Oxidative stress and successful antioxidant treatment in models of RYR1-related myopathy

Abstract

The skeletal muscle ryanodine receptor is an essential component of the excitation-contraction coupling apparatus. Mutations in RYR1 are associated with several congenital myopathies (termed RYR1-related myopathies) that are the most common non-dystrophic muscle diseases of childhood. Currently, no treatments exist for these disorders. Although the primary pathogenic abnormality involves defective excitation-contraction coupling, other abnormalities likely play a role in disease pathogenesis. In an effort to discover novel pathogenic mechanisms, we analysed two complementary models of RYR1-related myopathies, the relatively relaxed zebrafish and cultured myotubes from patients with RYR1-related myopathies. Expression array analysis in the zebrafish disclosed significant abnormalities in pathways associated with cellular stress. Subsequent studies focused on oxidative stress in relatively relaxed zebrafish and RYR1-related myopathy myotubes and demonstrated increased oxidant activity, the presence of oxidative stress markers, excessive production of oxidants by mitochondria and diminished survival under oxidant conditions. Exposure to the antioxidant N-acetylcysteine reduced oxidative stress and improved survival in the RYR1-related myopathies human myotubes ex vivo and led to significant restoration of aspects of muscle function in the relatively relaxed zebrafish, thereby confirming its efficacy in vivo. We conclude that oxidative stress is an important pathophysiological mechanism in RYR1-related myopathies and that N-acetylcysteine is a successful treatment modality ex vivo and in a vertebrate disease model. We propose that N-acetylcysteine represents the first potential therapeutic strategy for these debilitating muscle diseases.

Current and future therapeutic approaches to the congenital myopathies

“…Recent studies have indicated that oxidative stress may be one of the causes underlying MH, and that oxidative stress is elevated in muscle of patients with MH and RYR1-and SEPN1-related myopathies [33][34][35]. A study on patient muscle cells and the zebrafish "relatively relaxed" mutant, a model of human recessive RYR1-related myopathies lacking the skeletal muscle ryr1b isoform, demonstrated elevation of oxidative stress markers and excessive production of reactive oxygen species of probable mitochondrial origin.…”

Triadopathies: An Emerging Class of Skeletal Muscle Diseases

“…In a collaborative study [33], we (Dowling) uncovered increased cellular stress and aberrant oxidative stress in a zebrafish model of RYR1-related myopathy and in myotubes from patients with RYR1 mutations. Exposure of the zebrafish mutants to NAC improved muscle structure and motor endurance.…”

“…Recently, the zebrafish model has been utilized as an excellent preclinical vertebrate model for therapy identification [39]. This is well illustrated by the fact that NAC was identified as a potential therapeutic intervention using this system [33]. Additional studies using the existing zebrafish model (a recessive loss of expression mutant in ryr1b that most closely models RYR1-related CNM) will hopefully yield additional candidates.…”

Analysis of Oxidative Stress in Zebrafish Embryos

“…We recently demonstrate that statin treatment of zebrafish embryos induced oxidative stress that can be recovered by CoQ10 treatment 51 . Alternatively, the analysis of oxidative stress in the zebrafish animal model for human RYR1-related myopathies highlighted the role of the antioxidant drug NAC (N-acetyl cysteine) as a potential therapeutic approach for muscle disease 52 .…”