Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disease characterized by skeletal muscle wasting, weakness, and myotonia. DM1 is caused by the accumulation of CUG repeats, which alter the biological activities of RNA-binding proteins, including CUG-binding protein 1 (CUGBP1). CUGBP1 is an important skeletal muscle translational regulator that is activated by cyclin D3–dependent kinase 4 (CDK4). Here we show that mutant CUG repeats suppress Cdk4 signaling by increasing the stability and activity of glycogen synthase kinase 3β (GSK3β). Using a mouse model of DM1 (HSALR), we found that CUG repeats in the 3′ untranslated region (UTR) of human skeletal actin increase active GSK3β in skeletal muscle of mice, prior to the development of skeletal muscle weakness. Inhibition of GSK3β in both DM1 cell culture and mouse models corrected cyclin D3 levels and reduced muscle weakness and myotonia in DM1 mice. Our data predict that compounds normalizing GSK3β activity might be beneficial for improvement of muscle function in patients with DM1.
Karlie Jones, Christina Wei, Polina Iakova, Enrico Bugiardini, Christiane Schneider-Gold, Giovanni Meola, James Woodgett, James Killian, Nikolai A. Timchenko, Lubov T. Timchenko
ATR-X syndrome is a severe intellectual disability disorder caused by mutations in the ATRX gene. Many ancillary clinical features are attributed to CNS deficiencies, yet most patients have muscle hypotonia, delayed ambulation, or kyphosis, pointing to an underlying skeletal muscle defect. Here, we identified a cell-intrinsic requirement for Atrx in postnatal muscle growth and regeneration in mice. Mice with skeletal muscle–specific Atrx conditional knockout (Atrx cKO mice) were viable, but by 3 weeks of age presented hallmarks of underdeveloped musculature, including kyphosis, 20% reduction in body mass, and 34% reduction in muscle fiber caliber. Atrx cKO mice also demonstrated a marked regeneration deficit that was not due to fewer resident satellite cells or their inability to terminally differentiate. However, activation of Atrx-null satellite cells from isolated muscle fibers resulted in a 9-fold reduction in myoblast expansion, caused by delayed progression through mid to late S phase. While in S phase, Atrx colocalized specifically to late-replicating chromatin, and its loss resulted in rampant signs of genomic instability. These observations support a model in which Atrx maintains chromatin integrity during the rapid developmental growth of a tissue.
Michael S. Huh, Tina Price O’Dea, Dahmane Ouazia, Bruce C. McKay, Gianni Parise, Robin J. Parks, Michael A. Rudnicki, David J. Picketts
Cardiac natriuretic peptides (NP) are major activators of human fat cell lipolysis and have recently been shown to control brown fat thermogenesis. Here, we investigated the physiological role of NP on the oxidative metabolism of human skeletal muscle. NP receptor type A (NPRA) gene expression was positively correlated to mRNA levels of PPARγ coactivator-1α (PGC1A) and several oxidative phosphorylation (OXPHOS) genes in human skeletal muscle. Further, the expression of NPRA, PGC1A, and OXPHOS genes was coordinately upregulated in response to aerobic exercise training in human skeletal muscle. In human myotubes, NP induced PGC-1α and mitochondrial OXPHOS gene expression in a cyclic GMP–dependent manner. NP treatment increased OXPHOS protein expression, fat oxidation, and maximal respiration independent of substantial changes in mitochondrial proliferation and mass. Treatment of myotubes with NP recapitulated the effect of exercise training on muscle fat oxidative capacity in vivo. Collectively, these data show that activation of NP signaling in human skeletal muscle enhances mitochondrial oxidative metabolism and fat oxidation. We propose that NP could contribute to exercise training–induced improvement in skeletal muscle fat oxidative capacity in humans.
Stefan Engeli, Andreas L. Birkenfeld, Pierre-Marie Badin, Virginie Bourlier, Katie Louche, Nathalie Viguerie, Claire Thalamas, Emilie Montastier, Dominique Larrouy, Isabelle Harant, Isabelle de Glisezinski, Stefanie Lieske, Julia Reinke, Bibiana Beckmann, Dominique Langin, Jens Jordan, Cedric Moro
The native capacity of adult skeletal muscles to regenerate is vital to the recovery from physical injuries and dystrophic diseases. Currently, the development of therapeutic interventions has been hindered by the complex regulatory network underlying the process of muscle regeneration. Using a mouse model of skeletal muscle regeneration after injury, we identified hexamethylene bisacetamide inducible 1 (HEXIM1, also referred to as CLP-1), the inhibitory component of the positive transcription elongation factor b (P-TEFb) complex, as a pivotal regulator of skeletal muscle regeneration. Hexim1-haplodeficient muscles exhibited greater mass and preserved function compared with those of WT muscles after injury, as a result of enhanced expansion of satellite cells. Transplanted Hexim1-haplodeficient satellite cells expanded and improved muscle regeneration more effectively than WT satellite cells. Conversely, HEXIM1 overexpression restrained satellite cell proliferation and impeded muscle regeneration. Mechanistically, dissociation of HEXIM1 from P-TEFb and subsequent activation of P-TEFb are required for satellite cell proliferation and the prevention of early myogenic differentiation. These findings suggest a crucial role for the HEXIM1/P-TEFb pathway in the regulation of satellite cell–mediated muscle regeneration and identify HEXIM1 as a potential therapeutic target for degenerative muscular diseases.
Peng Hong, Kang Chen, Bihui Huang, Min Liu, Miao Cui, Inna Rozenberg, Brahim Chaqour, Xiaoyue Pan, Elisabeth R. Barton, Xian-Cheng Jiang, M.A.Q. Siddiqui
Dystroglycan is a transmembrane glycoprotein that links the extracellular basement membrane to cytoplasmic dystrophin. Disruption of the extensive carbohydrate structure normally present on α-dystroglycan causes an array of congenital and limb girdle muscular dystrophies known as dystroglycanopathies. The essential role of dystroglycan in development has hampered elucidation of the mechanisms underlying dystroglycanopathies. Here, we developed a dystroglycanopathy mouse model using inducible or muscle-specific promoters to conditionally disrupt fukutin (Fktn), a gene required for dystroglycan processing. In conditional Fktn-KO mice, we observed a near absence of functionally glycosylated dystroglycan within 18 days of gene deletion. Twenty-week-old KO mice showed clear dystrophic histopathology and a defect in glycosylation near the dystroglycan O-mannose phosphate, whether onset of Fktn excision driven by muscle-specific promoters occurred at E8 or E17. However, the earlier gene deletion resulted in more severe phenotypes, with a faster onset of damage and weakness, reduced weight and viability, and regenerating fibers of smaller size. The dependence of phenotype severity on the developmental timing of muscle Fktn deletion supports a role for dystroglycan in muscle development or differentiation. Moreover, given that this conditional Fktn-KO mouse allows the generation of tissue- and timing-specific defects in dystroglycan glycosylation, avoids embryonic lethality, and produces a phenotype resembling patient pathology, it is a promising new model for the study of secondary dystroglycanopathy.
Aaron M. Beedle, Amy J. Turner, Yoshiaki Saito, John D. Lueck, Steven J. Foltz, Marisa J. Fortunato, Patricia M. Nienaber, Kevin P. Campbell
Skeletal muscle injury activates adult myogenic stem cells, known as satellite cells, to initiate proliferation and differentiation to regenerate new muscle fibers. The skeletal muscle–specific microRNA miR-206 is upregulated in satellite cells following muscle injury, but its role in muscle regeneration has not been defined. Here, we show that miR-206 promotes skeletal muscle regeneration in response to injury. Genetic deletion of miR-206 in mice substantially delayed regeneration induced by cardiotoxin injury. Furthermore, loss of miR-206 accelerated and exacerbated the dystrophic phenotype in a mouse model of Duchenne muscular dystrophy. We found that miR-206 acts to promote satellite cell differentiation and fusion into muscle fibers through suppressing a collection of negative regulators of myogenesis. Our findings reveal an essential role for miR-206 in satellite cell differentiation during skeletal muscle regeneration and indicate that miR-206 slows progression of Duchenne muscular dystrophy.
Ning Liu, Andrew H. Williams, Johanna M. Maxeiner, Svetlana Bezprozvannaya, John M. Shelton, James A. Richardson, Rhonda Bassel-Duby, Eric N. Olson
Mutations in the E3 ubiquitin ligase tripartite motif-containing 32 (TRIM32) are responsible for the disease limb-girdle muscular dystrophy 2H (LGMD2H). Previously, we generated Trim32 knockout mice (Trim32–/– mice) and showed that they display a myopathic phenotype accompanied by neurogenic features. Here, we used these mice to investigate the muscle-specific defects arising from the absence of TRIM32, which underlie the myopathic phenotype. Using 2 models of induced atrophy, we showed that TRIM32 is dispensable for muscle atrophy. Conversely, TRIM32 was necessary for muscle regrowth after atrophy. Furthermore, TRIM32-deficient primary myoblasts underwent premature senescence and impaired myogenesis due to accumulation of PIAS4, an E3 SUMO ligase and TRIM32 substrate that was previously shown to be associated with senescence. Premature senescence of myoblasts was also observed in vivo in an atrophy/regrowth model. Trim32–/– muscles had substantially fewer activated satellite cells, increased PIAS4 levels, and growth failure compared with wild-type muscles. Moreover, Trim32–/– muscles exhibited features of premature sarcopenia, such as selective type II fast fiber atrophy. These results imply that premature senescence of muscle satellite cells is an underlying pathogenic feature of LGMD2H and reveal what we believe to be a new mechanism of muscular dystrophy associated with reductions in available satellite cells and premature sarcopenia.
Elena Kudryashova, Irina Kramerova, Melissa J. Spencer
Hypokalemic periodic paralysis (HypoPP) is an ion channelopathy of skeletal muscle characterized by attacks of muscle weakness associated with low serum K+. HypoPP results from a transient failure of muscle fiber excitability. Mutations in the genes encoding a calcium channel (CaV1.1) and a sodium channel (NaV1.4) have been identified in HypoPP families. Mutations of NaV1.4 give rise to a heterogeneous group of muscle disorders, with gain-of-function defects causing myotonia or hyperkalemic periodic paralysis. To address the question of specificity for the allele encoding the NaV1.4-R669H variant as a cause of HypoPP and to produce a model system in which to characterize functional defects of the mutant channel and susceptibility to paralysis, we generated knockin mice carrying the ortholog of the gene encoding the NaV1.4-R669H variant (referred to herein as R669H mice). Homozygous R669H mice had a robust HypoPP phenotype, with transient loss of muscle excitability and weakness in low-K+ challenge, insensitivity to high-K+ challenge, dominant inheritance, and absence of myotonia. Recovery was sensitive to the Na+/K+-ATPase pump inhibitor ouabain. Affected fibers had an anomalous inward current at hyperpolarized potentials, consistent with the proposal that a leaky gating pore in R669H channels triggers attacks, whereas a reduction in the amplitude of action potentials implies additional loss-of-function changes for the mutant NaV1.4 channels.
Fenfen Wu, Wentao Mi, Dennis K. Burns, Yu Fu, Hillery F. Gray, Arie F. Struyk, Stephen C. Cannon
IL-15 receptor α (IL-15Rα) is a component of the heterotrimeric plasma membrane receptor for the pleiotropic cytokine IL-15. However, IL-15Rα is not merely an IL-15 receptor subunit, as mice lacking either IL-15 or IL-15Rα have unique phenotypes. IL-15 and IL-15Rα have been implicated in muscle phenotypes, but a role in muscle physiology has not been defined. Here, we have shown that loss of IL-15Rα induces a functional oxidative shift in fast muscles, substantially increasing fatigue resistance and exercise capacity. IL-15Rα–knockout (IL-15Rα–KO) mice ran greater distances and had greater ambulatory activity than controls. Fast muscles displayed fatigue resistance and a slower contractile phenotype. The molecular signature of these muscles included altered markers of mitochondrial biogenesis and calcium homeostasis. Morphologically, fast muscles had a greater number of muscle fibers, smaller fiber areas, and a greater ratio of nuclei to fiber area. The alterations of physiological properties and increased resistance to fatigue in fast muscles are consistent with a shift toward a slower, more oxidative phenotype. Consistent with a conserved functional role in humans, a genetic association was found between a SNP in the IL15RA gene and endurance in athletes stratified by sport. Therefore, we propose that IL-15Rα has a role in defining the phenotype of fast skeletal muscles in vivo.
Emidio E. Pistilli, Sasha Bogdanovich, Fleur Garton, Nan Yang, Jason P. Gulbin, Jennifer D. Conner, Barbara G. Anderson, LeBris S. Quinn, Kathryn North, Rexford S. Ahima, Tejvir S. Khurana
MicroRNAs modulate cellular phenotypes by inhibiting expression of mRNA targets. In this study, we have shown that the muscle-specific microRNAs miR-133a-1 and miR-133a-2 are essential for multiple facets of skeletal muscle function and homeostasis in mice. Mice with genetic deletions of miR-133a-1 and miR-133a-2 developed adult-onset centronuclear myopathy in type II (fast-twitch) myofibers, accompanied by impaired mitochondrial function, fast-to-slow myofiber conversion, and disarray of muscle triads (sites of excitation-contraction coupling). These abnormalities mimicked human centronuclear myopathies and could be ascribed, at least in part, to dysregulation of the miR-133a target mRNA that encodes dynamin 2, a GTPase implicated in human centronuclear myopathy. Our findings reveal an essential role for miR-133a in the maintenance of adult skeletal muscle structure, function, bioenergetics, and myofiber identity; they also identify a potential modulator of centronuclear myopathies.
Ning Liu, Svetlana Bezprozvannaya, John M. Shelton, Madlyn I. Frisard, Matthew W. Hulver, Ryan P. McMillan, Yaru Wu, Kevin A. Voelker, Robert W. Grange, James A. Richardson, Rhonda Bassel-Duby, Eric N. Olson