Duchenne muscular dystrophy (DMD) is a prevalent and severe neuromuscular disorder resulting from the absence of dystrophin, leading to progressive muscle fiber degeneration. The absence of this key protein disrupts the dystrophin-associated protein complex (DAPC), resulting in the misregulation of multiple cellular pathways, including inflammation, impaired signalization, oxidative stress and calcium homeostasis. Given the complexity of the disease, robust cellular models are essential for investigating its underlying mechanisms.

In this study, our primary aim was to establish and characterize three distinct DMD-depleted myoblast models and to compare their pathological phenotypes. Our models included two immortalized cell lines. One was derived from a patient with a dystrophin gene deletion, and the other had DMD expression silenced using an inducible shRNA. A third model was obtained by transfecting primary myoblasts from healthy donors with a siDMD. We assessed their myotube formation and maturation, focusing on the expression of MEF2C by immunofluorescence, RT-qPCR and western blot, as well as structural integrity using α-actinin staining. All three models consistently exhibited a tendency toward decreased myotube width, although this trend was not statistically significant in two of the models. Furthermore, two of the models showed a significant decrease in MEF2C expression after 48 hours of differentiation. To evaluate membrane integrity and mechanosensitivity, we subjected the myotubes to osmotic stress. This revealed a consistent increase in intracellular calcium levels following hypotonic shock across all three models. Our findings demonstrate that these diverse cellular models recapitulate several key pathological features of DMD. Although one of the models exhibited a less pronounced phenotype, possibly due to a lower depletion of dystrophin, these models represent valuable tools for future investigations into the disease's mechanisms.