Skeletal Muscle - Latest Articles

Inhibition of CD26/DPP-IV enhances donor muscle cell engraftment and stimulates sustained donor cell proliferation

Maura Parker — 2012-02-16T00:00:00Z

Background: Transplantation of myogenic stem cells possesses great potential for long-term repair of dystrophic muscle. In murine-to-murine transplantation experiments, CXCR4 expression marks a population of adult murine satellite cells with robust engraftment potential in mdx mice, and CXCR4-positive murine muscle derived SP cells home more effectively to dystrophic muscle after intra-arterial delivery in mdx5cv mice. Together, these data suggest that CXCR4 plays an important role in donor cell engraftment. Therefore, we sought to translate these results to a clinically relevant canine-to-canine allogeneic transplant model for Duchenne muscular dystrophy (DMD), and determine if CXCR4 is important for donor cell engraftment. Methods: In this study, we used a canine-to-murine xenotransplantation model to quantitatively compare canine muscle cell engraftment, and test the most effective cell population and modulating factor in a canine model of DMD using allogeneic transplantation experiments. Results: We show that CXCR4 expressing cells are important for donor muscle cell engraftment, yet FACS sorted CXCR4-positive cells display decreased engraftment efficiency. However, diprotin A, a positive modulator of CXCR4-SDF-1 binding, significantly enhanced engraftment and stimulated sustained proliferation of donor cells in vivo. Furthermore, the canine-to-murine xenotransplantation model accurately predicted results in canine-to-canine muscle cell transplantation. Conclusions: Therefore, these results establish the efficacy of diprotin A in stimulating muscle cell engraftment, and highlight the pre-clinical utility of a xenotransplantation model in assessing the relative efficacy of muscle stem cell populations.

TAK-1/p38/NFkappaB signaling inhibits myoblast differentiation by increasing levels of Activin A

Anne-Ulrike Trendelenburg — 2012-02-07T00:00:00Z

Background: Skeletal-muscle differentiation is required for the regeneration of myofibers after injury. The differentiation capacity of satellite cells is impaired in settings of old age, which is at least one factor in the onset of sarcopenia, the age-related loss of skeletal-muscle mass and major cause of frailty. One important cause of impaired regeneration is increased levels of transforming growth factor (TGF)- accompanied by reduced Notch signaling. Pro-inflammatory cytokines are also upregulated in aging, which led us hypothesize that they might potentially contribute to impaired regeneration in sarcopenia. Thus, in this study, we further analyzed the muscle differentiation-inhibition pathway mediated by pro-inflammatory cytokines in human skeletal muscle cells (HuSKMCs). Methods: We studied the modulation of HuSKMC differentiation by the pro-inflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)- The grade of differentiation was determined by either imaging (fusion index) or creatine kinase (CK) activity, a marker of muscle differentiation. Secretion of TGF- proteins during differentiation was assessed by using a TGF--responsive reporter-gene assay and further identified by means of pharmacological and genetic inhibitors. In addition, signaling events were monitored by western blotting and reverse transcription PCR, both in HuSKMC cultures and in samples from a rat sarcopenia study. Results: The pro-inflammatory cytokines IL-1 and TNF- block differentiation of human myoblasts into myotubes. This anti-differentiation effect requires activation of TGF--activated kinase (TAK)-1. Using pharmacological and genetic inhibitors, the TAK-1 pathway could be traced to p38 and NFB. Surprisingly, the anti-differentiation effect of the cytokines required the transcriptional upregulation of Activin A, which in turn acted through its established signaling pathway: ActRII/ALK/SMAD. Inhibition of Activin A signaling was able to rescue human myoblasts treated with IL-1 or TNF-, resulting in normal differentiation into myotubes. Studies in aged rats as a model of sarcopenia confirmed that this pro-inflammatory cytokine pathway identified is activated during aging. Conclusions: In this study, we found an unexpected connection between cytokine andActivin signaling, revealing a new mechanism by which cytokines affect skeletal muscle, and establishing the physiologic relevance of this pathway in the impaired regeneration seen in sarcopenia.

TNFalpha- and tumor-induced skeletal muscle atrophy involves sphingolipid metabolism

Joffrey De Larichaudy — 2012-01-18T00:00:00Z

Background: Muscle atrophy associated with various pathophysiological conditions represents a major health problem, due to its contribution to the deterioration of patient status and its impact on mortality. Although the involvement of pro-inflammatory cytokines in this process is well-recognized, the role of sphingolipid metabolism alterations induced by the cytokines has received little attention. Results: We addressed this question both in vitro, using differentiated myotubes treated by TNFalpha, and in vivo, in a murine model of tumor-induced cachexia. Myotube atrophy induced by TNFalpha was accompanied by a substantial increase in cell ceramide levels, and could be mimicked by the addition of exogenous ceramides. It could be prevented by the addition of ceramide synthesis inhibitors that targeted either the de novo pathway (myriocin), or sphingomyelinases (GW4869 and 3-O-methylsphingomyelin). In the presence of TNFalpha, ceramide synthesis inhibitors significantly increased protein synthesis and decreased proteolysis. In parallel, they lowered the expression of both Atrogin-1 and LC3b genes, respectively involved in muscle protein degradation by proteasome, and autophagic proteolysis, and increased the proportion of inactive, phosphorylated Foxo3 transcription factor. Furthermore, these inhibitors increased the expression and/or phosphorylation levels of key factors regulating protein metabolism, including phospholipase D, an activator of mTOR, and the mTOR substrates S6K1 and Akt. In vivo, C26 carcinoma implantation induced a substantial increase of muscle ceramide, together with drastic muscle atrophy. Treatment of the animals by myriocin reduced the expression of atrogenes Foxo3 and Atrogin-1, and partially protected muscle tissue from atrophy. Conclusions: These results indicate that ceramide accumulation induced by TNFalpha or tumor development participates in the mechanism of muscle cell atrophy, and that sphingolipid metabolism can be a relevant target for pharmacological or nutritional interventions aiming at preserving muscle mass in pathological situations.

Skeletal Muscle - one year on

David Glass — 2012-01-05T00:00:00Z

No description available

Regulation of myotube formation by the actin-binding factor drebrin

Annalisa Mancini — 2011-12-08T00:00:00Z

Background: Myogenic differentiation involves cell-cycle arrest, activation of the muscle-specific transcriptome, and elongation, alignment and fusion of myoblasts into multinucleated myotubes. This process is controlled by promyogenic transcription factors and regulated by signaling pathways in response to extracellular cues. The p38 mitogen-activated protein kinase (p38 MAPK) pathway promotes the activity of several such transcription factors, including MyoD and MEF2, thereby controlling the muscle-specific transcription program. However, few p38-regulated genes that play a role in the regulation of myogenesis have been identified. Methods: RNA interference (RNAi), chemical inhibition and immunofluorescence approaches were used to assess the role of drebrin in differentiation of primary mouse myoblasts and C2C12 cells. Results: In a search for p38-regulated genes that promote myogenic differentiation, we identified Dbn1, which encodes the actin-binding protein drebrin. Drebrin is an F-actin side-binding protein that remodels actin to facilitate the change of filopodia into dendritic spines during synaptogenesis in developing neurons. Dbn1 mRNA and protein are induced during differentiation of primary mouse and C2C12 myoblasts, and induction is substantially reduced by the p38 MAPK inhibitor SB203580. Primary myoblasts and C2C12 cells depleted of drebrin by RNAi display reduced levels of myogenin and myosin heavy chain and form multinucleated myotubes very inefficiently. Treatment of myoblasts with BTP2, a small-molecule inhibitor of drebrin, produces a phenotype similar to that produced by knockdown of drebrin, and the inhibitory effects of BTP2 are rescued by expression of a mutant form of drebrin that is unable to bind BTP2. Drebrin in myoblasts is enriched in cellular projections and cell cortices and at regions of cell-cell contact, all sites where F-actin, too, was concentrated. Conclusions: Our findings reveal that Dbn1 expression is a target of p38 MAPK signaling during myogenesis and that drebrin promotes myoblast differentiation.

Dystrophin deficiency exacerbates skeletal muscle pathology in dysferlin-null mice

Renzhi Han — 2011-12-01T00:00:00Z

Background: Mutations in the genes coding for either dystrophin or dysferlin cause distinct forms of muscular dystrophy. Dystrophin links the cytoskeleton to the sarcolemma through direct interaction with beta-dystroglycan. This link extends to the extracellular matrix via beta-dystroglycan interacting with alpha-dystroglycan, which binds extracellular matrix proteins including laminin alpha2, agrin and perlecan that possess laminin globular (LG) domains. The absence of dystrophin disrupts this link leading to compromised muscle sarcolemmal integrity. Dysferlin, on the other hand, plays an important role in the Ca2+-dependent membrane repair of damaged sarcolemmal in skeletal muscle. Since dysferlin and dystrophin play different roles in maintaining muscle cell integrity, we hypothesize that disrupting sarcolemma integrity with dystrophin deficiency would exacerbate the pathology in dysferlin-null mice and allow further characterization of the role of dysferlin in skeletal muscle. Methods: To test this hypothesis, we generated mice lacking both dystrophin and dysferlin (DKO) by breeding mdx mice with dysferlin-null mice, and analyzed the effects of a combined deficiency of dysferlin and dystrophin on muscle pathology and sarcolemmal integrity. Results: The DKO mice exhibited more severe muscle pathology than either mdx or dysferlin-null mice and importantly, the onset of the muscle pathology is much earlier than dysferlin deficient mice. The DKO mice showed muscle pathology of various skeletal muscles, including the mandible muscles, as well as a greater number of regenerating muscle fibers, higher serum creatine kinase levels and elevated Evans blue dye uptake into skeletal muscles. Compromised dystrophin-glycoprotein complex (DGC) expression at the sarcolemma was demonstrated in both mdx and DKO muscles by immunofluorescence analyses. Lengthening contractions caused similar force deficits regardless of dysferlin expression. However, the rate of force recovery within 45 minutes following lengthening contractions was hampered in DKO muscles compared to mdx or dysferlin-null muscles, suggesting that dysferlin is required for the initial recovery from lengthening contractions-induced muscle injury of the DGC-compromised muscles. Conclusions: In summary, our study suggests that dysferlin-mediated membrane repair helps to limit the dystrophic changes in dystrophin-deficient skeletal muscle. Dystrophin deficiency unmasks the function of dysferlin in membrane repair during lengthening contractions. Finally, dystrophin/dysferlin deficient mice provide a very useful model to evaluate the effectiveness of therapies designed to treat dysferlin deficiency.

Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders.

Kamel Mamchaoui — 2011-11-01T00:00:00Z

Background: Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies. Methods: Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders. Results: The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both in vitro and in vivo after transplantation into regenerating muscle of immunodeficient mice. Conclusions: Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess in vivo the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.

PPARdelta regulates satellite cell proliferation and skeletal muscle regeneration

Alison Angione — 2011-11-01T00:00:00Z

Peroxisome proliferator-activated receptors (PPARs) are a class of nuclear receptors that play important roles in development and energy metabolism. Whereas PPARδ has been shown to regulate mitochondrial biosynthesis and slow-muscle fiber types, its function in skeletal muscle progenitors (satellite cells) is unknown. Since constitutive mutation of Pparδ leads to embryonic lethality, we sought to address this question by conditional knockout (cKO) of Pparδ using Myf5-Cre/Pparδflox/flox alleles to ablate PPARδ in myogenic progenitor cells. Although Pparδ-cKO mice were born normally and initially displayed no difference in body weight, muscle size or muscle composition, they later developed metabolic syndrome, which manifested as increased body weight and reduced response to glucose challenge at age nine months. Pparδ-cKO mice had 40% fewer satellite cells than their wild-type littermates, and these satellite cells exhibited reduced growth kinetics and proliferation in vitro. Furthermore, regeneration of Pparδ-cKO muscles was impaired after cardiotoxin-induced injury. Gene expression analysis showed reduced expression of the Forkhead box class O transcription factor 1 (FoxO1) gene in Pparδ-cKO muscles under both quiescent and regenerating conditions, suggesting that PPARδ acts through FoxO1 in regulating muscle progenitor cells. These results support a function of PPARδ in regulating skeletal muscle metabolism and insulin sensitivity, and they establish a novel role of PPARδ in muscle progenitor cells and postnatal muscle regeneration.

Transgenic overexpression of gamma-cytoplasmic actin protects against eccentric contraction-induced force loss in mdx mice

Kristen Baltgalvis — 2011-10-13T00:00:00Z

Background: γ-cytoplasmic (γ-cyto) actin levels are elevated in dystrophin-deficient mdx mouse skeletal muscle. The purpose of this study was to determine whether further elevation of γ-cyto actin levels improve or exacerbate the dystrophic phenotype of mdx mice. Methods: We transgenically overexpressed γ-cyto actin, specifically in skeletal muscle of mdx mice (mdx-TG), and compared skeletal muscle pathology and force-generating capacity between mdx and mdx-TG mice at different ages. We investigated the mechanism by which γ-cyto actin provides protection from force loss by studying the role of calcium channels and stretch-activated channels in isolated skeletal muscles and muscle fibers. Analysis of variance or independent t-tests were used to detect statistical differences between groups. Results: Levels of γ-cyto actin in mdx-TG skeletal muscle were elevated 200-fold compared to mdx skeletal muscle and incorporated into thin filaments. Overexpression of γ-cyto actin had little effect on most parameters of mdx muscle pathology. However, γ-cyto actin provided statistically significant protection against force loss during eccentric contractions. Store-operated calcium entry across the sarcolemma did not differ between mdx fibers compared to wild-type fibers. Additionally, the omission of extracellular calcium or the addition of streptomycin to block stretch-activated channels did not improve the force-generating capacity of isolated extensor digitorum longus muscles from mdx mice during eccentric contractions. Conclusions: The data presented in this study indicate that upregulation of γ-cyto actin in dystrophic skeletal muscle can attenuate force loss during eccentric contractions and that the mechanism is independent of activation of stretch-activated channels and the accumulation of extracellular calcium.

Level of muscle regeneration in limb-girdle muscular dystrophy type 2I relates to genotype and clinical severity

Thomas Krag — 2011-10-05T00:00:00Z

Background: The balance between muscle regeneration and ongoing degeneration is a relationship that greatly influences the progression of muscular dystrophy. Numerous factors may influence the muscle regeneration, but more information about the relationship between genotype, clinical severity and the ability to regenerate is needed. Methods: Muscle biopsies were obtained from the tibialis anterior muscle, and frozen sections were stained for general histopathological and immunohistological evaluation. Differences between groups were considered statistical significant at P < 0.05 using Student's unpaired t-test. Results: We found that all patients with limb-girdle muscular dystrophy type 2I (LGMD2I) had a large number of internally nucleated fibers, a sign of previous regeneration. The level of expression of muscle-specific developmental proteins, such as neonatal myosin heavy chain (nMHC) and myogenin, was related to the clinical severity. Additionally, we found that the majority of nMHC-positive fibers did not stain positively for utrophin in patients who were compound heterozygous for the L276I mutation, suggesting that the predominant form of regeneration in these patients is fiber repair rather than formation of new fibers. Double staining showed that many smaller nMHC-positive fibers were positive for antibodies against the glycosylation on α-dystroglycan, suggesting that such glycosylation may be a result of muscle regeneration. Conclusion: Severely affected patients with LGMD2I have a high level of muscle degeneration, which leads to a high rate of regeneration, but this is insufficient to change the imbalance between degeneration and regeneration, ultimately leading to progressive muscle wasting. Detailed information regarding the level and rate of muscle regeneration and potential obstructions of the regenerative pathway should be of use for future therapies involving satellite-cell activation.