[Truncated abstract] The pro-inflammatory cytokine tumour necrosis factor (TNF) has a critical role in skeletal muscle atrophy. The catabolic effect of TNF is partially due to abrogation of the anabolic insulin-like growth factor 1 (IGF-1) signalling pathway. However, the precise signalling events that lead to the loss of myofibrillar protein following activation of TNF receptor are unknown. The over arching aim of the study is to determine the mechanisms of by which TNF induces atrophy in differentiated muscles cells. To achieve this aim a series of experiments were performed to: 1) investigate the molecular events that lead to TNF mediated myofibre atrophy, 2) determine to what extent c-Jun N-terminal Kinase (JNK) signalling plays a part in TNF induced myotube atrophy, and in TNF-mediated inhibition of IGF-1 induced hypertrophy, and 3) use inhibitors of JNK to block the catabolic effects of TNF. 1) To investigate the molecular events that lead to TNF mediated myofibre atrophy, the experiments were conducted using C2C12 mouse myotube cultures and primary myotube cultures derived from FVB mice, and transgenic mice which over-express Class 2 IGF-1 Ea in skeletal muscles (IGF:C2). The treatment of mature C2C12 and FVB primary myotubes (respectively at 7 and 4 days after fusion medium) with 10 ng/mL of TNF for 3 days resulted in statistically significant myotube atrophy (decreased mean width). The observed TNF-mediated atrophy has not previously been demonstrated in tissue cultured myotubes. In contrast, addition of IGF-1 (20 ng/ml) to 7 day C2C12 myotubes for 3 days resulted in significant hypertrophy. ... The most suitable inhibitor was TAT-TIJIP and was thus used in subsequent studies. Inhibition of JNK activity by TAT-TIJIP was confirmed indirectly by detecting nuclear translocation of c- Jun, which is a downstream target of phosphorylated JNK. Immunohistochemical analyses showed nuclear localisation and phosphorylation of c-Jun in TNF treated myotubes. Nuclear localisation and phosphorylation of c-Jun was not observed in cultures pre-treated with TAT-TIJIP before TNF treatment, nor in the untreated control myotubes. 3) The use of JNK inhibitors to block the catabolic effects of TNF was tested using C2C12 and primary myotube cultures. Pre-treatment of C2C12 and primary FVB myotubes with the JNK inhibitor TAT-TIJIP, 30 min before TNF administration (for 3 days) prevented myotube atrophy. The mean width of myotubes pre-treated with TATTIJIP prior to TNF treatment closely resembled that of the control myotubes. Administration of TNF in combination with TAT-TIJIP for 3 days to C2C12 myotubes prevented myotube atrophy and unexpectedly resulted in hypertrophy when compared to the mean widths of untreated and TAT-TIJIP treated myotubes. This trend was also demonstrated in the FVB primary cultures. These combined results strongly support the role of JNK in TNF-mediated atrophy. Preliminary studies were carried out in vivo using the mdx mouse model of muscular dystrophy, TAT-TIJIP was administered via intraperitoneal injection to the mice for 3 days at a dose of 10 mg/ml, however the results form this study are inconclusive. These novel observations are of considerable interest to the field of muscle wasting because they demonstrate for the first time TNF-mediated myotube atrophy, the role of JNK in situations of TNF induced muscle atrophy, and explore the use of JNK inhibitors to prevent muscle atrophy.
|Qualification||Doctor of Philosophy|
|Publication status||Unpublished - 2009|