Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA153

Poster Communications

Reactive oxygen species mediate endoplasmic reticulum stress-induced mitochondrial dysfunction in human skeletal muscle cells

A. Thoma1, M. Lyon2, N. Al-Shanti1, R. Cooper2, A. P. Lightfoot1

1. School of Healthcare Science, Manchester Metropolitan University, Manchester, Greater Manchester, United Kingdom. 2. Musculoskeletal Biology, University of Liverpool, Liverpool, United Kingdom.

Myositis is an acquired autoimmune disease that causes profound skeletal muscle weakness and dysfunction (1). Historically thought to be predominantly immune-mediated, significant evidence now suggests that muscle weakness correlates poorly with the degree of inflammation. Moreover, weakness persists and in cases precedes inflammatory cell invasion into muscle. Chronic activation of the endoplasmic reticulum (ER) stress pathway is a characteristic hallmark of myositis and reported non-immune mediated mechanism involved in disease pathogenesis (2). ER stress is associated with modified reactive oxygen species (ROS) generation, altered mitochondrial bioenergetics, and oxidative damage (3); which is postulated as a potential mechanism involved in the underlying muscle weakness experienced by patients with myositis. In this study, we examine the impact of a superoxide dismutase/catalase mimetic (EUK-134) in an in vitro model of ER stress in skeletal muscle (4). Human skeletal muscle cells cultured in vitro were exposed to the ER stress inducer Tunicamycin (0.1mg/ml) with or without EUK-134 (2μM) for 24 hours. Quantitative PCR examined ER stress pathway activation and mitochondrial dynamics. Mitochondrial function was assessed using Seahorse extracellular flux analysis and mitochondrial membrane potential interrogated using the JC-1 fluorophore. ROS generation was measured using MitoSOX red (superoxide) alongside quantification of sulphydryl and oxidised glutathione (GSSG) content. Data were analysed for statistical significance by ANOVA and post hoc Tukey test. Tunicamycin induced upregulation in gene expression of ER stress markers Grp78, CHOP, XBP-1, and ERDJ4, which was significantly reduced in the presence of EUK-134 (p≤0.05). Data showed increased gene expression of mitochondrial-related markers, citrate synthase, FIS1, UCP-3, TFAM, DRP1, HSPA9, HSP60, SOD1, and MFN1, in response to Tunicamycin treatment (p≤0.05). A significant reduction was seen in gene expression of UCP-3, HSPA9, citrate synthase, SOD1, FIS1, and TFAM in the presence of EUK-134 (p≤0.05). Tunicamcyin induced declines in basal, maximal, and ATP-linked mitochondrial respiration, alongside increased proton leak and reduced mitochondrial membrane potential; effects mitigated by EUK-134 (p≤0.05). Tunicamycin increased mitochondrial superoxide and GSSG levels, and decreased overall sulphydryl content, an effect ameliorated in the presence of EUK-134. These findings suggest that the quenching of ROS generation using the superoxide dismutase/catalase mimetic EUK-134 can ameliorate aspects of ER stress-induced mitochondrial dysfunction. Overall, this reinforces a role for ROS in mediating ER stress effects, and that in instances of chronic ER stress, such as in myositis, targeting ROS generation may be an avenue for beneficial impact.

Where applicable, experiments conform with Society ethical requirements