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Polyunsaturated fatty acids (PUFAs) are the main components of cell membrane affecting its fluidity, signaling processes and play a vital role in muscle cell development. The effects of docosahexaenoic acid (DHA) on myogenesis are well known, while the effects of arachidonic acid (AA) are largely unclear. The purpose of this study is to evaluate the effect of two PUFAs (DHA and AA) on cell fate during myogenic processes, Wnt signaling and energy metabolism by using the C2C12 cells. The cells were treated with different concentrations of AA or DHA for 48 h during the differentiation period. PUFA treatment increased mRNA level of myogenic factor 5 ( Myf5 ), which is involved in early stage of myoblast proliferation. Additionally, PUFA treatment prevented myoblast differentiation, indicated by decreased myotube fusion index and differentiation index in parallel with reduced mRNA levels of myogenin ( MyoG ). After PUFA withdrawal, some changes in cell morphology and myosin heavy chain mRNA levels were still observed. Expression of genes associated with Wnt signaling pathway, and energy metabolism changed in PUFA treatment in a dose and time dependent manner. Our data suggests that PUFAs affect the transition of C2C12 cells from proliferation to differentiation phase by prolonging proliferation and preventing differentiation.

Myogenic regulators of muscle development, metabolism and growth differ between fish species in a context-specific manner. Commonly, the analysis of environmental influences on the expression of muscle-related gene regulators in teleosts is based on differences in swimming performance, feeding behaviour and stress-resistance, but the evaluation of behavioural phenotyping of immune and stress-related responsiveness in skeletal muscle is still scarce. Here we challenge proactive and reactive fingerlings of gilthead sea bream (Sparus aurata), one of the most commonly cultured species in the Mediterranean area, with highly pathogenic O1, O2α and O2β serotypes of Vibrio anguillarum, a widespread opportunistic pathogen of marine animals, to analyse skeletal muscle responses to bath vaccination. Transcripts related to inflammation (interleukin 1β, il1β; tumour necrosis factor-α, tnfα; and immunoglobulin M, igm), and muscle metabolism and growth (lipoprotein, lpl; myostatin, mstn-1; myogenin; and growth hormone receptors type I and II, ghr1 and ghr2, respectively) were analysed. Biochemical indicators of muscle metabolism and function (creatine kinase, CK, aspartate aminotransferase, AST; esterase activity, EA; total antioxidant status, TAC and glucose) were also determined. Our results indicate that proactive, but not reactive, fish respond to Vibrio vaccination by increasing the expression levels of mstn-1, myogenin and ghr2 transcripts at short-/medium- term (1 to 3 days' post vaccination). No effect of vaccination was observed in immune indicators or biochemical parameters in either phenotypes, except for elevated levels of EA in reactive fish one-week post vaccination. This suggests that behavioural divergence should be taken into account to evaluate the crosstalk between immune, metabolic and growth processes in muscle of immune-challenged fish., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Skeletal muscle has the remarkable capability to regenerate itself following injury. Adult myogenic stem cells (MSCs) are responsible for the repair and regeneration, and their activity is controlled by intrinsic and extrinsic factors. The aim of this study was to examine and compare the expression levels of Pax3, Pax7, MRF and p38 proteins during the course of regeneration and in different areas of the focal freeze-lesion damaged adult rat TA muscle. Using the focal freeze injury model, immunohistochemistry, laser-capture micro-dissection and Western blot analysis were performed. The results show that (1) in the severely damaged area, the focal freeze-lesion injury significantly activated Pax7 and myogenin expression within 7 days and down-regulated Pax3, MyoD and Myf-5 within 1 or 3 days, and (2) the level of the p38 protein was strongly and transiently up-regulated in the whole muscle on day 7 following injury, whereas the level of the pp38 protein was down-regulated within 3 days in the severely damaged and non-damaged areas. These findings indicate that the temporal (e.g. the time course of regeneration) and spatial (e.g. three zones created by the focal freeze-lesion) cues in a regenerating muscle have a significant impact on the activity of the adult MSCs., (© 2019 Anatomical Society.)

MyoD upstream noncoding RNA (MUNC) initiates in the distal regulatory region (DRR) enhancer of MYOD and is formally classified as an enhancer RNA (DRR eRNA ). MUNC is required for optimal myogenic differentiation, induces specific myogenic transcripts in trans ( MYOD , MYOGENIN , and MYH3 ), and has a functional human homolog. The vast majority of eRNAs are believed to act in cis primarily on their neighboring genes (1, 2), making it likely that MUNC action is dependent on the induction of MYOD RNA. Surprisingly, MUNC overexpression in MYOD -/- C2C12 cells induces many myogenic transcripts in the complete absence of MyoD protein. Genomewide analysis showed that, while many genes are regulated by MUNC in a MyoD-dependent manner, there is a set of genes that are regulated by MUNC, both upward and downward, independently of MyoD. MUNC and MyoD even appear to act antagonistically on certain transcripts. Deletion mutagenesis showed that there are at least two independent functional sites on the MUNC long noncoding RNA (lncRNA), with exon 1 more active than exon 2 and with very little activity from the intron. Thus, although MUNC is an eRNA of MYOD , it is also a trans -acting lncRNA whose sequence, structure, and cooperating factors, which include but are not limited to MyoD, determine the regulation of many myogenic genes., (Copyright © 2018 American Society for Microbiology.)

The enhancer regions of the myogenic master regulator MyoD give rise to at least two enhancer RNAs. Core enhancer eRNA ( CE eRNA) regulates transcription of the adjacent MyoD gene, whereas DRR eRNA affects expression of Myogenin in trans. We found that DRR eRNA is recruited at the Myogenin locus, where it colocalizes with Myogenin nascent transcripts. DRR eRNA associates with the cohesin complex, and this association correlates with its transactivating properties. Despite being expressed in undifferentiated cells, cohesin is not loaded on Myogenin until the cells start expressing DRR eRNA, which is then required for cohesin chromatin recruitment and maintenance. Functionally, depletion of either cohesin or DRR eRNA reduces chromatin accessibility, prevents Myogenin activation, and hinders muscle cell differentiation. Thus, DRR eRNA ensures spatially appropriate cohesin loading in trans to regulate gene expression., (Published by Elsevier Inc.)

Background: Tyrosine kinase inhibitors (TKIs) are effective therapies with demonstrated antineoplastic activity. Nilotinib is a second-generation FDA-approved TKI designed to overcome Imatinib resistance and intolerance in patients with chronic myelogenous leukemia (CML). Interestingly, TKIs have also been shown to be an efficient treatment for several non-malignant disorders such fibrotic diseases, including those affecting skeletal muscles., Methods: We investigated the role of Nilotinib on skeletal myogenesis using the well-established C2C12 myoblast cell line. We evaluated the impact of Nilotinib during the time course of skeletal myogenesis. We compared the effect of Nilotinib with the well-known p38 MAPK inhibitor SB203580. MEK1/2 UO126 and PI3K/AKT LY294002 inhibitors were used to identify the signaling pathways involved in Nilotinib-related effects on myoblast. Adult primary myoblasts were also used to corroborate the inhibition of myoblasts fusion and myotube-nuclei positioning by Nilotinib., Results: We found that Nilotinib inhibited myogenic differentiation, reducing the number of myogenin-positive myoblasts and decreasing myogenin and MyoD expression. Furthermore, Nilotinib-mediated anti-myogenic effects impair myotube formation, myosin heavy chain expression, and compromise myotube-nuclei positioning. In addition, we found that p38 MAPK is a new off-target protein of Nilotinib, which causes inhibition of p38 phosphorylation in a similar manner as the well-characterized p38 inhibitor SB203580. Nilotinib induces the activation of ERK1/2 and AKT on myoblasts but not in myotubes. We also found that Nilotinib stimulates myoblast proliferation, a process dependent on ERK1/2 and AKT activation., Conclusions: Our findings suggest that Nilotinib may have important negative effects on muscle homeostasis, inhibiting myogenic differentiation but stimulating myoblasts proliferation. Additionally, we found that Nilotinib stimulates the activation of ERK1/2 and AKT. On the other hand, we suggest that p38 MAPK is a new off-target of Nilotinib. Thus, there is a necessity for future studies to investigate the long-term effects of TKIs on skeletal muscle homeostasis, along with potential detrimental effects in cell differentiation and proliferation in patients receiving TKI therapies.

Background: Skeletal muscle satellite cell-derived myoblasts are mainly responsible for postnatal muscle growth and injury-induced regeneration. Many intracellular signaling pathways are essential for myogenic differentiation, while a number of kinases are involved in this modulation process. Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI) was identified as one of the key kinases involved in myogenic differentiation, but the underlying molecular mechanism is still unclear., Methods: PIP5K1α was quantified by quantitative reverse transcriptase PCR and western blot assay. Expression levels of myogenin and myosin heavy chain, which showed significant downregulation in PIP5K1α siRNA-mediated knockdown cells in western blot analysis, were confirmed by immunostaining. Phosphatidylinositol 4,5-bisphosphate in PIP5K1α siRNA-mediated knockdown cells was also measured by the PI(4,5)P2 Mass ELISA Kit. C2C12 cells were overexpressed with different forms of AKT, followed by western blot analysis on myogenin and myosin heavy chain, which reveals their function in myogenic differentiation. FLIPR assays are used to test the release of calcium in PIP5K1α siRNA-mediated knockdown cells after histamine or bradykinin treatment. Statistical significances between groups were determined by two-tailed Student's t test., Results: Since PIP5K1α was the major form in skeletal muscle, knockdown of PIP5K1α consistently inhibited myogenic differentiation while overexpression of PIP5K1α promoted differentiation and rescued the inhibitory effect of the siRNA. PIP5K1α was found to be required for AKT activation and calcium release, both of which were important for skeletal muscle differentiation., Conclusions: Taken together, these results suggest that PIP5K1α is an important regulator in myoblast differentiation.

Foxo4 and MyoG proteins regulate the transcription of numerous genes, including the E3 ubiquitin ligases MAFbx and MuRF1, which are activated in skeletal muscle under atrophy-inducing conditions. In the thirteen-lined ground squirrel, there is little muscle wasting that occurs during hibernation, a process characterized by bouts of torpor and arousal, despite virtual inactivity. Consequently, we were interested in studying the regulatory role of Foxo4 and MyoG on ubiquitin ligases throughout torpor-arousal cycles. Findings indicate that MAFbx and MuRF1 decreased during early torpor (ET) by 42% and 40%, respectively, relative to euthermic control (EC), although MuRF1 expression subsequently increased at late torpor (LT). The expression pattern of MyoG most closely resembled that of MAFbx, with levels decreasing during LT. In addition, the phosphorylation of Foxo4 at Thr-451 showed an initial increase during EN, followed by a decline throughout the remainder of the torpor-arousal cycle, suggesting Foxo4 inhibition. This trend was mirrored by inhibition of the Ras-Ral pathway, as the Ras and Ral proteins were decreased by 77% and 41% respectively, at ET. Foxo4 phosphorylation at S197 was depressed during entrance and torpor, suggesting Foxo4 nuclear localization, and possibly regulating the increase in MuRF1 levels at LT. These findings indicate that signaling pathways involved in regulating muscle atrophy, such as MyoG and Foxo4 through the Ras-Ral pathway, contribute to important muscle-specific changes during hibernation. Therefore, this data provides novel insight into the molecular mechanisms regulating muscle remodeling in a hibernator model., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Muscle regeneration is an important process for skeletal muscle growth and recovery. Repair of muscle damage is exquisitely programmed by cellular mechanisms inherent in myogenic stem cells, also known as muscle satellite cells. We demonstrated previously the involvement of homeobox transcription factors, SIX1, SIX4 and SIX5, in the coordinated proliferation and differentiation of isolated satellite cells in vitro. However, their roles in adult muscle regeneration in vivo remain elusive. To investigate SIX4 and SIX5 functions during muscle regeneration, we introduced knockout alleles of Six4 and Six5 into an animal model of Duchenne Muscular Dystrophy (DMD), mdx (Dmd(mdx) /Y) mice, characterized by frequent degeneration-regeneration cycles in muscles. A lower number of small myofibers, higher number of thick ones and lower serum creatine kinase and lactate dehydrogenase activities were noted in 50-week-old Six4(+/-) 5(+/-) Dmd(mdx) /Y mice than Dmd(mdx) /Y mice, indicating improvement of dystrophic phenotypes of Dmd(mdx) /Y mice. Higher proportions of cells positive for MYOD1 and MYOG (markers of regenerating myonuclei) and SIX1 (a marker of regenerating myoblasts and newly regenerated myofibers) in 12-week-old Six4(+/-) 5(+/-) Dmd(mdx) /Y mice suggested enhanced regeneration, compared with Dmd(mdx) /Y mice. Although grip strength was comparable in Six4(+/-) 5(+/-) Dmd(mdx) /Y and Dmd(mdx) /Y mice, treadmill exercise did not induce muscle weakness in Six4(+/-) 5(+/-) Dmd(mdx) /Y mice, suggesting higher regeneration capacity. In addition, Six4(+/-) 5(+/-) Dmd(mdx) /Y mice showed 33.8% extension of life span. The results indicated that low Six4 and Six5 gene dosage improved dystrophic phenotypes of Dmd(mdx) /Y mice by enhancing muscle regeneration, and suggested that SIX4 and SIX5 are potentially useful de novo targets in therapeutic applications against muscle disorders, including DMD., (© 2016 Japanese Society of Developmental Biologists.)

The therapeutic potential of mesenchymal stem cell-conditioned medium (MSC-CM) has been reported with various types of disease models. Here, we examine the therapeutic effect of umbilical cord MSC-CM (UCMSC-CM) on muscle-related disease, using a dexamethasone (Dex)-induced muscle atrophy in vitro model. The expressions of muscle atrophy-related proteins (MuRF-1 and MAFbx) and muscle-specific proteins (desmin and myogenin) were evaluated by Western blot analysis. The level of production of reactive oxygen species (ROS) was determined using a 2',7'-dichlorofluorescein diacetate (DCFDA) dye assay. The expression of antioxidant enzymes (copper/zinc-superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (MnSOD), glutathione peroxidase-1 (GPx-1), and catalase (CAT)) was verified by reverse transcription polymerase chain reaction (RT-PCR). When L6 cells were exposed to Dex, the expression of muscle atrophy-related proteins was increased by 50-70%, and the expression of muscle-specific proteins was in turn decreased by 23-40%. Conversely, when the L6 cells were co-treated with UCMSC-CM and Dex, the expression of muscle atrophy-related proteins was reduced in a UCMSC-CM dose-dependent manner and the expression of muscle-specific proteins was restored to near-normal levels. Moreover, ROS generation was effectively suppressed and the expression of antioxidant enzymes was recovered to a normal degree. These data imply that UCMSC-CM clearly has the potential to prevent muscle atrophy. Thus, our present study offers fundamental data on the potential treatment of muscle-related disease using UCMSC-CM.

Acetylcholine receptor (AChR) expression in innervated muscle is limited to the synaptic region. Neuron-induced electrical activity participates in this compartmentalization by promoting the repression of AChR expression in the extrasynaptic regions. Here, we show that the corepressor CtBP1 (C-terminal binding protein 1) is present on the myogenin promoter together with repressive histone marks. shRNA-mediated downregulation of CtBP1 expression is sufficient to derepress myogenin and AChR expression in innervated muscle. Upon denervation, CtBP1 is displaced from the myogenin promoter and relocates to the cytoplasm, while repressive histone marks are replaced by activating ones concomitantly to the activation of myogenin expression. We also observed that upon denervation the p21-activated kinase 1 (PAK1) expression is upregulated, suggesting that phosphorylation by PAK1 may be involved in the relocation of CtBP1. Indeed, preventing CtBP1 Ser158 phosphorylation induces CtBP1 accumulation in the nuclei and abrogates the activation of myogenin and AChR expression. Altogether, these findings reveal a molecular mechanism to account for the coordinated control of chromatin modifications and muscle gene expression by presynaptic neurons via a PAK1/CtBP1 pathway., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Several hormones and growth factors, including adipokines, play important roles during muscle development and regeneration. CTRP3, a paralog of adiponectin, is a member of the C1q and tumor necrosis factor-related protein (CTRP) superfamily. CTRP3 is a novel adipokine previously reported to reduce glucose output in hepatocytes and lower glucose levels in mice models. In the present study, we provide the first evidence for a physiological role of the CTRP3 in myogenesis using C2C12 myoblasts. CTRP3 was expressed in developing skeletal muscle tissues, and the expression level of CTRP3 was increased during myogenic differentiation of C2C12 cells. Recombinant CTRP3 (rCTRP3) promoted the proliferation of undifferentiated C2C12 myoblasts and this response required activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. In contrary, rCTRP3 inhibited myogenic differentiation and fusion of C2C12 cells by suppressing the expression of myogenic marker genes (myogenin and myosin heavy chain). CTRP3 mRNA expression was increased in C2C12 myoblasts treated with transforming growth factor-β3 (TGF-β3), suggesting that TGF-β3 is one of the extracellular factors regulating CTRP3 expression during myogenesis. These results indicate a novel physiological role for CTRP3 during skeletal myogenesis.

Klhl31 is a member of the Kelch-like family in vertebrates, which are characterized by an amino-terminal broad complex tram-track, bric-a-brac/poxvirus and zinc finger (BTB/POZ) domain, carboxy-terminal Kelch repeats and a central linker region (Back domain). In developing somites Klhl31 is highly expressed in the myotome downstream of myogenic regulators (MRF), and it remains expressed in differentiated skeletal muscle. In vivo gain- and loss-of-function approaches in chick embryos reveal a role of Klhl31 in skeletal myogenesis. Targeted mis-expression of Klhl31 led to a reduced size of dermomyotome and myotome as indicated by detection of relevant myogenic markers, Pax3, Myf5, myogenin and myosin heavy chain (MF20). The knock-down of Klhl31 in developing somites, using antisense morpholinos (MO), led to an expansion of Pax3, Myf5, MyoD and myogenin expression domains and an increase in the number of mitotic cells in the dermomyotome and myotome. The mechanism underlying this phenotype was examined using complementary approaches, which show that Klhl31 interferes with β-catenin dependent Wnt signaling. Klhl31 reduced the Wnt-mediated activation of a luciferase reporter in cultured cells. Furthermore, Klhl31 attenuated secondary axis formation in Xenopus embryos in response to Wnt1 or β-catenin. Klhl31 mis-expression in the developing neural tube affected its dorso-ventral patterning and led to reduced dermomyotome and myotome size. Co-transfection of a Wnt3a expression vector with Klhl31 in somites or in the neural tube rescued the phenotype and restored the size of dermomyotome and myotome. Thus, Klhl31 is a novel modulator of canonical Wnt signaling, important for vertebrate myogenesis. We propose that Klhl31 acts in the myotome to support cell cycle withdrawal and differentiation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Muscle abnormality could be a key reason for congenital clubfoot (CCF) deformity, which manifests itself during fetal development. FHL1 down-regulated expression is involved in the formation of skeletal muscle abnormalities in CCF and FHL1 gene mutations contribute to the development of some kinds of myopathies. Therefore, detecting dynamic expression of Fhl1 and other molecules (Hgf, MyoD1, Myogenin, and Myh4) that control limb muscle development in hind limbs of different gestational age will provide a foundation for further research on the molecular mechanism involves in the myopathies or CCF. The dynamic gene expression levels of Fhl1, Hgf, MyoD1, Myogenin, and Myh4 in the lower limbs of E16, E17, E19, and E20 rat embryos were examined by real-time RT-PCR. Immunofluorescence was used to detect formation of specific muscle fibers (fast or slow fibers) in distal E17 hind limbs. The expression levels of Fhl1, Hgf, MyoD1, Myogenin, and Myh4 were varying in hind limbs of different gestational age. Real-time PCR results showed that all the genes that control skeletal muscle development except for Fhl1 exhibited a peak in E17 lower limbs. Immunofluorescence results showed obviously positive fast-myosin in the distal E17 lower limbs and meanwhile slow-myosin had no apparently signals. E17 was a critical time point for terminal skeletal muscle differentiation in the lower limbs of rat embryos., (© 2014 APMIS. Published by John Wiley & Sons Ltd.)

Heat stress could promote skeletal muscle regeneration. But, in the regeneration process, effects of heat stress on myogenic cells and the regulating factors is unknown. Therefore, Influences of heat stress soon after injury on distribution of the myogenic cells and chronological changes in expression of MyoD and myogenin were examined. The first peak of MyoD expression was temporally correlated with the time when proliferating satellite cells began to appear, and the rapid decline of the MyoD expression from the first peak, with the appearance time of myoblasts, respectively in both the non-Heat and Heat groups. The first peak of myogenin expression was temporally correlated with the time when multinuclear cells began to form in the both groups. Due to the heat stress, proliferation and differentiation of myogenic cells and chronological changes in these factors were accelerated one day earlier than in the non-Heat group. As MyoD and myogenin are regulating factor of proliferation and differentiation, heat stress soon after the muscle injury could accelerate the proliferation and differentiation of myogenic cells and the expression of their regulating factors MyoD and myogenin.

In the present study, real time-polymerase chain reaction was applied to analyze the expression of IGF-I and MyoG genes in Hu sheep longissimus dorsi at different growth stages and their association with meat traits. Expression of the IGF-I gene in Hu sheep differed significantly between males and females at the two day-old (0.01 < P < 0.05), one-month old (0.01 < P < 0.05), and three month-old (P < 0.01) stages. IGF-I gene expression in male longissimus muscles was higher than that of females at all growth stages, except for the three month-old stage. There was no significant difference (P > 0.05) between males and females at any growth stage in expression of the MyoG gene. MyoG gene expression in male longissimus muscles tended to be higher than that of females at all growth stages, except for the six month-old stage. IGF-I gene expression was significantly and positively correlated with live weight (P < 0.01) and carcass weight (0.01< P < 0.05), and was non-significantly positively correlated with net meat weight (P > 0.05). In contrast, MyoG gene expression was non-significantly and positively correlated with live weight, carcass, and net meat weight (P > 0.05). Carcass traits showed highly significant positive correlations (P < 0.01). Furthermore, expressions of IGF-I and MyoG genes showed highly significant positive correlations (P < 0.01). We conclude that the expressions of IGF-I and MyoG genes are significantly and positively correlated with early muscle traits of Hu sheep.

The embryonic muscles of the axial skeleton and limbs take their origin from the dermomyotomes of the somites. During embryonic myogenesis, muscle precursors delaminate from the dermomyotome giving rise to the hypaxial and epaxial myotome. Mutant studies for myogenic regulatory factors have shown that the development of the hypaxial myotome differs from the formation of the epaxial myotome and that the development of the hypaxial myotome depends on the latter within the trunk region. The transcriptional networks that regulate the transition of proliferative dermomyotomal cells into the predominantly post-mitotic hypaxial myotome, as well as the eventual patterning of the myotome, are not fully understood. Similar transitions occurring during the development of the neural system have been shown to be controlled by the Atonal family of helix-loop-helix transcription factors. Here, we demonstrate that ATOH8, a member of the Atonal family, is expressed in a subset of embryonic muscle cells in the dermomyotome and myotome. Using the RNAi approach, we show that loss of ATOH8 in the lateral somites at the trunk level results in a blockage of differentiation and thus causes cells to be maintained in a predetermined state. Furthermore, we show that ATOH8 is also expressed in cultured C2C12 mouse myoblasts and becomes dramatically downregulated during their differentiation. We propose that ATOH8 plays a role during the transition of myoblasts from the proliferative phase to the differentiation phase and in the regulation of myogenesis in the hypaxial myotome of the trunk.

Background: Environmental factors affect stem cell differentiation. In addition to chemical factors, mechanical signals have been suggested to enhance myogenic differentiation of stem cells. Therefore, this study was undertaken to illustrate and compare the effect of chemical and mechanical stimuli on Myogenin (MyoG) and Myosin heavy chani 2 (Myh2) expression of mouse bone marrow-derived mesenchymal stem cells (BMSCs) and embryonic stem cells (ESCs)., Methods: After isolation and expansion of BMSCs and generation of embryoid bodies and spontaneous differentiation of ESCs, cells were examined in 4 groups: (1) control group: untreated cells; (2) chemical group: cells incubated in myogenic medium (5-azacythidine and horse serum for BMSCs, dimethyl sulfoxide (DMSO) and horse serum for ESCs) for 5 days; (3) mechanical group: cells exposed to uniaxial cyclic strain (8%, 1 Hz, 24 h) and (4) chemical + mechanical group: cells incubated in myogenic medium for 4 days and then exposed to uniaxial cyclic strain. Real-time PCR was used to examine the expression of MyoG and Myh2 as specific myogenic markers., Results: suggested that mechanical loading, as a single factor, could elevate MyoG and Myh2 expression. Combining chemical with mechanical factor increases expression and there was no significant difference in MyoG expression of ESCs- and MSCs-chemical + mechanical groups; however, Myh2 expression was significantly higher in ESCs-mechanical group than that in the same group of MSCs.

The complex process of skeletal muscle differentiation is organized by the myogenic regulatory factors (MRFs), Myf5, MyoD, Myf6, and myogenin, where myogenin plays a critical role in the regulation of the final stage of muscle differentiation. In an effort to investigate the role microRNAs (miRNAs) play in regulating myogenin, a bioinformatics approach was used and six miRNAs (miR-182, miR-186, miR-135, miR-491, miR-329, and miR-96) were predicted to bind the myogenin 3'-untranslated region (UTR). However, luciferase assays showed only miR-186 inhibited translation and 3'-UTR mutagenesis analysis confirmed this interaction was specific. Interestingly, the expression of miR-186 mirrored that of its host gene, ZRANB2, during development. Functional studies demonstrated that miR-186 overexpression inhibited the differentiation of C2C12 and primary muscle cells. Our findings therefore identify miR-186 as a novel regulator of myogenic differentiation.

This experiment was conducted to explore the biological functions of myogenin (MyoG) gene. MyoG gene was cloned from genome of Hu sheep by overlap extension PCR. Then, pEGFP-C1-MyoG and pcDNA3.0-MyoG fusion expression vectors was constructed and pEGFP-C1-MyoG vector had been transfected into NIH-3T3 cells by liposomes-mediated method, and MyoG was detected in vitro by RT-PCR,western blotting and its subcellular localization by EGFP marker. pcDNA3.0-MyoG was transfected into goat embryonic fibroblasts (GEF) cells in order to detect the myogenic function of MyoG in vitro. Then pEGFP-C1-MyoG plasmid was injected into the testes of sheep and goat, respectively, to produce the transgenic generation. The results showed that the length of MyoG coding region of Hu sheep was 675 bp, encoding 224 amino acids. Compared with goat, cattle, pig and rat, the sequence homology of sheep MyoG cDNA was 99.26, 97.04, 92.00, and 87.70 %, respectively. The bioinformatics prediction showed that MyoG protein contained a typical bHLH structure, but without a short signal peptide, revealing that MyoG protein might be a non-secretory protein. The result of RT-PCR and western blotting demonstrated that MyoG could be expressed successfully in the transfected cells in vitro and the MyoG protein was located in nucleus. The positive transfected GEF cells with pcDNA3.0-MyoG were found to express desmin protein. The positive rates of transgenic sheep and transgenic goat were 7.1 and 7.4 % in F1 generation, respectively. Conclusively, MyoG cDNA from Hu sheep had been cloned successfully. The subcellular localization and myogenic activity of MyoG were exactly detected on the basis of multiple biological analyses, which expanded our understanding of the biological function of MyoG.

Skeletal muscle fiber type composition is one of the important factors influencing muscle growth and meat quality. As a member of the myogenic transcription factors, myogenin (MyoG) is required for embryonic myoblast differentiation, but the expression of MyoG continues in mature muscle tissue of adult animals, especially in oxidative metabolic muscle, which suggests that MyoG may play a more extended role. Therefore, using MyoG gene transfer mice and C2C12 myoblasts as in vivo and in vitro models, respectively, we elected to study the role of MyoG in muscle fiber types and oxidative metabolism by using overexpression and siRNA suppression strategies. The overexpression of MyoG by DNA electroporation in mouse gastrocnemius muscle had no significant effect on fiber type composition but upregulated the mRNA expression (P<0.01) and enzyme activity (P<0.05) of oxidative succinic dehydrogenase (SDH). In addition, downregulation of the activity of the glycolytic enzymes lactate dehydrogenase (LDH, P<0.05) and pyruvate kinase (PK, P<0.05) was observed in MyoG gene transfer mice. In vitro experiments verified the results obtained in mice. Stable MyoG-transfected differentiating C2C12 cells showed higher mRNA expression levels of myosin heavy chain (MyHC) isoform IIX (P<0.01) and SDH (P<0.05), while the LDH mRNA was attenuated. The enzyme activities of SDH (P<0.01) and LDH (P<0.05) were similarly altered at the mRNA level. When MyoG was knocked down in C2C12 cells, MyHC IIX expression (P<0.05) was decreased, but the mRNA level (P<0.05) and the enzyme activity (P<0.05) of SDH were increased. Downregulating MyoG also increased the activity of the glycolytic enzymes PK (P<0.05) and hexokinase (HK, P<0.05). Based on those results, we concluded that MyoG barely changes the MyHC isoforms, except MyHC IIX, in differentiating myoblasts but probably influences the shift from glycolytic metabolism towards oxidative metabolism both in vivo and in vitro. These results contribute to further understand the role of MyoG in skeletal muscle energy metabolism and also help to explore the key genes that regulate meat quality., (© 2013.)

To confirm the entire developmental process and transition point of embryonic Pekin duck pectoral muscle, and to investigate the association between pectoral muscle development and their regulating genes, anatomical and morphological analyses of embryonic Pekin duck skeletal muscles were performed, and the expression patterns of its regulating genes were investigated. The anatomical analysis revealed that body weight increased with age, while increases in pectoral muscle weight nearly ceased after the embryo was 20 days of hatching (E20). The developmental morphological characteristics of Pekin duck pectoral muscle at the embryonic stage showed that E20 was the transition point (from proliferation to fusion) of Pekin duck pectoral muscle. The expression patterns of MRF4, MyoG, and MSTN indicated that E19 or E20 was the fastest point of pectoral muscle development and the crucial transition for Pekin duck pectoral muscle development during the embryonic stage. Together, these findings imply that E20 is the crucial transition point (from proliferation to fusion) of Pekin duck pectoral muscle and that there is no muscle fiber hypertrophy after E20. Results of this study provide further understanding of the developmental process and transition point of Pekin duck pectoral muscle during the embryo stage.

Aims: Myogenin (myf4) is a nuclear transcription factor that is considered to be a sensitive and highly specific marker for skeletal muscle differentiation. Following the identification of focal strong nuclear staining with myogenin in two fibroepithelial polyps of the lower female genital tract (the index cases), we stained a series of vulvovaginal spindle cell lesions with this marker in order to investigate how widespread myogenin staining is in these lesions., Methods and Results: Fibroepithelial polyps (n = 13), other vulvovaginal mesenchymal lesions (n = 21) and vulval or vaginal spindle cell squamous carcinomas (n = 4) were stained for myogenin. Apart from the index cases, all of the other cases were negative, except for one vaginal spindle cell squamous carcinoma, which showed focal weak nuclear immunoreactivity. Ten of 12 embryonal rhabdomyosarcomas of the lower female genital tract were myogenin-positive, as was a single vaginal rhabdomyoma., Conclusions: Our study illustrates that focal myogenin immunoreactivity occurs uncommonly in fibroepithelial polyps of the lower female genital tract. This may result in diagnostic confusion and misdiagnosis as a skeletal muscle neoplasm, especially the sarcoma botryoides variant of embryonal rhabdomyosarcoma., (© 2013 John Wiley & Sons Ltd.)

The hypothesis of this study was that 17β-estradiol (estradiol) stimulates turkey skeletal muscle growth by influencing myogenic satellite cell proliferation, differentiation, and the gene expression of selected proteins important in regulating growth and development. Increasing levels of estradiol were administered in basal medium containing additional nutrients. Female-derived pectoralis major (PM) satellite cell proliferation was stimulated by estradiol at a level of 10(-9)M following 4days of treatment. Male PM and biceps femoris (BF) satellite cell proliferation was increased at 10(-12)M estradiol. Turkey embryonic myoblast proliferation, however, decreased with 10(-9)M and 10(-5)M estradiol following 3days under these conditions. Estradiol had no effect on the differentiation of any of the 4 groups of cells. Likewise, glypican-1 expression was unaffected by estradiol treatment. MyoD expression decreased in male PM but not BF cells. MyoD expression in female PM cells and embryonic myoblasts were also unaffected by estradiol administration. Estradiol decreased myogenin expression in male satellite cells, but had no effect on female cells. There was a slight decrease in myogenin expression in embryonic myoblasts. The results demonstrate a direct effect of estradiol on avian satellite cell proliferation independent of glypican-1, and decreased expression of MyoD and myogenin in some myogenic cells, coinciding with increased cellular proliferation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Myogenic differentiation of skeletal muscle cells is characterized by a sequence of events that include activation of signal transducer and activator of transcription 3 (STAT3) and enhanced expression of its target gene Socs3. Autocrine effects of IL-6 may contribute to the activation of the STAT3-Socs3 cascade and thus to myogenic differentiation. The importance of IL-6 and STAT3 for the differentiation process was studied in C2C12 cells and in primary mouse wild-type and IL-6(-/-) skeletal muscle cells. In differentiating C2C12 myoblasts, the upregulation of IL-6 mRNA expression and protein secretion started after increased phosphorylation of STAT3 on tyrosine 705 and increased mRNA expression of Socs3 was observed. Knockdown of STAT3 and IL-6 mRNA in differentiating C2C12 myoblasts impaired the expression of the myogenic markers myogenin and MyHC IIb and subsequently myotube fusion. However, the knockdown of IL-6 did not prevent the induction of STAT3 tyrosine phosphorylation. The IL-6-independent activation of STAT3 was verified in differentiating primary IL-6(-/-) myoblasts. The phosphorylation of STAT3 and the expression levels of STAT3, Socs3, and myogenin during differentiation were comparable in the primary myoblasts independent of the genotype. However, IL-6(-/-) cells failed to induce MyHC IIb expression to the same level as in wild-type cells and showed reduced myotube formation. Supplementation of IL-6 could partially restore the fusion of IL-6(-/-) cells. These data demonstrate that IL-6 depletion during myogenic differentiation does not reduce the activation of the STAT3-Socs3 cascade, while IL-6 and STAT3 are both necessary to promote myotube fusion.

The biological function of selenium (Se) is mainly elicited through Se-containing proteins. Selenoprotein W (SelW), one member of the selenoprotein family, is essential for the normal function of the skeletal muscle system. To investigate the possible relationship of Se in the process of differentiation in chicken myoblasts and the expression of SelW, the cultured chicken embryonic myoblasts were incubated with sodium selenite at different concentrations for 72 h, and then the mRNA levels of SelW and myogenic regulatory factors (MRFs) in myoblasts were determined at 12, 24, 48, and 72 h, respectively. Furthermore, the correlation between SelW mRNA expression and MRF mRNA expression was assessed. The results showed that the sodium selenite medium enhanced the mRNA expression of SelW, Myf-5, MRF4, and myogenin in chicken myoblasts. The mRNA expression levels of MRFs were significantly correlated with those of SelW at 24, 48, and 72 h. These data demonstrate that Se is involved in the differentiation of chicken embryonic myoblasts, and SelW showed correlation with MRFs.

Gene expression profile in diaphragm in comparison to three principally different hindlimb muscles (soleus, red and white gastrocnemius) was studied using quantitative PCR. Expression levels of PGC-1alpha mRNA and myogenin mRNA in diaphragm were in accordance with its myosin phenotype and citrate synthase activity. However, diaphragm was characterised by atypically high content of MyoD mRNA as well as high content of IGF-1 mRNA and low content of myostatin mRNA. The latter two findings suggest high intensity of protein synthesis in diaphragm muscle fibers, although they have smaller cross sectional area than fibers in locomotor muscles.

In the nucleus of differentiated osteoblasts, the DNA-binding αNAC protein acts as a transcriptional coactivator of the Osteocalcin gene. Chromatin immunoprecipitation-microarray assays (ChIP-chip) showed that αNAC binds the Osteocalcin promoter but also identified the Myogenin promoter as an αNAC target. Here, we confirm these array data using quantitative ChIP and further detected that αNAC binds to these promoters in myoblasts. Since these genes are differentially regulated during osteoblastogenesis or myogenesis, these results suggest cell- and promoter-context specific functions for αNAC. We hypothesized that αNAC dynamically recruits corepressors to inhibit Myogenin expression in cells committing to the osteoblastic lineage or to inhibit Osteocalcin transcription in differentiating myoblasts. Using co-immunoprecipitation assays, we detected complexes between αNAC and the corepressors HDAC1 and HDAC3, in myoblasts and osteoblasts. Sequential ChIP confirmed HDAC1 recruitment by αNAC at the Osteocalcin and Myogenin promoters. Interaction with the corepressors was detectable in pre-osteoblasts and in myoblasts but disappeared as the cells differentiate. Treatment with an HDAC inhibitor caused de-repression of Osteocalcin expression in myoblasts. Overexpression of αNAC in myoblasts inhibits expression of Myogenin and differentiation. However, overexpression of an N-terminus truncated αNAC mutant allowed myoblasts to express Myogenin and differentiate, and this mutant did not interact with HDAC1 or HDAC3. This study identified an additional DNA-binding target and novel protein-protein interactions for αNAC. We propose that αNAC plays a role in regulating gene transcription during mesenchymal cell differentiation by differentially recruiting corepressors at target promoters., (Copyright © 2012 Elsevier B.V. All rights reserved.)

MyoD and myogenin (Myog) recognize sets of distinct but overlapping target genes and play different roles in skeletal muscle differentiation. MyoD is sufficient for near-full expression of early targets, while Myog can only partially enhance expression of MyoD-initiated late muscle genes. However, the way in which Myog enhances the expression of MyoD-initiated late muscle genes remains unclear. Here, we examine the effects of Myog on chromatin remodeling at late muscle gene promoters and their activation within chromatin environment. Chromatin immunoprecipitation (ChIP) assay showed that Myog selectively bound to the regulatory sequences of late muscle genes. Overexpression of Myog was found to overcome sodium butyrateinhibited chromatin at late muscle genes in differentiating C2C12 myoblasts, shifting the transcriptional activation of these genes to an earlier time period. Furthermore, overexpression of Myog led to increased hyperacetylation of core histone H4 in differentiating C2C12 myoblasts but not NIH3T3 fibroblasts, and hyperacetylated H4 was associated directly with the late muscle genes in differentiating C2C12, indicating that Myog can induce chromatin remodeling in the presence of MyoD. In addition, co-immunoprecipitation (CoIP) revealed that Myog was associated with the nuclear protein Brd4 in differentiating C2C12 myoblasts. Together, these results suggest that Myog enhances the expression of MyoD-initiated late muscle genes through MyoD-dependent ability of Myog to induce chromatin remodeling, in which Myog-Brd4 interaction may be involved.

The muscle growth in mammals is regulated by several growth factors including myostatin (MSTN), a member of the transforming growth factor-beta (TGF-beta) superfamily. To date, it is unknown in fish whether MSTN could have any effect on proliferation or differentiation of myogenic cells. Using culture of trout satellite cells, we showed that mstn1a and mstn1b mRNA are expressed in myoblasts and that their expression decreased in differentiating myoblasts. We also demonstrated that a treatment with huMSTN decreased the proliferation of IGF1-stimulated myoblasts in a dose-dependent manner. By contrast, treatment of myoblasts with 100 nM of huMSTN for three days, did not affect the percentage of positive cells for myogenin neither the percentage of nuclei in myosin positive cells. Moreover, our results clearly indicated that huMSTN treatment had no effect on MyoD and myogenin protein levels, which suggests that huMSTN did not strongly affect MyoD activity. In conclusion, we showed that huMSTN inhibited proliferation but not differentiation of trout myoblasts, probably resulting from a lack of huMSTN effect on MyoD activity. Altogether, these results show high interspecies differences in the function of MSTN., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Anabolic/androgenic steroids (AAS) are drugs that enhance muscle mass, and are often illegally utilized in athletes to improve their performances. Recent data suggest that the increased risk for amyotrophic lateral sclerosis (ALS) in male soccer and football players could be linked to AAS abuse. ALS is a motor neuron disease mainly occurring in sporadic (sALS) forms, but some familial forms (fALS) exist and have been linked to mutations in different genes. Some of these, in their wild type (wt) form, have been proposed as risk factors for sALS, i.e. superoxide dismutase 1 (SOD1) gene, whose mutations are causative of about 20% of fALS. Notably, SOD1 toxicity might occur both in motor neurons and in muscle cells. Using gastrocnemius muscles of mice overexpressing human mutant SOD1 (mutSOD1) at different disease stages, we found that the expression of a selected set of genes associated to muscle atrophy, MyoD, myogenin, atrogin-1, and transforming growth factor (TGF)β1, is up-regulated already at the presymptomatic stage. Atrogin-1 gene expression was increased also in mice overexpressing human wtSOD1. Similar alterations were found in axotomized mouse muscles and in cultured ALS myoblast models. In these ALS models, we then evaluated the pharmacological effects of the synthetic AAS nandrolone on the expression of the genes modified in ALS muscle. Nandrolone administration had no effects on MyoD, myogenin, and atrogin-1 expression, but it significantly increased TGFβ1 expression at disease onset. Altogether, these data suggest that, in fALS, muscle gene expression is altered at early stages, and AAS may exacerbate some of the alterations induced by SOD1 possibly acting as a contributing factor also in sALS., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Differentiation of skeletal muscle myoblasts involves activation of muscle-specific markers such as MyoD, Myf5, MRF4, and myogenin, followed by exit from the cell cycle, expression of structural proteins, and fusion into multinucleated myotubes. Cyclin D3 is upregulated during muscle differentiation, and expression of cyclin D3 in proliferating myoblasts causes early activation of myogenesis. In this study, we have identified the genes activated by cyclin D3 expression in C2C12 myoblasts and differentiated cells by real-time PCR analysis. Cyclin D3 expression induced faster differentiation kinetics and increase in levels of myogenic genes such as MyoD, Myf5, and myogenin at an early stage during the differentiation process, although long-term myogenic differentiation was not affected. Transcript levels of the transcription factor Pax7 that is expressed in muscle progenitors were enhanced by cyclin D3 expression in myoblasts. Components of a histone methyltransferase complex recruited by Pax7 to myogenic gene promoters were also regulated by cyclin D3. Further, the Pax7 promoter was upregulated in myoblasts expressing cyclin D3. Myoblasts that expressed cyclin D3 showed moderately higher levels of the cyclin-dependent kinase inhibitor p21 and were stalled in G2/M phase of the cell cycle. Our findings suggest that cyclin D3 primes myoblasts for differentiation by enhancing muscle specific gene expression and cell cycle exit., (Copyright © 2011 Wiley Periodicals, Inc.)

In adult muscles and under normal physiological conditions, satellite cells are found in a quiescent state but can be induced to enter the cell cycle by signals resulting from exercise, injury-induced muscle regeneration, or specific disease states. Once activated, satellite cells proliferate, self-renew, and differentiate to form myofibers. In the present study, we found that the zinc finger-containing factor Teashirt-3 (TSHZ3) was expressed in quiescent satellite cells of adult mouse skeletal muscles. We showed that following treatment with cardiotoxin TSHZ3 was strongly expressed in satellite cells of regenerating muscles. Moreover, immunohistochemical analysis indicated that TSHZ3 was expressed in both quiescent and activated satellite cells on intact myofibers in culture. TSHZ3 expression was maintained in myoblasts but disappeared with myotube formation. In C2C12 myoblasts, we showed that overexpression of Tshz3 impaired myogenic differentiation and promoted the down-regulation of myogenin (Myog) and up-regulation of paired-box factor 7 (Pax7). Moreover, knockdown experiments revealed a selective effect of Tshz3 on Myog regulation, and transcriptional reporter experiments indicated that TSHZ3 repressed Myog promoter. We identified the BRG1-associated factor 57 (BAF57), a subunit of the SWI/SNF complex, as a partner of TSHZ3. We showed that TSHZ3 cooperated with BAF57 to repress MYOD-dependent Myog expression. These results suggest a novel mechanism for transcriptional repression by TSHZ3 in which TSHZ3 and BAF57 cooperate to modulate MyoD activity on the Myog promoter to regulate skeletal muscle differentiation.

Background and Purpose: The bioflavonoid, hesperedin, promotes osteoblast differentiation in human mesenchymal stem cells, indicating an anabolic effect of hesperedin on bone metabolism. Murine bone marrow mesenchymal stem cells undergo myogenic differentiation as well as osteogenic differentiation. We therefore explored whether hesperedin modulates muscle cell differentiation., Experimental Approach: Myoblast C2C12 cells were differentiated into muscle cells in the presence or absence of hesperedin. The effects of hesperedin on myogenic differentiation were determined by analysing specific muscle markers in vitro using reporter gene assays, immunoblotting, RT-PCR and DNA pull-down assays. In vivo, the effects of hesperedin were assessed using the freeze injury-induced muscle regeneration model in mice and daily injections of hesperedin for 6 days., Key Results: Hesperedin promoted myogenic differentiation, in a dose-dependent manner, by increasing myogenin gene expression. MyoD-induced myogenin gene transcription was enhanced by hesperedin, as this bioflavonoid augmented the nuclear localization and myogenin promoter-binding of MyoD. In addition, hesperedin increased myogenin and muscle creatine kinase gene expression during myogenic differentiation from C3H10T1/2 mesenchymal stem cells in a MyoD-dependent manner and accelerated in vivo muscle regeneration induced by muscle injury., Conclusions and Implications: Our results demonstrate that hesperedin promoted myogenic differentiation in vitro and in vivo through activation of MyoD-mediated myogenin expression, suggesting a beneficial role in promoting muscle regeneration, following injury., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions. We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events. In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.5. Myogenin is a regulatory factor required for skeletal muscle differentiation. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

The effects of supplementation with creatine (Cr) and its analog, β-guanidinopropionic acid (β-GPA), on the differentiation of myoblasts and the numbers of nucleoli were studied in C2C12 cells. The cells were cultured in differentiation medium for 4 d. Then Cr (1 mM) or β-GPA (1 mM) was added to the cells, and the mixture was cultured for an additional 2 d. Although the number of myotubes was not different among the groups, myotube diameters and nuclear numbers in myotubes were increased by Cr and β-GPA treatment respectively. The expression of differentiation marker proteins, myogenin, and the myosine heavy chain, was increased in the β-GPA group. Supplementation with β-GPA also increased the percentage of p21 (inhibitor for cell cycle progression)-positive myoblasts. Supplementation with Cr inhibited the decrease in nucleoli numbers, whereas β-GPA increased nucleolar sizes in the myotubes. These results suggest that β-GPA supplementation stimulated the differentiation of myoblasts into multi-nucleated myotubes through induction of p21 expression.

Background: α-Syntrophin is a scaffolding protein linking signaling proteins to the sarcolemmal dystrophin complex in mature muscle. However, α-syntrophin is also expressed in differentiating myoblasts during the early stages of muscle differentiation. In this study, we examined the relationship between the expression of α-syntrophin and myogenin, a key muscle regulatory factor., Methods and Findings: The absence of α-syntrophin leads to reduced and delayed myogenin expression. This conclusion is based on experiments using muscle cells isolated from α-syntrophin null mice, muscle regeneration studies in α-syntrophin null mice, experiments in Sol8 cells (a cell line that expresses only low levels of α-syntrophin) and siRNA studies in differentiating C2 cells. In primary cultured myocytes isolated from α-syntrophin null mice, the level of myogenin was less than 50% that from wild type myocytes (p<0.005) 40 h after differentiation induction. In regenerating muscle, the expression of myogenin in the α-syntrophin null muscle was reduced to approximately 25% that of wild type muscle (p<0.005). Conversely, myogenin expression is enhanced in primary cultures of myoblasts isolated from a transgenic mouse over-expressing α-syntrophin and in Sol8 cells transfected with a vector to over-express α-syntrophin. Moreover, we find that myogenin mRNA is reduced in the absence of α-syntrophin and increased by α-syntrophin over-expression. Immunofluorescence microscopy shows that α-syntrophin is localized to the nuclei of differentiating myoblasts. Finally, immunoprecipitation experiments demonstrate that α-syntrophin associates with Mixed-Lineage Leukemia 5, a regulator of myogenin expression., Conclusions: We conclude that α-syntrophin plays an important role in regulating myogenesis by modulating myogenin expression.

Skeletal muscle growth is regulated by differential expression of myogenic regulatory factors (MRFs). We evaluated hyperplasia, hypertrophy and quantitative expression of MRFs MyoD and myogenin in 45, 90, 180, and 400 days post-hatching (dph) and adult pacu (Piaractus mesopotamicus) skeletal muscle. Transverse sections of white dorsal muscles were obtained to evaluate hypertrophy and hyperplasia. MyoD and myogenin gene expression was determined by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Pacu skeletal muscle had similar morphology at all stages. The highest and the lowest frequencies of fiber diameters <20 μm were found at the 45 dph and adult stages, respectively. Their frequency was similar in the 90, 180, and 400 dph stages. The highest percentage of >50 μm diameter fibers were found in 180 and 400 dph, and adult fish. Hyperplasia was the main mechanism observed in pacu skeletal muscle growth at 45dph; this declined through 90, 180, and 400 dph and remained low in adult fish; the latter presented hypertrophy as the main mechanism responsible for skeletal muscle growth. The high frequencies of 20-50 μm diameter fibers at 90, 180, and 400 dph can be related to intense hypertrophy. The mRNA levels for MyoD and myogenin were similar in 45, 90, and 400 dph and adult fish, peaking at 180 dph. The high MyoD expression at 180 dph can be related to intense myoblast proliferation and hyperplasia, while high myogenin expression can be related to intense myoblast differentiation and fusion during hypertrophy. MyoD and myogenin expression patterns in adults can respectively be associated with myoblast proliferation and differentiation, which both contribute to hypertrophy. Differential MyoD and myogenin expression in pacu white muscle probably is associated with differences in growth patterns during the stages analyzed. In this study, the 180 dph pacu could represent an interesting phase to investigate suitable strategies in commercial fish production focusing on skeletal muscle growth improvement to raise healthy, fast-growing fish., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

1. The role of satellite cells in muscle growth during development is well documented, but the involvement of these cells in muscle repair after contusion is less well known. In the present study, we investigated the time-course of satellite cell activity (from 3h to 7days) after contusion of rat gastrocnemius muscle using specific molecular markers for immunofluorescence and real-time polymerase chain reaction (PCR). 2. Inflammation of the injured muscle occurred within 6h, followed by disintegration of the damaged myofibres within 12h. Newly formed myofibres appeared by Day 7. 3. The number of MyoD-positive nuclei (activated satellite cells) in the injured muscle was significantly increased by 6h, reaching a maximum by 12h after contusion. However, the number of MyoD-positive nuclei decreased towards control levels by Day 7. Changes in the number of bromodeoxyuridine-labelled nuclei (proliferating satellite cells) paralleled the changes seen in the number of MyoD-positive nuclei. Conversely, expression of myogenin protein was not apparent until Day 3 and increased further by Day 7. Colabelling of MyoD and myogenin was seen in only a few cells. 4. The time-course of MyoD mRNA expression corresponded with MyoD protein expression. However, there were two peaks in myogenin mRNA expression: 6h and Day 7 after contusion. The second peak coincided with upregulation of myostatin mRNA levels. 5. The results of the present study suggest that contusion activates a homogeneous population of satellite cells to proliferate within 3days, followed by differentiation to form new myofibres. The latter may be regulated, in part, by myostatin., (© 2010 Blackwell Publishing Asia Pty Ltd.)

Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4(+/-)Six5(-/-) mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications., (Copyright © 2010 Elsevier Inc. All rights reserved.)

17beta-Estradiol (E(2)) mediates a wide variety of complex biological processes determining the growth and development of reproductive tract as well as nonreproductive tissues of male and female individuals. While E(2) effects on the reproductive system, bone, and cardiovascular system are quite well established, less is known about how it affects the physiology of other tissues. Skeletal muscle is a tissue that is expected to be E(2) responsive since both isoforms of estrogen receptor (ER-alpha and ER-beta) are expressed. Significant sex-related differences have been described in skeletal muscle, although the role played by E(2) and the mechanisms underlying it remain to be determined. Here, we demonstrate that E(2) increases the glucose transporter type 4 translocation at membranes as well as the expression of well-known differentiation markers of myogenesis (i.e., myogenin and myosin heavy chain) in rat myoblast cells (L6). These E(2)-induced effects require rapid extranuclear signals and the presence of ER-alpha, whereas no contribution of IGF-I receptor has been observed. In particular, ER-alpha-dependent Akt activation participates in regulating the first step of myogenic differentiation. Moreover, both receptors mediate the E(2)-induced activation of p38, which, in turn, affects the expression of myogenin and myosin heavy chain. All together, these data indicate that E(2) should be included in the list of skeletal muscle trophic factors.

The nuclear lamina and its associated proteins are important for nuclear structure and chromatin organization and also have been implicated in the regulation of cell signaling and gene expression. In this study we demonstrate that the lamina-associated nuclear envelope transmembrane protein NET37 is required for myogenic differentiation of C2C12 cells. NET37, a member of glycosidase family 31, is highly expressed in mouse skeletal muscle and is strongly up-regulated during C2C12 differentiation. By protease mapping we show that its glycosidase homology domain is located in the lumen of the nuclear envelope/endoplasmic reticulum. When NET37 is depleted from proliferating myoblasts by RNAi, myogenic differentiation is significantly impaired, and there is a concomitant delay in up-regulation of the late myogenic transcription factor myogenin. We expressed silencing-resistant NET37 mutated at a conserved residue in the glycosidase domain and found that this predicted catalytically inactive protein is unable to support myogenesis in cells depleted of wild type NET37. Therefore, the enzymatic function of NET37 appears to be important for myogenic differentiation. C2C12 cells depleted of NET37 have reduced activation of Akt after shifting to differentiation medium and are defective in insulin like growth factor-II (IGF-II) secretion, an autocrine/paracrine factor involved in Akt activation. We also observed that pro-IGF-II co-immunoprecipitates with NET37. Based on our results, we propose that NET37 has a role in IGF-II maturation in the secretory pathway during myoblast differentiation. The localization of NET37 at the nuclear envelope raises the possibility that it may coordinate myogenic events between the nuclear interior and the endoplasmic reticulum lumen via transmembrane communication.

We examined expression of four important members of myogenic regulatory factors (MRFs) in the myoblasts both at mRNA and protein levels, which were subjected to mechanical stretching in in vitro condition. Our results showed that MyoD expression existed both in the stretch and in the control group at all time periods of the mechanical stimulus. Myf-5 expressed only at early stage of the stretch group. Although mRNA and protein expressions of myogenin and MRF4 were detected both in the stretch and in the control group at 12 h after the stretching, their expressions were only shown in the stretch group at 24 h after the mechanical stimulus. However, at 36 and 48 h, none of the MRFs examined except MyoD appeared in both groups. Our results suggest that the MRFs are up-regulated upon mechanical stimulus and each member plays a different major role for either proliferation or differentiation of the myoblasts.

Rationale: Mechanical ventilation is known to induce ventilator-induced diaphragm dysfunction. Patients submitted to mechanical ventilation often receive massive doses of corticosteroids that may cause further deterioration of diaphragm function., Objectives: To examine whether the combination of 24 hours of controlled mechanical ventilation with corticosteroid administration would exacerbate ventilator-induced diaphragm dysfunction., Methods: Rats were randomly assigned to a group submitted to 24 hours of controlled mechanical ventilation receiving an intramuscular injection of saline or 80 mg/kg methylprednisolone, a group submitted to 24 hours of spontaneous breathing receiving saline, or methylprednisolone and a control group., Measurements and Main Results: The diaphragm force-frequency curve was shifted downward in the mechanical ventilation group, but this deleterious effect was prevented when corticosteroids were administered. Diaphragm cross-sectional area of type I fibers was similarly decreased in both mechanical ventilation groups while atrophy of type IIx/b fibers was attenuated after corticosteroid administration. The mechanical ventilation-induced reduction in diaphragm MyoD and myogenin protein expression was attenuated after corticosteroids. Plasma cytokine levels were unchanged while diaphragm lipid hydroperoxides were similarly increased in both mechanical ventilation groups. Diaphragmatic calpain activity was significantly increased in the mechanical ventilation group, but calpain activation was abated with corticosteroid administration. Inverse correlations were found between calpain activity and diaphragm force., Conclusions: A single high dose of methylprednisolone combined with controlled mechanical ventilation protected diaphragm function from the deleterious effects of controlled mechanical ventilation. Inhibition of the calpain system is most likely the mechanism by which corticosteroids induce this protective effect.

Calcineurin/NFAT signaling is involved in multiple aspects of skeletal muscle development and disease. The myogenic basic helix-loop-helix transcription factors, MyoD, myogenin, Myf5, and MRF4 specify the myogenic lineage. Here we show that calcineurin/NFAT (nuclear factor of activated T cells) signaling is required for primary myogenesis by transcriptional cooperation with the basic helix-loop-helix transcription factor MyoD. Calcineurin/NFAT signaling is involved in myogenin expression in differentiating myoblasts, where the myogenic regulatory factor MyoD synergistically cooperates with NFATc2/c3 at the myogenin promoter. Using gel shift and chromatin immunoprecipitation assays, we identified two conserved NFAT binding sites in the myogenin promoter that were occupied by NFATc3 upon skeletal muscle differentiation. The transcriptional integration between NFATc3 and MyoD is crucial for primary myogenesis in vivo, as myogenin expression is weak in myod:nfatc3 double null embryos, whereas myogenin expression is unaffected in embryos with null mutations for either factor alone. Thus, the combined findings provide a novel transcriptional paradigm for the first steps of myogenesis, where a calcineurin/NFATc3 pathway regulates myogenin induction in cooperation with MyoD during myogenesis.

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is characterized by muscle wasting and is caused by mutations in the LMNA gene encoding A-type lamins. Overexpression of the EDMD lamin A R453W mutation in C2C12 myoblasts impairs myogenic differentiation. We show here the influence of stable expression of the R453W and of the Dunnigan-type partial lipodystrophy R482W mutation of lamin A in C2C12 cells on transcription and epigenetic regulation of the myogenin (Myog) gene and on global chromatin organization. Expression of R453W-, but not R482W-lamin A, impairs activation of Myog and maintains a repressive chromatin state on the Myog promoter upon induction of differentiation, marked by H3 lysine (K) 9 dimethylation and failure to hypertrimethylate H3K4. Cells expressing WT-LaA also fail to hypertrimethylate H3K4. No defect occurs at the level of Myog promoter DNA methylation in any of the clones. Expression of R453W-lamin A and to a lesser extent R482W-lamin A in undifferentiated C2C12 cells redistributes H3K9me3 from pericentric heterochromatin. R453W-lamin A also elicits a redistribution of H3K27me3 from inactive X (Xi) and partial decondensation of Xi, but maintains Xist expression and coating of Xi, indicating that Xi remains inactivated. Our results argue that gene-specific and genome-wide chromatin rearrangements may constitute a molecular basis for laminopathies.

MicroRNA (miRNA) are important regulators of many biological processes, but the targets for most miRNA are still poorly defined. In this study, we profiled the expression of miRNA during myogenesis, from proliferating myoblasts through to terminally differentiated myotubes. Microarray results identified six significantly differentially expressed miRNA that were more than 2-fold different in myotubes. From this list, miRNA-26a (miR-26a), an up-regulated miRNA, was further examined. Overexpression of miR-26a in murine myogenic C2C12 cells induced creatine kinase activity, an enzyme that markedly increases during myogenesis. Further, myoD and myogenin mRNA expression levels were also up-regulated. These results suggest that increased expression of miR-26a promotes myogenesis. Through a bioinformatics approach, we identified the histone methyltransferase, Enhancer of Zeste homolog 2 (Ezh2), as a potential target of miR-26a. Overexpression of miR-26a suppressed the activity of a luciferase reporter construct fused with the 3'-untranslated region of Ezh2. In addition, miR-26a overexpression decreased Ezh2 mRNA expression. These results reveal a model of regulation during myogenesis whereby the up-regulation of miR-26a acts to post-transcriptionally repress Ezh2, a known suppressor of skeletal muscle cell differentiation.

Calcineurin activation ameliorates the dystrophic pathology of hindlimb muscles in mdx mice and decreases their susceptibility to contraction damage. In mdx mice, the diaphragm is more severely affected than hindlimb muscles and more representative of Duchenne muscular dystrophy. The constitutively active calcineurin Aalpha transgene (CnAalpha) was overexpressed in skeletal muscles of mdx (mdx CnAalpha*) mice to test whether muscle morphology and function would be improved. Contractile function of diaphragm strips and extensor digitorum longus and soleus muscles from adult mdx CnAalpha* and mdx mice was examined in vitro. Hindlimb muscles from mdx CnAalpha* mice had a prolonged twitch time course and were more resistant to fatigue. Because of a slower phenotype and a decrease in fiber cross-sectional area, normalized force was lower in fast- and slow-twitch muscles of mdx CnAalpha* than mdx mice. In the diaphragm, despite a slower phenotype and a approximately 35% reduction in fiber size, normalized force was preserved. This was likely mediated by the reduction in the area of the diaphragm undergoing degeneration (i.e., mononuclear cell and connective and adipose tissue infiltration). The proportion of centrally nucleated fibers was reduced in mdx CnAalpha* compared with mdx mice, indicative of improved myofiber viability. In hindlimb muscles of mdx mice, calcineurin activation increased expression of markers of regeneration, particularly developmental myosin heavy chain isoform and myocyte enhancer factor 2A. Thus activation of the calcineurin signal transduction pathway has potential to ameliorate the mdx pathophysiology, especially in the diaphragm, through its effects on muscle degeneration and regeneration and endurance capacity.

Certain patients suffering from chronic diseases such as AIDS or cancer experience a constant cellular secretion of tumor necrosis factor alpha and other pro-inflammatory cytokines that results in a continuous release of nitric oxide (*NO) to the bloodstream. One immediate consequence of the deleterious action of *NO is weight loss and the progressive destruction of muscular mass in a process known as cachexia. We have previously reported that caveolin-3, a specific marker of muscle cells, becomes down-regulated by the action of *NO on muscular myotubes. We describe herein that the changes observed in caveolin-3 levels are due to the alteration of the DNA binding activity of the muscular transcription factor myogenin. In the presence of *NO, the binding of transcription factors from cell nuclear extracts of muscular tissues to the E boxes present in the caveolin-3 promoter become substantially reduced. When we purified recombinant myogenin and treated it with *NO donors, we could detect its S-nitrosylation by three independent methods, suggesting that very likely one of the cysteine residues of the molecule is being modified. Given the role of myogenin as a regulatory protein that determines the level of multiple muscle genes expressed during late myogenesis, our results might represent a novel mode of regulation of muscle development under conditions of nitric oxide-mediated toxicity.

The widespread, massive loss of developing neurons in the central and peripheral nervous system of birds and mammals is generally considered to be an evolutionary adaptation. However, until recently, models for testing both the immediate and long-term consequences of preventing this normal cell loss have not been available. We have taken advantage of several methods for preventing neuronal death in vivo to ask whether rescued neurons [e.g., motoneurons (MNs)] differentiate normally and become functionally incorporated into the nervous system. Although many aspects of MN differentiation occurred normally after the prevention of cell death (including the expression of several motoneuron-specific markers, axon projections into the ventral root and peripheral nerves, ultrastructure, dendritic arborization, and afferent axosomatic synapses), other features of the neuromuscular system (MNs and muscle) were abnormal. The cell bodies and axons of MNs were smaller than normal, many MN axons failed to become myelinated or to form functional synaptic contacts with target muscles, and a subpopulation of rescued cells were transformed from alpha- to gamma-like MNs. Additionally, after the rescue of MNs in myogenin glial cell line-derived neurotrophic factor (MyoGDNF) transgenic mice, myofiber differentiation of extrafusal skeletal muscle was transformed and muscle physiology and motor behaviors were abnormal. In contrast, extrafusal myofiber phenotype, muscle physiology, and (except for muscle strength tests) motor behaviors were all normal after the rescue of MNs by genetic deletion of the proapoptotic gene Bax. However, there was an increase in intrafusal muscle fibers (spindles) in Bax knock-out versus both wild-type and MyoGDNF mice. Together, these data indicate that after the prevention of MN death, the neuromuscular system becomes transformed in novel ways to compensate for the presence of the thousands of excess cells.