Previous studies suggest that sepsis-induced muscle catabolism reflects ubiquitin-proteasome-dependent degradation of myofibrillar proteins regulated by glucocorticoids. Because intact myofibrils are not degraded by the proteasome, it is possible that actin and myosin are dissociated from the myofibrils before they are ubiquitinated and degraded by the proteasome. We will test the hypotheses: 1) sepsis results in glucocorticoid-mediated calcium/calpain-dependent Z-band disintegration and release of myofilaments in skeletal muscle; 2) sepsis results in increased N-end rule pathway-dependent ubiquitination and breakdown of muscle proteins and upregulated expression and activity of the ubiquitin- conjugating enzyme E2/14k and ubiquitin ligase E3alpha; 3) sepsis- induced muscle cachexia can be inhibited by proteasome blocker in vivo; 4) muscle cachexia in patients with sepsis is associated with increased expression and activity of calpains, release of myofilaments and upregulated protein breakdown in the N-end rule pathway. A septic model in rats consisting of cecal ligation and puncture is used in the majority of experiments. Total and myofibrillar protein breadkdown rates are measured in incubated muscles by determining net release of tyrosine and 3-methylhistidine respectively. Integrity of sarcomeric Z-bands is studied by electron microscopy. Gene and protein expression of calpain and calpastatin are determined by Northern and Western blot analysis, respectively. The role of calcium/calpain-dependent proteolysis is assessed by the effect of dantrolene and diltiazem on sepsis-induced morphologic and metabolic changes. The role of glucocorticoids in sepsis- induced changes in muscle calcium levels and release of myofilaments is determined by the glucocorticoid receptor antagonist RU38486. To test the role of the N-end rule pathway, expression and activity of E2/14k and E3alpha are determined and specific E3albha inhibitors are used in a cell- free system. Similar determinations are performed in muscle from patients with sepsis. The proposal is novel because it suggests that muscle cachexia during sepsis is caused by two distinct mechanisms, i.e., calcium/calpain-dependent release of myofilaments from the sarcomere followed by ubiquitination of myofilaments in the N-end rule pathway and subsequent degradation of ubiquitinated filaments by the 26S proteasome. The hypothesis implies two levels at which sepsis-induced muscle cachexia may be prevented/treated, i.e., inhibition of myofilament release by treatment with a calcium antagonist and inhibition of ubiquitin/proteasome-dependent degradation of the released myofilaments by a proteasome blocker.
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