Recent studies indicate that critically ill patients have profound respiratory muscle weakness. Infection (e.g. pneumonia) is common in these patients and likely an important contributor to the genesis of diaphragmatic weakness. The precise mechanisms by which infection induces respiratory muscle weakness remain, however, unclear. The purpose of the present proposal is to examine these issues in detail, testing our central hypothesis that caspase activation plays a critical role in initiating diaphragm dysfunction in infection. This theory will be evaluated in three groups of studies carried out in mice and C2C12 cells: Objective I studies will test the hypothesis that caspase activation mediates reductions in diaphragm and limb muscle force generation in acute infection/inflammation. Two models of infection/inflammation will be studied (endotoxin injection and pseudomonas pneumonia) in both wild type and transgenic animals with altered caspase pathway genes. Fluorogenic and Western techniques will be used to assess caspase activation indices. Both the effect of specific caspase inhibitors and the responses of caspase pathway gene knockout transgenics will be used to determine which caspase pathways mediate force reductions. Objective II will test the hypothesis that activated caspase alters force via effects on three cellular targets, i.e. specific contractile proteins, sarcolemmal membrane support proteins, and the calpain proteolytic system. These studies will use proteomic and single fiber techniques to identify specific contractile protein cleavage patterns, assays to assess cell integrity, and a variety of calpain activity assays to assess the downstream effects of caspase activation. Objective III will examine the upstream pathways of diaphragmatic caspase activation and test the specific hypotheses that both superoxide and nitric oxide radicals regulate inflammatory mediator induced caspase activation in muscle cells. These experiments will use both transgenic animal models and C2C12 cells to assess the effect of genetic and chemical inhibition of superoxide/nitric oxide on caspase responses. ? ?
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