Abstract
X-linked myotubular myopathy (XLMTM) is a life-threatening skeletal muscle disease caused by mutations in the MTM1 gene. XLMTM fibres display a population of nuclei mispositioned in the centre. In the present study, we aimed to explore whether positioning and overall distribution of nuclei affects cellular organization and contractile function, thereby contributing to muscle weakness in this disease. We also assessed whether gene therapy alters nuclear arrangement and function. We used tissue from human patients and animal models, including XLMTM dogs that had received increasing doses of recombinant AAV8 vector restoring MTM1 expression (rAAV8-cMTM1). We then used single isolated muscle fibres to analyze nuclear organization and contractile function. In addition to the expected mislocalization of nuclei in the centre of muscle fibres, a novel form of nuclear mispositioning was observed: irregular spacing between those located at the fibre periphery, and an overall increased number of nuclei, leading to dramatically smaller and inconsistent myonuclear domains. Nuclear mislocalization was associated with decreases in global nuclear synthetic activity, contractile protein content and intrinsic myofilament force production. A contractile deficit originating at the myofilaments, rather than mechanical interference by centrally positioned nuclei, was supported by experiments in regenerated mouse muscle. Systemic administration of rAAV8-cMTM1 at doses higher than 2.5 × 1013 vg kg−1 allowed a full rescue of all these cellular defects in XLMTM dogs. Altogether, these findings identify previously unrecognized pathological mechanisms in human and animal XLMTM, associated with myonuclear defects and contractile filament function. These defects can be reversed by gene therapy restoring MTM1 expression in dogs with XLMTM.
Highlights
The centronuclear myopathies (CNM) are a group of genetically and clinically heterogeneous early-onset muscle disorders, with phenotypes varying from severe cases with antenatal/neonatal onset and death in infancy if untreated, to mild cases with onset in adolescence or adulthood and a stable or only slowly progressive course
Myonuclear domain (MND) sizes were dramatically smaller in patients compared to controls (Fig. 2e)
We show that when MTM1 expression is restored by systemic injection of rAAV8cMTM1 (> 2.5 × 1013 vg.kg−1), these cellular and physiological defects are rescued in X-linked myotubular myopathy (XLMTM) dogs (Figs. 4–6)
Summary
The centronuclear myopathies (CNM) are a group of genetically and clinically heterogeneous early-onset muscle disorders, with phenotypes varying from severe cases with antenatal/neonatal onset and death in infancy if untreated, to mild cases with onset in adolescence or adulthood and a stable or only slowly progressive course. The precise pathogenesis of XLMTM remains uncertain, several observations, including altered excitation–contraction coupling, reduced muscle fibre size, neuromuscular junction (NMJ) abnormalities and alterations to autophagy have been proposed as contributing mechanisms [17, 18]. Skeletal muscle fibres are large cells filled with contractile filaments, and contain many nuclei (often termed myonuclei) [30]. Nascent fibres possess myonuclei that are in the centre or core of the fibre, buried among myofibrils; these translocate to the periphery where they remain throughout maturity. During muscle regeneration, nascent or partially repaired fibres possess internal nuclei, which translocate to the periphery [30]. Myonuclei are regularly distributed throughout the fibre, allowing the control of gene transcription for a defined volume of cytoplasm termed the myonuclear domain (MND). The maintenance of an optimal MND size is essential for the intrinsic force-generating capacity of myofibres and overall cellular function [22, 28]
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