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1
Knudsen, K. A., L. Smith, and S. McElwee. "Involvement of cell surface phosphatidylinositol-anchored glycoproteins in cell-cell adhesion of chick embryo myoblasts." Journal of Cell Biology 109, no. 4 (1989): 1779–86. http://dx.doi.org/10.1083/jcb.109.4.1779.
Full textAbstract:
During myogenesis myoblasts fuse to form multinucleate cells that express muscle-specific proteins. A specific cell-cell adhesion process precedes lipid bilayer union during myoblast fusion (Knudsen, K. A., and A. F. Horwitz. 1977. Dev. Biol. 58:328-338) and is mediated by cell surface glycoproteins (Knudsen, K. A., 1985. J. Cell Biol. 101:891-897). In this paper we show that myoblast adhesion and myotube formation are inhibited by treating fusion-competent myoblasts with phosphatidylinositol-specific phospholipase C (PI-PLC). The effect of PI-PLC on myoblast adhesion is dose dependent and inhibited by D-myo-inositol 1-monophosphate and the effect on myotube formation is reversible, suggesting a specific, nontoxic effect on myogenesis by the enzyme. A soluble form of adhesion-related glycoproteins is released from fusion-competent myoblasts by treatment with PI-PLC as evidenced by (a) the ability of phospholipase C (PLC)-released material to block the adhesion-perturbing activity of a polyclonal antiserum to intact myoblasts; and (b) the ability of PLC-released glycoprotein to stimulate adhesion-perturbing antisera when injected into mice. PI-PLC treatment of fusion-competent myoblasts releases an isoform of N-CAM into the supernate, suggesting that N-CAM may participate in mediating myoblast interaction during myogenesis.
2
Sun, Luguo, Kewei Ma, Haixia Wang, et al. "JAK1–STAT1–STAT3, a key pathway promoting proliferation and preventing premature differentiation of myoblasts." Journal of Cell Biology 179, no. 1 (2007): 129–38. http://dx.doi.org/10.1083/jcb.200703184.
Full textAbstract:
Skeletal muscle stem cell–derived myoblasts are mainly responsible for postnatal muscle growth and injury-induced muscle regeneration. However, the cellular signaling pathways controlling the proliferation and differentiation of myoblasts are not fully understood. We demonstrate that Janus kinase 1 (JAK1) is required for myoblast proliferation and that it also functions as a checkpoint to prevent myoblasts from premature differentiation. Deliberate knockdown of JAK1 in both primary and immortalized myoblasts induces precocious myogenic differentiation with a concomitant reduction in cell proliferation. This is caused, in part, by an accelerated induction of MyoD, myocyte enhancer–binding factor 2 (MEF2), p21Cip1, and p27Kip1, a faster down-regulation of Id1, and an increase in MEF2-dependent gene transcription. Downstream of JAK1, of all the signal transducer and activator of transcriptions (STATs) present in myoblasts, we find that only STAT1 knockdown promotes myogenic differentiation in both primary and immortalized myoblasts. Leukemia inhibitory factor stimulates myoblast proliferation and represses differentiation via JAK1–STAT1–STAT3. Thus, JAK1–STAT1–STAT3 constitutes a signaling pathway that promotes myoblast proliferation and prevents premature myoblast differentiation.
3
Jansen, Katie M., and Grace K. Pavlath. "Mannose receptor regulates myoblast motility and muscle growth." Journal of Cell Biology 174, no. 3 (2006): 403–13. http://dx.doi.org/10.1083/jcb.200601102.
Full textAbstract:
Myoblast fusion is critical for the formation, growth, and maintenance of skeletal muscle. The initial formation of nascent myotubes requires myoblast–myoblast fusion, but further growth involves myoblast–myotube fusion. We demonstrate that the mannose receptor (MR), a type I transmembrane protein, is required for myoblast–myotube fusion. Mannose receptor (MR)–null myotubes were small in size and contained a decreased myonuclear number both in vitro and in vivo. We hypothesized that this defect may arise from a possible role of MR in cell migration. Time-lapse microscopy revealed that MR-null myoblasts migrated with decreased velocity during myotube growth and were unable to migrate in a directed manner up a chemoattractant gradient. Furthermore, collagen uptake was impaired in MR-null myoblasts, suggesting a role in extracellular matrix remodeling during cell motility. These data identify a novel function for MR during skeletal muscle growth and suggest that myoblast motility may be a key aspect of regulating myotube growth.
4
Formigli, Lucia, Fabio Francini, Alessia Tani, et al. "Morphofunctional integration between skeletal myoblasts and adult cardiomyocytes in coculture is favored by direct cell-cell contacts and relaxin treatment." American Journal of Physiology-Cell Physiology 288, no. 4 (2005): C795—C804. http://dx.doi.org/10.1152/ajpcell.00345.2004.
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The success of cellular cardiomyoplasty, a novel therapy for the repair of postischemic myocardium, depends on the anatomical integration of the engrafted cells with the resident cardiomyocytes. Our aim was to investigate the interaction between undifferentiated mouse skeletal myoblasts (C2C12 cells) and adult rat ventricular cardiomyocytes in an in vitro coculture model. Connexin43 (Cx43) expression, Lucifer yellow microinjection, Ca2+ transient propagation, and electrophysiological analysis demonstrated that myoblasts and cardiomyocytes were coupled by functional gap junctions. We also showed that cardiomyocytes upregulated gap junctional communication and expression of Cx43 in myoblasts. This effect required direct cell-to-cell contact between the two cell types and was potentiated by treatment with relaxin, a cardiotropic hormone with potential effects on cardiac development. Analysis of the gating properties of gap junctions by dual cell patch clamping showed that the copresence of cardiomyocytes in the cultures significantly increased the transjunctional current and conductance between myoblasts. Relaxin enhanced this effect in both the myoblast-myoblast and myoblast-cardiomyocyte cell pairs, likely acting not only on gap junction formation but also on the electrical properties of the preexisting channels. Our findings suggest that myoblasts and cardiomyocytes interact actively through gap junctions and that relaxin potentiates the intercellular coupling. A potential role for gap junctional communication in favoring the intercellular exchange of regulatory molecules, including Ca2+, in the modulation of myoblast differentiation is discussed.
5
Knudsen, K. A. "The calcium-dependent myoblast adhesion that precedes cell fusion is mediated by glycoproteins." Journal of Cell Biology 101, no. 3 (1985): 891–97. http://dx.doi.org/10.1083/jcb.101.3.891.
Full textAbstract:
Presumptive myoblasts from explants of chick embryo pectoral muscle proliferate, differentiate, and fuse to form multinucleate myotubes. One event critical to multinucleate cell formation is the specific adhesion of myoblasts before union of their membranes. In the studies reported here five known inhibitors of myotube formation--trifluoperazine, sodium butyrate, chloroquine, 1,10 phenanthroline, and tunicamycin--were tested for their effect on the Ca++-dependent myoblast adhesion step. The first four inhibitors of myotube formation do not perturb myoblast adhesion but rather block fusion of aggregated cells, which suggests that these agents perturb molecular events required for the union of the lipid bilayers. By contrast, tunicamycin exerts its effect by inhibiting the myoblast adhesion step, thereby blocking myotube formation. The effect of tunicamycin can be blocked by a protease inhibitor, however, which implies that the carbohydrate residues protect the glycoproteins from proteolytic degradation rather than participate directly in cell-cell adhesion. Whereas trypsin treatment of myoblasts in the absence of Ca++ destroys the cells' ability to exhibit Ca++-dependent adhesion, the presence of Ca++ during trypsin treatment inhibits the enzyme's effect, which suggests that myoblast adhesion is mediated by a glycoprotein(s) that has a conformation affected by Ca++. Finally, myoblast adhesion is inhibited by an antiserum raised against fusion-competent myoblasts. The effect of the antiserum is blocked by a fraction from the detergent extract of pectoral muscle that binds to immobilized wheat germ agglutinin, which again suggests that glycoproteins mediate Ca++-dependent myoblast adhesion.
6
Fahime, E. El, M. Bouchentouf, B. F. Benabdallah, et al. "Tubulyzine®, a novel tri-substituted triazine, prevents the early cell death of transplanted myogenic cells and improves transplantation success." Biochemistry and Cell Biology 81, no. 2 (2003): 81–90. http://dx.doi.org/10.1139/o03-054.
Full textAbstract:
Myoblast transplantation (MT) is a potential therapeutic approach for several muscular dystrophies. A major limiting factor is that only a low percentage of the transplanted myoblasts survives the procedure. Recent advances regarding how and when the myoblasts die indicate that events preceding actual tissue implantation and during the first days after the transplantation are crucial. Myoseverin, a recently identified tri-substituted purine, was shown to induce in vitro the fission of multinucleated myotubes and affect the expression of a variety of growth factors, and immunomodulation, extracellular matrix-remodeling, and stress response genes. Since the effects of myoseverin are consistent with the activation of pathways involved in wound healing and tissue regeneration, we have investigated whether pretreatment and co-injection of myoblasts with Tubulyzine® (microtubule lysing triazine), an optimized myoseverin-like molecule recently identified from a triazine library, could reduce myoblast cell death following their transplantation and consequently improves the success of myoblast transplantation. In vitro, using annexin-V labeling, we showed that Tubulyzine (5 µM) prevents normal myoblasts from apoptosis induced by staurosporine (1 µM). In vivo, the pretreatment and co-injection of immortal and normal myoblasts with Tubulyzine reduced significantly cell death (assessed by the radio-labeled thymidine of donor DNA) and increased survival of myoblasts transplanted in Tibialis anterior (TA) muscles of mdx mice, thus giving rise to more hybrid myofibers compared to transplanted untreated cells. Our results suggest that Tubulyzine can be used as an in vivo survival factor to improve the myoblast-mediated gene transfer approach.Key words: myoblast survival, mdx mouse, myoblast transplantation, microtubule-binding molecule, cell death.
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Budzynska, Katarzyna, Katarzyna T. Bozyk, Klaudia Jarosinska, Anna Ziemiecka, Krzysztof Siemionow, and Maria Siemionow. "Developing Advanced Chimeric Cell Therapy for Duchenne Muscular Dystrophy." International Journal of Molecular Sciences 25, no. 20 (2024): 10947. http://dx.doi.org/10.3390/ijms252010947.
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Duchenne Muscular Dystrophy (DMD) is a lethal, X-linked disorder leading to muscle degeneration and premature death due to cardiopulmonary complications. Currently, there is no cure for DMD. We previously confirmed the efficacy of human Dystrophin-Expressing Chimeric (DEC) cells created via the fusion of myoblasts from normal and DMD-affected donors. The current study aimed to optimize the development of DEC therapy via the polyethylene glycol (PEG)-mediated fusion protocol of human myoblasts derived from normal, unrelated donors. The optimization of cell fusion assessed different factors influencing fusion efficacy, including myoblast passage number, the efficacy of PKH myoblast staining, the ratio of the single-stained myoblasts in the MIX, and PEG administration time. Additionally, the effect of PEG fusion procedure on cell viability was assessed. A correlation was found between the number of cells used for PKH staining and staining efficacy. Furthermore, the ratio of single-stained myoblasts in the MIX and PEG administration time correlated with fusion efficacy. There was no correlation found between the myoblast passage number and fusion efficacy. This study successfully optimized the myoblast fusion protocol for creation of human DEC cells, introducing DEC as a new Advanced Therapy Medicinal Product (ATMP) for DMD patients.
8
Entwistle, A., R. J. Zalin, S. Bevan, and A. E. Warner. "The control of chick myoblast fusion by ion channels operated by prostaglandins and acetylcholine." Journal of Cell Biology 106, no. 5 (1988): 1693–702. http://dx.doi.org/10.1083/jcb.106.5.1693.
Full textAbstract:
Chick myoblast fusion in culture was investigated using prostanoid synthesis inhibitors to delay spontaneous fusion. During this delay myoblast fusion could be induced by prostaglandin E1 (PGE1), by raising extracellular potassium and by addition of carbachol. Carbachol-induced fusion, but not PGE-induced fusion, was prevented by the acetylcholine receptor blocker alpha-bungarotoxin. Fusion induced by any of these agents was prevented by the Ca channel blockers lanthanum and D600. The threshold for potassium-induced fusion was 7-8 mM; maximal fusion occurred at 16-20 mM. Low extracellular potassium inhibited spontaneous fusion. Intracellular potassium in fusion competent myoblasts was 101 m-moles/l cell. Calcium flux measurements demonstrated that high potassium increased calcium permeability in fusion-competent myoblasts. A 30-s exposure to high potassium or PGE1 was sufficient to initiate myoblast fusion. Anion-exchange inhibitors (SITS and DIDS) delayed spontaneous myoblast fusion and blocked fusion induced by PGE1 but not carbachol. Blocking the acetylcholine receptor shifted the dose-response relation for PGE-induced fusion to higher concentrations. PGE1-induced fusion required chloride ions; carbachol-induced fusion required sodium ions. Provided calcium channels were available, potassium always induced fusion. We conclude that myoblasts possess at least three, independent pathways, each of which can initiate myoblast fusion and that the PGE-activated pathway and the acetylcholine receptor-activated pathway act synergistically. We suggest that fusion competent myoblasts have a high resting membrane potential and that fusion is controlled by depolarization initiated directly (potassium), by an increase in permeability to chloride ions (PGE), or by activation of the acetylcholine receptor (carbachol); depolarization triggers a rise in calcium permeability. The consequent increase in intracellular calcium initiates myoblast fusion.
9
Trudel, Geralyn C., and Paul C. Holland. "The glycoprotein-processing inhibitors bromoconduritol and N-methyl-1-deoxynojirimycin alter the adhesion phenotype of skeletal myoblasts." Biochemistry and Cell Biology 68, no. 12 (1990): 1411–18. http://dx.doi.org/10.1139/o90-204.
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Treatment of chick myoblasts with the glucosidase inhibitors bromoconduritol (BCD) or N-methyl-1-deoxynojirimycin (MDJN), but not the mannosidase I inhibitor 1-deoxymannojirimycin (ManDJN), decreased their rate of adhesion to fibronectin and laminin and increased their rate of adhesion to collagen types I and IV. The adhesion of chick myoblasts to fibronectin, collagen type IV, and laminin was predominantly mediated by β1-type integrin(s) as judged by inhibition of adhesion with the β1-specific monoclonal antibody JG22. Collagen binding in inhibitor-treated cells remained JG22-sensitive suggesting the inhibitors promote increased activity of a β1-type collagen-selective integrin. The effects of BCD, MDJN, and ManDJN on myoblast β1-integrin detectable at the myoblast cell surface with JG22 antibody correlated well with their effects on adhesion to fibronectin and laminin, and paralleled the previously reported effects of these agents on myogenesis. Interaction of integrin with the extracellular matrix appears to be required for myoblast terminal differentiation. We found that Mn2+ ions increased the adhesion of myoblasts to extracellular matrix proteins and antagonized the effect of BCD and MDJN on myoblast differentiation, supporting a role for cell–matrix interactions in myogenesis. Inhibition of myogenesis by BCD or MDJN was not reversed by growth under low serum conditions, suggesting these agents do not act by maintaining myoblasts in a proliferative state.Key words: myoblast, myogenesis, integrin, cell adhesion, glycoprotein processing.
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Entwistle, A., R. J. Zalin, A. E. Warner, and S. Bevan. "A role for acetylcholine receptors in the fusion of chick myoblasts." Journal of Cell Biology 106, no. 5 (1988): 1703–12. http://dx.doi.org/10.1083/jcb.106.5.1703.
Full textAbstract:
The role of acetylcholine receptors in the control of chick myoblast fusion in culture has been explored. Spontaneous fusion of myoblasts was inhibited by the nicotinic acetylcholine receptor antagonists alpha-bungarotoxin, Naja naja toxin and monoclonal antibody mcAb 5.5. The muscarinic antagonists QNB and n-methyl scopolamine were without effect. Atropine had no effect below 1 microM, where it blocks muscarinic receptors; at higher concentrations, when it blocks nicotinic receptors also, atropine inhibited myoblast fusion. The inhibitions imposed by acetylcholine receptor antagonists lasted for approximately 12 h; fusion stimulated by other endogenous substances then took over. The inhibition was limited to myoblast fusion. The increases in cell number, DNA content, the level of creatine phosphokinase activity (both total and muscle-specific isozyme) and the appearance of heavy chain myosin, which accompany muscle differentiation, followed a normal time course. Pre-fusion myoblasts, fusing myoblasts, and young myotubes specifically bound labeled alpha-bungarotoxin, indicating the presence of acetylcholine receptors. The nicotinic acetylcholine receptor agonist, carbachol, induced uptake of [14C]Guanidinium through the acetylcholine receptor. Myoblasts, aligned myoblasts and young myotubes expressed the synthetic enzyme Choline acetyltransferase and stained positively with antibodies against acetylcholine. The appearance of ChAT activity in myogenic cultures was prevented by treatment with BUDR; nonmyogenic cells in the cultures expressed ChAT at a level which was too low to account for the activity in myogenic cultures. We conclude that activation of the nicotinic acetylcholine receptor is part of the mechanism controlling spontaneous myoblast fusion and that myoblasts synthesize an endogenous, fusion-inducing agent that activates the nicotinic ACh receptor.
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Rout, Pratiti, Mathieu Preußner, and Susanne Filiz Önel. "Drosophila melanogaster: A Model System to Study Distinct Genetic Programs in Myoblast Fusion." Cells 11, no. 3 (2022): 321. http://dx.doi.org/10.3390/cells11030321.
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Muscle fibers are multinucleated cells that arise during embryogenesis through the fusion of mononucleated myoblasts. Myoblast fusion is a lifelong process that is crucial for the growth and regeneration of muscles. Understanding the molecular mechanism of myoblast fusion may open the way for novel therapies in muscle wasting and weakness. Recent reports in Drosophila and mammals have provided new mechanistic insights into myoblast fusion. In Drosophila, muscle formation occurs twice: during embryogenesis and metamorphosis. A fundamental feature is the formation of a cell–cell communication structure that brings the apposing membranes into close proximity and recruits possible fusogenic proteins. However, genetic studies suggest that myoblast fusion in Drosophila is not a uniform process. The complexity of the players involved in myoblast fusion can be modulated depending on the type of muscle that is formed. In this review, we introduce the different types of multinucleated muscles that form during Drosophila development and provide an overview in advances that have been made to understand the mechanism of myoblast fusion. Finally, we will discuss conceptual frameworks in cell–cell fusion in Drosophila and mammals.
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Gu, Lihong, Hongju Liu, Long Wang, et al. "Rapid high-throughput isolation and purification of chicken myoblasts based on deterministic lateral displacement microfluidic chips." PLOS ONE 19, no. 12 (2024): e0301309. https://doi.org/10.1371/journal.pone.0301309.
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Myoblasts are defined as stem cells containing skeletal muscle cell precursors. However, there are some challenges associated with the purification of myoblast samples, including long culture times and ease of bacterial contamination. In this study, we propose a microfluidic myoblast cell enrichment and purification platform based on the principle of deterministic lateral displacement (DLD). To achieve this, we designed a DLD chip with three outlets and tested it on 11-day-old (E11) Wenchang chicken pectoral muscle tissue. A cell suspension was prepared using the collagenase method, pretreated, and then passed into the designed DLD chip for myoblast enrichment and purification. In this study, the number of myoblasts and the diameter of myoblasts increased slowly before E9, and the diameter of myofibers decreased and the number of myofibers increased rapidly after E9. The period when the muscle fibers are most numerous is on the E12, and the period when the diameter of the muscle fibers begins to increase again after reaching its lowest point is also on the E12. After E12, the diameter of the muscle fibers increased and the number of muscle fibers decreased. At E12, myoblasts clustered and fused, and the proliferation of myoblasts was greatly reduced. E12 is both intact myoblasts and the most vigorous proliferation period, so the best time to determine isolation is E12. We attained a myoblast cell recovery rate of 80%, a target outlet collection purity of 99%, and a chip throughput of 50 μ m/min. In this paper, we innovate chips design according to specific geometries and functions for Wenchang chicken pectoral muscle tissue, so as to optimize the isolation and purification process of myoblasts. This study provides a novel and effective method for the isolation and purification of skeletal muscle myoblasts.
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Allen, David L., Daniel H. Teitelbaum, and Kotoku Kurachi. "Growth factor stimulation of matrix metalloproteinase expression and myoblast migration and invasion in vitro." American Journal of Physiology-Cell Physiology 284, no. 4 (2003): C805—C815. http://dx.doi.org/10.1152/ajpcell.00215.2002.
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We investigated the role of growth factors and fibronectin on matrix metalloproteinase (MMP) expression and on migration and invasion of mouse skeletal myoblasts in vitro. None of the growth factors tested significantly affected MMP-1 or MMP-2 activity as revealed by gelatin zymography, but both basic FGF (bFGF) and tumor necrosis factor (TNF)-α significantly increased MMP-9 activity (10- and 30-fold, respectively). The increase in secreted MMP-9 activity with TNF-α stimulation was due at least in part to an increase in MMP-9 gene transcription, because an MMP-9 promoter construct was approximately fivefold more active in TNF-α-treated myoblasts than in control myoblasts, as well as an increase in MMP-9 proteolytic activation. However, whereas fibronectin, bFGF, hepatocyte growth factor, and TGF-β1 significantly augmented migration of mouse myoblasts, TNF-α did not, nor did PDGF-BB or IGF-I. Fibronectin and bFGF also significantly augmented invasion of myoblasts across a Matrigel barrier, and plasmin cotreatment potentiated whereas N-acetyl cysteine suppressed the effects of bFGF and fibronectin on myoblast migration and invasion. Finally, transient transfection with an MMP-9 overexpression construct had only minimal effects on myoblast migration/invasion, whereas overexpression of either MMP-2 or MMP-1 significantly augmented myoblast migration and invasion. These observations support the hypothesis that MMP activity is a necessary component of growth factor-mediated myoblast migration but suggest that other consequences of growth factor signaling are also necessary for migration to occur.
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Qu, Zhuqing, Levent Balkir, Judith C. T. van Deutekom, Paul D. Robbins, Ryan Pruchnic, and Johnny Huard. "Development of Approaches to Improve Cell Survival in Myoblast Transfer Therapy." Journal of Cell Biology 142, no. 5 (1998): 1257–67. http://dx.doi.org/10.1083/jcb.142.5.1257.
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Myoblast transplantation has been extensively studied as a gene complementation approach for genetic diseases such as Duchenne Muscular Dystrophy. This approach has been found capable of delivering dystrophin, the product missing in Duchenne Muscular Dystrophy muscle, and leading to an increase of strength in the dystrophic muscle. This approach, however, has been hindered by numerous limitations, including immunological problems, and low spread and poor survival of the injected myoblasts. We have investigated whether antiinflammatory treatment and use of different populations of skeletal muscle–derived cells may circumvent the poor survival of the injected myoblasts after implantation. We have observed that different populations of muscle-derived cells can be isolated from skeletal muscle based on their desmin immunoreactivity and differentiation capacity. Moreover, these cells acted differently when injected into muscle: 95% of the injected cells in some populations died within 48 h, while others richer in desmin-positive cells survived entirely. Since pure myoblasts obtained from isolated myofibers and myoblast cell lines also displayed a poor survival rate of the injected cells, we have concluded that the differential survival of the populations of muscle-derived cells is not only attributable to their content in desmin-positive cells. We have observed that the origin of the myogenic cells may influence their survival in the injected muscle. Finally, we have observed that myoblasts genetically engineered to express an inhibitor of the inflammatory cytokine, IL-1, can improve the survival rate of the injected myoblasts. Our results suggest that selection of specific muscle-derived cell populations or the control of inflammation can be used as an approach to improve cell survival after both myoblast transplantation and the myoblast-mediated ex vivo gene transfer approach.
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Huard, Johnny, Geneviève Tremblay, Steve Verreault, Claude Labrecque, and Jacques P. Tremblay. "Utilization of an Antibody Specific for Human Dystrophin to Follow Myoblast Transplantation in Nude Mice." Cell Transplantation 2, no. 2 (1993): 113–18. http://dx.doi.org/10.1177/096368979300200204.
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Human myoblasts were transplanted in nude mice. The efficacy of these transplantations was analyzed using a monoclonal antibody (NCLDys3) specific for human dystrophin. This antibody did not reveal any dystrophin in nude mice that did not receive a human myoblast transplantation. However, about 30 days after a human myoblast transplantation, dystrophin-positive muscle fibers were observed. They were not abundant, and were present either in small clusters or isolated. This technique follows the fate of myoblast transplantation in animals that already have dystrophin, and distinguishes between new dystrophin-positive fibers due to the transplantation and the revertant fibers in mdx mice. Moreover, this technique does not require any labelling of the myoblasts before transplantation. It can also be used to detect dystrophin produced following the fusion of myoblasts transfected with the human dystrophin gene.
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Charlton, Carol A., William A. Mohler, Glenn L. Radice, Richard O. Hynes, and Helen M. Blau. "Fusion Competence of Myoblasts Rendered Genetically Null for N-Cadherin in Culture." Journal of Cell Biology 138, no. 2 (1997): 331–36. http://dx.doi.org/10.1083/jcb.138.2.331.
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Myoblast fusion is essential to muscle tissue development yet remains poorly understood. N-cadherin, like other cell surface adhesion molecules, has been implicated by others in muscle formation based on its pattern of expression and on inhibition of myoblast aggregation and fusion by antibodies or peptide mimics. Mice rendered homozygous null for N-cadherin revealed the general importance of the molecule in early development, but did not test a role in skeletal myogenesis, since the embryos died before muscle formation. To test genetically the proposed role of N-cadherin in myoblast fusion, we successfully obtained N-cadherin null primary myoblasts in culture. Fusion of myoblasts expressing or lacking N-cadherin was found to be equivalent, both in vitro by intracistronic complementation of lacZ and in vivo by injection into the muscles of adult mice. An essential role for N-cadherin in mediating the effects of basic fibroblast growth factor was also excluded. These methods for obtaining genetically homozygous null somatic cells from adult tissues should have broad applications. Here, they demonstrate clearly that the putative fusion molecule, N-cadherin, is not essential for myoblast fusion.
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Rando, T. A., and H. M. Blau. "Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy." Journal of Cell Biology 125, no. 6 (1994): 1275–87. http://dx.doi.org/10.1083/jcb.125.6.1275.
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The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity--suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.
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Yao, S. N., and K. Kurachi. "Implanted myoblasts not only fuse with myofibers but also survive as muscle precursor cells." Journal of Cell Science 105, no. 4 (1993): 957–63. http://dx.doi.org/10.1242/jcs.105.4.957.
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Intramuscular implanted myoblasts can fuse with existing myofibers. Here we report that implanted primary myoblasts marked with retroviral transgenes can also persist as muscle precursor cells. These cells can be recovered as viable myoblasts from muscles of recipient mice even months after myoblast implantation, and they can fully resume expression of the transgenes in culture. Upon re-implantation into muscles, they again not only fuse with existing myofibers, but also survive as muscle precursor cells in the tissue. These reserve myogenic cells should be able to contribute to host myofibers in muscle regeneration when the recombinant myofibers are damaged, providing an additional mechanism to maintain a persistent expression of transgenes delivered by myoblast-mediated gene transfer.
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Jurdana, Mihaela, Maja Cemazar, Katarina Pegan, and Tomaz Mars. "Effect of ionizing radiation on human skeletal muscle precursor cells." Radiology and Oncology 47, no. 4 (2013): 376–81. http://dx.doi.org/10.2478/raon-2013-0058.
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Abstract Background. Long term effects of different doses of ionizing radiation on human skeletal muscle myoblast proliferation, cytokine signalling and stress response capacity were studied in primary cell cultures. Materials and methods. Human skeletal muscle myoblasts obtained from muscle biopsies were cultured and irradiated with a Darpac 2000 X-ray unit at doses of 4, 6 and 8 Gy. Acute effects of radiation were studied by interleukin - 6 (IL-6) release and stress response detected by the heat shock protein (HSP) level, while long term effects were followed by proliferation capacity and cell death. Results. Compared with non-irradiated control and cells treated with inhibitor of cell proliferation Ara C, myoblast proliferation decreased 72 h post-irradiation, this effect was more pronounced with increasing doses. Post-irradiation myoblast survival determined by measurement of released LDH enzyme activity revealed increased activity after exposure to irradiation. The acute response of myoblasts to lower doses of irradiation (4 and 6 Gy) was decreased secretion of constitutive IL-6. Higher doses of irradiation triggered a stress response in myoblasts, determined by increased levels of stress markers (HSPs 27 and 70). Conclusions. Our results show that myoblasts are sensitive to irradiation in terms of their proliferation capacity and capacity to secret IL-6. Since myoblast proliferation and differentiation are a key stage in muscle regeneration, this effect of irradiation needs to be taken in account, particularly in certain clinical conditions.
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Jane, Derek T., Leslie C. Morvay, Francis Allen, Bonnie F. Sloane, and Michael J. Dufresne. "Selective inhibition of cathepsin B with cell-permeable CA074Me negatively affects L6 rat myoblast differentiation." Biochemistry and Cell Biology 80, no. 4 (2002): 457–65. http://dx.doi.org/10.1139/o02-134.
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Active cathepsin B, in concert with other cellular proteases, has been implicated in the catabolic restructuring associated with myotube formation during skeletal myoblast cell differentiation (i.e., myogenesis). We have examined this role in differentiating myoblasts using the cell-permeable, cathepsin B selective inhibitor CA074Me. Cathepsin B activity levels in differentiating L6 rat myoblasts treated with CA074Me were significantly lower than levels in control myoblasts. Inhibition of cathepsin B activity by CA074Me occurred at each stage of differentiation and was dose related. Myotube size and number and induced levels of fusion-related creatine phosphokinase activity and myosin heavy-chain protein were reduced from 30 to 50% in CA074Me-treated myoblasts. These reductions were also dose related. In contrast, CA074Me did not affect levels of myogenin, an early marker of myogenesis, or levels of cathepsin L type and myokinase activities, two nonspecific enzymes. The negative effects associated with CA074Me were reversed when the drug was removed. Collectively, these data suggest that active cathepsin B plays a role in myoblastmyoblast fusion and consequently may be necessary for the complete expression of those genes associated with the fusion process.Key words: cathepsin B, CA074Me, myogenesis.
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Milanesi, Anna, Jang-Won Lee, Nam-Ho Kim та ін. "Thyroid Hormone Receptor α Plays an Essential Role in Male Skeletal Muscle Myoblast Proliferation, Differentiation, and Response to Injury". Endocrinology 157, № 1 (2016): 4–15. http://dx.doi.org/10.1210/en.2015-1443.
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Abstract Thyroid hormone plays an essential role in myogenesis, the process required for skeletal muscle development and repair, although the mechanisms have not been established. Skeletal muscle develops from the fusion of precursor myoblasts into myofibers. We have used the C2C12 skeletal muscle myoblast cell line, primary myoblasts, and mouse models of resistance to thyroid hormone (RTH) α and β, to determine the role of thyroid hormone in the regulation of myoblast differentiation. T3, which activates thyroid hormone receptor (TR) α and β, increased myoblast differentiation whereas GC1, a selective TRβ agonist, was minimally effective. Genetic approaches confirmed that TRα plays an important role in normal myoblast proliferation and differentiation and acts through the Wnt/β-catenin signaling pathway. Myoblasts with TRα knockdown, or derived from RTH-TRα PV (a frame-shift mutation) mice, displayed reduced proliferation and myogenic differentiation. Moreover, skeletal muscle from the TRα1PV mutant mouse had impaired in vivo regeneration after injury. RTH-TRβ PV mutant mouse model skeletal muscle and derived primary myoblasts did not have altered proliferation, myogenic differentiation, or response to injury when compared with control. In conclusion, TRα plays an essential role in myoblast homeostasis and provides a potential therapeutic target to enhance skeletal muscle regeneration.
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Bach, Anne-Sophie, Sandrine Enjalbert, Franck Comunale, et al. "ADP-Ribosylation Factor 6 Regulates Mammalian Myoblast Fusion through Phospholipase D1 and Phosphatidylinositol 4,5-Bisphosphate Signaling Pathways." Molecular Biology of the Cell 21, no. 14 (2010): 2412–24. http://dx.doi.org/10.1091/mbc.e09-12-1063.
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Myoblast fusion is an essential step during myoblast differentiation that remains poorly understood. M-cadherin–dependent pathways that signal through Rac1 GTPase activation via the Rho-guanine nucleotide exchange factor (GEF) Trio are important for myoblast fusion. The ADP-ribosylation factor (ARF)6 GTPase has been shown to bind to Trio and to regulate Rac1 activity. Moreover, Loner/GEP100/BRAG2, a GEF of ARF6, has been involved in mammalian and Drosophila myoblast fusion, but the specific role of ARF6 has been not fully analyzed. Here, we show that ARF6 activity is increased at the time of myoblast fusion and is required for its implementation in mouse C2C12 myoblasts. Specifically, at the onset of myoblast fusion, ARF6 is associated with the multiproteic complex that contains M-cadherin, Trio, and Rac1 and accumulates at sites of myoblast fusion. ARF6 silencing inhibits the association of Trio and Rac1 with M-cadherin. Moreover, we demonstrate that ARF6 regulates myoblast fusion through phospholipase D (PLD) activation and phosphatidylinositol 4,5-bis-phosphate production. Together, these data indicate that ARF6 is a critical regulator of C2C12 myoblast fusion and participates in the regulation of PLD activities that trigger both phospholipids production and actin cytoskeleton reorganization at fusion sites.
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Skuk, Daniel, and Jacques P. Tremblay. "Complement Deposition and Cell Death after Myoblast Transplantation." Cell Transplantation 7, no. 5 (1998): 427–34. http://dx.doi.org/10.1177/096368979800700501.
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One of the problems limiting myoblast transplantation (MT) is the early death of the transplanted cells. Because complement can be fixed by myoblasts in vitro, and because it has the capacity to induce cell lysis, its possible role in the early death of transplanted myoblasts was investigated. CD1 mice and Macaca mulata monkeys were used as recipients for MT. In some mice, C3 was depleted before MT using Cobra Venom Factor. Mice were sacrificed during the first hour and up to 3 days after MT. Monkeys were biopsied 1 to 4 h after MT. Myoblast necrosis was assessed by the presence of intracellular calcium. Complement deposition was demonstrated by immunohistochemistry with anti-C3 and anti-C5b-9 neoantigen antibodies. In mice, C3 deposition was observed in damaged muscle fibers and in regions containing necrosed myoblasts. Complement depletion did not diminish the proportion of necrosed cells. In monkeys, only a small percentage of transplanted myoblasts showed C3 or C5b-9 deposition, mostly intracellular. Complement activation seems not to be implicated in directly damaging the transplanted cells, but seems secondary to cellular death. Taking into account its chemotactic functions, complement could be implicated in the migration of neutrophils and macrophages into the clusters of transplanted cells. © 1998 Elsevier Science Inc.
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Mohd Sahardi, Nur Fatin Nabilah, Faizul Jaafar, Mariam Firdhaus Mad Nordin, and Suzana Makpol. "Zingiber Officinale Roscoe Prevents Cellular Senescence of Myoblasts in Culture and Promotes Muscle Regeneration." Evidence-Based Complementary and Alternative Medicine 2020 (April 30, 2020): 1–13. http://dx.doi.org/10.1155/2020/1787342.
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Background. Ageing resulted in a progressive loss of muscle mass and strength. Increased oxidative stress in ageing affects the capacity of the myoblast to differentiate leading to impairment of muscle regeneration. Zingiber officinale Roscoe (ginger) has potential benefits in reversing muscle ageing due to its antioxidant property. This study aimed to determine the effect of ginger in the prevention of cellular senescence and promotion of muscle regeneration. Methods. Myoblast cells were cultured into young and senescent state before treated with different concentrations of ginger standardised extracts containing different concentrations of 6-gingerol and 6-shogaol. Analysis on cellular morphology and myogenic purity was carried out besides determination of SA-β-galactosidase expression and cell cycle profile. Myoblast differentiation was quantitated by determining the fusion index, maturation index, and myotube size. Results. Treatment with ginger extracts resulted in improvement of cellular morphology of senescent myoblasts which resembled the morphology of young myoblasts. Our results also showed that ginger treatment caused a significant reduction in SA-β-galactosidase expression on senescent myoblasts indicating prevention of cellular senescence, while cell cycle analysis showed a significant increase in the percentage of cells in the G0/G1 phase and reduction in the S-phase cells. Increased myoblast regenerative capacity was observed as shown by the increased number of nuclei per myotube, fusion index, and maturation index. Conclusions. Ginger extracts exerted their potency in promoting muscle regeneration as indicated by prevention of cellular senescence and promotion of myoblast regenerative capacity.
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Huang, Shiyuan, Xiaona Wang, Jiale Yu, et al. "LonP1 regulates mitochondrial network remodeling through the PINK1/Parkin pathway during myoblast differentiation." American Journal of Physiology-Cell Physiology 319, no. 6 (2020): C1020—C1028. http://dx.doi.org/10.1152/ajpcell.00589.2019.
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Myoblast differentiation is a crucial process for myogenesis. Mitochondria function as an energy-providing machine that is critical to this process, and mitochondrial dysfunction can prevent myoblasts from fusing into myotubes. However, the molecular mechanisms underlying the dynamic regulation of mitochondrial networks remain poorly understood. In the present study, we found that the PTEN induced kinase 1 (PINK1)/Parkin (an E3 ubiquitin-protein ligase) pathway is activated at the early stage of myoblast differentiation. Moreover, downregulation of mitofusin 2 (Mfn2) and increased dynamin-related protein 1 (Drp1) resulted in loosely formed mitochondria during this period. Furthermore, selective knockdown of the mitochondrial matrix protein Lon peptidase-1 (LonP1) at the early stage of myoblast differentiation induced mitochondrial depolarization and suppressed the PINK1/Parkin pathway and reduced Mfn2 and Drp1 levels, which blocked mitochondrial remodeling and myoblast differentiation. Overall, these data demonstrate that LonP1 promotes myoblast differentiation by regulating PINK1/Parkin-mediated mitochondrial remodeling.
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Guérette, B., D. Skuk, F. Célestin, et al. "Prevention by anti-LFA-1 of acute myoblast death following transplantation." Journal of Immunology 159, no. 5 (1997): 2522–31. http://dx.doi.org/10.4049/jimmunol.159.5.2522.
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Abstract Myoblast transplantation is a potential treatment for Duchenne muscular dystrophy. One of the problems possibly responsible for the limited success of clinical trials is the rapid death of the myoblasts after transplantation. To investigate this problem, myoblasts expressing beta-galactosidase were injected in the tibialis anterior muscles of mice. Beta-galactosidase activity was reduced by 74.7% after 3 days. Myoblast death observed at 3 days was reduced to 57.2% when the hosts were irradiated. This result suggested that host cells were contributing to this phenomenon. Transplantation in SCID and FK506-treated mice did not reduce cell death, indicating that mortality was not due to an acute specific reaction. In contrast, administration of the anti-LFA-1 (TIB-213) mAb markedly reduced myoblast death at 3 days without altering leukocyte tissue infiltration. We postulated that neutrophils were mediating myoblast mortality by an LFA-1-dependent mechanism. To test this hypothesis, IL-1beta-activated myoblasts were loaded with 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethylester) (DCFH), a marker for oxidative stress. Addition of neutrophils and zymosan-activated serum resulted in a time-dependent DCFH fluorescence; this neutrophil-induced oxidation was considerably inhibited by TIB-213. These results indicate that an effective control of the inflammatory reaction will be necessary for any new clinical trials of myoblast transplantation and suggest that neutrophil-mediated myoblast injury occurs by an LFA-1-dependent pathway.
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Balcerzak, D., S. Poussard, J. J. Brustis, et al. "An antisense oligodeoxyribonucleotide to m-calpain mRNA inhibits myoblast fusion." Journal of Cell Science 108, no. 5 (1995): 2077–82. http://dx.doi.org/10.1242/jcs.108.5.2077.
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Previous studies have led to the hypothesis of a possible role for m-calpain (EC 3.4.22.17) in myoblast fusion in culture in vitro. To support this hypothesis, an antisense strategy has been used with cultured primary rat myoblasts. Using an appropriate antisense oligodeoxyribonucleotide to m-calpain mRNA, an inhibition of myoblast fusion has been observed, the maximum being obtained when the cell culture was treated with 30 microM of oligomer. Synthesis of m-calpain was decreased by 48% while high concentrations of antisense oligonucleotide do not significantly affect myoblast proliferation. The specificity of m-calpain intervention during fusion has also been confirmed using antisense oligonucleotides to mu-calpain and p94 mRNAs, respectively.
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Konieczny, S. F., and C. P. Emerson. "Differentiation, not determination, regulates muscle gene activation: transfection of troponin I genes into multipotential and muscle lineages of 10T1/2 cells." Molecular and Cellular Biology 5, no. 9 (1985): 2423–32. http://dx.doi.org/10.1128/mcb.5.9.2423-2432.1985.
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Transcription of quail skeletal muscle troponin I (TnI) genes was examined after stable transfection into multipotential 10T1/2 mouse cells and into determined myoblast lineages derived by 5-azacytidine conversion. Transfected TnI and endogenous mouse muscle genes were inactive both in multipotential 10T1/2 and in proliferating myoblasts but were activated coordinately and to high levels when myoblast lineages differentiated, regardless of whether TnI genes were transfected before or after myoblast lineage determination. We conclude that the TnI gene contains evolutionarily conserved control sequences that activate its transcription in response to differentiation-specific regulatory signals. Myoblast lineage determination, therefore, does not appear to act directly on TnI and other muscle genes but likely establishes a regulatory control system that mediates expression of differentiation-specific transcription signals.
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Rushton, E., R. Drysdale, S. M. Abmayr, A. M. Michelson, and M. Bate. "Mutations in a novel gene, myoblast city, provide evidence in support of the founder cell hypothesis for Drosophila muscle development." Development 121, no. 7 (1995): 1979–88. http://dx.doi.org/10.1242/dev.121.7.1979.
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We have used mutations in the newly identified gene myoblast city to investigate the founder cell hypothesis of muscle development in Drosophila melanogaster. In embryos mutant for myoblast city the fusion of myoblasts into multinucleate muscles is virtually abolished. Nevertheless, a subset of the myoblasts develop specific muscle-like characteristics, including gene expression appropriate to particular muscles, migration to the appropriate part of the segment, correct position and orientation, and contact by motor neurons. We suggest that this subset of myoblasts represents the proposed muscle founder cells and we draw an analogy between these founder cells and the muscle pioneers described for grasshopper muscle development.
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Mesmer, O. T., and T. C. Lo. "Hexose transport in human myoblasts." Biochemical Journal 262, no. 1 (1989): 15–24. http://dx.doi.org/10.1042/bj2620015.
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The present investigation reports on the hexose transport properties of human myoblasts isolated from normal subjects and from patients with Duchenne muscular dystrophy (DMD). Similar to rat myoblast L6, normal human myoblasts possess a high- (HAHT) and a low- (LAHT) affinity hexose transport system. The non-metabolizable hexose analogue, 2-deoxyglucose, is preferentially taken up by HAHT. The transport of this analogue is the rate-limiting step in the uptake process. This human myoblast HAHT is also similar to that of the rat myoblast in its substrate specificity and in response to the energy uncouplers, cytochalasin B and phloretin. The human myoblast LAHT resembles that of rat myoblast in its insensitivity to energy uncouplers, and in its transport affinity and capacity for 3-O-methyl-D-glucose. Although DMD myoblasts resemble their normal counterpart in their ability to differentiate, they differ significantly in their hexose transport properties. In addition to HAHT and LAHT present in normal human myoblast, DMD myoblasts contain a super-high-affinity hexose transport system (SHAHT). SHAHT can be detected only at very low substrate concentrations. It differs from HAHT not only in its much higher transport affinity, but also in its response to the traditional hexose transport inhibitors. For example, SHAHT can be activated by cytochalasin B and phlorizin, whereas it is more sensitive to inhibition by phloretin. Unlike HAHT, energy uncouplers are found to be ineffective in inhibiting SHAHT. It should be mentioned that SHAHT cannot be detected in myoblasts isolated from patients with other types of myopathy. The present study serves to demonstrate that more than one hexose transport system is operating in human skeletal muscle cells, as found in other cell types.
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Cates, George A., Devki Nandan, Anne M. Brickenden, and Bishnu D. Sanwal. "Differentiation defective mutants of skeletal myoblasts altered in a gelatin-binding glycoprotein." Biochemistry and Cell Biology 65, no. 9 (1987): 767–75. http://dx.doi.org/10.1139/o87-100.
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We have previously described a myoblast cell surface glycoprotein of the molecular mass 46 000 (gp46), which is associated with myoblast differentiation. In this report we demonstrate that gp46 binds specifically to gelatin-Sepharose and in this respect is similar to a glycoprotein of the molecular mass 47 000, which has earlier been described as a cell surface localized protein in mouse parietal endoderm cells and in chick embryo fibroblasts. To ascertain the relationship of gp46 to myoblast differentiation, wild-type L6 myoblasts, as well as two concanavalin A (ConA) resistant, differentiation-negative, myoblast mutants (D-1 and C-8), were examined for gp46 expression. In the mutant designated D-1, which has a defect in dolichol mannosyl transferase, both mannose incorporation into gp46 and ConA binding to gp46 was reduced compared with L6, without markedly affecting the gelatin adhesion properties of gp46. Western blotting with a monoclonal antibody against gp46 was used to show that the expression of gp46 was normal in D-1 but was reduced in mutant C-8 compared with L6. Reduction occurred both in the plasma membrane and endoplasmic reticulum fractions of C-8 compared with wild-type L6. In L6 myoblasts, the expression of gp46 remained constant during myoblast replication and fusion but decreased markedly postfusion. In the nonfusing myoblast mutants D-1 and C-8 and in wild-type L6 cells that were prevented from fusing by treatment with 5-bromo-2′-deoxyuridine, the expression of gp46 remained invariant. We suggest that collagen interactions, mediated by gp46, are important for normal rat skeletal muscle differentiation.
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Önel, Susanne-Filiz. "Actin regulators take the reins in Drosophila myoblast fusion." Open Life Sciences 4, no. 1 (2009): 11–18. http://dx.doi.org/10.2478/s11535-008-0059-9.
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AbstractSkeletal muscle formation, growth and repair depend on myoblast fusion events. Therefore, in-depth understanding of the underlying molecular mechanisms controlling these events that ultimately lead to skeletal muscle formation may be fundamental for developing new therapies for tissue repair. To this end, the greatest advances in furthering understanding myoblast fusion has been made in Drosophila. Recent studies have shown that transient F-actin structures, so-called actin plugs or foci, are known to form at the site of contacting myoblasts. Indeed, actin regulators of the WASP family that control the activation of the Arp2/3 complex and thereby branched F-actin formation have been demonstrated to be crucial for myoblast fusion. Myoblast-specific cell adhesion molecules seem to be involved in the recruitment of WASP family members to the site of myoblast fusion and form a Fusion-Restricted Myogenic-Adhesive Structure (FuRMAS). Currently, the exact role of the FuRMAS is not completely understood. However, recent studies indicate that WASP-dependent F-actin regulation is required for fusion pore formation as well as for the correct integration of fusing myoblasts into the growing muscle. In this review, I discuss latest cellular studies, and recent genetic and biochemical analyses on actin regulation during myoblast fusion.
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Mege, R. M., D. Goudou, C. Diaz, et al. "N-cadherin and N-CAM in myoblast fusion: compared localisation and effect of blockade by peptides and antibodies." Journal of Cell Science 103, no. 4 (1992): 897–906. http://dx.doi.org/10.1242/jcs.103.4.897.
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The expression and distribution of two cell adhesion molecules, N-cadherin and N-CAM, at the surface of cultured leg muscle cells from 11-day-old chicken embryos were studied and compared. N-cadherin, which was expressed by fusing myoblasts, was down-regulated on old myotubes while N-CAM was still present. Both molecules, as viewed by confocal microscopy, appeared to have coaccumulated at the areas of contact between fusing myoblasts. However, immunogold electron microscopy did not reveal significant colocalization of N-cadherin and N-CAM, and their segregation after antibody-induced patching suggested the absence of direct interactions between N-cadherin and N-CAM. The role of the Ca2+ dependent cell adhesion molecule N-cadherin in myogenesis was investigated. Myoblast fusion was inhibited (1) with a synthetic peptide containing the H-A-V sequence and (2) with a monoclonal anti-N-cadherin antibody, demonstrating that N-cadherin-mediated cell adhesion is required for myoblast fusion. Under the same conditions no effect of anti-N-CAM antibodies was observed. Taken together these observations suggest that N-cadherin, acting independently from N-CAM, is a major cell adhesion molecule involved in embryonic myoblast fusion in vitro.
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Kagawa, Yuki, and Masahiro Kino-oka. "An in silico prediction tool for the expansion culture of human skeletal muscle myoblasts." Royal Society Open Science 3, no. 10 (2016): 160500. http://dx.doi.org/10.1098/rsos.160500.
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Regenerative therapy using autologous skeletal myoblasts requires a large number of cells to be prepared for high-level secretion of cytokines and chemokines to induce good regeneration of damaged regions. However, myoblast expansion culture is hindered by a reduction in growth rate owing to cellular quiescence and differentiation, therefore optimization is required. We have developed a kinetic computational model describing skeletal myoblast proliferation and differentiation, which can be used as a prediction tool for the expansion process. In the model, myoblasts migrate, divide, quiesce and differentiate as observed during in vitro culture. We assumed cell differentiation initiates following cell–cell attachment for a defined time period. The model parameter values were estimated by fitting to several predetermined experimental datasets. Using an additional experimental dataset, we confirmed validity of the developed model. We then executed simulations using the developed model under several culture conditions and quantitatively predicted that non-uniform cell seeding had adverse effects on the expansion culture, mainly by reducing the existing ratio of proliferative cells. The proposed model is expected to be useful for predicting myoblast behaviours and in designing efficient expansion culture conditions for these cells.
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Ceci, Roberta, Mariateresa Maldini, Piergiorgio La Rosa, et al. "The Effect of Moringa oleifera Leaf Extract on C2C12 Myoblast Proliferation and Redox Status Under Oxidative Insult." Antioxidants 13, no. 12 (2024): 1460. http://dx.doi.org/10.3390/antiox13121460.
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Skeletal muscle tissue can regenerate after damage through the action of satellite cells, which proliferate as myoblasts when activated. Oxidative stress, marked by high rates of reactive oxygen species (e.g., hydrogen peroxide, H2O2), impairs this process by increasing myoblast cell death. Moringa oleifera leaf extract (MOLE), known for its antioxidant properties, was tested for its protective effects on C2C12 myoblasts under oxidative stress. We assessed MOLE’s impact on total antioxidant capacity (TAC), glutathione homeostasis (GSH/GSSG), cell viability, and wound recovery. The metabolomic analysis of MOLE using an LC-MSMS ZenoTOF 7600 mass spectrometry system identified key compounds, including peculiar glucosinolates (42.1%) and flavonoids (18.8%), as well as phenolic acids (4.5%) and other significant metabolites (34.6%; among them, amino acids, vitamins, and fatty acids). H2O2 disrupted myoblast redox balance and caused cell death, but MOLE treatment restored the GSH/GSSG ratio, improved TAC, and increased cell viability. Additionally, MOLE promoted faster wound closure in myoblasts exposed to H2O2. These findings suggest that MOLE can protect C2C12 myoblasts by restoring redox balance and enhancing recovery under oxidative stress.
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Youm, Tae Hyun, Sun-Hee Woo, Eun-Soo Kwon, and Sung Sup Park. "NADPH Oxidase 4 Contributes to Myoblast Fusion and Skeletal Muscle Regeneration." Oxidative Medicine and Cellular Longevity 2019 (November 18, 2019): 1–12. http://dx.doi.org/10.1155/2019/3585390.
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Myoblast fusion is an essential step in skeletal muscle development and regeneration. NADPH oxidase 4 (Nox4) regulates cellular processes such as proliferation, differentiation, and survival by producing reactive oxygen species (ROS). Insulin-like growth factor 1 induces muscle hypertrophy via Nox4, but its function in myoblast fusion remains elusive. Here, we report a ROS-dependent role of Nox4 in myoblast differentiation. Regenerating muscle fibers after injury by cardiotoxin had a lower cross-sectional area in Nox4-knockout (KO) mice than myofibers in wild-type (WT) mice. Diameters and fusion index values of myotubes differentiated from Nox4-KO primary myoblasts were significantly lower than those of myotubes derived from WT myoblasts. However, no difference was observed in the differentiation index and expression of MyoD, myogenin, and myosin heavy chain 3 (MHC) between KO and WT myotubes. The decreased fusion index was also observed during differentiation of primary myoblasts and C2C12 cells with suppressed Nox4 expression. In contrast, in C2C12 cells overexpressing Nox4, the fusion index was increased, whereas the differentiation index and MHC and myogenin protein expression were not affected compared to control. Interestingly, the expression of myomaker (Tmem8c), a fusogenic protein that controls myoblast fusion, was reduced in Nox4-knockdown C2C12 cells. The myomaker expression level was proportional to the cellular ROS level, which was regulated by of Nox4 expression level. These results suggests that Nox4 contributes to myoblast fusion, possibly through the regulation of myomaker expression via ROS production, and that Nox4-dependent ROS may promote skeletal muscle regeneration and growth.
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Sundaram, M., J. Yochem, and M. Han. "A Ras-mediated signal transduction pathway is involved in the control of sex myoblast migration in Caenorhabditis elegans." Development 122, no. 9 (1996): 2823–33. http://dx.doi.org/10.1242/dev.122.9.2823.
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Sex myoblast migration in the Caenorhabditis elegans hermaphrodite represents a simple, genetically amenable model system for studying how cell migration is regulated during development. Two separable components of sex myoblast guidance have been described: a gonad-independent mechanism sufficient for the initial anterior migration to the mid-body region, and a gonad-dependent mechanism required for precise final positioning (J. H. Thomas, M. J. Stern and H. R. Horvitz (1990) Cell 62, 1041–1052). Here, we demonstrate a role for a Ras-mediated signal transduction pathway in controlling sex myoblast migration. Loss-of-function mutations in let-60 ras, ksr-1, lin-45 raf, let-537/mek-2 or sur-1/mpk-1 cause defects in sex myoblast final positions that resemble those seen in gonad-ablated animals, while constitutively active let-60 ras(G13E) trans-genes allow fairly precise positioning to occur in the absence of the gonad. A mosaic analysis demonstrated that let-60 ras is required within the sex myoblasts to control proper positioning. Our results suggest that gonadal signals normally stimulate let-60 ras activity in the sex myoblasts, thereby making them competent to sense or respond to positional cues that determine the precise endpoint of migration. let-60 ras may have additional roles in sex myoblast guidance as well. Finally, we have also investigated genetic interactions between let-60 ras and other genes important for sex myoblast migration, including egl-15, which encodes a fibroblast growth factor receptor tyrosine kinase (D. L. DeVore, H. R. Horvitz and M. J. Stern (1995) Cell 83, 611–623). Since mutations reducing Ras pathway activity cause a different phenotype than those reducing egl-15 activity and since constitutive Ras activity only partially suppresses the migration defects of egl-15 mutants, we argue that let-60 ras and egl-15 do not act together in a single linear pathway.
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Pedrotty, Dawn M., Jennifer Koh, Bryce H. Davis, Doris A. Taylor, Patrick Wolf, and Laura E. Niklason. "Engineering skeletal myoblasts: roles of three-dimensional culture and electrical stimulation." American Journal of Physiology-Heart and Circulatory Physiology 288, no. 4 (2005): H1620—H1626. http://dx.doi.org/10.1152/ajpheart.00610.2003.
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Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty in attempts to regenerate cardiac muscle tissue by injection of cells into damaged myocardium. In some studies, muscle tissue within myoblast implant sites may be morphologically similar to cardiac muscle. We hypothesized that identifiable aspects of the cardiac milieu may contribute to growth and development of implanted myoblasts in vivo. To test this hypothesis, we designed a novel in vitro system to mimic some aspects of the electrical and biochemical environment of native myocardium. This system enabled us to separate the three-dimensional (3-D) electrical and biochemical signals that may be involved in myoblast proliferation and plasticity. Myoblasts were grown on 3-D polyglycolic acid mesh scaffolds under control conditions, in the presence of cardiac-like electrical current fluxes, or in the presence of culture medium that had been conditioned by mature cardiomyocytes. Cardiac-like electrical current fluxes caused increased myoblast number in 3-D culture, as determined by DNA assay. The increase in cell number was due to increased cellular proliferation and not differences in apoptosis, as determined by proliferating cell nuclear antigen and TdT-mediated dUTP nick-end labeling. Cardiomyocyte-conditioned medium also significantly increased myoblast proliferation. Expression of transcription factors governing differentiation along skeletal or cardiac lineages was evaluated by immunoblotting. Although these assays are qualitative, no changes in differentiation state along skeletal or cardiac lineages were observed in response to electrical current fluxes. Furthermore, from these experiments, conditioned medium did not appear to alter the differentiation state of skeletal myoblasts. Hence, cardiac milieu appears to stimulate proliferation but does not affect differentiation of skeletal myoblasts.
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Yamashita, Aline M. S., Maryana T. C. Ancillotti, Luciana P. Rangel, et al. "Balance between S-nitrosylation and denitrosylation modulates myoblast proliferation independently of soluble guanylyl cyclase activation." American Journal of Physiology-Cell Physiology 313, no. 1 (2017): C11—C26. http://dx.doi.org/10.1152/ajpcell.00140.2016.
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Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form S-nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of S-nitrosoglutathione reductase (GSNOR) during myogenesis is understood. Here, we used primary cultures of chick embryonic skeletal muscle cells to investigate whether changes in intracellular RSNO alter proliferation and fusion of myoblasts in the presence and absence of cGMP. Cultures were grown to fuse most of the myoblasts into myotubes, with and without S-nitrosocysteine (CysNO), 8-Br-cGMP, DETA-NO, or inhibitors for NO synthase (NOS), GSNOR, soluble guanylyl cyclase (sGC), or a combination of these, followed by analysis of GSNOR activity, protein expression, RSNO, cGMP, and cell morphology. Although the activity of GSNOR increased progressively over 72 h, inhibiting GSNOR (by GSNOR inhibitor – GSNORi – or by knocking down GSNOR with siRNA) produced an increase in RSNO and in the number of myoblasts and fibroblasts, accompanied by a decrease in myoblast fusion index. This was also detected with CysNO supplementation. Enhanced myoblast number was proportional to GSNOR inhibition. Effects of the GSNORi and GSNOR knockdown were blunted by NOS inhibition, suggesting their dependence on NO synthesis. Interestingly, GSNORi and GSNOR knockdown reversed the attenuated proliferation obtained with sGC inhibition in myoblasts, but not in fibroblasts. Hence myoblast proliferation is enhanced by increasing RSNO, and regulated by GSNOR activity, independently of cGMP production and signaling.
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Dhanyasi, Nagaraju, Dagan Segal, Eyal Shimoni, et al. "Surface apposition and multiple cell contacts promote myoblast fusion in Drosophila flight muscles." Journal of Cell Biology 211, no. 1 (2015): 191–203. http://dx.doi.org/10.1083/jcb.201503005.
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Fusion of individual myoblasts to form multinucleated myofibers constitutes a widely conserved program for growth of the somatic musculature. We have used electron microscopy methods to study this key form of cell–cell fusion during development of the indirect flight muscles (IFMs) of Drosophila melanogaster. We find that IFM myoblast–myotube fusion proceeds in a stepwise fashion and is governed by apparent cross talk between transmembrane and cytoskeletal elements. Our analysis suggests that cell adhesion is necessary for bringing myoblasts to within a minimal distance from the myotubes. The branched actin polymerization machinery acts subsequently to promote tight apposition between the surfaces of the two cell types and formation of multiple sites of cell–cell contact, giving rise to nascent fusion pores whose expansion establishes full cytoplasmic continuity. Given the conserved features of IFM myogenesis, this sequence of cell interactions and membrane events and the mechanistic significance of cell adhesion elements and the actin-based cytoskeleton are likely to represent general principles of the myoblast fusion process.
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Ehrlich, Melanie, Kenneth C. Ehrlich, Michelle Lacey, et al. "Epigenetics of Genes Preferentially Expressed in Dissimilar Cell Populations: Myoblasts and Cerebellum." Epigenomes 8, no. 1 (2024): 4. http://dx.doi.org/10.3390/epigenomes8010004.
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While studying myoblast methylomes and transcriptomes, we found that CDH15 had a remarkable preference for expression in both myoblasts and cerebellum. To understand how widespread such a relationship was and its epigenetic and biological correlates, we systematically looked for genes with similar transcription profiles and analyzed their DNA methylation and chromatin state and accessibility profiles in many different cell populations. Twenty genes were expressed preferentially in myoblasts and cerebellum (Myob/Cbl genes). Some shared DNA hypo- or hypermethylated regions in myoblasts and cerebellum. Particularly striking was ZNF556, whose promoter is hypomethylated in expressing cells but highly methylated in the many cell populations that do not express the gene. In reporter gene assays, we demonstrated that its promoter’s activity is methylation sensitive. The atypical epigenetics of ZNF556 may have originated from its promoter’s hypomethylation and selective activation in sperm progenitors and oocytes. Five of the Myob/Cbl genes (KCNJ12, ST8SIA5, ZIC1, VAX2, and EN2) have much higher RNA levels in cerebellum than in myoblasts and displayed myoblast-specific hypermethylation upstream and/or downstream of their promoters that may downmodulate expression. Differential DNA methylation was associated with alternative promoter usage for Myob/Cbl genes MCF2L, DOK7, CNPY1, and ANK1. Myob/Cbl genes PAX3, LBX1, ZNF556, ZIC1, EN2, and VAX2 encode sequence-specific transcription factors, which likely help drive the myoblast and cerebellum specificity of other Myob/Cbl genes. This study extends our understanding of epigenetic/transcription associations related to differentiation and may help elucidate relationships between epigenetic signatures and muscular dystrophies or cerebellar-linked neuropathologies.
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Guérette, Benoit, Isabelle Asselin, Daniel Skuk, Mark Entman, and Jacques P. Tremblay. "Control of Inflammatory Damage by Anti-Lfa-1: Increase Success of Myoblast Transplantation." Cell Transplantation 6, no. 2 (1997): 101–7. http://dx.doi.org/10.1177/096368979700600203.
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Myoblast transplantation is a potential treatment for Duchenne Muscular Dystrophy. This article confirms by experiments in mice that one problem that has limited the success of clinical trials of this procedure is a rapid (within 3 days) inflammatory reaction which kills most of the injected myoblasts. The death of the transplanted myoblasts can be prevented by treating the host with a mAb against LFA-1. This led to a 27-fold increase in the number of muscle fibers expressing a reporter gene present in the donor myoblasts when the host is also adequately immunosuppressed with FK506. Therefore, both the nonspecific inflammatory reaction and the specific immune response should be adequately controlled following myoblast transplantation.
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Shu, Lili, and Peter J. Houghton. "The mTORC2 Complex Regulates Terminal Differentiation of C2C12 Myoblasts." Molecular and Cellular Biology 29, no. 17 (2009): 4691–700. http://dx.doi.org/10.1128/mcb.00764-09.
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ABSTRACT Rapamycin, a selective inhibitor of mTORC1 signaling, blocks terminal myoblast differentiation. We found that downregulation of rictor, a component of the mTORC2 complex, but not downregulation of raptor, a component of the mTORC1 complex, prevented terminal differentiation (fusion) of C2C12 myoblasts. Both rapamycin and rictor downregulation suppressed the phosphorylation of AKT(S473), and rapamycin treatment of C2C12 myoblasts disrupted the mTORC2 complex. Importantly, downregulation of rictor inhibited TORC2 signaling without inhibiting mTORC1 signaling, suggesting that inhibition of mTORC1 by rapamycin may not be the cause of arrested differentiation. In support of this, expression of a phosphomimetic mutant AKT(S473D) in rictor-deficient cells rescued myoblast fusion even in the presence of rapamycin. mTORC2 signaling to AKT appears necessary for downregulation of the Rho-associated kinase (ROCK1) that occurs during myogenic differentiation. Rapamycin treatment prevented ROCK1 inactivation during differentiation, while suppression of ROCK1 activity during differentiation and myoblast fusion was restored through expression of AKT(S473D), even in the presence of rapamycin. Further, the ROCK inhibitor Y-27632 restored terminal differentiation in rapamycin-treated myoblasts. These results provide the first evidence of a specific role for mTORC2 signaling in terminal myogenic differentiation.
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Chinni, C., M. R. de Niese, A. L. Jenkins, R. N. Pike, S. P. Bottomley, and E. J. Mackie. "Protease-activated receptor-2 mediates proliferative responses in skeletal myoblasts." Journal of Cell Science 113, no. 24 (2000): 4427–33. http://dx.doi.org/10.1242/jcs.113.24.4427.
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Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved by proteases within the N terminus, exposing a new tethered ligand that binds and activates the receptor. Activators of PAR-2 include trypsin and mast cell tryptase. Skeletal myoblasts are known to express PAR-1, a thrombin receptor. The current study was undertaken to determine whether myoblasts express PAR-2. Primary neonatal rat and mouse skeletal myoblast cultures were shown to express PAR-2 in polymerase chain reaction and immunocytochemical studies. Expression of PAR-2 was also demonstrated by immunohistochemistry in developing mouse skeletal muscle in vivo. Trypsin or a synthetic peptide corresponding to the rat PAR-2 tethered ligand caused a dose-dependent elevation in intracellular calcium in cultured rat myoblasts, with an EC(50) of 13 nM or 56 microM, respectively. Studies aimed at identifying the function of PAR-2 in myoblasts demonstrated no effect of the receptor-activating peptide on survival or fusion in serum-deprived myoblasts. The PAR-2-activating peptide did, however, stimulate proliferation of serum-deprived myoblasts. These results demonstrate that skeletal muscle cells express PAR-2, activation of which leads to stimulation of myoblast proliferation.
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Shi, Jun, Pengpeng Bi, Jimin Pei, et al. "Requirement of the fusogenic micropeptide myomixer for muscle formation in zebrafish." Proceedings of the National Academy of Sciences 114, no. 45 (2017): 11950–55. http://dx.doi.org/10.1073/pnas.1715229114.
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Skeletal muscle formation requires fusion of mononucleated myoblasts to form multinucleated myofibers. The muscle-specific membrane proteins myomaker and myomixer cooperate to drive mammalian myoblast fusion. Whereas myomaker is highly conserved across diverse vertebrate species, myomixer is a micropeptide that shows relatively weak cross-species conservation. To explore the functional conservation of myomixer, we investigated the expression and function of the zebrafish myomixer ortholog. Here we show that myomixer expression during zebrafish embryogenesis coincides with myoblast fusion, and genetic deletion of myomixer using CRISPR/Cas9 mutagenesis abolishes myoblast fusion in vivo. We also identify myomixer orthologs in other species of fish and reptiles, which can cooperate with myomaker and substitute for the fusogenic activity of mammalian myomixer. Sequence comparison of these diverse myomixer orthologs reveals key amino acid residues and a minimal fusogenic peptide motif that is necessary for promoting cell–cell fusion with myomaker. Our findings highlight the evolutionary conservation of the myomaker–myomixer partnership and provide insights into the molecular basis of myoblast fusion.
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Yao, C. C., B. L. Ziober, A. E. Sutherland, D. L. Mendrick, and R. H. Kramer. "Laminins promote the locomotion of skeletal myoblasts via the alpha 7 integrin receptor." Journal of Cell Science 109, no. 13 (1996): 3139–50. http://dx.doi.org/10.1242/jcs.109.13.3139.
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The alpha 7 beta 1 integrin is specifically expressed by skeletal and cardiac muscles, and its expression and alternative mRNA splicing at the cytoplasmic domain are developmentally regulated. We analyzed the role of alpha 7 integrin in mediating myoblast adhesion and motility on different laminin isoforms. Mouse C2C12 and MM14 myoblast cell lines were found by flow cytometry and immunoprecipitation to express high levels of the alpha 7 integrin. Overall expression of alpha 7 increased as the C2C12 myoblasts differentiated; myoblasts expressed only the alpha 7B cytoplasmic variant whereas in differentiating myotubes alpha 7A increased markedly. Function-perturbing monoclonal antibodies generated to alpha 7 integrin efficiently blocked both adhesion and migration of MM14 and C2C12 mouse myoblasts on laminin 1. Other studies with MM14 myoblasts showed that alpha 7 is also a receptor for laminin 2/4 (human placental merosins) but not for epithelial-cell-specific laminin 5. Blocking antibody to alpha 7 only partially inhibited adhesion to laminin 2/4 but almost completely blocked motility on this substrate. Finally, to assess the potential role of the alpha 7 cytoplasmic domain, CHO cells were stably transfected to expressed chimeric alpha 5 cDNA constructs containing the wild-type alpha 5 or the alpha 7A or alpha 7B cytoplasmic domain; all forms of the integrin showed identical activities for adhesion, migration, proliferation, and matrix assembly on fibronectin substrates. These results established that alpha 7 beta 1 receptor can promote myoblast adhesion and motility on a restricted number of laminin isoforms and may be important in myogenic precursor recruitment during regeneration and differentiation.
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Chen, Ming, Hua-Ju Li, Qiuwen Fang, Tena G. Goodwin, J. Ann Florendo, and Peter K. Law. "Dystrophin Cytochemistry in Mdx Mouse Muscles Injected with Labeled Normal Myoblasts." Cell Transplantation 1, no. 1 (1992): 17–22. http://dx.doi.org/10.1177/096368979200100105.
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A new technique enables correlation of dystrophin expression with the location of donor versus host nuclei in the same sections of mdx mouse muscle injected with normal myoblasts. Myoblasts from C57BL/6J mice or from humans were labeled with 0.01% fluoro-gold (FG) in Dulbecco's Modified Eagles Medium (DMEM) for 16 h at 37°C before myoblast transfer. About 3 × 104 myoblasts were injected into the quadriceps muscles of mdx mice immunosuppressed with cyclosporine A (CsA). At 11, 21, or 25 days after myoblast transfer, injected muscles were dissected out and sectioned. These mouse sections were processed for dystrophin and then labeled with a fluorescent nucleus counterstain, 5 μg% Hoechst 33342 in phosphate-buffered saline (PBS), for 10 min at room temperature. Fluoro-gold labeling corresponding with Hoechst 33342 staining indicated survival of normal nuclei in dystrophic muscle. Dystrophin was found in the sarcolemma of myofibers containing FG-labeled nuclei but not of myofibers containing only Hoechst 33342-labeled nuclei. Control muscle samples showed neither FG labeling nor dystrophin. This study demonstrates that the donor human and mouse myoblasts survived and developed in host mouse muscles for at least 25 days after myoblast transfer, and that the localization of their normal nuclei correlates with dystrophin expression in muscle fibers of immunosuppressed mdx host mice.
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Mezil, Yasmeen, Joyce Obeid, Inna Ushcatz, Sandeep Raha, and Brian W. Timmons. "The Effects of Exercise Serum From Prepubertal Girls and Women on In Vitro Myoblast and Osteoblast Proliferation and Differentiation." Pediatric Exercise Science 33, no. 2 (2021): 82–89. http://dx.doi.org/10.1123/pes.2020-0206.
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Purpose: In girls and women, the authors studied the effects of an acute bout of low-impact, moderate-intensity exercise serum on myoblast and osteoblast proliferation in vitro. Methods: A total of 12 pre/early pubertal girls (8–10 y old) and 12 women (20–30 y old) cycled at 60% VO2max for 1 hour followed by 1-hour recovery. Blood samples were collected at rest, mid-exercise, end of exercise, mid-recovery, and end of recovery. C2C12 myoblasts and MC3T3E1 osteoblasts were incubated with serum from each time point for 1 hour, then monitored for 24 hours (myoblasts) or 36 hours (osteoblasts) to examine proliferation. Cells were also monitored for 6 days (myoblasts) to examine myotube formation and 21 days (osteoblasts) to examine mineralization. Results: Exercise did not affect myoblast or osteoblast proliferation. Girls exhibited lower cell proliferation relative to women at end of exercise (osteoblasts, P = .041; myoblasts, P = .029) and mid-recovery (osteoblasts, P = .010). Mineralization was lower at end of recovery relative to rest (P = .014) in both girls and women. Myotube formation was not affected by exercise or group. Conclusion: The systemic environment following one acute bout of low-impact moderate-intensity exercise in girls and women does not elicit osteoblast or myoblast activity in vitro. Differences in myoblast and osteoblast proliferation between girls and women may be influenced by maturation.
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Bellido, Teresita, and Ricardo Boland. "Stimulation of Myoblast Membrane Protein Synthesis by 25-Hydroxy-Vitamin D 3." Zeitschrift für Naturforschung C 44, no. 9-10 (1989): 807–12. http://dx.doi.org/10.1515/znc-1989-9-1019.
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Abstract The effects of 25-hydroxy-vitamin D3 (25 OHD3) on myoblast protein synthesis were studied in connection with its role on muscle cell phosphate metabolism . The sterol markedly increased leucine incorporation into total cell proteins in cultured chick embryo myoblasts. This enhancement was greater than that produced by 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) and occurred prior to a significant stimulation of cell phosphate accumulation. Maximum effects of 25 OHD3 (8 h) on myoblast phosphate uptake were suppressed by cycloheximide indicating that they are mediated by de novo protein synthesis. At a similar treatment period, labelling of myoblasts with [3H]leucine (control) and [14C]leucine (+ 25 OHD3) followed by co-electrophoresis of total protein extracts on SDS-PAGE and isoelectrofocusing gels revealed that the sterol selectively affects the synthesis of proteins of 20 kDa and 50 kDa. These macromolecules were recovered in the microsomal fraction after differential centrifugation of homogenates. Further fractionation of myoblast microsomes on sucrose density gradients show ed co-localization of the 50 kDa and 20 kDa proteins with microsomal subfractions which preferentially bind [3H -alpha]bungarotoxin, suggesting that the proteins induced by 25 OHD3 are associated to plasma membranes and may play a role in the effects of the sterol on cell phosphate uptake.
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Budai, Zsófia, Nour Al-Zaeed, Péter Szentesi, et al. "Impaired Skeletal Muscle Development and Regeneration in Transglutaminase 2 Knockout Mice." Cells 10, no. 11 (2021): 3089. http://dx.doi.org/10.3390/cells10113089.
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Skeletal muscle regeneration is triggered by local inflammation and is accompanied by phagocytosis of dead cells at the injury site. Efferocytosis regulates the inflammatory program in macrophages by initiating the conversion of their inflammatory phenotype into the healing one. While pro-inflammatory cytokines induce satellite cell proliferation and differentiation into myoblasts, growth factors, such as GDF3, released by healing macrophages drive myoblast fusion and myotube growth. Therefore, improper efferocytosis may lead to impaired muscle regeneration. Transglutaminase 2 (TG2) is a versatile enzyme participating in efferocytosis. Here, we show that TG2 ablation did not alter the skeletal muscle weights or sizes but led to the generation of small size myofibers and to decreased grip force in TG2 null mice. Following cardiotoxin-induced injury, the size of regenerating fibers was smaller, and the myoblast fusion was delayed in the tibialis anterior muscle of TG2 null mice. Loss of TG2 did not affect the efferocytic capacity of muscle macrophages but delayed their conversion to Ly6C−CD206+, GDF3 expressing cells. Finally, TG2 promoted myoblast fusion in differentiating C2C12 myoblasts. These results indicate that TG2 expressed by both macrophages and myoblasts contributes to proper myoblast fusion, and its ablation leads to impaired muscle development and regeneration in mice.