The antioxidant EUK-134 mitigates ER stress-induced mitochondrial dysfunction in human skeletal muscle cells [Abstract]

Lyon, Max, Al-Shanti, Nasser, Lightfoot, AP and Cooper, Robert (2018) The antioxidant EUK-134 mitigates ER stress-induced mitochondrial dysfunction in human skeletal muscle cells [Abstract]. In: British Society for Rheumatology, 01 May 2018 - 03 May 2018, Liverpool, England.

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Abstract

Background: Myositis is a rare, acquired autoimmune disease spectrum, which causes variable levels of skeletal muscle weakness, and thus disability. Infiltration of immune cells (CD4+/CD8+ T-cells) into muscle is an important pathogenic feature of myositis. However, there is evidence that the magnitude of this inflammatory cell infiltration often correlate only poorly with the degree of muscle weakness experienced by patients. Moreover, in e.g. immune-mediated necrotising myopathy patients, muscle weakness typically occurs in the absence of inflammatory cell infiltrations. Murine models of myositis have also demonstrated that weakness may actually precede inflammatory cell infiltrations. Non-immune cell mediated mechanisms have thus been reported to explain this component of muscle weakness, and the endoplasmic reticulum (ER) stress pathway has been suggested to play a key role in mediating this weakness component. Chronic ER stress pathway activation is characteristically observed in the muscle of myositis patients. ER stress pathway activation is also associated with an increased generation of reactive oxygen species (ROS), to potentially damage contractile proteins, as well as with an induced mitochondrial dysfunction. We have thus tested whether ER stress invoked mitochondrial dysfunction in normal human muscle cells could be pharmacologically ameliorated by use of the antioxidant EUK-134 to quench ROS effects. Methods: Immortalised normal adult human skeletal myoblasts were grown in culture and exposed to the ER stress-inducing molecule Tunicamcyin, without or in the presence of the antioxidant EUK-134, a superoxide dismutase/catalase mimetic. ER stress pathway and mitochondrial gene expressions were examined by use of qPCR. Mitochondrial function was assessed using Seahorse extracellular flux analysis, and ATP levels measured using a bioluminescence assay. Results: Treatment of adult human skeletal muscle cells with Tunicamycin induced significant increases in gene expression levels of the ER stress markers: Grp78, ATF4, ATF6 and spliced XBP-1. These increases were all reduced in the presence of EUK-134. ER stress pathway activation also resulted in a significant decline in overall basal, maximal and non-mitochondrial respiration levels – which were all prevented by EUK-134. Total ATP levels were significantly depressed following ER stress-induction, but were maintained at basal levels during ER stress by EUK-134. Gene expressions of the mitochondrial fission markers DRP1 and FIS1 were elevated upon ER stress-induction, but were maintained at basal levels during ER stress by EUK-134. Conclusions: These findings suggest that ER stress-induction in normal adult human muscle cells disrupts mitochondrial function, to depress cellular respiration rates. Pharmacological intervention with the antioxidant EUK-134 clearly and significantly reduces ER stress pathway activation, and thus protects overall mitochondrial function. An antioxidant-based therapeutic strategy may thus represent a worthwhile additional therapeutic intervention in myositis patients with persisting weakness in the absence of inflammatory cell infiltrations. The authors would like to thank University of Liverpool and Manchester Metropolitan University for generous financial support.