http://www.skeletalmusclejournal.com The latest research articles published by Skeletal Muscle 2013-05-16T00:00:00Z Background: During the past decade, several animal studies have demonstrated that in addition to local cells, cells from the bone marrow (BM) possess the ability to contribute to regeneration of injured skeletal muscle tissue. In addition, in mice, regular physical activity has been displayed to be a sufficient stimulus for BM-derived cell contribution to the muscle, indicating that this is part of the ongoing physiological remodeling of skeletal muscle. However, whether BM-derived cells participate in human skeletal muscle remodeling is not known. To this end, we analyzed the incorporation of BM-derived cells in healthy human skeletal muscle in women transplanted with male BM. Methods: Skeletal muscle biopsies were obtained from the m. vastus lateralis of women transplanted with male donor hematopoietic stem cells 6 to 12 years earlier. Healthy women served as controls. Immunohistochemical staining for skeletal muscle fibers, satellite cells (SCs) or endothelial cells (ECs) combined with fluorescent in situ hybridization (FISH) of X and Y chromosomes was used to identify cells of BM origin within the biopsies. Three dimensional confocal imaging was performed to demonstrate colocalization of Y chromosome and DAPI within muscle fibers. To further investigate whether BM-derived cells incorporate into the SC niche, myoblasts were extracted from the biopsies from the transplanted women, cultured, and analyzed using XY FISH and immunocytochemistry. Results: Three dimensional confocal imaging indisputably demonstrated colocalization of Y chromosome and DAPI within muscle fibers. Some Y chromosomes were found within centrally located nuclei. No Y chromosomes were detected in CD56+ SCs in the tissue sections nor in the myoblasts cultured from the extracted SCs. Y chromosome+ ECs were found in all sections from the transplanted subjects. No Y chromosomes were found in the skeletal muscle biopsies obtained from healthy control women. Conclusions: We demonstrate that BM-derived cells contribute to skeletal muscle fibers and ECs. Our results support that BM contribution to skeletal muscle occurs via direct fusion to muscle fibers, and that the contributing cells derive from the hematopoietic lineage. Thus, the present findings encourage further studies of the importance of this process for the physiological adaptation occurring throughout life. http://www.skeletalmusclejournal.com/content/3/1/12 Anna Strömberg Monika Jansson Helene Fischer Eric Rullman Hans Hägglund Thomas Gustafsson Skeletal Muscle 2013, null:12 2013-05-16T00:00:00Z doi:10.1186/2044-5040-3-12 /content/figures/2044-5040-3-12-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 12 2013-05-16T00:00:00Z PDF Background: A sophisticated level of coordinated gene expression is necessary for skeletal muscle fibers to obtain their unique functional identities. We have previously shown that the transcription factor Sox6 plays an essential role in coordinating muscle fiber type differentiation by acting as a transcriptional suppressor of slow fiber-specific genes. Currently, mechanisms regulating the activity of Sox6 in skeletal muscle and how these mechanisms affect the fiber phenotype remain unknown. Methods: Yeast two-hybrid screening was used to identify binding partners of Sox6 in muscle. Small interfering RNA (siRNA)-mediated knockdown of one of the Sox6 binding proteins, Trip12, was used to determine its effect on Sox6 activity in C2C12 myotubes using quantitative analysis of fiber type-specific gene expression. Results: We found that the E3 ligase Trip12, a HECT domain E3 ubiquitin ligase, recognizes and polyubiquitinates Sox6. Inhibiting Trip12 or the 26S proteasome activity resulted in an increase in Sox6 protein levels in C2C12 myotubes. This control of Sox6 activity in muscle cells via Trip12 ubiquitination has significant phenotypic outcomes. Knockdown of Trip12 in C2C12 myotubes led to upregulation of Sox6 protein levels and concurrently to a decrease in slow fiber-specific Myh7 expression coupled with an increased expression in fast fiber-specific Myh4. Therefore, regulation of Sox6 cellular levels by the ubiquitin-proteasome system can induce identity-changing alterations in the expression of fiber type-specific genes in muscle cells. Conclusions: Based on our data, we propose that in skeletal muscle, E3 ligases have a significant role in regulating fiber type-specific gene expression, expanding their importance in muscle beyond their well-established role in atrophy. http://www.skeletalmusclejournal.com/content/3/1/11 Chung-Il An Edward Ganio Nobuko Hagiwara Skeletal Muscle 2013, null:11 2013-05-10T00:00:00Z doi:10.1186/2044-5040-3-11 /content/figures/2044-5040-3-11-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 11 2013-05-10T00:00:00Z PDF Background: During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear. Methods: We have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line. Results: Analyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls. Conclusion: Our results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated. http://www.skeletalmusclejournal.com/content/3/1/10 Sean Gross Peter Rotwein Skeletal Muscle 2013, null:10 2013-05-02T00:00:00Z doi:10.1186/2044-5040-3-10 /content/figures/2044-5040-3-10-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 10 2013-05-02T00:00:00Z PDF Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and an X-linked recessive, progressive muscle wasting disease caused by the absence of a functional dystrophin protein. Dystrophin has a structural role as a cytoskeletal stabilization protein and protects cells against contraction-induced damage. Dystrophin also serves a signaling role through mechanotransduction of forces and localization of neuronal nitric oxide synthase (nNOS), which produces nitric oxide (NO) to facilitate vasorelaxation. In DMD, the signaling defects produce inadequate tissue perfusion caused by functional ischemia due to a diminished ability to respond to shear stress induced endothelium-dependent dilation. Additionally, the structural defects seen in DMD render myocytes with an increased susceptibility to mechanical stress. The combination of both defects is necessary to generate myocyte damage, which induces successive rounds of myofiber degeneration and regeneration, loss of calcium homeostasis, chronic inflammatory response, fibrosis, and myonecrosis. In individuals with DMD, these processes inevitably cause loss of ambulation shortly after the first decade and an abbreviated life with death in the third or fourth decade due to cardio-respiratory anomalies. There is no known cure for DMD, and although the culpable gene has been identified for more than twenty years, research on treatments has produced few clinically relevant results. Several recent studies on novel DMD therapeutics are vascular targeted and focused on attenuating the inherent functional ischemia. One approach improves vasorelaxation capacity through pharmaceutical inhibition of either phosphodiesterase 5 (PDE5) or angiotensin-converting enzyme (ACE). Another approach increases the density of the underlying vascular network by inducing angiogenesis, and this has been accomplished through either direct delivery of vascular endothelial growth factor (VEGF) or by downregulating the VEGF decoy-receptor type 1 (VEGFR-1 or Flt-1). The pro-angiogenic approaches also seem to be pro-myogenic and could resolve the age-related decline in satellite cell (SC) quantity seen in mdx models through expansion of the SC juxtavascular niche. Here we review these four vascular targeted treatment strategies for DMD and discuss mechanisms, proof of concept, and the potential for clinical relevance associated with each therapy. http://www.skeletalmusclejournal.com/content/3/1/9 James Ennen Mayank Verma Atsushi Asakura Skeletal Muscle 2013, null:9 2013-04-23T00:00:00Z doi:10.1186/2044-5040-3-9 /content/figures/2044-5040-3-9-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 9 2013-04-23T00:00:00Z XML Background: Transcription factor overexpression is common in biological experiments and transcription factor amplification is associated with many cancers, yet few studies have directly compared the DNA-binding profiles of endogenous versus overexpressed transcription factors. Methods: We analyzed MyoD ChIP-seq data from C2C12 mouse myotubes, primary mouse myotubes, and mouse fibroblasts differentiated into muscle cells by overexpression of MyoD and compared the genome-wide binding profiles and binding site characteristics of endogenous and overexpressed MyoD. Results: Overexpressed MyoD bound to the same sites occupied by endogenous MyoD and possessed the same E-box sequence preference and co-factor site enrichments, and did not bind to new sites with distinct characteristics. Conclusions: Our data demonstrate a robust fidelity of transcription factor binding sites over a range of expression levels and that increased amounts of transcription factor increase the binding at physiologically bound sites. http://www.skeletalmusclejournal.com/content/3/1/8 Zizhen Yao Abraham Fong Yi Cao Walter Ruzzo Robert Gentleman Stephen Tapscott Skeletal Muscle 2013, null:8 2013-04-08T00:00:00Z doi:10.1186/2044-5040-3-8 /content/figures/2044-5040-3-8-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 8 2013-04-08T00:00:00Z PDF Background: Kruppel-like factor 6 (KLF6) has been recently identified as a MEF2D target gene involved in neuronal cell survival. In addition, KLF6 and TGFbeta have been shown to regulate each other's expression in non-myogenic cell types. Since MEF2D and TGFbeta also fulfill crucial roles in skeletal myogenesis, we wanted to identify whether KLF6 functions in a myogenic context. Methods: KLF6 protein expression levels and promoter activity were analyzed using standard cellular and molecular techniques in cell culture. Results: We found that KLF6 and MEF2D are co-localized in the nuclei of mononucleated but not multinucleated myogenic cells and, that the MEF2 cis element is a key component of the KLF6 promoter region. In addition, TGFbeta potently enhanced KLF6 protein levels and this effect was repressed by pharmacological inhibition of Smad3. Interestingly, pharmacological inhibition of MEK/ERK (1/2) signaling resulted in re-activation of the differentiation program in myoblasts treated with TGFbeta, which is ordinarily repressed by TGFbeta treatment. Conversely, MEK/ERK (1/2) inhibition had no effect on TGFbeta-induced KLF6 expression whereas Smad3 inhibition negated this effect, together supporting the existence of two separable arms of TGFbeta signaling in myogenic cells. Loss of function analysis using siRNA-mediated KLF6 depletion resulted in enhanced myogenic differentiation whereas TGFbeta stimulation of myoblast proliferation was reduced in KLF6 depleted cells. Conclusions: Collectively these data implicate KLF6 in myoblast proliferation and survival in response to TGFbeta with consequences for our understanding of muscle development and a variety of muscle pathologies. http://www.skeletalmusclejournal.com/content/3/1/7 Mathew Dionyssiou Jahan Salma Mariya Bevzyuk Stephanie Wales Lusine L John McDermott Skeletal Muscle 2013, null:7 2013-04-02T00:00:00Z doi:10.1186/2044-5040-3-7 /content/figures/2044-5040-3-7-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 7 2013-04-02T00:00:00Z PDF Background: Skeletal muscle mass is determined by the balance between protein synthesis and degradation. Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of protein translation and has been implicated in the control of muscle mass. Inactivation of mTORC1 by skeletal muscle-specific deletion of its obligatory component raptor results in smaller muscles and a lethal dystrophy. Moreover, raptor-deficient muscles are less oxidative through changes in the expression PGC-1α, a critical determinant of mitochondrial biogenesis. These results suggest that activation of mTORC1 might be beneficial to skeletal muscle by providing resistance to muscle atrophy and increasing oxidative function. Here, we tested this hypothesis by deletion of the mTORC1 inhibitor tuberous sclerosis complex (TSC) in muscle fibers.MethodSkeletal muscles of mice with an acute or a permanent deletion of raptor or TSC1 were examined using histological, biochemical and molecular biological methods. Response of the muscles to changes in mechanical load and nerve input was investigated by ablation of synergistic muscles or by denervation . Results: Genetic deletion or knockdown of raptor, causing inactivation of mTORC1, was sufficient to prevent muscle growth and enhance muscle atrophy. Conversely, short-term activation of mTORC1 by knockdown of TSC induced muscle fiber hypertrophy and atrophy-resistance upon denervation, in both fast tibialis anterior (TA) and slow soleus muscles. Surprisingly, however, sustained activation of mTORC1 by genetic deletion of Tsc1 caused muscle atrophy in all but soleus muscles. In contrast, oxidative capacity was increased in all muscles examined. Consistently, TSC1-deficient soleus muscle was atrophy-resistant whereas TA underwent normal atrophy upon denervation. Moreover, upon overloading, plantaris muscle did not display enhanced hypertrophy compared to controls. Biochemical analysis indicated that the atrophy response of muscles was based on the suppressed phosphorylation of PKB/Akt via feedback inhibition by mTORC1 and subsequent increased expression of the E3 ubiquitin ligases MuRF1 and atrogin-1/MAFbx. In contrast, expression of both E3 ligases was not increased in soleus muscle suggesting the presence of compensatory mechanisms in this muscle. Conclusions: Our study shows that the mTORC1- and the PKB/Akt-FoxO pathways are tightly interconnected and differentially regulated depending on the muscle type. These results indicate that long-term activation of the mTORC1 signaling axis is not a therapeutic option to promote muscle growth because of its strong feedback induction of the E3 ubiquitin ligases involved in protein degradation. http://www.skeletalmusclejournal.com/content/3/1/6 C Bentzinger Shuo Lin Klaas Romanino Perrine Castets Maitea Guridi Serge Summermatter Christoph Handschin Lionel Tintignac Michael Hall Markus Rüegg Skeletal Muscle 2013, null:6 2013-03-06T00:00:00Z doi:10.1186/2044-5040-3-6 /content/figures/2044-5040-3-6-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 6 2013-03-06T00:00:00Z XML Background: The Wnts are secreted proteins that play important roles in skeletal myogenesis, muscle fiber type diversification, neuromuscular junction formation and muscle stem cell function. How Wnt proteins orchestrate such diverse activities remains poorly understood. Canonical Wnt signaling stabilizes β-catenin, which subsequently translocate to the nucleus to activate the transcription of TCF/LEF family genes. Methods: We employed TCF-reporter mice and performed analysis of embryos and of muscle groups. We further isolated fetal myoblasts and performed cell and molecular analyses. Results: We found that canonical Wnt signaling is strongly activated during fetal myogenesis and weakly activated in adult muscles limited to the slow myofibers. Muscle-specific transgenic expression of a stabilized β-catenin protein led to increased oxidative myofibers and reduced muscle mass, suggesting that canonical Wnt signaling promotes slow fiber types and inhibits myogenesis. By TCF-luciferase reporter assay, we identified Wnt-1 and Wnt-3a as potent activators of canonical Wnt signaling in myogenic progenitors. Consistent with in vivo data, constitutive overexpression of Wnt-1 or Wnt-3a inhibited the proliferation of both C2C12 and primary myoblasts. Surprisingly, Wnt-1 and Wnt-3a overexpression up-regulated BMP-4, and inhibition of BMP-4 by shRNA or recombinant Noggin protein rescued the myogenic inhibitory effect of Wnt-1 and Wnt-3a. Importantly, Wnt-3a or BMP-4 recombinant proteins promoted slow myosin heavy chain expression during myogenic differentiation of fetal myoblasts. Conclusions: These results demonstrate a novel interaction between canonical Wnt and BMP signaling that induces myogenic differentiation towards slow muscle phenotype. http://www.skeletalmusclejournal.com/content/3/1/5 Kazuki Kuroda Shihuan Kuang Taketo Makoto Michael Rudnicki Skeletal Muscle 2013, null:5 2013-03-05T00:00:00Z doi:10.1186/2044-5040-3-5 /content/figures/2044-5040-3-5-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 5 2013-03-05T00:00:00Z XML Background: The clinical features of myofibrillar myopathies display a wide phenotypic heterogeneity. To this date, no studies have evaluated this parameter due to the absence of pertinent animal models. By studying two mutants of desmin, which induce subtle phenotypic differences in patients, we address this issue using an animal model based on the use of adeno-associated virus (AAV) vectors carrying mutated desmin cDNA. Methods: After preparation of the vectors, they were injected directly into the tibialis anterior muscles of C57BL/6 mice to allow expression of wild-type (WT) or mutated (R406W or E413K) desmin. Measurements of maximal force were carried out on the muscle in situ and then the injected muscles were analyzed to determine the structural consequences of the desmin mutations on muscle structure (microscopic observations, histology and immunohistochemistry). Results: Injection of AAV carrying WT desmin results in the expression of exogenous desmin in 98% of the muscle fibers without any pathological or functional perturbations. Exogenous WT and endogenous desmin are co-localized and no differences were observed compared to non-injected muscle. Expression of desmin mutants in mouse muscles induce morphological changes of muscle fibers (irregular shape and size) and the appearance of desmin accumulations around the nuclei (for R406W) or in subsarcolemmal regions of fibers (for E413K). These accumulations seem to occur and disrupt the Z-line, and a strong regeneration was observed in muscle expressing the R406W desmin, which is not the case for E413K. Moreover, both mutants of desmin studied here induce a decrease in muscle force generation capacity. Conclusions: In this study we show that AAV-mediated expression of desmin mutants in mouse muscles recapitulate the aggregation features, the decrease in contractile function and the morphological changes observed in patients with myofibrillar myopathy. More importantly, our results suggest that the R406W desmin mutant induces a robust muscle regeneration, which is not the case for the E413K mutant. This difference could help to explain the phenotypic differences observed in patients. Our results highlight the heterogeneous pathogenic mechanisms between different desmin mutants and open the way for new advances in the study of myofibrillar myopathies. http://www.skeletalmusclejournal.com/content/3/1/4 Pierre Joanne Oussama Chourbagi Christophe Hourdé Arnaud Ferry Gillian Butler-Browne Patrick Vicart Julie Dumonceaux Onnik Agbulut Skeletal Muscle 2013, null:4 2013-02-20T00:00:00Z doi:10.1186/2044-5040-3-4 /content/figures/2044-5040-3-4-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 4 2013-02-20T00:00:00Z XML Background: The complexity of the skeletal muscle and the identification of numerous human disease-causing mutations in its constitutive proteins make it an interesting tissue for proteomic studies aimed at understanding functional relationships of interacting proteins in both health and diseases.MethodWe undertook a large-scale study using two-hybrid screens and a human skeletal-muscle cDNA library to establish a proteome-scale map of protein-protein interactions centered on proteins involved in limb-girdle muscular dystrophies (LGMD). LGMD is a group of more than 20 different neuromuscular disorders that principally affect the proximal pelvic and shoulder girdle muscles.Results and conclusionThe interaction network we unraveled incorporates 1018 proteins connected by 1492 direct binary interactions and includes 1420 novel protein-protein interactions. Computational, experimental and literature-based analyses were performed to assess the overall quality of this network. Interestingly, LGMD proteins were shown to be highly interconnected, in particular indirectly through sarcomeric proteins. In-depth mining of the LGMD-centered interactome identified new candidate genes for orphan LGMDs and other neuromuscular disorders. The data also suggest the existence of functional links between LGMD2B/dysferlin and gene regulation, between LGMD2C/γ-sarcoglycan and energy control and between LGMD2G/telethonin and maintenance of genome integrity. This dataset represents a valuable resource for future functional investigations. http://www.skeletalmusclejournal.com/content/3/1/3 Gaëlle Blandin Sylvie Marchand Karine Charton Nathalie Danièle Evelyne Gicquel Jean-Baptiste Boucheteil Azéddine Bentaib Laetitia Barrault Daniel Stockholm Marc Bartoli Isabelle Richard Skeletal Muscle 2013, null:3 2013-02-15T00:00:00Z doi:10.1186/2044-5040-3-3 /content/figures/2044-5040-3-3-toc.gif Skeletal Muscle 2044-5040 ${item.volume} 3 2013-02-15T00:00:00Z XML