Differential Muscle Hypertrophy Is Associated with Satellite Cell Numbers and Akt Pathway Activation Following Activin Type IIB Receptor Inhibition in Mtm1 p.R69C Mice

X-linked myotubular myopathy is a congenital myopathy caused by deficiency of myotubularin. Patients often present with severe perinatal weakness, requiring mechanical ventilation to prevent death from respiratory failure. We recently reported that an activin receptor type IIB inhibitor produced hypertrophy of type 2b myofibers and modest increases of strength and life span in the severely myopathic Mtm1δ4 mouse model of X-linked myotubular myopathy. We have now performed a similar study in the less severely symptomatic Mtm1 p.R69C mouse in hopes of finding greater treatment efficacy. Activin receptor type IIB inhibitor treatment of Mtm1 p.R69C animals produced behavioral and histological evidence of hypertrophy in gastrocnemius muscles but not in quadriceps or triceps. The ability of the muscles to respond to activin receptor type IIB inhibitor treatment correlated with treatment-induced increases in satellite cell number and several muscle-specific abnormalities of hypertrophic signaling. Treatment-responsive Mtm1 p.R69C gastrocnemius muscles displayed lower levels of phosphorylated ribosomal protein S6 and higher levels of phosphorylated eukaryotic elongation factor 2 kinase than were observed in Mtm1 p.R69C quadriceps muscle or in muscles from wild-type littermates. Hypertrophy in the Mtm1 p.R69C gastrocnemius muscle was associated with increased levels of phosphorylated ribosomal protein S6. Our findings indicate that muscle-, fiber type-, and mutation-specific factors affect the response to hypertrophic therapies that will be important to assess in future therapeutic trials.X-linked myotubular myopathy (XLMTM) is a severe form of congenital myopathy with an estimated incidence of 1:50,000 male births that most often presents with severe perinatal weakness and respiratory failure.^1,2 Many patients with XLMTM die within the first year of life despite the use of mechanical ventilation, and no treatments approved by the Food and Drug Administration are available. XLMTM is caused by mutations in the gene that encodes myotubularin (MTM1), which is a phosphoinositide phosphatase thought to be involved in endosomal trafficking, cytoskeletal organization, apoptosis, and/or maintenance of the sarcoplasmic reticulum/T-tubular system within myofibers.^3–8 Muscle biopsies from patients with XLMTM display excessively small fibers with increased numbers of fibers that contain central nuclei and central aggregation of organelles.⁹ Although the number of centrally nucleated fibers bears little relationship to a patient''s prognosis, there is a clear correlation between the degree of fiber smallness at birth and the severity of the patients'' disease.¹⁰ Two murine models of myotubularin deficiency are used, the severely symptomatic Mtm1δ4 (also referred to as Mtm1 knockout in prior studies^3,11,12) and the moderately symptomatic Mtm1 p.R69C mice,¹³ both of which display weakness and myofiber smallness and similar pathology to that seen in XLMTM.Because of the relationship between myofiber size and symptomatic severity in patients with XLMTM and in Mtm1δ4 mice, we had previously hypothesized that correction of myofiber smallness in myotubularin deficiency would greatly improve strength. Inhibitors of myostatin or nonfunctional decoys of its receptor, the activin type IIB receptor (ActRIIB), can be used to inhibit this negative regulator of myofiber size, leading to myofiber hypertrophy. Myostatin binds to (and signals through) the ActRIIB to activate the transforming growth factor-β pathway, which prevents progression through the cell cycle and down-regulates several key processes related to myofiber hypertrophy.^14,15 We recently reported a trial of ActRIIB-mFC in Mtm1δ4 mice, which produced 17% extension of life span, with transient increases in weight, forelimb grip strength, myofiber size, and myofiber hypertrophy restricted to type 2b myofibers in Mtm1δ4 animals.¹² Interestingly, ActRIIB-mFc produces hypertrophy in all muscle fiber types in wild-type (WT) mice,^12,16 which suggests that myotubularin deficiency interferes with the activation of hypertrophic pathways in oxidative fibers.We hypothesized that the transience of the therapeutic effects observed in treated Mtm1δ4 mice may have been related to the severity of the disease, so we have now repeated this study in the less severely affected Mtm1 p.R69C mouse.¹³ Surprisingly, treatment of Mtm1 p.R69C mice did not produce significant increases in animal weight or grip strength, and treatment-induced myofiber hypertrophy was only observed in the Mtm1 p.R69C gastrocnemius muscles. The ability of these muscles to respond to ActRIIB-mFC treatment correlated with treatment-induced increases in satellite cell number and several muscle-specific abnormalities of hypertrophic signaling. The main difference between treatment-responsive (gastrocnemius) and treatment-resistant (quadriceps) muscles in Mtm1 p.R69C mice was related to low levels of phosphorylated ribosomal protein 6 (p-rpS6) and high levels of eukaryotic elongation factor 2 kinase (eEF2K) in the treatment-responsive gastrocnemius muscle that were not observed in other Mtm1 p.R69C muscles or in WT mice. rpS6 and eEF2K are terminal signaling molecules of the insulinlike growth factor-1/Akt and extracellular signal-related kinase (ERK) pathways that are involved in the fine-tuning of global protein synthesis, with a role in the determination of cell size that remains unclear (reviewed in Meyuhas¹⁷). Our findings indicate that the response to hypertrophic agents does not always correlate with activities of known hypertrophic pathways, such as the Akt pathway, but unexpectedly varies both by muscle type and fiber type and in XLMTM is affected by the nature of the Mtm1 mutation. These results highlight that there is much we still do not understand about the control of muscle size and emphasize the importance of evaluating multiple muscle and fiber types in future trials of hypertrophic therapies.     

AAV-mediated intramuscular delivery of myotubularin corrects the myotubular myopathy phenotype in targeted murine muscle and suggests a function in plasma membrane homeostasis

Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent occurrence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insight into its localization and function. We show that a substantial proportion of myotubularin associates with the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibers. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle.     

Myotubularin-deficient myoblasts display increased apoptosis, delayed proliferation, and poor cell engraftment

X-linked myotubular myopathy is a severe congenital myopathy caused by deficiency of the lipid phosphatase, myotubularin. Recent studies of human tissue and animal models have discovered structural and physiological abnormalities in myotubularin-deficient muscle, but the impact of myotubularin deficiency on myogenic stem cells within muscles is unclear. In the present study, we evaluated the viability, proliferative capacity, and in vivo engraftment of myogenic cells obtained from severely symptomatic (Mtm1δ4) myotubularin-deficient mice. Mtm1δ4 muscle contains fewer myogenic cells than wild-type (WT) littermates, and the number of myogenic cells decreases with age. The behavior of Mtm1δ4 myoblasts is also abnormal, because they engraft poorly into C57BL/6/Rag1null/mdx5cv mice and display decreased proliferation and increased apoptosis compared with WT myoblasts. Evaluation of Mtm1δ4 animals at 21 and 42 days of life detected fewer satellite cells in Mtm1δ4 muscle compared with WT littermates, and the decrease in satellite cells correlated with progression of disease. In addition, analysis of WT and Mtm1δ4 regeneration after injury detected similar abnormalities of satellite cell function, with fewer satellite cells, fewer dividing cells, and increased apoptotic cells in Mtm1δ4 muscle. These studies demonstrate specific abnormalities in myogenic cell number and behavior that may relate to the progression of disease in myotubularin deficiency, and may also be used to develop in vitro assays by which novel treatment strategies can be assessed.     

Modeling the human MTM1 p.R69C mutation in murine Mtm1 results in exon 4 skipping and a less severe myotubular myopathy phenotype

X-linked myotubular myopathy (MTM) is a severe neuromuscular disease of infancy caused by mutations of MTM1, which encodes the phosphoinositide lipid phosphatase, myotubularin. The Mtm1 knockout (KO) mouse has a severe phenotype and its short lifespan (8 weeks) makes it a challenge to use as a model in the testing of certain preclinical therapeutics. Many MTM patients succumb early in life, but some have a more favorable prognosis. We used human genotype-phenotype correlation data to develop a myotubularin-deficient mouse model with a less severe phenotype than is seen in Mtm1 KO mice. We modeled the human c.205C>T point mutation in Mtm1 exon 4, which is predicted to introduce the p.R69C missense change in myotubularin. Hemizygous male Mtm1 p.R69C mice develop early muscle atrophy prior to the onset of weakness at 2 months. The median survival period is 66 weeks. Histopathology shows small myofibers with centrally placed nuclei. Myotubularin protein is undetectably low because the introduced c.205C>T base change induced exon 4 skipping in most mRNAs, leading to premature termination of myotubularin translation. Some full-length Mtm1 mRNA bearing the mutation is present, which provides enough myotubularin activity to account for the relatively mild phenotype, as Mtm1 KO and Mtm1 p.R69C mice have similar muscle phosphatidylinositol 3-phosphate levels. These data explain the basis for phenotypic variability among human patients with MTM1 p.R69C mutations and establish the Mtm1 p.R69C mouse as a valuable model for the disease, as its less severe phenotype will expand the scope of testable preclinical therapies.     

Identification of novel mutations in the MTM1 gene causing severe and mild forms of X-linked myotubular myopathy.

X-linked myotubular myopathy (XLMTM) is a congenital muscular disease characterized by severe hypotonia and generalized muscle weakness, leading in most cases to early postnatal death. The gene responsible for the disease, MTM1, encodes a dual specificity phosphatase, named myotubularin, which is highly conserved throughout evolution. To date, 139 MTM1 mutations in independent patients have been reported, corresponding to 93 different mutations. In this report we describe the identification of 21 mutations (14 novel) in XLMTM patients. Seventeen mutations are associated with a severe phenotype in males, with death occurring mainly before the first year of life. However, four mutations-three missense (R241C, I225T, and novel mutation P179S) and one single-amino acid deletion (G294del)-were found in patients with a much milder phenotype. These patients, while having a severe hypotonia at birth, are still alive at the age of 4, 7, 13, and 15 years, respectively, and display mild to moderate muscle weakness.     

Mutations in the MTM1 gene implicated in X-linked myotubular myopathy. ENMC International Consortium on Myotubular Myopathy. European Neuro- Muscular Center

X-linked recessive myotubular myopathy (XLMTM) is characterized by severe hypotonia and generalized muscle weakness, with impaired maturation of muscle fibres. The gene responsible, MTM1, was identified recently by positional cloning, and encodes a protein (myotubularin) with a tyrosine phosphatase domain (PTP). Myotubularin is highly conserved through evolution and defines a new family of putative tyrosine phosphatases in man. We report the identification of MTM1 mutations in 55 of 85 independent patients screened by single-strand conformation polymorphism for all the coding sequence. Large deletions were observed in only three patients. Five point mutations were found in multiple unrelated patients, accounting for 27% of the observed mutations. The possibility of detecting mutations and determining carrier status in a disease with a high proportion of sporadic cases is of importance for genetic counselling. More than half of XLMTM mutations are expected to inactivate the putative enzymatic activity of myotubularin, either by truncation or by missense mutations affecting the predicted PTP domain. Additional mutations are missenses clustered in two regions of the protein. Most of these affect amino acids conserved in the homologous yeast and Caenorhabditis elegans proteins, thus indicating the presence of other functional domains.      

Targeting the activin type IIB receptor to improve muscle mass and function in the mdx mouse model of Duchenne muscular dystrophy

The activin receptor type IIB (ActRIIB) is a transmembrane receptor for transforming growth factor-β superfamily members, including myostatin, that are involved in the negative regulation of skeletal muscle mass. We tested the translational hypothesis that blocking ligand binding to ActRIIB for 12 weeks would stimulate skeletal muscle growth and improve muscle function in the mdx mouse. ActRIIB was targeted using a novel inhibitor comprised of the extracellular portion of the ActRIIB fused to the Fc portion of murine IgG (sActRIIB), at concentrations of 1.0 and 10.0 mg/kg(-1) body weight. After 12 weeks of treatment, the 10.0 mg/kg(-1) dose caused a 27% increase in body weight with a concomitant 33% increase in lean muscle mass. Absolute force production of the extensor digitorum longus muscle ex vivo was higher in mice after treatment with either dose of sActRIIB, and the specific force was significantly higher after the lower dose (1.0 mg/kg(-1)), indicating functional improvement in the muscle. Circulating creatine kinase levels were significantly lower in mice treated with sActRIIB, compared with control mice. These data show that targeting the ActRIIB improves skeletal muscle mass and functional strength in the mdx mouse model of DMD, providing a therapeutic rationale for use of this molecule in treating skeletal myopathies.     

Increased IGF-1 in muscle modulates the phenotype of severe SMA mice

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by the mutation of the survival motor neuron 1 (SMN1) gene and deficiency of the SMN protein. Severe SMA mice have abnormal motor function and small, immature myofibers early in development suggesting that SMN protein deficiency results in retarded muscle growth. Insulin-like growth factor 1 (IGF-1) stimulates myoblast proliferation, induces myogenic differentiation and generates myocyte hypertrophy in vitro and in vivo. We hypothesized that increased expression of IGF-1 specifically in skeletal muscle would attenuate disease features of SMAΔ7 mice. SMAΔ7 mice overexpressing a local isoform of IGF-1 (mIGF-1) in muscle showed enlarged myofibers and a 40% increase in median survival compared with mIGF-1-negative SMA littermates (median survival = 14 versus 10 days, respectively, log-rank P = 0.025). Surprisingly, this was not associated with a significant improvement in motor behavior. Treatment of both mIGF-1(NEG) and mIGF-1(POS) SMA mice with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in a further extension of survival and improved motor behavior, but the combination of mIGF-1 and TSA treatment was not synergistic. These results show that increased mIGF-1 expression restricted to muscle can modulate the phenotype of SMA mice indicating that therapeutics targeted to muscle alone should not be discounted as potential disease-modifying therapies in SMA. IGF-1 may warrant further investigation in mild SMA animal models and perhaps SMA patients.     

Germline mosaicism in X-linked myotubular myopathy

X-linked myotubular myopathy (XLMTM; OMIM310400) is a congenital muscle disorder characterized by severe hypotonia and respiratory insufficiency. The disorder was mapped to Xq28 by linkage studies and the MTM1 gene was isolated by positional cloning. The gene product is a 603 amino acid protein named myotubularin. A small domain in its sequence shows high homology to a consensus active site of tyrosine phosphatases, a diverse class of proteins involved in signal transduction, control of cell growth, and differentiation. In this report, two brothers affected with XLMTM are shown to have a point mutation (G1187A) in exon 11 of the MTM1 gene. Surprisingly, their mother does not have this mutation in her lymphocytes. Therefore, she likely has a germline mosaicism. As this is the third report of germline mosaicism in XLMTM, the finding has important implications for genetic counseling.     

Skeletal muscle in the diabetic mouse: histochemical and morphometric analysis

Despite the extensive literature concerning the neuropathy associated with diabetes, only limited information describes changes in the associated muscle. The objective of this study was to evaluate the histochemical and morphometric characteristics of diabetic muscle in the C57BL/KsJ db-m strain of mouse. The histochemical analysis of myofiber type for the diabetic mouse revealed that the extensor digitorum longus muscle consisted of 53.1% type 2a, 46.0% type 2b, and 0.9% type 1 myofibers, a significant shift from the percentages found in the nondiabetic litter mates (44.4% type 2a, 55.6% type 2b, no type 1). Computer-assisted morphometric analysis of myofiber size by fiber type indicated a significant difference in myofiber size for the type 2b fibers in muscles from diabetic mice. Similarly, there was a shift in the fiber size distribution to include a greater number of small type 2b myofibers when compared to controls. Skeletal muscle from diabetic mice exhibited a significant change in the percentage of fiber types, with an increase in the number of type 2a fibers, a fiber type grouping that implies possible denervation and reinnervation, and a decrease in myofiber size. These findings may explain why some diabetic patients complain of muscle weakness.     

X-linked myotubular myopathy in a family with two infant siblings: a case with MTM1 mutation

X-linked myotubular myopathy (XLMTM) is a rare congenital muscle disorder, caused by mutations in the MTM1 gene. Affected male infants present severe hypotonia, and generalized muscle weakness, and the disorder is most often complicated by respiratory failure. Herein, we describe a family with 2 infants with XLMTM which was diagnosed by gene analysis and muscle biopsy. In both cases, histological findings of muscle showed severely hypoplastic muscle fibers with centrally placed nuclei. From the family gene analysis, the Arg486STOP mutation in the MTM1 gene was confirmed.     

Degenerative and regenerative features of myofibers differ among skeletal muscles in a murine model of muscular dystrophy

Skeletal muscle myofibers constantly undergo degeneration and regeneration. Histopathological features of 6 skeletal muscles (cranial tibial [CT], gastrocnemius, quadriceps femoris, triceps brachii [TB], lumbar longissimus muscles, and costal part of the diaphragm [CPD]) were compared using C57BL/10ScSn-Dmd ^mdx (mdx) mice, a model for muscular dystrophy versus control, C57BL/10 mice. Body weight and skeletal muscle mass were lower in mdx mice than the control at 4 weeks of age; these results were similar at 6–30 weeks. Additionally, muscular lesions were observed in all examined skeletal muscles in mdx mice after 4 weeks, but none were noted in the controls. Immunohistochemical staining revealed numerous paired box 7-positive satellite cells surrounding the embryonic myosin heavy chain-positive regenerating myofibers, while the number of the former and staining intensity of the latter decreased as myofiber regeneration progressed. Persistent muscular lesions were observed in skeletal muscles of mdx mice between 4 and 14 weeks of age, and normal myofibers decreased with age. Number of muscular lesions was lowest in CPD at all ages examined, while the ratio of normal myofibers was lowest in TB at 6 weeks. In CT, TB, and CPD, Iba1-positive macrophages, the main inflammatory cells in skeletal muscle lesions, showed a significant positive correlation with the appearance of regenerating myofibers. Additionally, B220-positive B-cells showed positive and negative correlation with regenerating and regenerated myofibers, respectively. Our data suggest that degenerative and regenerative features of myofibers differ among skeletal muscles and that inflammatory cells are strongly associated with regenerative features of myofibers in mdx mice.     

Myotubularin, a phosphatase deficient in myotubular myopathy, acts on phosphatidylinositol 3-kinase and phosphatidylinositol 3-phosphate pathway

Myotubular myopathy (MTM1) is an X-linked disease, characterized by severe neonatal hypotonia and generalized muscle weakness, with pathological features suggesting an impairment in maturation of muscle fibres. The MTM1 gene encodes a protein (myotubularin) with a phosphotyrosine phosphatase consensus. It defines a family of at least nine genes in man, including the antiphosphatase hMTMR5/Sbf1 and hMTMR2, recently found mutated in a recessive form of Charcot-Marie-Tooth disease. Myotubularin shows a dual specificity protein phosphatase activity in vitro. We have performed an in vivo test of tyrosine phosphatase activity in Schizosaccharomyces pombe, indicating that myotubularin does not have a broad specificity tyrosine phosphatase activity. Expression of active human myotubularin inhibited growth of S.pombe and induced a vacuolar phenotype similar to that of mutants of the vacuolar protein sorting (VPS) pathway and notably of mutants of VPS34, a phosphatidylinositol 3-kinase (PI3K). In S.pombe cells deleted for the endogenous MTM homologous gene, expression of human myotubularin decreased the level of phosphatidylinositol 3-phosphate (PI3P). We have created a substrate trap mutant which shows relocalization to plasma membrane projections (spikes) in HeLa cells and was inactive in the S.pombe assay. This mutant, but not the wild-type or a phosphatase site mutant, was able to immunoprecipitate a VPS34 kinase activity. Wild-type myotubularin was also able to directly dephosphorylate PI3P and PI4P in vitro. Myotubularin may thus decrease PI3P levels by down-regulating PI3K activity and by directly degrading PI3P.     

Laminin-111 Restores Regenerative Capacity in a Mouse Model for α7 Integrin Congenital Myopathy

Mutations in the α7 integrin gene cause congenital myopathy characterized by delayed developmental milestones and impaired mobility. Previous studies in dystrophic mice suggest the α7β1 integrin may be critical for muscle repair. To investigate the role that α7β1 integrin plays in muscle regeneration, cardiotoxin was used to induce damage in the tibialis anterior muscle of α7 integrin-null mice. Unlike wild-type muscle, which responded rapidly to repair damaged myofibers, α7 integrin-deficient muscle exhibited defective regeneration. Analysis of Pax7 and MyoD expression revealed a profound delay in satellite cell activation after cardiotoxin treatment in α7 integrin-null animals when compared with wild type. We have recently demonstrated that the muscle of α7 integrin-null mice exhibits reduced laminin-α2 expression. To test the hypothesis that loss of laminin contributes to the defective muscle regeneration phenotype observed in α7 integrin-null mice, mouse laminin-111 (α1, β1, γ1) protein was injected into the tibialis anterior muscle 3 days before cardiotoxin-induced injury. The injected laminin-111 protein infiltrated the entire muscle and restored myogenic repair and muscle regeneration in α7 integrin-null muscle to wild-type levels. Our data demonstrate a critical role for a laminin-rich microenvironment in muscle repair and suggest laminin- 111 protein may serve as an unexpected and novel therapeutic agent for patients with congenital myopathies.     

Altered expression of ARPP protein in skeletal muscles of patients with muscular dystrophy, congenital myopathy and spinal muscular atrophy.

OBJECTIVES: Ankyrin-repeated protein with PEST and a proline-rich region (ARPP) is a recently identified protein with 4 ankyrin-repeated motifs in its central portion. Type 1 myofibers of skeletal muscle express high levels of ARPP. Recently, we have found that ARPP expression was induced in mouse denervated skeletal muscle. This led us to hypothesize that ARPP expression might be induced in skeletal muscle under some pathological conditions. METHODS: In this study, we performed immunohistochemical analysis of ARPP expression in biopsy specimens of muscle tissue from 15 patients with muscular dystrophies (MDs), 13 with congenital myopathies and 11 with spinal muscular atrophies (SMAs). RESULTS: The ARPP expression levels of all the specimens from MD patients appeared to be lower than control muscle levels. In contrast, the specimens from the 13 patients with congenital myopathies were all ARPP positive. We also found increased numbers of ARPP-positive myofibers in patients with congenital myopathies, and these myofibers co-expressed the slow myosin heavy chain. Indeed, it has been reported that type 1 myofibers are predominant in patients with congenital myopathies, suggesting that increased numbers of ARPP-positive myofibers in such patients may be associated with increased numbers of type 1 fibers. In patients with SMAs, we found that ARPP-positive myofibers tended to be distributed in groups. As grouped myofibers have been reported to result from the process of denervation, innervation and subsequent denervation of re-innervated myofibers, the grouped ARPP-positive myofibers in SMA patients may result from denervation of the motor units. CONCLUSIONS: These findings suggest that evaluation of ARPP may be helpful for the histological diagnosis of muscle diseases.     

Myosatellite cells,growth, and regeneration in murine dystrophic muscle: A quantitative study

Patterns of growth and regeneration in 2-, 4-, 8-, and 17-week-old murine dystrophic (129 ReJ dy/dy) extensor digitorum longus muscles have been determined. Necrosis and myofiber loss, hypertrophy, and regeneration result in a reduced population of myofibers whose diameter distribution is more extensive than that found in the extensor digitorum longus muscles of age-matched normal mice. At the onset of dystrophic symptoms (2 weeks postnatal), the ratio of myosatellite cell nuclei to the total sublaminal nuclear population (myonuclei + myosatellite cells) is similar to that found in 2-week-old control muscles. The frequency of finding myosatellite cells decreases with age in both control and dystrophic muscles. Myosatellite cells account for 11%, 6%, 5%, and 3% of the total sublaminal nuclear population in control muscle and 12%, 8%, 6%, and 5% of the total sublaminal nuclear population in dystrophic muscle at 2, 4, 8, and 17 weeks, respectively. No preferential association of myosatellite cells with myofibers of a particular diameter is found in control muscle or in the two youngest dystrophic groups. At 8 and 17 weeks, myosatellite cells are less frequently encountered on small-diameter, regenerating myofibers of dystrophic muscle, and they are preferentially associated with large diameter, hypertrophied myofibers. The labeling index of myosatellite cells decreases with age in both normal and dystrophic muscle. At all ages the myosatellite cell labeling index is higher in dystrophic muscle (23%, 7%, 5%, and 2% at 2, 4, 8, and 17 weeks, respectively) than in normal muscle (5%, < 1% at 2 and 4 weeks, respectively), with no labeled myosatellite cells being found in 8- and 17-week-old normal muscles. It is suggested that the magnitude of the regenerative response of dystrophic murine muscle decreases with age and that this factor may be responsible for the inability of the regenerative response of dystrophic muscle to keep pace with the rapid muscle deterioration.     

Branched myofibers in long-term whole muscle transplants: a quantitative study

Orthotopic transplants of whole extensor digitorum longus muscles were performed on six 4-6-week-old 129 ReJ mice. One hundred days posttransplantation, the animals were killed and the regenerated muscles were processed for electron microscopy. The grafts contained polygonal-shaped myofibers with persistent central nuclei, organized into discrete muscle fascicles. No central area of fatty infiltration or fibrosis was observed. The mean number of myofibers in a regenerating transplanted muscle, as determined from an ultrathin section taken from the graft's widest girth, was 631 (SEM = +/- 59), a reduction of approximately 32% from that found in age-matched control muscle (Ontell et al., 1983). By following the myofibers in spaced, serial ultrathin sections along their length, it was found that the branched, regenerating myofibers found in immature grafts of normal muscle (Ontell et al., 1982) persisted in stabilized, long-term transplanted muscle. The frequency of branching was determined by following each fiber found at the widest girths of four of the grafts in spaced, serial ultrathin sections (15-micron intervals) for approximately 2% of the total length of the grafts. Over this distance, 6.6% of the fibers were involved in the branching phenomenon. The persistence of branched fibers in long-term grafts and the frequency with which the branching phenomenon was found to occur may have physiological consequences and should be investigated.     

Genetic Background Affects Properties of Satellite Cells and mdx Phenotypes

Duchenne muscular dystrophy (DMD) is the most common lethal genetic disorder of children. The mdx (C57BL/10 background, C57BL/10-mdx) mouse is a widely used model of DMD, but the histopathological hallmarks of DMD, such as the smaller number of myofibers, accumulation of fat and fibrosis, and insufficient regeneration of myofibers, are not observed in adult C57BL/10-mdx except for in the diaphragm. In this study, we showed that DBA/2 mice exhibited decreased muscle weight, as well as lower myofiber numbers after repeated degeneration–regeneration cycles. Furthermore, the self-renewal efficiency of satellite cells of DBA/2 is lower than that of C57BL/6. Therefore, we produced a DBA/2-mdx strain by crossing DBA/2 and C57BL/10-mdx. The hind limb muscles of DBA/2-mdx mice exhibited lower muscle weight, fewer myofibers, and increased fat and fibrosis, in comparison with C57BL/10-mdx. Moreover, remarkable muscle weakness was observed in DBA/2-mdx. These results indicate that the DBA/2-mdx mouse is a more suitable model for DMD studies, and the efficient satellite cell self-renewal ability of C57BL/10-mdx might explain the difference in pathologies between humans and mice.Duchenne muscular dystrophy (DMD) is a progressive and lethal X-linked muscular disorder caused by mutations in the dystrophin gene.¹ The dystrophin gene encodes a 427-kDa cytoskeletal protein that forms the dystrophin/glycoprotein complex at the sarcolemma with α- and β-dystroglycans, α-, β-, γ-, and δ-sarcoglycans, and other molecules, and links the cytoskeleton of myofibers to the extracellular matrix in skeletal muscle.^2,3 The lack of dystrophin in the sarcolemma disturbs the assembly of the dystrophin/glycoprotein complex and causes instability of the muscle membrane, leading to muscle degeneration and myofiber loss. The histopathological hallmarks of DMD include degeneration, necrosis, accumulation of fat and fibrosis, and insufficient regeneration of myofibers accompanied by a loss of myofibers.⁴ Therefore, the manifestations of DMD are considered to result from an imbalance between degeneration and regeneration.The function and structure of dystrophin has been elucidated by studies of a variety of dystrophin-deficient animals. Among these animal models, the mdx mouse (the correct nomenclature is C57BL/10-Dmd^mdx), first described in1984, is the most prolific. A spontaneous mutation (mdx) arose in an inbred colony of C57BL/10 mice, which have a high level of serum pyruvate kinase.⁵ The muscle pathology of the mice includes active fiber necrosis, cellular infiltration, a wide range of fiber sizes, and numerous centrally nucleated regenerating fibers. However, in contrast to DMD, replacement of muscle with fat and fibrosis is not prominent, and no losses of muscle fiber and muscle weight are observed in the skeletal muscle of mdx mice except in the diaphragm.^6,7 In contrast, most of the limb muscles of the mdx mouse maintain hypertrophy and increased skeletal muscle mass throughout much of their life span.⁸ One reason for the difference between DMD and mdx is explained by the up-regulation of expression of utrophin, a homolog of dystrophin.^9,10 Another reason has been supposed to be the excellent regeneration capacity of mdx compared with DMD. However, this hypothesis has not been verified.Regeneration of skeletal muscle depends on the competence of muscle satellite cells. Muscle satellite cells, which account for 2 to 5% of the total nuclei in adult skeletal muscle, play a major role in muscle regeneration.¹¹ Under normal conditions, satellite cells are found external to the myofiber plasma membrane and beneath the muscle basal lamina,¹² and they are mitotically quiescent in adult skeletal muscle.¹³ When activated by muscle damage, satellite cells proliferate, differentiate, fuse with each other or injured myofibers, and eventually regenerate mature myofibers. During the regenerative processes, satellite cells not only produce large amounts of muscle, but also renew themselves to maintain their own population.¹⁴ In fact, it is reported that the satellite cell pool of C57BL/10 continues to respond efficiently even when the skeletal muscle is subjected to as many as 50 cycles of severe damage.¹⁵ Therefore, it is thought that maintenance of the satellite cell pool is indispensable to retain the long-term regenerative potential for skeletal muscle injury, including in muscular dystrophies.To investigate genetic differences in long-term regeneration potential, we first induced repeated degeneration–regeneration cycles in four inbred strains of mice. Among these strains, C57BL/6, a widely used strain akin to C57BL/10, was tolerant of repeated injury. This is consistent with the results of C57BL/10 previously described.¹⁵ In contrast, among four inbred strains, DBA/2 mice exhibited the most remarkable skeletal muscle loss and impaired regeneration after repeated injury. Importantly, the self-renewal potential of DBA/2 satellite cells was significantly lower than that of C57BL/6. In addition, in vitro colony formation and proliferation assays indicated that intrinsic difference between C57BL/6 and DBA/2 satellite cells exist. Finally, we crossed the mdx genotype with the DBA/2 for more than five generations. At the fifth backcross, the mice are not yet fully congenic (D2.B10-DMD^mdx), and thus we refer to them as DBA/2-mdx hereafter. We investigated their phenotypes. Intriguingly, severe loss of skeletal muscle weight, decreased myofiber number, increased fat and fibrosis volume, and apparent muscle weakness were observed in the DBA/2-mdx mice. These results indicate that the intrinsic genetic program affects the properties of satellite cells, and DBA/2-mdx will be a more useful model of DMD than C57BL/10-mdx. It is also speculated that the high self-renewal potential of C57BL/10 satellite cells might explain the difference in pathologies between humans and mice.     

MTM1 mutations in X-linked myotubular myopathy

X-linked myotubular myopathy (XLMTM; MIM# 310400) is a severe congenital muscle disorder caused by mutations in the MTM1 gene. This gene encodes a dual-specificity phosphatase named myotubularin, defining a large gene family highly conserved through evolution (which includes the putative anti-phosphatase Sbf1/hMTMR5). We report 29 mutations in novel cases, including 16 mutations not described before. To date, 198 mutations have been identified in unrelated families, accounting for 133 different disease-associated mutations which are widespread throughout the gene. Most point mutations are truncating, but 26% (35/133) are missense mutations affecting residues conserved in the Drosophila ortholog and in the homologous MTMR1 gene. Three recurrent mutations affect 17% of the patients, and a total of 21 different mutations were found in several independent families. The frequency of female carriers appears higher than expected (only 17% are de novo mutations). While most truncating mutations cause the severe and early lethal phenotype, some missense mutations are associated with milder forms and prolonged survival (up to 54 years).