PUFA Treatment Affects C2C12 Myocyte Differentiation, Myogenesis Related Genes and Energy Metabolism.

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.

Divergent personalities influence the myogenic regulatory genes myostatin, myogenin and ghr2 transcript responses to Vibrio anguillarum vaccination in fish fingerlings (Sparus aurata).

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 O2ß serotypes of Vibrio anguillarum, a widespread opportunistic pathogen of marine animals, to analyse skeletal muscle responses to bath vaccination. Transcripts related to inflammation (interleukin 1ß, il1ß; 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.

Spatial and temporal changes in myogenic protein expression by the microenvironment after freeze injury.

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.

MUNC, an Enhancer RNA Upstream from the MYOD Gene, Induces a Subgroup of Myogenic Transcripts in trans Independently of MyoD.

MyoD upstream noncoding RNA (MUNC) initiates in the distal regulatory region (DRR) enhancer of MYOD and is formally classified as an enhancer RNA (DRReRNA). 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.

A Muscle-Specific Enhancer RNA Mediates Cohesin Recruitment and Regulates Transcription In trans.

The enhancer regions of the myogenic master regulator MyoD give rise to at least two enhancer RNAs. Core enhancer eRNA (CEeRNA) regulates transcription of the adjacent MyoD gene, whereas DRReRNA affects expression of Myogenin in trans. We found that DRReRNA is recruited at the Myogenin locus, where it colocalizes with Myogenin nascent transcripts. DRReRNA 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 DRReRNA, which is then required for cohesin chromatin recruitment and maintenance. Functionally, depletion of either cohesin or DRReRNA reduces chromatin accessibility, prevents Myogenin activation, and hinders muscle cell differentiation. Thus, DRReRNA ensures spatially appropriate cohesin loading in trans to regulate gene expression.

PIP5K1α promotes myogenic differentiation via AKT activation and calcium release.

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.

Nilotinib impairs skeletal myogenesis by increasing myoblast proliferation.

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.

Inhibition of skeletal muscle atrophy during torpor in ground squirrels occurs through downregulation of MyoG and inactivation of Foxo4.

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.

Low Six4 and Six5 gene dosage improves dystrophic phenotype and prolongs life span of mdx mice.

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.

Umbilical cord mesenchymal stem cell-conditioned media prevent muscle atrophy by suppressing muscle atrophy-related proteins and ROS generation.

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.

PAK1 and CtBP1 Regulate the Coupling of Neuronal Activity to Muscle Chromatin and Gene Expression.

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.

A novel adipokine C1q/TNF-related protein 3 is expressed in developing skeletal muscle and controls myoblast proliferation and differentiation.

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-ß3 (TGF-ß3), suggesting that TGF-ß3 is one of the extracellular factors regulating CTRP3 expression during myogenesis. These results indicate a novel physiological role for CTRP3 during skeletal myogenesis.

Klhl31 attenuates ß-catenin dependent Wnt signaling and regulates embryo 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 ß-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 ß-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.

Comparing the effect of uniaxial cyclic mechanical stimulation and chemical factors on myogenin and Myh2 expression in mouse embryonic and bone marrow derived mesenchymal stem cells.

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.

Effect of heat stress soon after muscle injury on the expression of MyoD and myogenin during regeneration process.

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.

Developmental changes in IGF-I and MyoG gene expression and their association with meat traits in sheep.

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.

Dynamic expression of molecules that control limb muscle development including Fhl1 in hind limbs of different gestational age.

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.

Cloning and expression of MyoG gene from Hu sheep and identification of its myogenic specificity.

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.

miR-186 inhibits muscle cell differentiation through myogenin regulation.

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.

ATOH8, a regulator of skeletal myogenesis in the hypaxial myotome of the trunk.

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.