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Mutations in the MTM1 gene encoding myotubularin cause X-linked myotubular myopathy (XLMTM), a well-defined subtype of human centronuclear myopathy. Seven male Labrador Retrievers, age 14-26 wk, were clinically evaluated for generalized weakness and muscle atrophy. Muscle biopsies showed variability in fiber size, centrally placed nuclei resembling fetal myotubes, and subsarcolemmal ringed and central dense areas highlighted with mitochondrial specific reactions. Ultrastructural studies confirmed the centrally located nuclei, abnormal perinuclear structure, and mitochondrial accumulations. Wild-type triads were infrequent, with most exhibiting an abnormal orientation of T tubules. MTM1 gene sequencing revealed a unique exon 7 variant in all seven affected males, causing a nonconservative missense change, p.N155K, which haplotype data suggest derives from a recent founder in the local population. Analysis of a worldwide panel of 237 unaffected Labrador Retrievers and 59 additional control dogs from 25 other breeds failed to identify this variant, supporting it as the pathogenic mutation. Myotubularin protein levels and localization were abnormal in muscles from affected dogs, and expression of GFP-MTM1 p.N155K in COS-1 cells showed that the mutant protein was sequestered in proteasomes, where it was presumably misfolded and prematurely degraded. These data demonstrate that XLMTM in Labrador Retrievers is a faithful genetic model of the human condition. congenital myopathy | myotubularin | necklace fibers | canine myopathy | animal model doi/ 10.1073/pnas.1003677107

Skeletal muscle contraction is triggered by the excitation-contraction (E-C) coupling machinery residing at the triad, a membrane structure formed by the juxtaposition of T-tubules and sarcoplasmic reticulum (SR) cisternae. The formation and maintenance of this structure is key for muscle function but is not well characterized. We have investigated the mechanisms leading to X-linked myotubular myopathy (XLMTM), a severe congenital disorder due to loss of function mutations in the MTM1 gene, encoding myotubularin, a phosphoinositide phosphatase thought to have a role in plasma membrane homeostasis and endocytosis. Using a mouse model of the disease, we report that Mtm/-deficient muscle fibers have a decreased number of triads and abnormal longitudinally oriented T-tubules. In addition, SR [Ca.sup.2+] release elicited by voltage-clamp depolarizations is strongly depressed in myotubularin-deficient muscle fibers, with myoplasmic [Ca.sup.2+] removal and SR [Ca.sup.2+] content essentially unaffected. At the molecular level, Mtmldeficient myofibers exhibit a 3-fold reduction in type 1 ryanodine receptor (RyR1) protein level. These data reveal a critical role of myotubularin in the proper organization and function of the E-C coupling machinery and strongly suggest that defective RyR1mediated SR [Ca.sup.2+] release is responsible for the failure of muscle function in myotubular myopathy. myotubular myopathy | triad www.pnas.org/cgi/doi/ 10.1073/pnas.0900705106

Myotubularin is a ubiquitously expressed phosphatase that acts on phosphatidylinositol 3-monophosphate [PI(3)P], a lipid implicated in intracellular vesicle trafficking and autophagy. It is encoded by the MTM1 gene, which is mutated in X-linked myotubular myopathy (XLMTM), a muscular disorder characterized by generalized hypotonia and muscle weakness at birth leading to early death of most affected males. The disease was proposed to result from an arrest in myogenesis, as the skeletal muscle from patients contains hypotrophic fibers with centrally located nuclei that resemble fetal myotubes. To understand the physiopathological, mechanism of XLMTM, we have generated mice lacking myotubularin by homologous recombination. These mice are viable, but their lifespan is severely reduced. They develop a generalized and progressive myopathy starting at around 4 weeks of age, with amyotrophy and accumulation of central nuclei in skeletal muscle fibers leading to death at 6-14 weeks. Contrary to expectations, we show that muscle differentiation in knockout mice occurs normally. We provide evidence that fibers with centralized myonuclei originate mainly from a structural maintenance defect affecting myotubularin-deficient muscle rather than a regenerative process. In addition, we demonstrate, through a conditional gene-targeting approach, that skeletal muscle is the primary target of murine XLMTM pathology. These mutant mice represent animal models for the human disease and will be a valuable tool for understanding the physiological role of myotubularin.

Mutations in the gene encoding the phosphoinositide 3-phosphatase myotubularin (MTM1) are responsible for a pediatric disease of skeletal muscle named myotubular myopathy (XLMTM). Muscle fibers from MTM1-deficient mice present defects in excitation–contraction (EC) coupling likely responsible for the disease-associated fatal muscle weakness. However, the mechanism leading to EC coupling failure remains unclear. During normal skeletal muscle EC coupling, transverse (t) tubule depolarization triggers sarcoplasmic reticulum (SR) Ca2+ release through ryanodine receptor channels gated by conformational coupling with the t-tubule voltage-sensing dihydropyridine receptors. We report that MTM1 deficiency is associated with a 60% depression of global SR Ca2+ release over the full range of voltage sensitivity of EC coupling. SR Ca2+ release in the diseased fibers is also slower than in normal fibers, or delayed following voltage activation, consistent with the contribution of Ca2+-gated ryanodine receptors to EC coupling. In addition, we found that SR Ca2+ release is spatially heterogeneous within myotubularin-deficient muscle fibers, with focally defective areas recapitulating the global alterations. Importantly, we found that pharmacological inhibition of phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity rescues the Ca2+ release defects in isolated muscle fibers and increases the lifespan and mobility of XLMTM mice, providing proof of concept for the use of PtdIns 3-kinase inhibitors in myotubular myopathy and suggesting that unbalanced PtdIns 3-kinase activity plays a critical role in the pathological process. [ABSTRACT FROM AUTHOR]

Mutations in the gene encoding the phosphoinositide 3-phosphatase myotubularin (MTM1) are responsible for a pediatric disease of skeletal muscle named myotubular myopathy (XLMTM). Muscle fibers from MTM1-deficient mice present defects in excitation-contraction (EC) coupling likely responsible for the disease-associated fatal muscle weakness. However, the mechanism leading to EC coupling failure remains unclear. During normal skeletal muscle EC coupling, transverse (t) tubule depolarization triggers sarcoplasmic reticulum (SR) Ca 2+ release through ryanodine receptor channels gated by conformational coupling with the t-tubule voltage-sensing dihydropyridine receptors. We report that MTM1 deficiency is associated with a 60% depression of global SR Ca 2+ release over the full range of voltage sensitivity of EC coupling. SR Ca 2+ release in the diseased fibers is also slower than in normal fibers, or delayed following voltage activation, consistent with the contribution of Ca 2+ -gated ryanodine receptors to EC coupling. In addition, we found that SR Ca 2+ release is spatially heterogeneous within myotubularin-deficient muscle fibers, with focally defective areas recapitulating the global alterations. Importantly, we found that pharmacological inhibition of phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity rescues the Ca 2+ release defects in isolated muscle fibers and increases the lifespan and mobility of XLMTM mice, providing proof of concept for the use of PtdIns 3-kinase inhibitors in myotubular myopathy and suggesting that unbalanced PtdIns 3-kinase activity plays a critical role in the pathological process., Competing Interests: M.W.L. receives research funding support from Audentes Therapeutics, Solid GT, and Demeter Therapeutics; is a member of the Scientific Advisory Board of Audentes Therapeutics; and was recently a consultant for Sarepta Therapeutics. A.B.-B. is a scientific advisor of Audentes Therapeutics.