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Journal articles on the topic 'Myoblasts'
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1
Ruiz-Gómez, Mar, Nikola Coutts, Maximiliano L. Suster, Matthias Landgraf, and Michael Bate. "myoblasts incompetent encodes a zinc finger transcription factor required to specify fusion-competent myoblasts in Drosophila." Development 129, no. 1 (January 1, 2002): 133–41. http://dx.doi.org/10.1242/dev.129.1.133.
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We report a new gene, myoblasts incompetent, essential for normal myogenesis and myoblast fusion in Drosophila. myoblasts incompetent encodes a putative zinc finger transcription factor related to vertebrate Gli proteins and to Drosophila Cubitus interruptus. myoblasts incompetent is expressed in immature somatic and visceral myoblasts. Expression is predominantly in fusion-competent myoblasts and a loss-of-function mutation in myoblasts incompetent leads to a failure in the normal differentiation of these cells and a complete lack of myoblast fusion. In the mutant embryos, founder myoblasts differentiate normally and form mononucleate muscles, but genes that are specifically expressed in fusion-competent cells are not activated and the normal downregulation of twist expression in these cells fails to occur. In addition, fusion-competent myoblasts fail to express proteins characteristic of the general pathway of myogenesis such as myosin and Dmef2. Thus myoblasts incompetent appears to function specifically in the general pathway of myogenesis to control the differentiation of fusion-competent myoblasts.
2
Sun, Luguo, Kewei Ma, Haixia Wang, Fang Xiao, Yan Gao, Wei Zhang, Kepeng Wang, Xiang Gao, Nancy Ip, and Zhenguo Wu. "JAK1–STAT1–STAT3, a key pathway promoting proliferation and preventing premature differentiation of myoblasts." Journal of Cell Biology 179, no. 1 (October 1, 2007): 129–38. http://dx.doi.org/10.1083/jcb.200703184.
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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
McCaig, Colin D. "Myoblasts and notochord influence the orientation of somitic myoblasts from Xenopus laevis." Development 93, no. 1 (April 1, 1986): 121–31. http://dx.doi.org/10.1242/dev.93.1.121.
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The orientation of developing myoblasts extending a bipolar axis in the presence of explanted myoblasts or whole notochord has been studied in vitro. Myoblasts tended to elongate perpendicular to the lines of diffusion of substances from these tissues. A slow-release source of agar impregnated with medium conditioned by segmented somitic myoblast or notochord also caused myoblasts to elongate perpendicular to the lines of diffusion from the source. Medium conditioned by neural tube cells or unsegmented mesoderm cells did not influence the orientation of myoblasts. It is concluded that somites and notochord release diffusible substances in vitro which are capable of directing the orientation of developing myoblasts. In vivo, a somite-derived material could play a role in determining the direction of myoblast elongation in the presomitic mesoderm. An interaction between somite and notochord-derived secretions couldinfluence the rotation of presomitic myoblasts to form a segmented somite.
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Haugk, Kathleen L., Heather-Marie P. Wilson, Karen Swisshelm, and LeBris S. Quinn. "Insulin-Like Growth Factor (IGF)-Binding Protein-Related Protein-1: An Autocrine/Paracrine Factor That Inhibits Skeletal Myoblast Differentiation but Permits Proliferation in Response to IGF1." Endocrinology 141, no. 1 (January 1, 2000): 100–110. http://dx.doi.org/10.1210/endo.141.1.7235.
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Abstract Skeletal myogenic cells respond to the insulin-like growth factors (IGF-I and IGF-II) by differentiating or proliferating, which are mutually exclusive pathways. What determines which of these responses to IGF skeletal myoblast undergo is unclear. IGF-binding protein-related protein 1 (IGFBP-rP1) is a secreted protein with close homology to the IGF-binding proteins (IGFBPs) in the N-terminal region. IGFBP-rP1, previously called mac25 and IGFBP-7, is highly expressed in C2 skeletal myoblasts during the proliferative phase, but is down-regulated during myoblast differentiation. To determine the role of IGFBP-rP1 in myogenesis, IGFBP-rP1 was overexpressed in C2 myoblasts using a retroviral vector. Western blots indicated that the resulting C2-rP1 myoblasts secreted approximately 27-fold higher levels of IGFBP-rP1 than control C2-LX myoblasts that were transduced with a control vector (LXSN). Compared with C2-LX myoblasts, the differentiation responses of C2-rP1 myoblasts to IGF-I, IGF-II, insulin, and des(1–3)IGF-I were significantly reduced (P < 0.05). However, proliferation responses of C2-rP1 and C2-LX myoblasts to these same factors were not significantly different. Exposure of control C2-LX myoblasts to factors secreted by C2-rP1 myoblasts using a transwell coculture system reduced C2-LX myoblast differentiation significantly (P < 0.05). Experiments with the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 suggested that IGFBP-rP1 inhibits a MAPK-dependent differentiation pathway. In confirmation of this idea, levels of phosphorylated extracellular signal-regulated kinase-2 (a MAPK) were reduced in C2-rP1 myoblasts compared with those in C2-LX myoblasts. These findings indicate that IGFBP-rP1 may function as an autocrine/paracrine factor that specifies the proliferative response to the IGFs in myogenesis.
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Fahime, E. El, M. Bouchentouf, B. F. Benabdallah, D. Skuk, J. F. Lafreniere, Y. T. Chang, and J. P. Tremblay. "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 (April 1, 2003): 81–90. http://dx.doi.org/10.1139/o03-054.
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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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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 (June 15, 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.
7
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 (October 1, 1989): 1779–86. http://dx.doi.org/10.1083/jcb.109.4.1779.
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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.
8
BARNOY, Sivia, Lia SUPINO-ROSIN, and Nechama S. KOSOWER. "Regulation of calpain and calpastatin in differentiating myoblasts: mRNA levels, protein synthesis and stability." Biochemical Journal 351, no. 2 (October 10, 2000): 413–20. http://dx.doi.org/10.1042/bj3510413.
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Calpain (Ca2+-dependent intracellular protease)-induced proteolysis has been considered to play a role in myoblast fusion to myotubes. We found previously that calpastatin (the endogenous inhibitor of calpain) diminishes transiently during myoblast differentiation. To gain information about the regulation of calpain and calpastatin in differentiating myoblasts, we evaluated the stability and synthesis of calpain and calpastatin, and measured their mRNA levels in L8 myoblasts. We show here that µ-calpain and m-calpain are stable, long-lived proteins in both dividing and differentiating L8 myoblasts. Calpain is synthesized in differentiating myoblasts, and calpain mRNA levels do not change during differentiation. In contrast, calpastatin (though also a long-lived protein in myoblasts), is less stable in differentiating myoblasts than in the dividing cells, and its synthesis is inhibited upon initiation of differentiation. Inhibition of calpastatin synthesis is followed by a diminution in calpastatin mRNA levels. A similar calpastatin mRNA diminution is observed upon drug-induced inhibition of protein translation. On the other hand, transforming growth factor β (which inhibits differentiation) allows calpastatin synthesis and prevents the diminution in calpastatin mRNA. The overall results suggest that at the onset of myoblast differentiation, calpastatin is regulated mainly at the level of translation and that an inhibition of calpastatin synthesis leads to the decrease in its mRNA stability. The existing calpastatin then diminishes, resulting in decreased calpastatin activity in the fusing myoblasts, allowing calpain activation and protein degradation required for fusion.
9
Mesmer, O. T., and T. C. Lo. "Hexose transport in human myoblasts." Biochemical Journal 262, no. 1 (August 15, 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.
10
Fang, Qiuwen, Ming Chen, Hua-ju Li, Tena G. Goodwin, and Peter K. Law. "Vital marker for muscle nuclei in myoblast transfer." Canadian Journal of Physiology and Pharmacology 69, no. 1 (January 1, 1991): 49–52. http://dx.doi.org/10.1139/y91-008.
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A new method is developed using Fluoro-Gold (FG) as a vital stain to label the nuclei of donor myoblasts in myoblast transfer studies. In vitro incubation with 0.01% FG for 16 h resulted in 100% nuclei labelling. Intensive fluorescence persisted following 9 days of subculture, when the human myoblasts were injected into the quadriceps of mouse recipients immunosuppressed with cyclosporine. Injected muscles showed mosaicism of host and donor nuclei 25 days after injection, indicating (i) survival and fusion among donor myoblasts, and (ii) fusion between host and donor cells. FG labelling was not observed in control muscles injected with an equal volume of FG-labelled dead myoblasts, 0.01% FG medium, or phosphate-buffered saline.Key words: Fluoro-Gold, nucleus vital stain, myoblast culture and transplant.
11
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 (May 1, 1988): 1703–12. http://dx.doi.org/10.1083/jcb.106.5.1703.
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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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Formigli, Lucia, Fabio Francini, Alessia Tani, Roberta Squecco, Daniele Nosi, Lucia Polidori, Silvia Nistri, 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 (April 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.
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Badu-Mensah, Agnes, Paola Valinski, Hemant Parsaud, James J. Hickman, and Xiufang Guo. "Hyperglycemia Negatively Affects IPSC-Derived Myoblast Proliferation and Skeletal Muscle Regeneration and Function." Cells 11, no. 22 (November 18, 2022): 3674. http://dx.doi.org/10.3390/cells11223674.
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Diabetic myopathy is a co-morbidity diagnosed in most diabetes mellitus patients, yet its pathogenesis is still understudied, which hinders the development of effective therapies. This project aimed to investigate the effect of hyperglycemia on human myoblast physiology, devoid of other complicating factors, by utilizing human myoblasts derived from induced pluripotent stem cells (iPSCs), in a defined in vitro system. IPSC-derived myoblasts were expanded under three glucose conditions: low (5 mM), medium (17.5 mM) or high (25 mM). While hyperglycemic myoblasts demonstrated upregulation of Glut4 relative to the euglycemic control, myoblast proliferation demonstrated a glucose dose-dependent impedance. Further cellular analysis revealed a retarded cell cycle progression trapped at the S phase and G2/M phase and an impaired mitochondrial function in hyperglycemic myoblasts. Terminal differentiation of these hyperglycemic myoblasts resulted in significantly hypertrophic and highly branched myotubes with disturbed myosin heavy chain arrangement. Lastly, functional assessment of these myofibers derived from hyperglycemic myoblasts demonstrated comparatively increased fatigability. Collectively, the hyperglycemic myoblasts demonstrated deficient muscle regeneration capability and functionality, which falls in line with the sarcopenia symptoms observed in diabetic myopathy patients. This human-based iPSC-derived skeletal muscle hyperglycemic model provides a valuable platform for mechanistic investigation of diabetic myopathy and therapeutic development.
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Milanesi, Anna, Jang-Won Lee, Nam-Ho Kim, Yan-Yun Liu, An Yang, Sargis Sedrakyan, Andrew Kahng, et al. "Thyroid Hormone Receptor α Plays an Essential Role in Male Skeletal Muscle Myoblast Proliferation, Differentiation, and Response to Injury." Endocrinology 157, no. 1 (January 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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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 (December 15, 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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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 (April 1, 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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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 (July 1, 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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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 (March 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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Vilchinskaya, N. A., T. M. Mirzoev, and B. S. Shenkman. "The Maintenance of AMPK Activity Eliminates Abnormally Accelerated Differentiation of Primary Myoblasts Isolated from Atrophied Rat Soleus Muscle." Российский физиологический журнал им И М Сеченова 109, no. 4 (April 1, 2023): 502–16. http://dx.doi.org/10.31857/s086981392304012x.
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Mechanical unloading of skeletal muscles leads to the development of atrophic processes and a decrease in the total number of satellite cells (SCs) that are involved in muscle regeneration. In vitro studies revealed an increased differentiation of myoblasts derived from rat soleus muscle after an unloading-induced decrease in AMP-activated protein kinase (AMPK). AMPK is necessary for the activation of SCs and also participates in the regulation of myoblast proliferation and differentiation. It can be assumed that a decrease in the activity of AMPK after mechanical unloading can contribute to the acceleration of myoblast differentiation. The main purpose of this study was to elucidate a possible role of AMPK in the regulation of differentiation of myoblasts isolated from rat soleus muscle after mechanical unloading. To test this hypothesis, a specific AMPK activator, AICAR, was used to prevent a decrease in AMPK activity during differentiation of myoblasts isolated from rat soleus muscle after 7-day unloading. Immunocytochemistry, PCR-RT and Western blotting were used to assess changes during myoblast differentiation. In differentiating myoblasts derived from the unloaded soleus muscle there was a significant decrease in AMPK (Thr172) and ACC (Ser 79) phosphorylation levels, an increase in myotube differentiation index, myoblast fusion factors and the expression of myogenic regulatory factors (MRF). Furthermore, there was a decrease in the expression of slow myosin heavy chains (MyHC) and an increase in the expression of fast MyHC isoforms. AICAR treatment of differentiating myoblasts obtained from the unloaded soleus muscle prevented a decrease in AMPK and ACC phosphorylation, returned the expression levels of MRF and fast isoforms of MyHC to the control levels as well as maintained the expression of slow MyHC. Thus, abnormally accelerated differentiation of myoblasts isolated from atrophied rat soleus muscle can be compensated by maintaining the control levels of AMPK activity using AICAR.
20
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 (December 1, 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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Jin, P., M. Rahm, L. Claesson-Welsh, C. H. Heldin, and T. Sejersen. "Expression of PDGF A-chain and beta-receptor genes during rat myoblast differentiation." Journal of Cell Biology 110, no. 5 (May 1, 1990): 1665–72. http://dx.doi.org/10.1083/jcb.110.5.1665.
Full textAbstract:
L6J1 rat myoblasts and rat skeletal muscle were studied for expression of mRNAs encoding PDGF A-chain, PDGF B-chain, PDGF alpha-receptor, and PDGF beta-receptor during in vitro and in vivo myoblast differentiation. RNA blot hybridizations demonstrated expression of the PDGF A-chain gene and the PDGF beta-receptor gene in L6J1 myoblasts and in crude muscle tissue isolated from developing rats. Transcripts of the PDGF A-chain were identified at all examined stages of in vitro and in vivo myogenic differentiation. Expression of the PDGF beta-receptor gene decreased in differentiated myotubes of L6J1 cells and in rat adult muscle tissue. Receptor binding assays demonstrated specific binding of PDGF-BB, but not -AA, to exponentially proliferating L6J1 myoblasts and to terminally differentiated L6J1 myotubes. The binding per cell nucleus was higher in exponentially proliferating myoblasts than in differentiated L6J1 myotubes. In serum free medium PDGF-BB was shown to increase c-fos protooncogene immunoreactivity in L6J1 myoblasts. In the presence of 0.5% FCS, PDGF-BB increased DNA synthesis in L6J1 myoblasts, while PDGF-AA showed no such effect. Differentiation, as monitored by myotube formation, was reduced in PDGF-BB-treated cultures. The possible role of PDGF in myoblast proliferation and differentiation is discussed.
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Lee, Nicole K. L., Jarrod P. J. Skinner, Jeffrey D. Zajac, and Helen E. MacLean. "Ornithine decarboxylase is upregulated by the androgen receptor in skeletal muscle and regulates myoblast proliferation." American Journal of Physiology-Endocrinology and Metabolism 301, no. 1 (July 2011): E172—E179. http://dx.doi.org/10.1152/ajpendo.00094.2011.
Full textAbstract:
The aim of this study is to determine if the Odc1 gene, which encodes ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is directly regulated by the androgen receptor (AR) in skeletal muscle myoblasts and if Odc1 regulates myoblast proliferation and differentiation. We previously showed that expression of Odc1 is decreased in muscle from AR knockout male mice. In this study, we show in vivo that Odc1 expression is also decreased >60% in muscle from male muscle-specific AR knockout mice. In normal muscle homeostasis, Odc1 expression is regulated by age and sex, reflecting testosterone levels, as muscle of adult male mice expresses high levels of Odc1 compared with age-matched females and younger males. In vitro, expression of Odc1 is 10- and 1.5-fold higher in proliferating mouse C2C12 and human skeletal muscle myoblasts, respectively, than in differentiated myotubes. Dihydrotestosterone increases Odc1 levels 2.7- and 1.6-fold in skeletal muscle cell myoblasts after 12 and 24 h of treatment, respectively. Inhibition of ODC activity in C2C12 myoblasts by α-difluoromethylornithine decreases myoblast number by 40% and 66% following 48 and 72 h of treatment, respectively. In contrast, overexpression of Odc1 in C2C12 myoblasts results in a 27% increase in cell number vs. control when cells are grown under differentiation conditions for 96 h. This prolonged proliferation is associated with delayed differentiation, with reduced expression of the differentiation markers myogenin and Myf6 in Odc1-overexpressing cells. In conclusion, androgens act via the AR to upregulate Odc1 in skeletal muscle myoblasts, and Odc1 promotes myoblast proliferation and delays differentiation.
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Knudsen, K. A. "The calcium-dependent myoblast adhesion that precedes cell fusion is mediated by glycoproteins." Journal of Cell Biology 101, no. 3 (September 1, 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.
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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 (August 1, 1993): 957–63. http://dx.doi.org/10.1242/jcs.105.4.957.
Full textAbstract:
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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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 (March 1997): 101–7. http://dx.doi.org/10.1177/096368979700600203.
Full textAbstract:
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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Artero, Ruben D., Irinka Castanon, and Mary K. Baylies. "The immunoglobulin-like protein Hibris functions as a dose-dependent regulator of myoblast fusion and is differentially controlled by Ras and Notch signaling." Development 128, no. 21 (November 1, 2001): 4251–64. http://dx.doi.org/10.1242/dev.128.21.4251.
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Hibris (Hbs) is a transmembrane immunoglobulin-like protein that shows extensive homology to Drosophila Sticks and stones (Sns) and human kidney protein Nephrin. Hbs is expressed in embryonic visceral, somatic and pharyngeal mesoderm among other tissues. In the somatic mesoderm, Hbs is restricted to fusion competent myoblasts and is regulated by Notch and Ras signaling pathways. Embryos that lack or overexpress hbs show a partial block of myoblast fusion, followed by abnormal muscle morphogenesis. Abnormalities in visceral mesoderm are also observed. In vivo mapping of functional domains suggests that the intracellular domain mediates Hbs activity. Hbs and its paralog, Sns, co-localize at the cell membrane of fusion-competent myoblasts. The two proteins act antagonistically: loss of sns dominantly suppresses the hbs myoblast fusion and visceral mesoderm phenotypes, and enhances Hbs overexpression phenotypes. Data from a P-homed enhancer reporter into hbs and co-localization studies with Sns suggest that hbs is not continuously expressed in all fusion-competent myoblasts during the fusion process. We propose that the temporal pattern of hbs expression within fusion-competent myoblasts may reflect previously undescribed functional differences within this myoblast population.
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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 (August 1, 2002): 457–65. http://dx.doi.org/10.1139/o02-134.
Full textAbstract:
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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Son, Boram, Seong Ho Lee, Seyoung Hong, Miji Kwon, Jinmyoung Joo, Kwang Suk Lim, and Hee Ho Park. "Direct Conversion of Bovine Dermal Fibroblasts into Myotubes by Viral Delivery of Transcription Factor bMyoD." Applied Sciences 12, no. 9 (May 6, 2022): 4688. http://dx.doi.org/10.3390/app12094688.
Full textAbstract:
Direct reprogramming of somatic cells to myoblasts and myotubes holds great potential for muscle development, disease modeling and regenerative medicine. According to recent studies, direct conversion of fibroblasts to myoblasts was performed by using a transcription factor, myoblast determination protein (MyoD), which belongs to a family of myogenic regulatory factors. Therefore, MyoD is considered to be a key driver in the generation of induced myoblasts. In this study, we compared the direct phenotypic conversion of bovine dermal fibroblasts (BDFs) into myoblasts and myotubes by supplementing a transcription factor, bovine MyoD (bMyoD), in the form of recombinant protein or the bMyoD gene, through retroviral vectors. As a result, the delivery of the bMyoD gene to BDFs was more efficient for inducing reprogramming, resulting in direct conversion to myoblasts and myotubes, when compared with protein delivery. BDFs cultured with retrovirus encoding bMyoD increased myogenic gene expression, such as MyoG, MYH3 and MYMK. In addition, the cells expressed myoblast or myotube-specific marker proteins, MyoG and Desmin, respectively. Our findings provide an informative tool for the myogenesis of domestic-animal-derived somatic cells via transgenic technology. By using this method, a new era of regenerative medicine and cultured meat is expected.
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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 (May 1, 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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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 (April 2005): H1620—H1626. http://dx.doi.org/10.1152/ajpheart.00610.2003.
Full textAbstract:
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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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 (January 1992): 17–22. http://dx.doi.org/10.1177/096368979200100105.
Full textAbstract:
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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Ng, Stephen K., and Keith E. Lewis. "Characteristics of myoblasts isolated from golden Syrian and dystrophic (strain CHF-146) hamsters." Canadian Journal of Biochemistry and Cell Biology 63, no. 7 (July 1, 1985): 730–36. http://dx.doi.org/10.1139/o85-091.
Full textAbstract:
A method for dissociating and culturing myoblast cells from normal (golden Syrian) and myopathic (CHF-146) hind leg muscles of 8- to 10-day-old hamsters has been described. We have compared the fusion ability of the myoblasts from normal tissue with that of myopathic myoblasts and, further, we have studied this process with respect to gelatin-coated and uncoated plastic culture dishes. Our data show that myoblasts from normal tissue grows well on both surfaces, although fusion is enhanced when these cells are grown on a gelatin-coated surface. Myoblasts from dystrophic tissue, however, appear to have an impaired ability to adhere to an uncoated surface, although fusion is enhanced when these myoblasts are grown on coated plates. The fusion percentage of the dystrophic cells is consistently lower than that of myoblasts from normal tissue. At low concentrations of 5-azacytidine tested, there seems to be no enhancement of fusion capability in either the normal or dystrophic lines.
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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 (May 1, 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.
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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 (September 7, 1998): 1257–67. http://dx.doi.org/10.1083/jcb.142.5.1257.
Full textAbstract:
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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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 (December 1, 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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Ceci, Roberta, Guglielmo Duranti, Stefano Giuliani, Marianna Nicoletta Rossi, Ivan Dimauro, Stefania Sabatini, Paolo Mariottini, and Manuela Cervelli. "The Impact of Spermidine on C2C12 Myoblasts Proliferation, Redox Status and Polyamines Metabolism under H2O2 Exposure." International Journal of Molecular Sciences 23, no. 19 (September 20, 2022): 10986. http://dx.doi.org/10.3390/ijms231910986.
Full textAbstract:
A central feature of the skeletal muscle is its ability to regenerate through the activation, by environmental signals, of satellite cells. Once activated, these cells proliferate as myoblasts, and defects in this process profoundly affect the subsequent process of regeneration. High levels of reactive oxygen species such as hydrogen peroxide (H2O2) with the consequent formation of oxidized macromolecules increase myoblasts’ cell death and strongly contribute to the loss of myoblast function. Recently, particular interest has turned towards the beneficial effects on muscle of the naturally occurring polyamine spermidine (Spd). In this work, we tested the hypothesis that Spd, upon oxidative challenge, would restore the compromised myoblasts’ viability and redox status. The effects of Spd in combination with aminoguanidine (Spd-AG), an inhibitor of bovine serum amine oxidase, on murine C2C12 myoblasts treated with a mild dose of H2O2 were evaluated by analyzing: (i) myoblast viability and recovery from wound scratch; (ii) redox status and (iii) polyamine (PAs) metabolism. The treatment of C2C12 myoblasts with Spd-AG increased cell number and accelerated scratch wound closure, while H2O2 exposure caused redox status imbalance and cell death. The combined treatment with Spd-AG showed an antioxidant effect on C2C12 myoblasts, partially restoring cellular total antioxidant capacity, reducing the oxidized glutathione (GSH/GSSG) ratio and increasing cell viability through a reduction in cell death. Moreover, Spd-AG administration counteracted the induction of polyamine catabolic genes and PA content decreased due to H2O2 challenges. In conclusion, our data suggest that Spd treatment has a protective role in skeletal muscle cells by restoring redox balance and promoting recovery from wound scratches, thus making myoblasts able to better cope with an oxidative insult.
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Broholm, Christa, Claus Brandt, Ninna S. Schultz, Anders R. Nielsen, Bente K. Pedersen, and Camilla Scheele. "Deficient leukemia inhibitory factor signaling in muscle precursor cells from patients with type 2 diabetes." American Journal of Physiology-Endocrinology and Metabolism 303, no. 2 (July 15, 2012): E283—E292. http://dx.doi.org/10.1152/ajpendo.00586.2011.
Full textAbstract:
The cytokine leukemia-inhibitory factor (LIF) is expressed by skeletal muscle and induces proliferation of muscle precursor cells, an important feature of skeletal muscle maintenance and repair. We hypothesized that muscle precursor cells from patients with type 2 diabetes had a deficient response to LIF. The mRNA and protein expressions of LIF and its receptor (LIFR) were measured in skeletal muscle biopsies from healthy individuals and patients with type 2 diabetes by use of qPCR and Western blot. LIF signaling and response were studied following administration of recombinant LIF and siRNA knockdown of suppressor of cytokine signaling (SOCS)3 in myoblast cultures established from healthy individuals and patients with type 2 diabetes. Myoblast proliferation rate was assessed by bromodeoxyuridine incorporation. LIF and LIFR proteins were increased in both muscle tissue and cultured myoblasts from diabetic patients. Nonetheless, in the diabetic myoblasts, LIF-induced phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3 was impaired. The deficient response to LIF administration in the diabetic myoblasts was further emphasized by a lack of increase in LIF-stimulated cell proliferation and a decreased LIF-stimulated induction of the proliferation-promoting factors cyclin D1, JunB, and c-myc. SOCS3 protein was upregulated in diabetic myoblasts, and knockdown of SOCS3 rescued LIF-induced gene expression in diabetic myoblasts, whereas neither STAT1 or STAT3 signaling nor proliferation rate was affected. In conclusion, although LIF and LIFR proteins were increased in muscle tissue and myoblasts from diabetic patients, LIF signaling and LIF-stimulated cell proliferation were impaired in diabetic myoblasts, suggesting a novel mechanism by which muscle function is compromised in diabetes.
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Vilchinskaya, N. A., O. V. Turtikova, T. M. Mirzoev, and B. S. Shenkman. "DIFFERENTIATION OF MYOBLASTS ISOLATED FROM RAT'S M. SOLEUS FOLLOWING TAIL-SUSPENSION IS ACCOMPANIED BY P27KIP1 DEPHOSPHORYLATION AND AGGRAVATION OF APOPTOSIS." Aerospace and Environmental Medicine 57, no. 6 (2023): 44–51. http://dx.doi.org/10.21687/0233-528x-2023-57-6-44-51.
Full textAbstract:
Apoptosis and its control during differentiation of primary myoblasts isolated from rat soleus muscle after 7-d tail suspension were investigated. Primary myoblasts were first subjected to myogenic differentiation. TUNEL labeling of double-strand DNA breaks was applied to detect apoptotic cells during myoblast differentiation. Western blot was used to determine the apoptosis markers and a number of signaling molecules, i.e. protein BAX, activated caspase-3, phospho-AMPK (Thr172), phospho-AKT (Ser 437), phospho-p27Kip1 (Thr198), phospho-p27Kip1 (Thr157). PCR analysis was used to investigate the expression of pro-apoptotic markers in myoblasts. The investigation showed enhancement of apoptosis as well as АМРК and p27Kip1 dephosphorylation in the process of differentiation of myoblasts taken from m. soleus of tail-suspended rats.
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Labrecque, C., J. Huard, G. Dansereau, N. Albert, and J. P. Tremblay. "In vitro bromodeoxyuridine labeling of nuclei: application to myotube hybridization." Journal of Histochemistry & Cytochemistry 39, no. 10 (October 1991): 1421–26. http://dx.doi.org/10.1177/39.10.1940314.
Full textAbstract:
Rat myoblast nuclei were labeled with various concentrations of bromodeoxyuridine (BrdU), an analogue of thymidine, for 24 or 48 hr. Almost every myoblast was labeled with BrdU at concentrations between 10(-7) M and 10(-5) M. When the cells were labeled with 0.5 microM or more, the percentage of labeled cells remained over 90% and 80% at 2 and 5 days, respectively. However, when the cells were labeled with BrdU concentration lower than 10(-7) M the percentage of labeled nuclei decreased more rapidly with time. The BrdU-labeled cells were mixed with an unlabeled population to determine whether their capacity to fuse was reduced. At a BrdU concentration of 0.5 x 10(-6) M, labeled myoblasts fused to a similar extent as unlabeled myoblasts, and a high percentage of marked cells were still perceptively labeled after 5 days. In contrast, the fusion capacity of myoblasts incubated with more than 10(-6) M BrdU was inhibited after only few rounds of DNA synthesis. These myoblasts were eventually able to fuse, however, when the BrdU diminished in the DNA due to cell division. These results indicate that labeling with BrdU at a concentration of 0.5 x 10(-6) M and an incorporation time of 48 hr is optimal to obtain perceptible immunocytochemical staining without affecting myoblast fusion. Such BrdU immunolabeling could be used as a nuclear marker for hybridization studies.
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Pavlath, G. K., T. A. Rando, and H. M. Blau. "Transient immunosuppressive treatment leads to long-term retention of allogeneic myoblasts in hybrid myofibers." Journal of Cell Biology 127, no. 6 (December 15, 1994): 1923–32. http://dx.doi.org/10.1083/jcb.127.6.1923.
Full textAbstract:
Normal and genetically engineered skeletal muscle cells (myoblasts) show promise as drug delivery vehicles and as therapeutic agents for treating muscle degeneration in muscular dystrophies. A limitation is the immune response of the host to the transplanted cells. Allogeneic myoblasts are rapidly rejected unless immunosuppressants are administered. However, continuous immunosuppression is associated with significant toxic side effects. Here we test whether immunosuppressive treatment, administered only transiently after allogeneic myoblast transplantation, allows the long-term survival of the transplanted cells in mice. Two immunosuppressive treatments with different modes of action were used: (a) cyclosporine A (CSA); and (b) monoclonal antibodies to intracellular adhesion molecule-1 and leukocyte function-associated molecule-1. The use of myoblasts genetically engineered to express beta-galactosidase allowed quantitation of the survival of allogeneic myoblasts at different times after cessation of the immunosuppressive treatments. Without host immunosuppression, allogeneic myoblasts were rejected from all host strains tested, although the relative time course differed as expected for low and high responder strains. The allogeneic myoblasts initially fused with host myofibers, but these hybrid cells were later destroyed by the massive immunological response of the host. However, transient immunosuppressive treatment prevented the hybrid myofiber destruction and led to their long-term retention. Even four months after the cessation of treatment, the hybrid myofibers persisted and no inflammatory infiltrate was present in the tissue. Such long-term survival indicates that transient immunosuppression may greatly increase the utility of myoblast transplantation as a therapeutic approach to the treatment of muscle and nonmuscle disease.
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Rochat, Anne, Anne Fernandez, Marie Vandromme, Jeàn-Pierre Molès, Triston Bouschet, Gilles Carnac, and Ned J. C. Lamb. "Insulin and Wnt1 Pathways Cooperate to Induce Reserve Cell Activation in Differentiation and Myotube Hypertrophy." Molecular Biology of the Cell 15, no. 10 (October 2004): 4544–55. http://dx.doi.org/10.1091/mbc.e03-11-0816.
Full textAbstract:
During ex vivo myoblast differentiation, a pool of quiescent mononucleated myoblasts, reserve cells, arise alongside myotubes. Insulin/insulin-like growth factor (IGF) and PKB/Akt-dependent phosphorylation activates skeletal muscle differentiation and hypertrophy. We have investigated the role of glycogen synthase kinase 3 (GSK-3) inhibition by protein kinase B (PKB)/Akt and Wnt/β-catenin pathways in reserve cell activation during myoblast differentiation and myotube hypertrophy. Inhibition of GSK-3 by LiCl or SB216763, restored insulin-dependent differentiation of C2ind myoblasts in low serum, and cooperated with insulin in serum-free medium to induce MyoD and myogenin expression in C2ind myoblasts, quiescent C2 or primary human reserve cells. We show that LiCl treatment induced nuclear accumulation of β-catenin in C2 myoblasts, thus mimicking activation of canonical Wnt signaling. Similarly to the effect of GSK-3 inhibitors with insulin, coculturing C2 reserve cells with Wnt1-expressing fibroblasts enhanced insulin-stimulated induction of MyoD and myogenin in reserve cells. A similar cooperative effect of LiCl or Wnt1 with insulin was observed during late ex vivo differentiation and promoted increased size and fusion of myotubes. We show that this synergistic effect on myotube hypertrophy involved an increased fusion of reserve cells into preexisting myotubes. These data reveal insulin and Wnt/β-catenin pathways cooperate in muscle cell differentiation through activation and recruitment of satellite cell-like reserve myoblasts.
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Mott, David M., Cristen Hoyt, Rael Caspari, Karen Stone, Richard Pratley, and Clifton Bogardus. "Palmitate oxidation rate and action on glycogen synthase in myoblasts from insulin-resistant subjects." American Journal of Physiology-Endocrinology and Metabolism 279, no. 3 (September 1, 2000): E561—E569. http://dx.doi.org/10.1152/ajpendo.2000.279.3.e561.
Full textAbstract:
Elevated plasma lipid and nonesterified fatty acid concentrations reduce insulin-mediated glucose disposal in skeletal muscle. Cultured myoblasts from 21 subjects were studied for rates of palmitate oxidation and the effect of palmitate on glycogen synthase activity at the end of an 18-h incubation in serum- and glucose-free media. Oxidation rates of 40 μM palmitate in cultured myoblasts correlated with the fasting glucose ( r = 0.71, P = 0.001), log fasting insulin ( r = 0.52, P = 0.03), and insulin-mediated glucose storage rate ( r = −0.50, P = 0.04) of the muscle donors. Myoblast glycogen synthase activity can be regulated by 240 μM palmitate, but the changes are associated with the basal respiratory quotient and not with the insulin resistance of the muscle donor. These results indicate that myoblasts producing elevated palmitate oxidation rates in vitro can be used to identify skeletal muscle abnormalities which are primary contributors to insulin resistance in vivo. Effects of 240 μM palmitate on myoblast glycogen synthase activity appear to be mechanistically different from the relationship between myoblast palmitate oxidation rates and insulin resistance of the muscle donor.
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Tomaz da Silva, Meiricris, Aniket S. Joshi, Micah B. Castillo, Tatiana E. Koike, Anirban Roy, Preethi H. Gunaratne, and Ashok Kumar. "Fn14 promotes myoblast fusion during regenerative myogenesis." Life Science Alliance 6, no. 12 (October 9, 2023): e202302312. http://dx.doi.org/10.26508/lsa.202302312.
Full textAbstract:
Skeletal muscle regeneration involves coordinated activation of an array of signaling pathways. Fibroblast growth factor–inducible 14 (Fn14) is a bona fide receptor for the TWEAK cytokine. Levels of Fn14 are increased in the skeletal muscle of mice after injury. However, the cell-autonomous role of Fn14 in muscle regeneration remains unknown. Here, we demonstrate that global deletion of the Fn14 receptor in mice attenuates muscle regeneration. Conditional ablation of Fn14 in myoblasts but not in differentiated myofibers of mice inhibits skeletal muscle regeneration. Fn14 promotes myoblast fusion without affecting the levels of myogenic regulatory factors in the regenerating muscle. Fn14 deletion in myoblasts hastens initial differentiation but impairs their fusion. The overexpression of Fn14 in myoblasts results in the formation of myotubes having an increased diameter after induction of differentiation. Ablation of Fn14 also reduces the levels of various components of canonical Wnt and calcium signaling both in vitro and in vivo. Forced activation of Wnt signaling rescues fusion defects in Fn14-deficient myoblast cultures. Collectively, our results demonstrate that Fn14-mediated signaling positively regulates myoblast fusion and skeletal muscle regeneration.
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Guérette, B., D. Skuk, F. Célestin, C. Huard, F. Tardif, I. Asselin, B. Roy, et al. "Prevention by anti-LFA-1 of acute myoblast death following transplantation." Journal of Immunology 159, no. 5 (September 1, 1997): 2522–31. http://dx.doi.org/10.4049/jimmunol.159.5.2522.
Full textAbstract:
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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Sastry, S. K., M. Lakonishok, D. A. Thomas, J. Muschler, and A. F. Horwitz. "Integrin alpha subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation." Journal of Cell Biology 133, no. 1 (April 1, 1996): 169–84. http://dx.doi.org/10.1083/jcb.133.1.169.
Full textAbstract:
The role of integrins in muscle differentiation was addressed by ectopic expression of integrin alpha subunits in primary quail skeletal muscle, a culture system particularly amenable to efficient transfection and expression of exogenous genes. Ectopic expression of either the human alpha5 subunit or the chicken alpha6 subunit produced contrasting phenotypes. The alpha5-transfected myoblasts remain in the proliferative phase and are differentiation inhibited even in confluent cultures. In contrast, myoblasts that overexpress the alpha6 subunit exhibit inhibited proliferation and substantial differentiation. Antisense suppression of endogenous quail alpha6 expression inhibits myoblast differentiation resulting in sustained proliferation. These effects of ectopic alpha subunit expression are mediated, to a large extent, by the cytoplasmic domains. Ectopic expression of chimeric alpha subunits, alpha5ex/6cyto and alpha6ex/5cyto, produced phenotypes opposite to those observed with ectopic alpha5 or alpha6 expression. Myoblasts that express alpha5ex/6cyto show decreased proliferation while differentiation is partially restored. In contrast, the alpha6ex/5cyto transfectants remain in the proliferative phase unless allowed to become confluent for at least 24 h. Furthermore, expression of human alpha5 subunit cytoplasmic domain truncations, before and after the conserved GFFKR motif, shows that this sequence is important in alpha5 regulation of differentiation. Ectopic alpha5 and alpha6 expression also results in contrasting responses to the mitogenic effects of serum growth factors. Myoblasts expressing the human alpha5 subunit differentiate only in the absence of serum while differentiation of untransfected and alpha6-transfected myoblasts is insensitive to serum concentration. Addition of individual, exogenous growth factors to alpha5-transfected myoblasts results in unique responses that differ from their effects on untransfected cells. Both bFGF or TGFbeta inhibit the serum-free differentiation of alpha5-transfected myoblasts, but differ in that bFGF stimulates proliferation whereas TGF-beta inhibits it. Insulin or TGF-alpha promote proliferation and differentiation of alpha5-transfected myoblasts; however, insulin alters myotube morphology. TGF-alpha or PDGF-BB enhance muscle alpha-actinin organization into myofibrils, which is impaired in differentiated alpha5 cultures. With the exception of TGF-alpha, these growth factor effects are not apparent in untransfected myoblasts. Finally, myoblast survival under serum-free conditions is enhanced by ectopic alpha5 expression only in the presence of bFGF and insulin while TGF-alpha and TGF-beta promote survival of untransfected myoblasts. Our observations demonstrate (1) a specificity for integrin alpha subunits in regulating myoblast proliferation and differentiation; (2) that the ratio of integrin expression can affect the decision to proliferate or differentiate; (3) a role for the alpha subunit cytoplasmic domain in mediating proliferative and differentiative signals; and (4) the regulation of proliferation, differentiation, cytoskeletal assembly, and cell survival depend critically on the expression levels of different integrins and the growth factor environment in which the cells reside.
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Saini, Amarjit, Linda Björkhem-Bergman, Johan Boström, Mats Lilja, Michael Melin, Karl Olsson, Lena Ekström, et al. "Impact of vitamin D and vitamin D receptor TaqI polymorphism in primary human myoblasts." Endocrine Connections 8, no. 7 (July 2019): 1070–81. http://dx.doi.org/10.1530/ec-19-0194.
Full textAbstract:
The CC genotype of the vitamin D receptor (VDR) polymorphism TaqI rs731236 has previously been associated with a higher risk of developing myopathy compared to TT carriers. However, the mechanistic role of this polymorphism in skeletal muscle is not well defined. The effects of vitamin D on patients genotyped for the VDR polymorphism TaqI rs731236, comparing CC and TT carriers were evaluated. Primary human myoblasts isolated from 4 CC carriers were compared with myoblasts isolated from four TT carriers and treated with vitamin D in vitro. A dose-dependent inhibitory effect on myoblast proliferation and differentiation was observed concurrent with modifications of key myogenic regulatory factors. RNA sequencing revealed a vitamin D dose–response gene signature enriched with a higher number of VDR-responsive elements (VDREs) per gene. Interestingly, the greater the expression of muscle differentiation markers in myoblasts, the more pronounced was the vitamin D-mediated response to suppress genes associated with myogenic fusion and myotube formation. This novel finding provides a mechanistic explanation to the inconsistency regarding previous reports of the role of vitamin D in myoblast differentiation. No effects in myoblast proliferation, differentiation or gene expression were related to CC vs TT carriers. Our findings suggest that the VDR polymorphism TaqI rs731236 comparing CC vs TT carriers did not influence the effects of vitamin D on primary human myoblasts and that vitamin D inhibits myoblast proliferation and differentiation through key regulators of cell cycle progression. Future studies need to employ strategies to identify the primary responses of vitamin D that drive the cellular response towards quiescence.
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Yamashita, Aline M. S., Maryana T. C. Ancillotti, Luciana P. Rangel, Marcio Fontenele, Cicero Figueiredo-Freitas, Ana C. Possidonio, Carolina P. Soares, Martha M. Sorenson, Claudia Mermelstein, and Leonardo Nogueira. "Balance between S-nitrosylation and denitrosylation modulates myoblast proliferation independently of soluble guanylyl cyclase activation." American Journal of Physiology-Cell Physiology 313, no. 1 (July 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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Steyn, Paul J., Kevin Dzobo, Robert I. Smith, and Kathryn H. Myburgh. "Interleukin-6 Induces Myogenic Differentiation via JAK2-STAT3 Signaling in Mouse C2C12 Myoblast Cell Line and Primary Human Myoblasts." International Journal of Molecular Sciences 20, no. 21 (October 24, 2019): 5273. http://dx.doi.org/10.3390/ijms20215273.
Full textAbstract:
Postnatal muscle growth and exercise- or injury-induced regeneration are facilitated by myoblasts. Myoblasts respond to a variety of proteins such as cytokines that activate various signaling cascades. Cytokines belonging to the interleukin 6 superfamily (IL-6) influence myoblasts’ proliferation but their effect on differentiation is still being researched. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway is one of the key signaling pathways identified to be activated by IL-6. The aim of this study was to investigate myoblast fate as well as activation of JAK-STAT pathway at different physiologically relevant IL-6 concentrations (10 pg/mL; 100 pg/mL; 10 ng/mL) in the C2C12 mouse myoblast cell line and primary human myoblasts, isolated from eight young healthy male volunteers. Myoblasts’ cell cycle progression, proliferation and differentiation in vitro were assessed. Low IL-6 concentrations facilitated cell cycle transition from the quiescence/Gap1 (G0/G1) to the synthesis (S-) phases. Low and medium IL-6 concentrations decreased the expression of myoblast determination protein 1 (MyoD) and myogenin and increased proliferating cell nuclear antigen (PCNA) expression. In contrast, high IL-6 concentration shifted a larger proportion of cells to the pro-differentiation G0/G1 phase of the cell cycle, substantiated by significant increases of both MyoD and myogenin expression and decreased PCNA expression. Low IL-6 concentration was responsible for prolonged JAK1 activation and increased suppressor of cytokine signaling 1 (SOCS1) protein expression. JAK-STAT inhibition abrogated IL-6-mediated C2C12 cell proliferation. In contrast, high IL-6 initially increased JAK1 activation but resulted in prolonged JAK2 activation and elevated SOCS3 protein expression. High IL-6 concentration decreased interleukin-6 receptor (IL-6R) expression 24 h after treatment whilst low IL-6 concentration increased IL-6 receptor (IL-6R) expression at the same time point. In conclusion, this study demonstrated that IL-6 has concentration- and time-dependent effects on both C2C12 mouse myoblasts and primary human myoblasts. Low IL-6 concentration induces proliferation whilst high IL-6 concentration induces differentiation. These effects are mediated by specific components of the JAK/STAT/SOCS pathway.
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Miller, J. B., and F. E. Stockdale. "Developmental regulation of the multiple myogenic cell lineages of the avian embryo." Journal of Cell Biology 103, no. 6 (December 1, 1986): 2197–208. http://dx.doi.org/10.1083/jcb.103.6.2197.
Full textAbstract:
The developmental regulation of myoblasts committed to fast, mixed fast/slow, and slow myogenic cell lineages was determined by analyzing myotube formation in high density and clonal cultures of myoblasts isolated from chicken and quail embryos of different ages. To identify cells of different myogenic lineages, myotubes were analyzed for content of fast and slow classes of myosin heavy chain (MHC) isoforms by immunocytochemistry and immunoblotting using specific monoclonal antibodies. Myoblasts from the hindlimb bud, forelimb bud, trunk, and pectoral regions of the early chicken embryo and hindlimb bud of the early quail embryo (days 3-6 in ovo) were committed to three distinct lineages with 60-90% of the myoblasts in the fast lineage, 10-40% in the mixed fast/slow lineage, and 0-3% in the slow lineage depending on the age and species of the myoblast donor. In contrast, 99-100% of the myoblasts in the later embryos (days 9-12 in ovo) were in the fast lineage. Serial subculturing from a single myoblast demonstrated that commitment to a particular lineage was stably inherited for over 30 cell doublings. When myoblasts from embryos of the same age were cultured, the percentage of muscle colonies of the fast, fast/slow, and slow types that formed in clonal cultures was the same as the percentage of myotubes of each of these types that formed in high density cultures, indicating that intercellular contact between myoblasts of different lineages did not affect the type of myotube formed. An analysis in vivo showed that three types of primary myotubes--fast, fast/slow, and slow--were also found in the chicken thigh at day 7 in ovo and that synthesis of both the fast and slow classes of MHC isoforms was concomitant with the formation of primary myotubes. On the basis of these results, we propose that in the avian embryo, there is an early phase of muscle fiber formation in which primary myotubes with differing MHC contents are formed from myoblasts committed to three intrinsically different primary myogenic lineages independent of innervation and a later phase in which secondary myotubes are formed from myoblasts in a single, secondary myogenic lineage with maturation and maintenance of fiber diversity dependent on innervation.
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Knoll, Jasmin, Bastian Amend, Tanja Abruzzese, Niklas Harland, Arnulf Stenzl, and Wilhelm K. Aicher. "Production of Proliferation- and Differentiation-Competent Porcine Myoblasts for Preclinical Studies in a Porcine Large Animal Model of Muscular Insufficiency." Life 14, no. 2 (January 31, 2024): 212. http://dx.doi.org/10.3390/life14020212.
Full textAbstract:
Muscular insufficiency is observed in many conditions after injury, chronic inflammation, and especially in elderly populations. Causative cell therapies for muscle deficiencies are not state of the art. Animal models to study the therapy efficacy are, therefore, needed. We developed an improved protocol to produce myoblasts suitable for pre-clinical muscle therapy studies in a large animal model. Myoblasts were isolated from the striated muscle, expanded by employing five different protocols, and characterized on transcript and protein expression levels to determine procedures that yielded optimized regeneration-competent myoblasts and multi-nucleated myotubes. We report that swine skeletal myoblasts proliferated well under improved conditions without signs of cellular senescence, and expressed significant levels of myogenic markers including Pax7, MyoD1, Myf5, MyoG, Des, Myf6, CD56 (p ≤ 0.05 each). Upon terminal differentiation, myoblasts ceased proliferation and generated multi-nucleated myotubes. Injection of such myoblasts into the urethral sphincter complex of pigs with sphincter muscle insufficiency yielded an enhanced functional regeneration of this muscle (81.54% of initial level) when compared to the spontaneous regeneration in the sham controls without myoblast injection (67.03% of initial level). We conclude that the optimized production of porcine myoblasts yields cells that seem suitable for preclinical studies of cell therapy in a porcine large animal model of muscle insufficiency.