Duchenne muscular dystrophy is driven by defective membrane repair and annexin-A2 dysregulation in skeletal muscle

Background Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, which encodes dystrophin in skeletal muscle cells. Although the role of dystrophin as a structural protein is well known, the cellular processes underlying myofiber degeneration are still not fully understood. Despite advances from studies in murine models, these models do not fully replicate the human pathology.

We investigated sarcolemmal 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.

We identified defective membrane repair in DMD skeletal muscle cells, independent of increased membrane fragility, by evaluating resealing capacity in control and DMD derived-patient cell lines using both a shear stress assay (N = l2, p < 0.000l) and a laser irradiation assay (N = 3, p < 0.000l). Analyses performed on human DMD muscle biopsies (N = l0) further confirmed this defect, demonstrating massive intracellular IgG uptake (p < 0.000l) together with altered annexin expression profiles. While mechanical stress induces the upregulation of annexin A5 (ANXA5, p < 0.0l) and A6 (ANXA6, p < 0.05) in healthy skeletal muscle cells - suggesting an adaptive response to membrane damage, given the annexin family’s central role in membrane repair - we observed dysregulated expression patterns of these proteins in DMD cells. Notably, ANXAl (p < 0.05) and ANXA2 (p < 0.0l) were not only significantly overexpressed but also aberrantly localized to the extracellular space, a putative consequence of defective membrane repair. Since extracellular ANXA2 has been associated with adipocyte accumulation in the muscle tissue of patients with dysferlinopathy, a similar pathological mechanism may be at play in DMD.

Our findings propose that ANXA2 contributes to muscle degeneration in DMD and highlight it as a potential therapeutic target to prevent adipogenesis and muscle loss. Competing Interest Statement The authors have declared no competing interest. Footnotes 1.To assess whether membrane fragility is preferentially associated with slow or fast myofibers, we performed additional coimmunostaining for IgG and fast myosin heavy chain on DMD muscle biopsies. These analyses showed that IgG accumulation occurs in both type I and type II myofibers without any apparent preference, indicating comparable membrane fragility in the two fiber types. 2.To analyze dysferlin expression in DMD cells, considering that ANXA2 is overexpressed in these cells, we re-examined the Western blot membranes to assess dysferlin expression in the DMD patients. Overall, dysferlin levels were significantly higher in DMD biopsies than in controls (mean ratio = 1.63), consistent with previous findings (Vontzalidis et al., 2014). In addition, we analyzed dysferlin expression in DMD muscle cell lines compared with control cells at various stages of differentiation, from undifferentiated myoblasts to differentiated myotubes. These analyses confirmed the upregulation of dysferlin in DMD cells. 3.To examine dystrophin expression in control myoblasts differentiated for 24 hours that were used to evaluate membrane resealing capacity, we conducted additional experiments to analyze dystrophin expression in control and DMD muscle cell lines across different stages of differentiation, from undifferentiated myoblasts to differentiated myotubes. In control cells, dystrophin was detectable at all stages, including undifferentiated and 24hour differentiated myoblasts, with expression increasing approximately twofold in myotubes. As expected, DMD muscle cells lacked dystrophin at every stage examined. 4.To demonstrate a defect of membrane repair in DMD muscle cells, we performed membrane repair assays on fully differentiated myotubes using a laser ablation based assay. DMD myotubes exhibited impaired membrane repair compared with controls, confirming the deficiency in membrane resealing in DMD muscle cells. 5.We tempered the conclusion that extracellular ANXA2 contributes to adipogenic differentiation since we cannot, at this stage, establish a direct causal link between extracellular ANXA2 and adipocyte accumulation. Other factors released from damaged fibers may also contribute to FAP adipogenic differentiation, we have clarified this point in the manuscript. 6.The analysis of dysferlin expression in DMD biopsies was carried out by Nathalie Deburgrave and France Leturcq from the Cochin Institute in Paris. The analyses of dysferlin and dystrophin expression in control and DMD cell lines, as well as the membrane repair assays performed on differentiated myotubes using laser ablation, were conducted by Romain Carmeille, who has recently joined my team as an assistant professor. All of them have been added as co-authors of the manuscript. Abreviation - ANXA2 - Annexin-A2; - DMD - Duchenne muscular dystrophy; - FAP - fibro-adipogenic progenitors; - IgG - immunoglobulin type G; - MD - muscular dystrophies; - PI - propidium iodide.