Duchenne muscular dystrophy is associated with defective membrane repair and dysregulation of annexin-A1/A2 in skeletal muscle

Abstract

Background: Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, which encodes dystrophin in skeletal muscle cells. Despite extensive knowledge of dystrophin function, the cellular mechanisms responsible for progressive muscle degeneration remain poorly understood. Methods: We investigated sarcolemma integrity, membrane repair capacity, and annexin protein expression in DMD patient muscle biopsies and human skeletal muscle cell lines using immunohistochemistry, both shear stress-based and laser irradiation injury assays, Western blotting, and live-cell imaging of GFP-tagged annexins. To assess whether extracellular annexin A2 (ANXA2) is associated with disease severity, we further analyzed muscle biopsies from patients with Becker muscular dystrophy (BMD) and compared the mdx and D2-mdx mouse models. Results: We identified defective membrane repair in DMD skeletal muscle cells, independent of increased membrane fragility, by evaluating resealing capacity in control and DMD patient-derived cell lines using both a shear stress assay (N = 12, p < 0.0001) and a laser irradiation assay (N = 3, p < 0.0001). Analyses performed on human DMD muscle biopsies (N = 10) further confirmed this defect, demonstrating massive intracellular IgG uptake (p < 0.0001) together with altered annexin expression profiles. While mechanical stress induces the upregulation of annexin A5 (ANXA5, p < 0.01) and A6 (ANXA6, p < 0.05) in healthy skeletal muscle cells - suggesting an adaptive response to membrane damage - we observed dysregulated expression patterns of these proteins in DMD cells. Notably, ANXA1 (p < 0.05) and ANXA2 (p < 0.01) were not only significantly overexpressed but also aberrantly localized to the extracellular space, a putative consequence of defective membrane repair. Importantly, extracellular ANXA2 was progressively increased with disease severity, showing intermediate levels in BMD (N = 3, p < 0.05) compared with DMD biopsies and greater accumulation in D2-mdx than in mdx mice (N = 3, p < 0.05), identifying extracellular ANXA2 as a marker closely associated with dystrophinopathy severity. Conclusions: Our findings identify defective membrane repair as a central pathogenic mechanism in human DMD and indicate that the extracellular accumulation of ANXA2 results from impaired membrane repair and is associated with disease severity. Together, these findings suggest that extracellular annexins may contribute to disease progression and represent potential therapeutic targets, provided that their extracellular functions can be selectively inhibited without compromising their essential intracellular role in membrane repair.