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Skeletal muscle wasting in the critically ill with sepsis is intrinsically linked to the elevations in circulating levels of cytokines characteristic to systemic inflammation and this effect may be exacerbated in older people. A key cytokine in this hyper- inflammatory state is TNF -a. Muscle is a plastic organ which adapts to stresses, including systemic inflammation in a number of ways, including the increased expression of Heat Shock Proteins (HSPs) and increased expression and release of cytokines (myokines). The release of HSPs by non-muscle cells has been described, but the extracellular function of HSPs is poorly understood. The aims of this thesis were to determine the effect of TNF-a on HSP and cytokine release by muscle; to elucidate the mechanism of HSP release from skeletal muscle, to identify the autocrine and paracrine signalling properties of eHSPs and to determine the impact of ageing on the ability of skeletal muscle to act as an endocrine organ. C2C12 myotubes were treated with TNF-a and cells and media were examined for HSP and cytokine content. The mechanisms of HSP and cytokine release were examined which included exosomal and golgi-mediated processes. The paracrine effects of muscle-derived HSP60 were determined by treatment of myotubes or SaOs-2 osteoblast cells with HSP60 at concentrations comparable to serum levels reported in critically ill patients. To determine the effect of TNF-a on the release of HSP60 and cytokines from muscles of old mice, isolated muscle fibres from the FDB of adult and old mice were treated with TNF-a. Treatment ofC2C12 myotubes with TNF-a resulted in increased content ofHSPlO, HSP60 and HSP70 and several cytokines. TNF-a treatment resulted in the specific release of HSP60 as well as IL-6, MCP-l, KC and RANTES from myotubes into the cell culture media. Inhibitor studies demonstrated that the release of HSP60 occurred via exosomes whereas cytokines were released by golgi-mediated transport. Treatment of myotubes with HSP60 resulted in the release of IL-6, MCP-l and RANTES by muscle cells but had no effect on markers of bone formation in SaOs-2 osteoblast cells. Treatment of C2C12 myotubes or SaOs-2 osteoblast cells with muscle-derived exosomes had no effect on cytokine release or markers of bone formation respectively. Comparison of the amount of JiSP60 released by muscle in exosomes to levels reported in pathological states sugpest that muscle is not a major source of HSP60. In contrast, comparison of the levels of cytokines released by muscle suggest that muscle is likely to be a major source of these cytokines in the critically ill. Treatment of isolated fibres from the FDB muscle of adult WT mice with TNF-a resulted in the significant increase in HSP60, IL-6 and KC in the media. In contrast, no effect was seen on the media content of HSP60 following treatment of isolated fibres from old WT mice although the media content of cytokines was comparable to that from fibres of adult WT mice. Data suggest that muscle can act as a significant source of cytokines in response to elevated levels of TNF -a and this may have significant implications for treatment of the critically ill. Muscle can release HSP60 as part of an exosomal process although the levels of HSP60 released are lower than those required to activate cytokine production and release by muscle or osteoblast cells. In contrast, muscle can act as a significant contributing source of cytokines in response to elevated levels of HSP60 derived from other cell types. |
