Background/Purpose: The symmetrical proximal muscle weakness typical of myositis often fails to improve completely with any treatment, due to irreversible muscle fibre degeneration. Although inflammatory cell infiltration is a primary feature of myositis, increasing evidence suggests that muscle weakness correlates poorly with the degree of infiltration (Englund et al. 2001; Li et al. 2004), and in fact may precede inflammatory cell infiltrates (Nagaraju et al. 2001). Research suggests that non-immune cell-mediated mechanisms contribute to muscle weakness, including activation of the ER stress response which is associated with muscle fibre dysfunction and damage (Nagaraju et al. 2005; Yoshida 2007). The mechanisms which mediate ER stress-induced muscle dysfunction in myositis remain unelucidated. However, studies suggest that interplay between the ER stress response, mitochondrial dysfunction and oxidative damage may be involved(Yuzefovych et al. 2013; Cao et al. 2014). Targeted transgenic up-regulation of molecular chaperones, termed Heat Shock Proteins (HSPs), specifically HSP70, attenuates muscle dysfunction and oxidative damage in muscle of old rodents (McArdle et al. 2004; Broome et al. 2006). Similarly, pharmacological increases in HSP70 content of muscle using 17-N-allylamino-17-demethoxygeldanamycin (17AAG), provided an enhanced functional recovery of muscle following exercise-induced damage (Kayani et al. 2008). We hypothesised that, in myositis, ER stress induces mitochondrial dysfunction and oxidative damage, which is a major non-immune cell mediated factor contributing to muscle weakness. We further hypothesised  that targeted pharmacological up-regulation of HSP70 could provide a therapeutic strategy to protect muscle fibres in myositis. Methods : C2C12 myoblasts were grown in standard cell culture conditions (5% CO₂, 37^oC) and differentiated to myotubes in growth media (DMEM) supplemented with 2% horse serum. Myotubes were treated with 1mg/ml Tunicamycin to induce ER stress, in the prescence and abscence of 17AAG (0.1mg/ml) for a period of 24 hours. Cells were harvested and oxygen consumption assessed using a clark electrode (Hansatech Instruments), in the presence of electron transport chain (ETC) substrates and inhibitors: Succintate/Rotenone and Glutamate/Malate to determine the respiratory control ratio (RCR) and Phosphate/Oxygen (P:O) ratio. ATP generation was quantified using bioluminescence assay (Roche). Specific ER stress markers were measured using SDS-PAGE/western blotting and qPCR. Myotube morphology changes were assessed using light microscopy. Results :  Activation of the ER stress response in C2C12 myotubes resulted in mitochondrial dysfunction, evidenced by declines in RCR, P:O ratio and in ATP generation. Cells treated with Tunicamycin in the presence of 17AAG showed full preservation of mitochondrial function and ATP generation. ER stress-induced atrophy of C2C12 myotubes was prevented by the presence of 17AAG. Conclusion : Data demonstrate that pharmacological up-regulation of HSP70 provides protection against ER stress-induced mitochondrial dysfunction and atrophy in C2C12 myotubes.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/endoplasmic-reticulum-er-stress-induced-mitochondrial-dysfunction-and-atrophy-can-be-prevented-by-pharmacological-upregulation-of-heat-shock-protein-70-hsp-in-cultured-murine-myotubes/