Membrane Repair Deficit in Facioscapulohumeral Muscular Dystrophy

Abstract

Deficits in plasma membrane repair have been identified in dysferlinopathy and Duchenne Muscular Dystrophy, and contribute to progressive myopathy. Although Facioscapulohumeral Muscular Dystrophy (FSHD) shares clinicopathological features with these muscular dystrophies, it is unknown if FSHD is characterized by plasma membrane repair deficits. Therefore, we exposed immortalized human FSHD myoblasts, immortalized myoblasts from unaffected siblings, and myofibers from a murine model of FSHD (FLExDUX4) to focal, pulsed laser ablation of the sarcolemma. Repair kinetics and success were determined from the accumulation of intracellular FM1-43 dye post-injury. We subsequently treated FSHD myoblasts with a DUX4-targeting antisense oligonucleotide (AON) to reduce DUX4 expression, and with the antioxidant Trolox to determine the role of DUX4 expression and oxidative stress in membrane repair. Compared to unaffected myoblasts, FSHD myoblasts demonstrate poor repair and a greater percentage of cells that failed to repair, which was mitigated by AON and Trolox treatments. Similar repair deficits were identified in FLExDUX4 myofibers. This is the first study to identify plasma membrane repair deficits in myoblasts from individuals with FSHD, and in myofibers from a murine model of FSHD. Our results suggest that DUX4 expression and oxidative stress may be important targets for future membrane-repair therapies.

Pathomechanisms and biomarkers in facioscapulohumeral muscular dystrophy: roles of DUX4 and PAX7

“…Impaired muscle oxygenation in FSHD may also contribute to ROS sensitivity (Olivier et al , 2016 ; Wilson et al , 2018 ). Other molecular mechanisms underlying oxidative stress sensitivity include p21 upregulation (Winokur et al , 2003a ), increased HIF1α (Tsumagari et al , 2011 ; Banerji et al , 2015 ; Banerji et al , 2017 ; Lek et al , 2020 ), mitochondrial dysfunction (Turki et al , 2012 ; Banerji et al , 2019 ), RAGE‐NF‐κB signalling (Macaione et al , 2007 ) and membrane repair deficits (Bittel et al , 2020 ). Compensatory mechanisms for oxidative stress are also enhanced in FSHD, an example being increased catalase to remove H 2 O 2 (Turki et al , 2012 ; Yao et al , 2014 ).…”

Interplay between mitochondria and reactive oxygen and nitrogen species in metabolic adaptation to hypoxia in facioscapulohumeral muscular dystrophy: potential therapeutic targets

“…Lipid peroxidation has been found in FSHD patient muscles [42], and there are membrane repair deficits in FSHD myoblasts and DUX4 expressing murine myofibres ex vivo, which are alleviated through antioxidant treatment [71]. Membrane lipid peroxidation is a comparably specific oxidative mechanism and it is unclear how more general oxidative/nitrosative protein modifications affect muscle function in FSHD.…”

“…Involvement of DUX4-induced oxidative stress as a negative regulator of FSHD myogenesis has mainly been shown in DUX4 overexpression systems, where antioxidants can alleviate myopathic phenotypes [28,44,71], but rarely in FSHD patient-derived cellular models, marked by much lower and sporadic DUX4 expression. In addition, conventional, non-targeted antioxidants have been investigated in all but one study [57].…”

“…Conventional, non-targeted antioxidant treatment can rescue aspects of FSHD pathology in vitro [44,53,71] and in vivo [55,56], although with only moderate therapeutic efficiency. In addition, antioxidant rescue studies in vitro have only been performed under ambient O2 levels, so potential therapeutic effects on metabolic switching under low O2 availability are unknown.…”

Genetic Approaches for the Treatment of Facioscapulohumeral Muscular Dystrophy

“…Focusing on exon 3-targeting gapmers, we saw improvements in muscle cell fusion and size, as well as minimal to no effects on potential off-target genes in vitro. A separate study also found that treatment with one of the 2′-MOE gapmers increased membrane repair in immortalized patient myoblasts (Bittel et al, 2020). One gapmer from each chemistry was also tested in the FLExDUX4 model, which carry a stably integrated, Creinducible DUX4 transgene (Jones and Jones, 2018).…”