Aberrant lysosomal dynamics disrupt myogenesis via mTORC1 signalling in X-linked myotubular myopathy.

X-linked myotubular myopathy is a severe congenital muscle disorder caused by pathogenic variants in the MTM1 gene, which encodes the phosphoinositide phosphatase myotubularin. Muscle biopsies from patients with X-linked myotubular myopathy exhibit distinctive histopathological features, including small, rounded myofibres with centrally located nuclei, indicating a developmental defect in muscle maturation. While earlier studies have indicated that myotubularin dysfunction causes dysregulation of mechanistic target of rapamycin complex 1 (mTORC1) signalling, the underlying mechanisms and phenotypic impact on human muscle cells remain poorly understood. Currently, there are no approved therapies available for the treatment of this disorder. In this study, we established an induced pluripotent stem cell-based model of X-linked myotubular myopathy using two pairs of isogenic induced pluripotent stem cells: healthy-control versus MTM1-knockout and patient-derived versus gene-corrected induced pluripotent stem cells. Through MyoD-inducible myogenic differentiation, this model successfully recapitulates the key pathological features of X-linked myotubular myopathy, including elevated phosphatidylinositol-3-phosphate levels, hyperactivation of mTORC1 signalling, and increased expression of integrin-β1 and dynamin 2. We identified impaired lysosomal dynamics as a novel pathogenic mechanism in X-linked myotubular myopathy. Our induced pluripotent stem cell-derived X-linked myotubular myopathy myotubes exhibited an abnormal redistribution of lysosomes, with peripheral accumulation, leading to abnormally activated mTORC1 signalling. FYCO1 knockdown, a key regulator of lysosomal trafficking, ameliorated this hyperactivation of mTORC1 signalling. Comprehensive transcriptome analysis revealed distinct gene expression patterns associated with altered mTORC1 signalling and lysosomal localisation in X-linked myotubular myopathy myotubes. Network analysis suggested the central role of the mTORC1 signalling pathway and its connections to disrupted muscle development and differentiation. To investigate the influence of mTORC1 signalling and myotubularin deficiency on myogenic differentiation, we established two mouse myoblast models: one with constitutively activated mTORC1 signalling and another with Mtm1 knockout. Increased mTORC1 signalling in mouse myoblasts impaired myogenic differentiation, and this impairment was reversed by mTORC1 inhibitor rapamycin. Notably, rapamycin treatment also ameliorated the impaired myogenic differentiation observed in Mtm1-knockout mouse myoblasts, supporting the causative role of mTORC1 hyperactivation in X-linked myotubular myopathy pathogenesis. In conclusion, our findings establish the first human cell model of XLMTM, revealing that myotubularin deficiency leads to impaired lysosomal dynamics, which in turn causes mTORC1 dysregulation, a critical factor in the early stage of myogenic differentiation in X-linked myotubular myopathy. These findings provide new insights into the pathogenesis of X-linked myotubular myopathy and suggest that targeting mTORC1 signalling may be a promising therapeutic strategy for this debilitating disorder.