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Title: The molecular and cellular aspects of muscle degeneration and regeneration
Author: Saini, Amarjit
ISNI:       0000 0004 2748 2230
Awarding Body: Manchester Metropolitan University
Current Institution: Manchester Metropolitan University
Date of Award: 2008
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The concept of skeletal muscle homeostasis - often viewed as the net balance between two separate processes, namely protein degradation and protein synthesis - are not occurring independently of each other, but are finely co-ordinated by a web of intricate signalling networks (Nader, 2005). Such signalling networks are in charge of executing environmental and cellular cues that ultimately determine whether muscle proteins are synthesised or degraded. Prolonged elevations of proinflammatory cytokines are closely associated with muscle wasting that occurs during the sarcopenia of ageing and in cachectic AIDS and cancer patients (Strle et a/. 2007). These clinical disorders occur along with a decline in IGF-I anabolic activity, which is consistent with in vitro findings in muscle progenitor cells (Strle et a/. 2007). Very low concentrations ofTNF-a (0.01-1") inhibit IGF-I-induced protein synthesis (Broussard et a/. 2003; Strle et al. 2004) and expression of the critical muscle differentiation factors, MyoD (Strle et a/., 2004) and myogenin (Broussard et al. 2003; Strle et a/. 2004). Potential treatments that might overcome TNF-a-induced hormone resistance in myoblasts are unknown. Increased activation of the IGF/insulin pathway is an attractive target for combating many of the cachectic conditions associated with muscle wasting. Using rodent skeletal muscle cell lines we have investigated TNF-a/IGF-I interactions, in an attempt to mimic and understand mechanisms underlying the wasting process. We hypothesised that treatment of mouse myoblasts with TNF-a at specific doses ranging from high (20') to low (1.25") would result in dose-dependent block of differentiation and induction of apoptosis and that subsequent IGF-I co-incubations would stimulate myoblast survival and myotube formation. Objectives were to ascertain signalling pathways underpinning these outcomes. In contrast to our hypothesis, a novel role of IGF-I has been identified whereby eo-incubation of skeletal muscle C2 cells with IGF-I (1.5') and a non- apoptotic dose of TNF-a (1.25"; sufficient to block differentiation) unexpectedly were shown to facilitate a significant four-fold increase in myoblast death (P < 0.05). Specificity of the apoptotic potential of this growth factor was confirmed when neither bFGF-2 nor PDGF-BB (10 or 30' and 1.25 or 5 ng.rnl", respectively) were able to reveal the apoptotic potential of low dose TNF-a. By contrast, but in line with our II hypothesis, dosing with 10" TNF-a resulted in a block of differentiation and initiation of apoptosis, which was rescued by IGF-1. Preliminary signalling studies suggest that MAPK activation rather than the caspases are involved in the induction of death associated with low dose TNF-a (1.25 ng.mrl)/IGF-I incubation and therefore blocking the caspases would be without effect in this circumstance. The PI(3)K pathway is involved in the survival effects of high TNF-a (10 ng.mrl)/IGF co-incubations. Importantly, the rescue of death (regardless of the means required) did not facilitate differentiation and did not rescue the block of expression of IGF-ll or IGFBP-5 (produced by skeletal myoblasts as early events in their terminal differentiation and associated with preventing cell death) in our models. Using array technology we further established potential insulin survival and apoptotic genes that were upregulated in the above conditions and confirmed their expression with qRT-PCR. Of these genes three were selected to conduct gene silencing experiments. The gene silencing studies were effective in reducing expression of Adrald, Birc2 and Sirtl. Our findings suggest that inhibition of Adrald leads to an increase in myoblast death in conditions that are associated with myoblast survival and include basal conditions. This novel finding indicates Adrald expression to be essential for the general maintenance of myoblasts. This may be due to the multiple signalling pathways which the al-ARs regulate which include the PI(3)K-Akt pathway that is associated with growth and anti-apoptosis. Birc2 expression, which is upregulated in our cell model under conditions of myotoxic stress showed no significant effect on myoblast survival when suppressed. Associated with inhibition of apoptosis, it was hypothesised that inhibition of Birc2 would result in an increase in myoblast death however levels of damage were comparable to control myoblasts. Recent articles have stated that Birc, only when overexpressed above physiological levels, is associated with anti-apoptosis and consequently have proposed an alternative nomenclature that names the family after its distinctive structural feature, the BIR, rather than by inhibitor of apoptosis proteins lAPs (Silke & Vaux, 200 l; for review Srinivasula & Ashwell, 2008). Finally Sirtl, similar to Birc2 was highly expressed in conditions that induced the greatest incidence of myoblast death. Subsequent inhibition resulted in further increase in death which was not observed under basal conditions where myoblasts received DM alone. Unlike Adrald, this implicates Sirtl expression as a III survival mechanism which is specific for conditions associated with myotoxic stress. The mammalian Sirtl deacetylase was originally shown to modulate life-span in various species. However, the molecular mechanisms by which Sirtl increases longevity and with regard to the present study, survival, are largely unknown. In mammalian cells, Sirtl appears to control the cellular response to stress by regulating the FOXO family of Forkhead transcription factors. The FOXO family members are negatively regulated by the PI(3)K-Akt signalling pathway. Mammalian FOXOs control various biological functions, including cell cycle arrest, differentiation, repair of damaged DNA and apoptosis. Because the ability to regulate apoptosis and repair damage is correlated with increased organismal longevity and survival in many species these particular functions of FOXO transcription factors may be relevant to Sirtl ability to control longevity These experiments in myoblasts show that IGF-I (') can facilitate apoptosis in the presence of non-a pop to tic doses ofTNF-a (\ which appears to depend not only on the upregulation of specific apoptosis genes (potentially downstream of MAPK) but also on the suppression of survival factors IGF-ll and IGFBP-5 which may also lie downstream of MAPK. These studies highlight the complex regulation of cell survival and cell death at the signalling level, as a consequence of interactions of one cytokine, TNF-a, and one growth factor, IGF-I. More information regarding the pathways involved in regulating their expression and activity will be necessary to fully understand the action of these molecules.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available