A 3D skeletal muscle system for disease modelling and secretome profiling of Duchenne muscular dystrophy

Background: Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disease characterized by progressive muscle degeneration, exhaustion of the muscle stem cell pool, and extensive fibrotic remodelling, ultimately leading to loss of function and reduced quality of life. Although conventional cultures and mouse models have provided valuable insights into the pathogenesis of DMD, their mild phenotypes and prolonged disease progression require large sample sizes and lengthy experimental timelines. In turn, the field lacks experimental models that recapitulate the complexity of the dystrophic muscle phenotype in vitro for disease modelling or drug screening. Methods: We developed a three-dimensional (3D) construct of dystrophic skeletal muscle using a scaffold-free approach, starting from primary cells isolated from the mdx4cv mouse strain, a widely used model of Duchenne muscular dystrophy (DMD). To assess the pathological fidelity of the DMD 3D model, we conducted a thorough morphological and functional characterization. Taking advantage of the controlled and isolated nature of the system, we explored the paracrine role of the derived muscle-extracellular vesicles (EVs), investigating their potential contribution to disease progression. Results: The heterogeneous 3D skeletal muscle model of DMD faithfully reproduced the hallmark pathological features observed in patient-derived muscle tissue, including progressive muscle degeneration, fibrotic remodelling, and defective regenerative capacity. Furthermore, it enabled mechanistic investigations of muscle-derived EVs, revealing their ability to propagate both regenerative and catabolic signals. Conclusions: This 3D model provides a physiologically relevant and reproducible tool for studying the molecular mechanisms underlying DMD and evaluating potential therapeutic interventions, reducing the use of animal models. Its capacity to replicate key aspects of the muscle pathology holds significant potential for identifying novel biomarkers and therapeutic targets, with broad implications for translational research.