http://www.skeletalmusclejournal.com The latest research articles published by Skeletal Muscle 2012-05-07T00:00:00Z Background: The use of the Cre/loxP system for gene targeting has been proven to be a powerful tool for understanding gene function. The purpose of this study was to create and characterize an inducible, skeletal muscle-specific Cre transgenic mouse strain. Methods: To achieve skeletal muscle-specific expression, the human alpha-skeletal actin promoter was used to drive expression of a chimeric Cre recombinase containing two mutated estrogen receptor ligand-binding domains. Results: Western blot analysis, PCR and beta-galactosidase staining confirmed that Cre-mediated recombination was restricted to limb and craniofacial skeletal muscles only after tamoxifen administration. Conclusions: A transgenic mouse was created that allows inducible, gene targeting of floxed genes in adult skeletal muscle of different developmental origins. This new mouse will be of great utility to the skeletal muscle community. http://www.skeletalmusclejournal.com/content/2/1/8 John McCarthy Ratchakrit Srikuea Tyler Kirby Charlotte Peterson Karyn Esser Skeletal Muscle 2012, null:8 2012-05-07T00:00:00Z doi:10.1186/2044-5040-2-8 /content/figures/2044-5040-2-8-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 8 2012-05-07T00:00:00Z PDF Background: Similar to replicating myoblasts, many rhabdomyosarcoma cells express the myogenic determination gene MyoD. In contrast to myoblasts, rhabdomyosarcoma cells do not make the transition from a regulative growth phase to terminal differentiation. Previously we demonstrated that the forced expression of MyoD with its E-protein dimerization partner was sufficient to induce differentiation and suppress multiple growth-promoting genes, suggesting that the dimer was targeting a switch that regulated the transition from growth to differentiation. Our data also suggested that a balance between various inhibitory transcription factors and MyoD activity kept rhabdomyosarcomas trapped in a proliferative state. Methods: Potential myogenic co-factors were tested for their ability to drive differentiation in rhabdomyosarcoma cell culture models, and their relation to MyoD activity determined through molecular biological experiments. Results: Modulation of the transcription factors RUNX1 and ZNF238 can induce differentiation in rhabdomyosarcoma cells and their activity is integrated, at least in part, through the activation of miR-206, which acts as a genetic switch to transition the cell from a proliferative growth phase to differentiation. The inhibitory transcription factor MSC also plays a role in controlling miR-206, appearing to function by occluding a binding site for MyoD in the miR-206 promoter. Conclusions: These findings support a network model composed of coupled regulatory circuits with miR-206 functioning as a switch regulating the transition from one stable state (growth) to another (differentiation). http://www.skeletalmusclejournal.com/content/2/1/7 Kyle MacQuarrie Zizhen Yao Janet Young Yi Cao Stephen Tapscott Skeletal Muscle 2012, null:7 2012-04-29T00:00:00Z doi:10.1186/2044-5040-2-7 /content/figures/2044-5040-2-7-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 7 2012-04-29T00:00:00Z PDF Background: Mice lacking MyoD exhibit delayed skeletal muscle regeneration and markedly enhanced numbers of satellite cells. Myoblasts isolated from MyoD-/- myoblasts proliferate more rapidly than wild type myoblasts, display a dramatic delay in differentiation, and continue to incorporate BrdU after serum withdrawal. Methods: Primary myoblasts isolated from wild type and MyoD-/- mutant mice were examined by microarray analysis and further characterized by cell and molecular experiments in cell culture. Results: We found that NF-kappaB, a key regulator of cell-cycle withdrawal and differentiation, aberrantly maintains nuclear localization and transcriptional activity in MyoD-/- myoblasts. As a result, expression of cyclin D is maintained during serum withdrawal, inhibiting expression of muscle-specific genes and progression through the differentiation program. Sustained nuclear localization of cyclin E, and a concomitant increase in cdk2 activity maintains S-phase entry in MyoD-/- myoblasts even in the absence of mitogens. Importantly, this deficit was rescued by forced expression of IkappaBalphaSR, a non-degradable mutant of IkappaBalpha, indicating that inhibition of NF-kappaB is sufficient to induce terminal myogenic differentiation in the absence of MyoD. Conclusion: MyoD-induced cytoplasmic relocalization of NF-kappaB is an essential step in linking cell-cycle withdrawal to the terminal differentiation of skeletal myoblasts. These results provide important insight into the unique functions of MyoD in regulating the switch from progenitor proliferation to terminal differentiation. http://www.skeletalmusclejournal.com/content/2/1/6 Maura Parker Julia von Maltzahn Nadine Bakkar Ban Al-Joubori Jeff Ishibashi Denis Guttridge Michael Rudnicki Skeletal Muscle 2012, null:6 2012-04-27T00:00:00Z doi:10.1186/2044-5040-2-6 /content/figures/2044-5040-2-6-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 6 2012-04-27T00:00:00Z PDF Background: The p38α mitogen-activated protein kinase (MAPK) is a critical mediator of myoblast differentiation, and does so in part through the phosphorylation and regulation of several transcription factors and chromatin remodelling proteins. However, whether p38α is involved in processes other than gene regulation during myogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear. Methods: To further characterise the involvement of p38α during myoblast differentiation, we developed and applied a simple technique for identifying relevant in vivo kinase substrates and their phosphorylation sites. In addition to identifying substrates for one kinase, the technique can be used in vitro to compare multiple kinases in the same experiment, and we made use of this to study the substrate specificities of the p38α and β isoforms. Results: Applying the technique to p38α resulted in the identification of seven in vivo phosphorylation sites on six proteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An in vitro comparison with p38β revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation. Conclusion: Our results suggest p38α has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38β and other isoforms cannot compensate for its absence. The substrate-finding approach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and for uncovering the deeper mechanisms of phosphorylation-dependent cell signalling. http://www.skeletalmusclejournal.com/content/2/1/5 James Knight Ruijun Tian Robin Lee Fangjun Wang Ariane Beauvais Hanfa Zou Lynn Megeney Anne-Claude Gingras Tony Pawson Daniel Figeys Rashmi Kothary Skeletal Muscle 2012, null:5 2012-03-06T00:00:00Z doi:10.1186/2044-5040-2-5 /content/figures/2044-5040-2-5-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 5 2012-03-06T00:00:00Z XML 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. http://www.skeletalmusclejournal.com/content/2/1/4 Maura Parker Carol Loretz Ashlee Tyler Lauren Snider Rainer Storb Stephen Tapscott Skeletal Muscle 2012, null:4 2012-02-16T00:00:00Z doi:10.1186/2044-5040-2-4 /content/figures/2044-5040-2-4-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 4 2012-02-16T00:00:00Z XML 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)-β 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)-1α and tumor necrosis factor (TNF)-α The grade of differentiation was determined by either imaging (fusion index) or creatine kinase (CK) activity, a marker of muscle differentiation. Secretion of TGF-β proteins during differentiation was assessed by using a TGF-β-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-1α and TNF-α block differentiation of human myoblasts into myotubes. This anti-differentiation effect requires activation of TGF-β-activated kinase (TAK)-1. Using pharmacological and genetic inhibitors, the TAK-1 pathway could be traced to p38 and NFκB. 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-1β or TNF-α, 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 and Activin 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. http://www.skeletalmusclejournal.com/content/2/1/3 Anne-Ulrike Trendelenburg Angelika Meyer Carsten Jacobi Jerome Feige David Glass Skeletal Muscle 2012, null:3 2012-02-07T00:00:00Z doi:10.1186/2044-5040-2-3 /content/figures/2044-5040-2-3-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 3 2012-02-07T00:00:00Z XML Background: Muscle atrophy associated with various pathophysiological conditions represents a major health problem, because of its contribution to the deterioration of patient status and its effect 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 with TNF-α, and in vivo in a murine model of tumor-induced cachexia. Myotube atrophy induced by TNF-α 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 the sphingomyelinases (GW4869 and 3-O-methylsphingomyelin). In the presence of TNF-α, ceramide-synthesis inhibitors significantly increased protein synthesis and decreased proteolysis. In parallel, they lowered the expression of both the Atrogin-1 and LC3b genes, involved in muscle protein degradation by proteasome and in autophagic proteolysis, respectively, 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 mammalian target of rapamycin (mTOR), and the mTOR substrates S6K1 and Akt. In vivo, C26 carcinoma implantation induced a substantial increase in muscle ceramide, together with drastic muscle atrophy. Treatment of the animals with myriocin reduced the expression of the atrogenes Foxo3 and Atrogin-1, and partially protected muscle tissue from atrophy. Conclusions: Ceramide accumulation induced by TNF-α or tumor development participates in the mechanism of muscle-cell atrophy, and sphingolipid metabolism is a logical target for pharmacological or nutritional interventions aiming at preserving muscle mass in pathological situations. http://www.skeletalmusclejournal.com/content/2/1/2 Joffrey De Larichaudy Alessandra Zufferli Filippo Serra Andrea Isidori Fabio Naro Kevin Dessalle Marine Desgeorges Monique Piraud David Cheillan Hubert Vidal Etienne Lefai Georges Nemoz Skeletal Muscle 2012, null:2 2012-01-18T00:00:00Z doi:10.1186/2044-5040-2-2 /content/figures/2044-5040-2-2-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 2 2012-01-18T00:00:00Z XML No description available http://www.skeletalmusclejournal.com/content/2/1/1 David Glass Kevin Campbell Michael Rudnicki Skeletal Muscle 2012, null:1 2012-01-05T00:00:00Z doi:10.1186/2044-5040-2-1 /content/figures/2044-5040-2-1-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 1 2012-01-05T00:00:00Z XML 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. http://www.skeletalmusclejournal.com/content/1/1/36 Annalisa Mancini Dario Sirabella Weijia Zhang Hiroyuki Yamazaki Tomoaki Shirao Robert Krauss Skeletal Muscle 2011, null:36 2011-12-08T00:00:00Z doi:10.1186/2044-5040-1-36 /content/figures/2044-5040-1-36-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 36 2011-12-08T00:00:00Z XML 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 β-dystroglycan. This link extends to the extracellular matrix by β-dystroglycan's interaction with α-dystroglycan, which binds extracellular matrix proteins, including laminin α2, agrin and perlecan, that possess laminin globular 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 sarcolemma in skeletal muscle. Because dysferlin and dystrophin play different roles in maintaining muscle cell integrity, we hypothesized that disrupting sarcolemmal 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 our hypothesis, we generated dystrophin/dysferlin double-knockout (DKO) mice 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 mice or dysferlin-null mice, and, importantly, the onset of the muscle pathology occurred much earlier than it did in 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. 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 muscles or dysferlin-null muscles, suggesting that dysferlin is required for the initial recovery from lengthening contraction-induced muscle injury of the dystrophin-glycoprotein complex-compromised muscles. Conclusions: The results of our study suggest 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. Dystrophin/dysferlin-deficient mice provide a very useful model with which to evaluate the effectiveness of therapies designed to treat dysferlin deficiency. http://www.skeletalmusclejournal.com/content/1/1/35 Renzhi Han Erik Rader Jennifer Levy Dimple Bansal Kevin Campbell Skeletal Muscle 2011, null:35 2011-12-01T00:00:00Z doi:10.1186/2044-5040-1-35 Dysferlin and dystrophin deficiencies in skeletal muscle Dystrophin deficiency unmasks the function of dysferlin in membrane repair during lengthening contractions. This dysferlin-mediated membrane repair limits the dystrophic changes in dystrophin-deficient skeletal muscle. /content/figures/2044-5040-1-35-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 35 2011-12-01T00:00:00Z XML