Previous studies suggest that sepsis-induced muscle wasting is associated with increased muscle calcium levels and that glucocorticoids are the predominant mediator of sepsis-induced muscle proteolysis. The overall hypothesis of the current project is that glucocorticoids increase muscle calcium levels and that sepsis and glucocorticoids increase muscle proteolysis through calcium-dependent mechanisms. This is tested in 3 specific aims.
Specific Aim 1 tests the hypothesis that sepsis and glucocorticoids increase muscle calcium levels and calcium-calmodulin protein kinase II (CaMK II) and calpain activities.
Specific Aim 2 tests the hypothesis that sepsis- and glucocorticoid-induced activation of the muscle wasting-associated transcription factors C/EBPp and 8 and NF-kB is regulated by increased calcium levels and activation of CaMK II and calpains.
Specific Aim 3 tests the hypothesis that sepsis- and glucocorticoid-induced muscle proteolysis and upregulation of the ubiquitin ligases atrogin-1 arid MuRF1 are regulated by increased calcium levels and activation of CaMK II and calpains. Studies are performed in two models of muscle wasting, i.e., in septic rats and in dexamethasone-treated cultured myotubes. In several previous studies, we have found that sepsis in rats induced by cecal ligation and puncture (CLP) and treatment of cultured myotubes with dexamethasone result in increased protein degradation and upregulated gene expression of the ubiquitin-proteasome-proteolytic pathway. The role of calcium is examined by using the calcium chelator BAPTA or the calcium """"""""antagonists"""""""" verapamil and dantrolene. The roles of CaMK II and calpains are tested by using specific inhibitors or by transfecting myocytes with plasmids expressing the enzymes or the natural calpain inhibitor calpastatin. The project is important because it will provide information about early and initiating mechanisms of sepsis- and glucocorticoid-induced muscle wasting, if our hypothesis is correct, calcium-regulated mechanisms may become important therapeutic targets in the prevention and treatment of muscle wasting in sepsis and other catabolic conditions. Relevance: Patients with sepsis, cancer, or injury suffer loss of muscle mass. Muscle wasting in these and other catabolic conditions contributes significantly to morbidity and mortality. The proposed experiments will define the role of calcium and calcium-regulated mechanisms in the development of muscle-wasting in sepsis. Increased understanding of the mechanisms driving muscle wasting will help improve prevention and treatment of this debilitating condition. ? ? ?
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