MicroRNA-27a promotes myoblast proliferation by targeting myostatin

MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. However, the role of miRNAs in myoblast proliferation remains poorly understood. Here we found that the expression of miR-27a was increased during proliferation of C2C12 myoblasts. Moreover, overexpression of miR-27a in C2C12 cells promoted myoblast proliferation by reducing the expression of myostatin, a critical inhibitor of skeletal myogenesis. In addition, the miR-27a was confirmed to target myostatin 3'UTR by a luciferase reporter analysis. Together, these results suggest that miR-27a promotes myoblast proliferation through targeting myostatin.     

A lncRNA promotes myoblast proliferation by up-regulating GH1

Long noncoding RNAs (lncRNAs) are key regulatory factors for gene expression in a variety of biological processes; however, the role of lncRNAs in muscle formation and development is poorly understood, particularly in cattle. Here, we identified a highly expressed lncRNA in muscle, lncYYW, by high-throughput sequencing in bovine longissimus, scapular, intercostal, and gluteus muscles. The expression of lncYYW increased gradually during myoblast differentiation. Overexpression of lncYYW increased the number of cells in the DNA synthesis (S) stage of the cell cycle and upregulated the expression of two well-established myogenic markers, myogenin and myosin heavy chain. A microarray analysis showed that lncYYW positively regulates the expression of growth hormone 1 and its downstream genes, AKT1 and PIK3CD, in bovine myoblasts. This discovery provides a good foundation for further study of the mechanism of action of lncYYW during bovine myoblast development. Taken together, our results reveal a novel lncRNA associated with bovine myoblast proliferation and differentiation. This lncRNA will play a crucial and critical role in future studies of bovine muscle development.     

Enhancement of C2C12 myoblast proliferation and differentiation by GASP-2, a myostatin inhibitor

BackgroundGASP-2 is a secreted multi-domain glycoprotein known as a specific inhibitor of myostatin and GDF-11. Here we investigate the role of GASP-2 on myogenesis and the effect of its glycosylation on its activity.MethodsGASP-2 overexpression or knockdown by shRNAs were carried out on C2C12 myoblasts cells. In silico analysis of GASP-2 protein was performed to identify its glycosylation sites. We produced a mouse recombinant GASP-2 protein in a prokaryotic system to obtain a fully deglycosylated protein allowing us to study the importance of this post-translational modification on GASP-2 activity.ResultsBoth mature and deglycosylated GASP-2 proteins increase C2C12 proliferation and differentiation by inhibiting the myostatin pathway. In silico and western-blot analyses revealed that GASP-2 presents one consensus sequence for N-glycosylation and six potential sites of mucin-type O-glycosylation.ConclusionsGASP-2 promotes myogenesis and thus independently of its glycosylation.General significanceThis is the first report demonstrating that GASP-2 promotes proliferation and differentiation of myoblasts by inhibiting the canonical pathway of myostatin.     

Human myostatin negatively regulates human myoblast growth and differentiation

Myostatin, a member of the transforming growth factor-β superfamily, has been implicated in the potent negative regulation of myogenesis in murine models. However, little is known about the mechanism(s) through which human myostatin negatively regulates human skeletal muscle growth. Using human primary myoblasts and recombinant human myostatin protein, we show here that myostatin blocks human myoblast proliferation by regulating cell cycle progression through targeted upregulation of p21. We further show that myostatin regulates myogenic differentiation through the inhibition of key myogenic regulatory factors including MyoD, via canonical Smad signaling. In addition, we have for the first time demonstrated the capability of myostatin to regulate the Notch signaling pathway during inhibition of human myoblast differentiation. Treatment with myostatin results in the upregulation of Hes1, Hes5, and Hey1 expression during differentiation; moreover, when we interfere with Notch signaling, through treatment with the γ-secretase inhibitor L-685,458, we find enhanced myotube formation despite the presence of excess myostatin. Therefore, blockade of the Notch pathway relieves myostatin repression of differentiation, and myostatin upregulates Notch downstream target genes. Immunoprecipitation studies demonstrate that myostatin treatment of myoblasts results in enhanced association of Notch1-intracellular domain with Smad3, providing an additional mechanism through which myostatin targets and represses the activity of the myogenic regulatory factor MyoD. On the basis of these results, we suggest that myostatin function and mechanism of action are very well conserved between species, and that myostatin regulation of postnatal myogenesis involves interactions with numerous downstream signaling mediators, including the Notch pathway.     

Ursolic acid directly promotes protein accretion in myotubes but does not affect myoblast proliferation

Ursolic acid (UA) has been recently proposed as a potential candidate for the treatment of muscle wasting conditions because of its protein sparring/anabolic effects. Despite this finding, it is unknown whether this response is the consequence of a direct effect on the muscle fibre or if it is mediated by neural or other systemic factors. In the present study, we sought to determine if UA has direct effects in skeletal muscle cells, whether it can increase myoblast proliferation and whether UA can become myotoxic at higher doses. Our results demonstrate that UA directly promoted protein accretion in cultured myotubes but did not modulate myoblast proliferation. At higher doses, UA compromised cell viability in both myoblasts and myotubes. We conclude that the anabolic properties of UA seen in vivo and in vitro are likely a direct effect on the muscle cell, but at higher doses, the benefits decline in favour of a myotoxic outcome.     

Mitochondrial-dependent regulation of myoblast proliferation

The aim of the present study was to determine whether mitochondrial activity could regulate myoblast proliferation. We demonstrate that an increase in mitochondrial activity of L6E9 myoblasts can be easily obtained by simply raising extracellular pyruvate concentration in the culture dish. Under this condition, L6E9 myoblasts underwent a rapid growth arrest in G1 + S phases concomitant to a marked cellular hypertrophy. No sign of myoblast fusion was evident. This was accompanied by the down-regulation of proliferating cell nuclear antigen expression and an increase in p21 expression. Mitochondrial biogenesis was also stimulated, as indicated by a twofold increase in mitochondrial content. These cells exhibited a large increase in the production of reactive oxygen species that could contribute to the observed phenotypic alterations. However, exposure of pyruvate-treated cells to antioxidants did not reverse growth arrest. Similarly, exposure of control cells to oxidants did not induce growth arrest. Our observations suggest that mitochondrial activity appears to play a central role in regulating myoblast proliferation. They also argue strongly in favor of a retrograde communication establishing a mitochondrial control of nuclear gene expression that could be modulated by mitochondrial activity.     

GRAF1 promotes ferlin-dependent myoblast fusion

Myoblast fusion (a critical process by which muscles grow) occurs in a multi-step fashion that requires actin and membrane remodeling; but important questions remain regarding the spatial/temporal regulation of and interrelationship between these processes. We recently reported that the Rho-GAP, GRAF1, was particularly abundant in muscles undergoing fusion to form multinucleated fibers and that enforced expression of GRAF1 in cultured myoblasts induced robust fusion by a process that required GAP-dependent actin remodeling and BAR domain-dependent membrane sculpting. Herein we developed a novel line of GRAF1-deficient mice to explore a role for this protein in the formation/maturation of myotubes in vivo. Post-natal muscles from GRAF1-depleted mice exhibited a significant and persistent reduction in cross-sectional area, impaired regenerative capacity and a significant decrease in force production indicative of lack of efficient myoblast fusion. A significant fusion defect was recapitulated in isolated myoblasts depleted of GRAF1 or its closely related family member GRAF2. Mechanistically, we show that GRAF1 and 2 facilitate myoblast fusion, at least in part, by promoting vesicle-mediated translocation of fusogenic ferlin proteins to the plasma membrane.     

Evolutionary dynamics of endogenous Jaagsiekte sheep retroviruses proliferation in the domestic sheep,mouflon and Pyrenean chamois

The oncogenic exogenous Jaagsiekte sheep retrovirus (JSRV), responsible for ovine pulmonary adenocarcinoma, has several endogenous counterparts termed enJSRVs. Although many of these elements have been inactivated over time by the accumulation of deleterious mutations or internal recombination leading to solo long terminal repeat (LTR) formation, several members of enJSRVs have been identified as nearly intact and probably represent recent integration events. To determine the level of enJSRV polymorphism in the sheep population and related species, we have undertaken a study by characterizing enJSRVs copies and independent integration sites in six domestic sheep and two wild species of the sheep lineage. enJSRVs copies were detected by amplifying the env-LTR region by PCR, and for the detection of the insertion sites, we used two approaches: (1) an in silico approach based on the recently published Sheep Reference Genome Assembly (OARv3.0) and (2) an experimental approach based on PCR suppression and inverse PCR techniques. In total, 103 enJSRV sequences were generated across 10 individuals and enJSRV integrations were found on 11 of the 28 sheep chromosomes. These findings suggest that there are still uncharacterized enJSRVs, and that some of the integration sites are variable among the different species, breeds of the same species, subspecies and geographic locations.     

Silencing myostatin gene by RNAi in sheep embryos

Myostatin (MSTN) gene is described as a negative regulator of the skeletal muscle growth. Controlling MSTN gene expression by genetic manipulation could accelerate the muscle growth and meat production of livestock animals. In the present study, several siRNAs targeting sheep MSTN gene were designed and their interfering efficiency was evaluated in vitro. The present study showed that one of the siRNAs, PSL1, could down-regulate the expression of MSTN significantly. PSL1 was ligated into lentivirus vector, GP-Supersilencing, to construct a siRNA expression lentivirus vector. Fibroblast cells were infected by lentivirus particles and positive cells were isolated by flow cytometry. Nucleus of the positive cell was transferred into enucleated oocytes of sheep. The present study showed that 99.4% of the sorted cells displayed green fluorescence. After enucleation of oocytes with microinjection, about 20% of reconstructed embryos can be developed into morulas, and strong green fluorescence could be observed using a fluorescence microscope. This method can be available to produce transgenic cell line for somatic cell nucleus transfer for transgenic animals.     

Sleeping Beauty-mediated knockdown of sheep myostatin by RNA interference

Myostatin is a negative regulator of skeletal muscle growth. Myostatin dysfunction therefore offers a strategy for promoting animal muscle growth in livestock production. Knockdown of myostatin was achieved by combining RNA interference and the Sleeping Beauty (SB) transposon system in sheep cells. Four targeting sites of sheep myostatin were designed and measured for myostatin silencing in sheep fetal fibroblasts by real-time PCR. The sh3 construct induced significant decrease of myostatin gene expression by 90% (P < 0.05). Myostatin silencing induced by SB-mediated sh3 was further tested in stably transfected cells. SB transposition increased the integration frequency of genes into sheep genomes and mediated a more efficient myostatin knockdown than random integration of sh3. We suggest that SB-mediated shRNA provides a novel potential tool for gene knockdown in the donor cells of animal cloning.     

The critical role of myostatin in differentiation of sheep myoblasts

Myostatin [MSTN, also known as growth differentiation factor 8 (GDF8)], is an inhibitor of skeletal muscle growth. Blockade of MSTN function has been reported to result in increased muscle mass in mice. However, its role in myoblast differentiation in farm animals has not been determined. In the present study, we sought to determine the role of MSTN in the differentiation of primary sheep myoblasts. We found that ectopic overexpression of MSTN resulted in lower fusion index in sheep myoblasts, which indicated the repression of myoblast differentiation. This phenotypic change was reversed by shRNA knockdown of the ectopically expressed MSTN in the cells. In contrast, shRNA knockdown of the endogenous MSTN resulted in induction of myogenic differentiation. Additional studies revealed that the induction of differentiation by knocking down the ectopically or endogenously expressed MSTN was accompanied by up-regulation of MyoD and myogenin, and down-regulation of Smad3. Our results demonstrate that MSTN plays critical role in myoblast differentiation in sheep, analogous to that in mice. This study also suggests that shRNA knockdown of MSTN could be a potentially promising approach to improve sheep muscle growth, so as to increase meat productivity.     

Cloning and sequence analysis of myostatin promoter in sheep.

To better understand the structure and function of the myostatin's gene promoter region in sheep, we cloned and sequenced a 1.517 kb fragment containing the 5'-regulatory region of the sheep myostatin gene (GenBank accession number is AY918121). The promoter sequence consists of three TATA boxes, one CAAT box, and eight putative E-boxes. Some putative muscle growth response elements for Octamer-binding factor 1(Octamer), Activator protein 1(AP1), Growth factor independence 1 zinc finger protein (Gfi-1B), Myocyte enhancer factor 2 (MEF2), Muscle-specific Mt binding site (MTBF), Glucocorticoid response elements (GRE) and Progesterone receptor binding site (PRE) were detected. Some of the motifs are conserved as compared to with that in the goat, bovine and porcine myostatin promoters. However, some differences were also found.     

Control of myoblast proliferation with a synthetic ligand

Skeletal myoblast grafts can form contractile tissue to replace scar and repair injured myocardium. Although potentially therapeutic, generating reproducible and sufficiently large grafts remains a challenge. To control myoblast proliferation in situ, we created a chimeric receptor composed of a modified FK506-binding protein (F36V) fused with the fibroblast growth factor receptor-1 cytoplasmic domain. Mouse MM14 myoblasts were transfected with this construct and treated with AP20187, a dimeric F36V ligand, to induce receptor dimerization. Transfected myoblasts proliferated in response to dimerizer (comparable with basic fibroblast growth factor (bFGF) treatment), whereas the dimerizer had no effect on non-transfected cells. Similar to bFGF treatment, dimerizer treatment blocked myotube formation and myosin heavy chain expression and stimulated mitogen-activated protein (MAP) kinase phosphorylation in transfected cells. Non-transfected cells differentiated normally and showed no MAP kinase phosphorylation with dimerizer treatment. Furthermore, myoblasts treated with dimerizer for 30 days in culture reduced MAP kinase phosphorylation, withdrew from the cell cycle, and differentiated normally upon drug withdrawal, demonstrating reversibility of the effect. Thus, forced dimerization of the fibroblast growth factor receptor-1 cytoplasmic domain reproduces critical aspects of bFGF signaling in myoblasts. We hypothesize that in vivo administration of AP20187 following myoblast grafting may allow control over graft size and ultimately improve cardiac function.