Recent studies indicate that sepsis elicits severe reductions in respiratory muscle force generating capacity that are oxygen free radical mediated. The cells and metabolic pathways responsible for generating free radicals in the respiratory muscles during development of sepsis have not, however, been determined, and the specific subcellular alterations accounting for sepsis-related contractile dysfunction have not been identified. The purpose of the studies in this proposal is to investigate these issues. Three groups of experiments will be performed using rat models: Objective I studies will test the hypothesis that contraction-related superoxide generation by diaphragm myocytes is upregulated in sepsis. The investigators will employ novel techniques of detecting tissue superoxide elaboration to demonstrate greater superoxide production by septic muscles and to determine the cellular pathways responsible for this heightened free radical generation. As part of this work, they will also determine if muscle superoxide generation is modulated by PLA2, and if sepsis results in cytokine-mediated upregulation of this pathway. Objective II studies will determine if neutrophils infiltrate the diaphragm in sepsis and provide a second source of reactive oxygen species. The investigators will examine the effect of inhibiting neutrophil function (i.e., blocking free radical generation or neutrophil attachment) on endotoxin-induced diaphragmatic dysfunction, and will examine the interaction between superoxide ions and nitric oxide in mediating neutrophil-induced muscle damage. Objective III will examine the subcellular biochemical and physiological alterations elicited within the diaphragm during the development of sepsis. The investigators will characterize the distribution of sepsis-induced lipid peroxidation and protein nitrosylation within the diaphragm, and will use skinned muscle preparations to examine contractile protein and SR function. Finally, they will characterize the effects of sepsis on mitochondrial function. In each study the investigators will determine if nitric oxide and/or superoxide dependent processes are responsible for producing identified abnormalities. The preliminary studies provide the first evidence that PLA2 modulates superoxide generation and that sepsis induces free radical mediated alterations in contractile protein function. These data suggest that the proposed experiments should provide important information regarding the pathogenesis of sepsis-induced muscle dysfunction.
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