order to examine changes in early and late phases of sepsis, the CLP rats will be studied 8-10 hours (early CLP sepsis) and 16- 20 hours (Late CLP sepsis) after the CLP procedure, and the IAA septic rats will be studied before and after they are injected with non-lethal low doses of shock producing agents endotoxin, TNF and IL-1. The specific objectives of the study are: 1) to characterize and evaluate skeletal muscle's net protein catabolic response, 2) to assess skeletal muscle's cellular calcium regulation, 3) to ascertain relationship between cellular calcium regulation and net protein degradation in skeletal muscle, and 4) to investigate calcium linked activation of proteinases as the cellular mechanism of net proteolysis in skeletal muscle in initial and late phase of septic injury. Protein degradation and syntheses will be studies using both in vitro and in vivo techniques. To evaluate calcium regulatory functions, calcium flux across sarcolemma and sarcoplasmic reticulum will be determine along with measurements of cytosolic free calcium ion levels. The cytosolic calcium levels will be measure using fluorescent fura-2 and indo-1. The relationship between protein metabolic activities and cellular calcium regulation in skeletal muscle will be evaluated by modifying cellular calcium regulation in soleus muscle during incubation with calcium ionophores (A23187, ionomycin), calcium agonist (Bay K8644), or varying extracellular calcium. The proposes studies will also evaluate the efficacy of calcium blockers in the prevention of septic-injury-induced alterations in skeletal muscle protein metabolism when blockers are administered to septic rats. Finally, skeletal muscle proteinases will be evaluated to ascertain if they are activated in the septic rat muscles. If successful, these studies will contribute to an understanding of the relationships between intracellular calcium regulation and protein metabolism in the skeletal muscle during septic injury and will provide a rational basis for therapeutic interventions to prevent unabated skeletal muscle catabolism in the septic patient. The proposed experiments will examine the potential role of calcium entry blockers as agents which could be used to attenuate/prevent septic-related skeletal muscle catabolism.
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