Background: Facioscapulohumeral muscular dystrophy (FSHD) is caused by transcriptional de-repression of the double homeobox 4 (DUX4) transcription factor. We previously showed that FSHD myoblasts have significant delays in plasma membrane (PM) repair compared to healthy myoblasts. However, the molecular mechanism of this deficit is not clear.
Approach: We used single cell RNA sequencing in n=2 FSHD and healthy sibling myoblast donor pairs to identify differential transcriptional responses to PM injury. Ferroptotic stress was identified using FerroOrange (labile ferrous iron) and BODIPYTM 581/591 (lipid peroxidation) staining in myoblasts, and via meta-analysis of ferroptosis biomarker gene expression in FSHD vs healthy muscle biopsies. The effect of rising (via ferrous ammonium sulfate) and lowering (via 2,2′-Bipyridyl) cytoplasmic iron levels, and the effect of inhibiting ferroptosis (via Ferrostatin-1) on plasma membrane repair was examined using scrape-injury and laser ablation assays.
Results: FSHD myoblasts show dysregulation of genes in the ferroptosis cell death signaling pathway in response to injury, and features of ferroptotic stress – including elevated labile iron and lipid peroxidation post-injury. The expression of ferroptosis biomarkers is elevated in FSHD muscle biopsies, and is predictive of the presence of inflammation and the degree of fatty infiltration in FSHD skeletal muscle. Increasing labile iron and lipid peroxidation worsens membrane repair in FSHD myoblasts, while treatment with the iron chelator 2,2′-Bipyridyl and the ferroptosis inhibitor ferrostatin-1 improve repair.
Conclusion: This study is the first to identify signs of ferroptotic stress in human FSHD myoblasts and demonstrates the potential benefit of iron chelation and ferroptosis inhibition on membrane repair capacity in FSHD.