Journal articles on the topic 'Primary muscle myoblasts'
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1
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.
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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.
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Intramuscular implanted myoblasts can fuse with existing myofibers. Here we report that implanted primary myoblasts marked with retroviral transgenes can also persist as muscle precursor cells. These cells can be recovered as viable myoblasts from muscles of recipient mice even months after myoblast implantation, and they can fully resume expression of the transgenes in culture. Upon re-implantation into muscles, they again not only fuse with existing myofibers, but also survive as muscle precursor cells in the tissue. These reserve myogenic cells should be able to contribute to host myofibers in muscle regeneration when the recombinant myofibers are damaged, providing an additional mechanism to maintain a persistent expression of transgenes delivered by myoblast-mediated gene transfer.
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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.
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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.
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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.
6
Duxson, M. J., Y. Usson, and A. J. Harris. "The origin of secondary myotubes in mammalian skeletal muscles: ultrastructural studies." Development 107, no. 4 (December 1, 1989): 743–50. http://dx.doi.org/10.1242/dev.107.4.743.
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The distribution of secondary myotubes and undifferentiated mononucleated cells (presumed to be myoblasts) within foetal IVth lumbrical muscles of the rat was analyzed with serial section electron microscopy. In all myotube clusters for which the innervation zone was located, every secondary myotube overlapped the end-plate region of the primary myotube. No secondary myotubes were ever demonstrated to occur at a distance from the primary myotube innervation zone. This indicates that new secondary myotubes begin to form only in the innervation zone of the muscle. Some young secondary myotubes made direct contact with a nerve terminal, but we cannot say if this is true for all developing secondary myotubes. Myoblasts were not clustered near the innervation zone, but were uniformly distributed throughout the muscle. Myoblasts were frequently interposed between a primary and a secondary myotube, in equally close proximity to both cell membranes. We conclude that specificity in myoblast-myotube fusion does not depend on restrictions in the physical distribution of myoblasts within the muscle, and therefore must reflect more subtle mechanisms for intercellular recognition.
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Langendorf, Eva K., Pol M. Rommens, Philipp Drees, and Ulrike Ritz. "Dexamethasone Inhibits the Pro-Angiogenic Potential of Primary Human Myoblasts." International Journal of Molecular Sciences 22, no. 15 (July 26, 2021): 7986. http://dx.doi.org/10.3390/ijms22157986.
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Tissue regeneration depends on the complex processes of angiogenesis, inflammation and wound healing. Regarding muscle tissue, glucocorticoids (GCs) inhibit pro-inflammatory signalling and angiogenesis and lead to muscle atrophy. Our hypothesis is that the synthetic GC dexamethasone (dex) impairs angiogenesis leading to muscle atrophy or inhibited muscle regeneration. Therefore, this study aims to elucidate the effect of dexamethasone on HUVECs under different conditions in mono- and co-culture with myoblasts to evaluate growth behavior and dex impact with regard to muscle atrophy and muscle regeneration. Viability assays, qPCR, immunofluorescence as well as ELISAs were performed on HUVECs, and human primary myoblasts seeded under different culture conditions. Our results show that dex had a higher impact on the tube formation when HUVECs were maintained with VEGF. Gene expression was not influenced by dex and was independent of cells growing in a 2D or 3D matrix. In co-culture CD31 expression was suppressed after incubation with dex and gene expression analysis revealed that dex enhanced expression of myogenic transcription factors, but repressed angiogenic factors. Moreover, dex inhibited the VEGF mediated pro angiogenic effect of myoblasts and inhibited expression of angiogenic inducers in the co-culture model. This is the first study describing a co-culture of human primary myoblast and HUVECs maintained under different conditions. Our results indicate that dex affects angiogenesis via inhibition of VEGF release at least in myoblasts, which could be responsible not only for the development of muscle atrophy after dex administration, but also for inhibition of muscle regeneration after vascular damage.
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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.
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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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Wang, Xiaotong, Junyuan Lin, Zhenhai Jiao, Li Zhang, Dongxue Guo, Lilong An, Tingting Xie, and Shudai Lin. "Circular RNA circIGF2BP3 Promotes the Proliferation and Differentiation of Chicken Primary Myoblasts." International Journal of Molecular Sciences 24, no. 21 (October 24, 2023): 15545. http://dx.doi.org/10.3390/ijms242115545.
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The quality and quantity of animal meat are closely related to the development of skeletal muscle, which, in turn, is determined by myogenic cells, including myoblasts and skeletal muscle satellite cells (SMSCs). Circular RNA, an endogenous RNA derivative formed through specific reverse splicing in mRNA precursors, has the potential to influence muscle development by binding to miRNAs or regulating gene expression involved in muscular growth at the transcriptional level. Previous high-throughput sequencing of circRNA in chicken liver tissue revealed a circular transcript, circIGF2BP3, derived from the gene encoding insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3). In this study, we confirmed the presence of the natural circular molecule of circIGF2BP3 through an RNase R enzyme tolerance assay. RT-qPCR results showed high circIGF2BP3 expression in the pectoral and thigh muscles of Yuexi frizzled feather chickens at embryonic ages 14 and 18, as well as at 7 weeks post-hatch. Notably, its expression increased during embryonic development, followed by a rapid decrease after birth. As well as using RT-qPCR, Edu, CCK-8, immunofluorescence, and Western blot techniques, we demonstrated that overexpressing circIGF2BP3 could promote the proliferation and differentiation of chicken primary myoblasts through upregulating genes such as proliferating cell nuclear antigen (PCNA), cyclin D1 (CCND1), cyclin E1 (CCNE1), cyclin dependent kinase 2 (CDK2), myosin heavy chain (MyHC), myoblast-determining 1 (MyoD1), myogenin (MyoG), and Myomaker. In conclusion, circIGF2BP3 promotes the proliferation and differentiation of myoblasts in chickens. This study establishes a foundation for further investigation into the biological functions and mechanisms of circIGF2BP3 in myoblasts proliferation and differentiation.
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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.
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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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Charlton, Carol A., William A. Mohler, Glenn L. Radice, Richard O. Hynes, and Helen M. Blau. "Fusion Competence of Myoblasts Rendered Genetically Null for N-Cadherin in Culture." Journal of Cell Biology 138, no. 2 (July 28, 1997): 331–36. http://dx.doi.org/10.1083/jcb.138.2.331.
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Myoblast fusion is essential to muscle tissue development yet remains poorly understood. N-cadherin, like other cell surface adhesion molecules, has been implicated by others in muscle formation based on its pattern of expression and on inhibition of myoblast aggregation and fusion by antibodies or peptide mimics. Mice rendered homozygous null for N-cadherin revealed the general importance of the molecule in early development, but did not test a role in skeletal myogenesis, since the embryos died before muscle formation. To test genetically the proposed role of N-cadherin in myoblast fusion, we successfully obtained N-cadherin null primary myoblasts in culture. Fusion of myoblasts expressing or lacking N-cadherin was found to be equivalent, both in vitro by intracistronic complementation of lacZ and in vivo by injection into the muscles of adult mice. An essential role for N-cadherin in mediating the effects of basic fibroblast growth factor was also excluded. These methods for obtaining genetically homozygous null somatic cells from adult tissues should have broad applications. Here, they demonstrate clearly that the putative fusion molecule, N-cadherin, is not essential for myoblast fusion.
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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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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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Youm, Tae Hyun, Sun-Hee Woo, Eun-Soo Kwon, and Sung Sup Park. "NADPH Oxidase 4 Contributes to Myoblast Fusion and Skeletal Muscle Regeneration." Oxidative Medicine and Cellular Longevity 2019 (November 18, 2019): 1–12. http://dx.doi.org/10.1155/2019/3585390.
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Myoblast fusion is an essential step in skeletal muscle development and regeneration. NADPH oxidase 4 (Nox4) regulates cellular processes such as proliferation, differentiation, and survival by producing reactive oxygen species (ROS). Insulin-like growth factor 1 induces muscle hypertrophy via Nox4, but its function in myoblast fusion remains elusive. Here, we report a ROS-dependent role of Nox4 in myoblast differentiation. Regenerating muscle fibers after injury by cardiotoxin had a lower cross-sectional area in Nox4-knockout (KO) mice than myofibers in wild-type (WT) mice. Diameters and fusion index values of myotubes differentiated from Nox4-KO primary myoblasts were significantly lower than those of myotubes derived from WT myoblasts. However, no difference was observed in the differentiation index and expression of MyoD, myogenin, and myosin heavy chain 3 (MHC) between KO and WT myotubes. The decreased fusion index was also observed during differentiation of primary myoblasts and C2C12 cells with suppressed Nox4 expression. In contrast, in C2C12 cells overexpressing Nox4, the fusion index was increased, whereas the differentiation index and MHC and myogenin protein expression were not affected compared to control. Interestingly, the expression of myomaker (Tmem8c), a fusogenic protein that controls myoblast fusion, was reduced in Nox4-knockdown C2C12 cells. The myomaker expression level was proportional to the cellular ROS level, which was regulated by of Nox4 expression level. These results suggests that Nox4 contributes to myoblast fusion, possibly through the regulation of myomaker expression via ROS production, and that Nox4-dependent ROS may promote skeletal muscle regeneration and growth.
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Pääsuke, Reedik, Margus Eimre, Andres Piirsoo, Nadežda Peet, Liidia Laada, Lumme Kadaja, Mart Roosimaa, et al. "Proliferation of Human Primary Myoblasts Is Associated with Altered Energy Metabolism in Dependence on AgeingIn VivoandIn Vitro." Oxidative Medicine and Cellular Longevity 2016 (2016): 1–10. http://dx.doi.org/10.1155/2016/8296150.
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Background. Ageing is associated with suppressed regenerative potential of muscle precursor cells due to decrease of satellite cells and suppressive intramuscular milieu on their activation, associated with ageing-related low-grade inflammation. The aim of the study was to characterize the function of oxidative phosphorylation (OXPHOS), glycolysis, adenylate kinase (AK), and creatine kinase (CK) mediated systems in young and older individuals.Materials and Methods. Myoblasts were cultivated from biopsies taken by transcutaneous conchotomy from vastus lateralis muscle in young (20–29 yrs,n=7) and older (70–79 yrs,n=7) subjects. Energy metabolism was assessed in passages 2 to 6 by oxygraphy and enzyme analysis.Results. In myoblasts of young and older subjects the rate of OXPHOS decreased during proliferation from passages 2 to 6. The total activities of CK and AK decreased. Myoblasts of passage 2 cultivated from young muscle showed higher rate of OXPHOS and activities of CK and AK compared to myoblasts from older subjects while hexokinase and pyruvate kinase were not affected by ageing.Conclusions. Proliferation of myoblastsin vitrois associated with downregulation of OXPHOS and energy storage and transfer systems. Ageingin vivoexerts an impact on satellite cells which results in altered metabolic profile in favour of the prevalence of glycolytic pathways over mitochondrial OXPHOS of myoblasts.
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Cusella-De Angelis, M. G., S. Molinari, A. Le Donne, M. Coletta, E. Vivarelli, M. Bouche, M. Molinaro, S. Ferrari, and G. Cossu. "Differential response of embryonic and fetal myoblasts to TGF beta: a possible regulatory mechanism of skeletal muscle histogenesis." Development 120, no. 4 (April 1, 1994): 925–33. http://dx.doi.org/10.1242/dev.120.4.925.
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Embryonic and fetal skeletal myoblasts were grown in culture in the presence of TGF beta. Under the conditions employed, TGF beta inhibited differentiation of fetal but not of embryonic myoblasts. To investigate the possible relevance of these data to skeletal muscle histogenesis in vivo, we studied the proliferation/differentiation state of mesodermal cells in the proximal region of the limb bud at the time of primary fiber formation. BrdU labeling and immunostaining for myosin heavy chains revealed that very few mesodermal cells enter the S phase of the cycle when differentiated primary fibers first appear. However, a few hours later, many cells in S phase surround newly formed muscle fibers, suggesting that the latter may be a source of mitogens for undifferentiated myoblasts. Co-culture experiments supported this hypothesis, showing that medium conditioned by fiber-containing explants can stimulate myoblast proliferation. Taken together these data suggested a possible mechanism for the regulation of muscle fiber formation. The model assumes that fibers form in the proximal region of the limb bud, where TGF beta is known to be present, and BrdU labeling experiments did not reveal cells in S phase. It is conceivable that non-dividing embryonic myoblasts (which do not respond to TGF beta) can undergo differentiation, while fetal myoblasts are inhibited by TGF beta. Once formed, primary fibers may stimulate a new wave of proliferation in fetal myoblasts, in order to expand the pool of cells needed to form secondary fibers.(ABSTRACT TRUNCATED AT 250 WORDS)
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Miller, S. C., H. Ito, H. M. Blau, and F. M. Torti. "Tumor necrosis factor inhibits human myogenesis in vitro." Molecular and Cellular Biology 8, no. 6 (June 1988): 2295–301. http://dx.doi.org/10.1128/mcb.8.6.2295-2301.1988.
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We examined the effects of human recombinant tumor necrosis factor-alpha (TNF) on human primary myoblasts. When added to proliferating myoblasts, TNF inhibited the expression of alpha-cardiac actin, a muscle-specific gene whose expression is observed at low levels in human myoblasts. TNF also inhibited muscle differentiation as measured by several parameters, including cell fusion and the expression of other muscle-specific genes, such as alpha-skeletal actin and myosin heavy chain. Muscle cells were sensitive to TNF in a narrow temporal window of differentiation. Northern (RNA) blot and immunofluorescence analyses revealed that human muscle gene expression became unresponsive to TNF coincident with myoblast differentiation. When TNF was added to differentiated myotubes, there was no effect on muscle gene expression. In contrast, TNF-inducible mRNAs such as interferon beta-2 still responded, suggesting that the signal mediated by TNF binding to its receptor had no effect on muscle-specific genes after differentiation.
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Miller, S. C., H. Ito, H. M. Blau, and F. M. Torti. "Tumor necrosis factor inhibits human myogenesis in vitro." Molecular and Cellular Biology 8, no. 6 (June 1988): 2295–301. http://dx.doi.org/10.1128/mcb.8.6.2295.
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We examined the effects of human recombinant tumor necrosis factor-alpha (TNF) on human primary myoblasts. When added to proliferating myoblasts, TNF inhibited the expression of alpha-cardiac actin, a muscle-specific gene whose expression is observed at low levels in human myoblasts. TNF also inhibited muscle differentiation as measured by several parameters, including cell fusion and the expression of other muscle-specific genes, such as alpha-skeletal actin and myosin heavy chain. Muscle cells were sensitive to TNF in a narrow temporal window of differentiation. Northern (RNA) blot and immunofluorescence analyses revealed that human muscle gene expression became unresponsive to TNF coincident with myoblast differentiation. When TNF was added to differentiated myotubes, there was no effect on muscle gene expression. In contrast, TNF-inducible mRNAs such as interferon beta-2 still responded, suggesting that the signal mediated by TNF binding to its receptor had no effect on muscle-specific genes after differentiation.
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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.
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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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Irintchev, A., J. D. Rosenblatt, M. J. Cullen, M. Zweyer, and A. Wernig. "Ectopic skeletal muscles derived from myoblasts implanted under the skin." Journal of Cell Science 111, no. 22 (November 15, 1998): 3287–97. http://dx.doi.org/10.1242/jcs.111.22.3287.
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We investigated the potential of cultured myoblasts to generate skeletal muscle in an ectopic site. Myoblasts from a clonal cell line or from expanded primary cultures were injected under the skin of the lumbar region of adult syngenic Balb/c mice. One to 7 weeks after injection, distinct muscles, of greater mass in mice injected with clonal myoblasts (6–78 mg, n=37) than in mice injected with primary myoblasts (1–7 mg, n=26), had formed between the subcutaneous panniculus carnosus muscle and the trunk muscles of host animals. These ectopic muscles exhibited spontaneous and/or electrically-evoked contractions after the second week and, when stimulated directly in vitro, isometric contractile properties similar to those of normal muscles. Histological, electron microscopical and tissue culture examination of these muscles revealed their largely mature morphology and phenotype. The fibres, most of which were branched, were contiguous, aligned and capillarised, exhibited normal sarcormeric protein banding patterns, and expressed muscle-specific proteins, including desmin, dystrophin, and isoforms of developmental and adult myosin heavy chain. Enveloping each fibre was a basal lamina, beneath which lay quiescent satellite cells, which could be stimulated to produce new muscle in culture. Presence of endplates (revealed by alpha-bungarotoxin and neurofilament staining), and the eventual loss of expression of neural cell adhesion molecule and extrasynaptic acetylcholine receptors, indicated that some fibres were innervated. That these muscle fibres were of implanted-cell origin was supported by the finding of Y-chromosome and a lack of dystrophin in ectopic muscles formed after subcutaneous injection of, respectively, male myoblasts into female mice and dystrophin-deficient (mdx) myoblasts into normal C57Bl/10 muscle. Our results demonstrate that an organised, functional muscle can be generated de novo from a disorganised mass of myoblasts implanted in an extramuscular subcutaneous site, whereby the host contributes significantly in providing support tissues and innervation. Our observations are also consistent with the idea that myogenic cells behave like tissue-specific stem cells, generating new muscle precursor (satellite) cells as well as mature muscle. Subcutaneous implantation of myoblasts may have a range of useful applications, from the study of myogenesis to the delivery of gene products.
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Komarova, Margarita Y., Sergey V. Rozhkov, Oksana A. Ivanova, Olga V. Turtikova, Timur M. Mirzoev, Renata I. Dmitrieva, Boris S. Shenkman, and Natalia A. Vilchinskaya. "Cultured Myoblasts Derived from Rat Soleus Muscle Show Altered Regulation of Proliferation and Myogenesis during the Course of Mechanical Unloading." International Journal of Molecular Sciences 23, no. 16 (August 15, 2022): 9150. http://dx.doi.org/10.3390/ijms23169150.
Full textAbstract:
The structure and function of soleus muscle fibers undergo substantial remodeling under real or simulated microgravity conditions. However, unloading-induced changes in the functional activity of skeletal muscle primary myoblasts remain poorly studied. The purpose of our study was to investigate how short-term and long-term mechanical unloading would affect cultured myoblasts derived from rat soleus muscle. Mechanical unloading was simulated by rat hindlimb suspension model (HS). Myoblasts were purified from rat soleus at basal conditions and after 1, 3, 7, and 14 days of HS. Myoblasts were expanded in vitro, and the myogenic nature was confirmed by their ability to differentiate as well as by immunostaining/mRNA expression of myogenic markers. The proliferation activity at different time points after HS was analyzed, and transcriptome analysis was performed. We have shown that soleus-derived myoblasts differently respond to an early and later stage of HS. At the early stage of HS, the proliferative activity of myoblasts was slightly decreased, and processes related to myogenesis activation were downregulated. At the later stage of HS, we observed a decrease in myoblast proliferative potential and spontaneous upregulation of the pro-myogenic program.
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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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Simon, Liz, Nicole LeCapitaine, Paul Berner, Curtis Vande Stouwe, Jason C. Mussell, Timothy Allerton, Stefany D. Primeaux, et al. "Chronic binge alcohol consumption alters myogenic gene expression and reduces in vitro myogenic differentiation potential of myoblasts from rhesus macaques." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 306, no. 11 (June 1, 2014): R837—R844. http://dx.doi.org/10.1152/ajpregu.00502.2013.
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Chronic alcohol abuse is associated with skeletal muscle myopathy. Previously, we demonstrated that chronic binge alcohol (CBA) consumption by rhesus macaques accentuates skeletal muscle wasting at end-stage of simian immunodeficiency virus (SIV) infection. A proinflammatory, prooxidative milieu and enhanced ubiquitin proteasome activity were identified as possible mechanisms leading to loss of skeletal muscle. The possibility that impaired regenerative capacity, as reflected by the ability of myoblasts derived from satellite cell (SCs) to differentiate into myotubes has not been examined. We hypothesized that the inflammation and oxidative stress in skeletal muscle from CBA animals impair the differentiation capacity of myoblasts to form new myofibers in in vitro assays. We isolated primary myoblasts from the quadriceps femoris of rhesus macaques that were administered CBA or isocaloric sucrose (SUC) for 19 mo. Proliferation and differentiation potential of cultured myoblasts were examined in vitro. Myoblasts from the CBA group had significantly reduced PAX7, MYOD1, MYOG, MYF5, and MEF2C expression. This was associated with decreased myotube formation as evidenced by Jenner-Giemsa staining and myonuclei fusion index. No significant difference in the proliferative ability, cell cycle distribution, or autophagy was detected between myoblasts isolated from CBA and SUC groups. Together, these results reflect marked dysregulation of myoblast myogenic gene expression and myotube formation, which we interpret as evidence of impaired skeletal muscle regenerative capacity in CBA-administered macaques. The contribution of this mechanism to alcoholic myopathy warrants further investigation.
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Nie, Yaping, Shufang Cai, Renqiang Yuan, Suying Ding, Xumeng Zhang, Luxi Chen, Yaosheng Chen, and Delin Mo. "Zfp422 promotes skeletal muscle differentiation by regulating EphA7 to induce appropriate myoblast apoptosis." Cell Death & Differentiation 27, no. 5 (November 4, 2019): 1644–59. http://dx.doi.org/10.1038/s41418-019-0448-9.
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Abstract Zinc finger protein 422 (Zfp422) is a widely expressed zinc finger protein that serves as a transcriptional factor to regulate downstream gene expression, but until now, little is known about its roles in myogenesis. We found here that Zfp422 plays a critical role in skeletal muscle development and regeneration. It highly expresses in mouse skeletal muscle during embryonic development. Specific knockout of Zfp422 in skeletal muscle impaired embryonic muscle formation. Satellite cell-specific Zfp422 deletion severely inhibited muscle regeneration. Myoblast differentiation and myotube formation were suppressed in Zfp422-deleted C2C12 cells, isolated primary myoblasts, and satellite cells. Chromatin Immunoprecipitation Sequencing (ChIP-Seq) revealed that Zfp422 regulated ephrin type-A receptor 7 (EphA7) expression by binding an upstream 169-bp DNA sequence, which was proved to be an enhancer of EphA7. Knocking EphA7 down in C2C12 cells or deleting Zfp422 in myoblasts will inhibit cell apoptosis which is required for myoblast differentiation. These results indicate that Zfp422 is essential for skeletal muscle differentiation and fusion, through regulating EphA7 expression to maintain proper apoptosis.
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Robson, L. G., and S. M. Hughes. "The distal limb environment regulates MyoD accumulation and muscle differentiation in mouse-chick chimaeric limbs." Development 122, no. 12 (December 1, 1996): 3899–910. http://dx.doi.org/10.1242/dev.122.12.3899.
Full textAbstract:
Differentiation of muscle and cartilage within developing vertebrate limbs occurs in a proximodistal progression. To investigate the cues responsible for regulating muscle pattern, mouse myoblasts were implanted into early chick wings prior to endogenous chick muscle differentiation. Fetal myogenic cells originating from transgenic mice carrying a lacZ reporter were readily detected in vivo after implantation and their state of differentiation determined with species-specific antibodies to MyoD and myosin heavy chain. When mouse myogenic cells are implanted at the growing tip of early stage 21 limbs MyoD expression is suppressed and little differentiation of the mouse cells is detected initially. At later stages ectopically implanted mouse cells come to lie within muscle masses, re-express MyoD and differentiate in parallel with differentiating chick myoblasts. However, if mouse cells are implanted either proximally at stage 21 or into the limb tip at stage 24, situations in which mouse cells encounter endogenous differentiating chick myoblasts earlier, MyoD suppression is not detected and a higher proportion of mouse cells differentiate. Mouse cells that remain distal to endogenous differentiating myogenic cells are more likely to remain undifferentiated than myoblasts that lie within differentiated chick muscle. Undifferentiated distal mouse cells are still capable of differentiating if explanted in vitro, suggesting that myoblast differentiation is inhibited in vivo. In vitro, MyoD is suppressed in primary mouse myoblasts by the addition of FGF2 and FGF4 to the culture media. Taken together, our data suggest that the inhibition of myogenic differentiation in the distal limb involves MyoD suppression in myoblasts, possibly through an FGF-like activity.
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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.
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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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Gros, Katarina, Urška Matkovič, Giulia Parato, Katarina Miš, Elisa Luin, Annalisa Bernareggi, Marina Sciancalepore, Tomaž Marš, Paola Lorenzon, and Sergej Pirkmajer. "Neuronal Agrin Promotes Proliferation of Primary Human Myoblasts in an Age-Dependent Manner." International Journal of Molecular Sciences 23, no. 19 (October 4, 2022): 11784. http://dx.doi.org/10.3390/ijms231911784.
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Neuronal agrin, a heparan sulphate proteoglycan secreted by the α-motor neurons, promotes the formation and maintenance of the neuromuscular junction by binding to Lrp4 and activating muscle-specific kinase (MuSK). Neuronal agrin also promotes myogenesis by enhancing differentiation and maturation of myotubes, but its effect on proliferating human myoblasts, which are often considered to be unresponsive to agrin, remains unclear. Using primary human myoblasts, we determined that neuronal agrin induced transient dephosphorylation of ERK1/2, while c-Abl, STAT3, and focal adhesion kinase were unresponsive. Gene silencing of Lrp4 and MuSK markedly reduced the BrdU incorporation, suggesting the functional importance of the Lrp4/MuSK complex for myoblast proliferation. Acute and chronic treatments with neuronal agrin increased the proliferation of human myoblasts in old donors, but they did not affect the proliferation of myoblasts in young donors. The C-terminal fragment of agrin which lacks the Lrp4-binding site and cannot activate MuSK had a similar age-dependent effect, indicating that the age-dependent signalling pathways activated by neuronal agrin involve the Lrp4/MuSK receptor complex as well as an Lrp4/MuSK-independent pathway which remained unknown. Collectively, our results highlight an age-dependent role for neuronal agrin in promoting the proliferation of human myoblasts.
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Vilchinskaya, Natalia A., Sergey V. Rozhkov, Olga V. Turtikova, Timur M. Mirzoev, and Boris S. Shenkman. "AMPK Phosphorylation Impacts Apoptosis in Differentiating Myoblasts Isolated from Atrophied Rat Soleus Muscle." Cells 12, no. 6 (March 16, 2023): 920. http://dx.doi.org/10.3390/cells12060920.
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Regrowth of atrophied myofibers depends on muscle satellite cells (SCs) that exist outside the plasma membrane. Muscle atrophy appears to result in reduced number of SCs due to apoptosis. Given reduced AMP-activated protein kinase (AMPK) activity during differentiation of primary myoblasts derived from atrophic muscle, we hypothesized that there may be a potential link between AMPK and susceptibility of differentiating myoblasts to apoptosis. The aim of this study was to estimate the effect of AMPK activation (via AICAR treatment) on apoptosis in differentiating myoblasts derived from atrophied rat soleus muscle. Thirty rats were randomly assigned to the following two groups: control (C, n = 10) and 7-day hindlimb suspension (HS, n = 20). Myoblasts derived from the soleus muscles of HS rats were divided into two parts: AICAR-treated cells and non-treated cells. Apoptotic processes were evaluated by using TUNEL assay, RT-PCR and WB. In differentiating myoblasts derived from the atrophied soleus, there was a significant decrease (p < 0.05) in AMPK and ACC phosphorylation in parallel with increased number of apoptotic nuclei and a significant upregulation of pro-apoptotic markers (caspase-3, -9, BAX, p53) compared to the cells derived from control muscles. AICAR treatment of atrophic muscle-derived myoblasts during differentiation prevented reductions in AMPK and ACC phosphorylation as well as maintained the number of apoptotic nuclei and the expression of pro-apoptotic markers at the control levels. Thus, the maintenance of AMPK activity can suppress enhanced apoptosis in differentiating myoblasts derived from atrophied rat soleus muscle.
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Torrente, Y., E. El Fahime, N. J. Caron, R. Del Bo, M. Belicchi, F. Pisati, J. P. Tremblay, and N. Bresolin. "Tumor Necrosis Factor-α (TNF-α) Stimulates Chemotactic Response in Mouse Myogenic Cells." Cell Transplantation 12, no. 1 (January 2003): 91–100. http://dx.doi.org/10.3727/000000003783985115.
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Migration of transplanted myogenic cells occurs during both embryogenesis and regeneration of skeletal muscles and is important for successful myoblast transplantation, but little is known about factors that promote chemotaxis of these cells. Tumor necrosis factor-α (TNF-α) is known to induce chemotactic effect on several cell types. In this study, we investigated its influence on the in vitro and in vivo motility of C2C12 and primary myoblasts. In the in vitro test performed in the blind-well Boyden chambers, we showed that TNF-α (50–400 U/ml) significantly enhanced the ability of myogenic cells to migrate. The dose–response curve for this factor was bell shaped, with maximum activity in the 200 U/ml range. In the in vivo test, intramuscular administration of TNF-α was performed by an Alzet pump connected to a perforated polyethylene microtube inserted in the tibialis anterior (TA) of CD1 mice. In these experiments, myoblasts were injected under the muscle epimysium. The recipient mice were immunosuppressed with FK506. Our results showed that, 5 days after myoblast transplantation, cells migrated further in the muscles infused with TNF-α than in the muscles not exposed to TNF-α. TNF-α not only has a chemotactic activity but may also modify cell migration via its action on matrix metalloproteinase (MMP) expression. The proteolytic activities of the MMPs secreted in the muscles were thus also assessed by gelatin zymography. The results showed an increased of MMP-2 and MMP-9 transcripts in the TNF-α-infused muscles injected with myogenic cells. Myoblast migration during transplantation may be enhanced by overlapping gradients of several effector molecules such as TNF-α, interferon-γ (INF-γ), and interleukins, released at the site of muscle injury. We propose that TNF-α may promote myoblast migration directly through chemotactic activity and indirectly by enhancing MMP activity at the site of muscle injury.
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Rauen, Melanie, Dandan Hao, Aline Müller, Eva Mückter, Leo Cornelius Bollheimer, and Mahtab Nourbakhsh. "Free Fatty Acid Species Differentially Modulate the Inflammatory Gene Response in Primary Human Skeletal Myoblasts." Biology 10, no. 12 (December 12, 2021): 1318. http://dx.doi.org/10.3390/biology10121318.
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Age-related loss of skeletal muscle is associated with obesity and inflammation. In animal models, intramuscular fat deposits compromise muscle integrity; however, the relevant fat components that mediate muscular inflammation are not known. Previously, we hypothesized that free fatty acids (FFAs) may directly induce inflammatory gene expression in skeletal muscle cells of obese rats. Here, we examined this hypothesis in primary human skeletal myoblasts (SkMs) using multiplex expression analysis of 39 inflammatory proteins in response to different FFA species. Multiplex mRNA quantification confirmed that the IL6, IL1RA, IL4, LIF, CXCL8, CXCL1, CXCL12 and CCL2 genes were differentially regulated by saturated and unsaturated C16 or C18 FFAs. Fluorescence staining revealed that only saturated C16 and C18 strongly interfere with myoblast replication independent of desmin expression, mitochondrial abundance and oxidative activity. Furthermore, we addressed the possible implications of 71 human receptor tyrosine kinases (RTKs) in FFA-mediated effects. Phosphorylated EphB6 and TNK2 were associated with impaired myoblast replication by saturated C16 and C18 FFAs. Our data suggest that abundant FFA species in human skeletal muscle tissue may play a decisive role in the progression of sarcopenic obesity by affecting inflammatory signals or myoblast replication.
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Li, Yi-Ping. "TNF-α is a mitogen in skeletal muscle." American Journal of Physiology-Cell Physiology 285, no. 2 (August 2003): C370—C376. http://dx.doi.org/10.1152/ajpcell.00453.2002.
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Emerging evidence suggests that tumor necrosis factor (TNF)-α plays a role in muscle repair. To determine whether TNF-α modulates satellite cell proliferation, the current study evaluated TNF-α effects on DNA synthesis in primary myoblasts and on satellite cell activation in adult mouse muscle. Exposure to recombinant TNF-α increased total DNA content in rat primary myoblasts dose-dependently over a 24-h period and increased the number of primary myoblasts incorporating 5-bromo-2′-deoxyuridine (BrdU) during a 30-min pulse labeling. Systemic injection of TNF-α stimulated BrdU incorporation by satellite cells in muscles of adult mice, whereas no BrdU was incorporated by satellite cells in control mice. TNF-α stimulated serum response factor (SRF) binding to the serum response element (SRE) present in the c- fos gene promoter and stimulated reporter gene expression controlled by the same element. Our data suggest that TNF-α activates satellite cells to enter the cell cycle and accelerates G1-to-S phase transition, and these actions may involve activation of early response genes via SRF.
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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.
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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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Dong, Xiuxue, Yu Cheng, Lingyun Qiao, Xin Wang, Cuiping Zeng, and Yanping Feng. "Male-Biased gga-miR-2954 Regulates Myoblast Proliferation and Differentiation of Chicken Embryos by Targeting YY1." Genes 12, no. 9 (August 27, 2021): 1325. http://dx.doi.org/10.3390/genes12091325.
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Previous studies have shown that gga-miR-2954 was highly expressed in the gonads and other tissues of male chickens, including muscle tissue. Yin Yang1 (YY1), which has functions in mammalian skeletal muscle development, and was predicted to be a target gene of gga-miR-2954. The purpose of this study was to investigate whether gga-miR-2954 plays a role in skeletal muscle development by targeting YY1, and evaluate its function in the sexual dimorphism development of chicken muscle. Here, all the temporal and spatial expression profiles in chicken embryonic muscles showed that gga-miR-2954 is highly expressed in males and mainly localized in cytoplasm. Gga-miR-2954 exhibited upregulated expression of in vitro myoblast differentiation stages. Next, through the overexpression and loss-of-function experiments performed in chicken primary myoblasts, we found that gga-miR-2954 inhibited myoblast proliferation but promoted differentiation. During myogenesis, gga-miR-2954 could suppress the expression of YY1, which promoted myoblast proliferation and inhibited the process of myoblast cell differentiation into multinucleated myotubes. Overall, these findings reveal a novel role of gga-miR-2954 in skeletal muscle development through its function of the myoblast proliferation and differentiation by suppressing the expression of YY1. Moreover, gga-miR-2954 may contribute to the sex difference in chicken muscle development.
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Vilquin, J. T., I. Kinoshita, B. Roy, M. Goulet, E. Engvall, F. Tomé, M. Fardeau, and J. P. Tremblay. "Partial laminin alpha2 chain restoration in alpha2 chain-deficient dy/dy mouse by primary muscle cell culture transplantation." Journal of Cell Biology 133, no. 1 (April 1, 1996): 185–97. http://dx.doi.org/10.1083/jcb.133.1.185.
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Laminin-2 is a component of skeletal and cardiac basal lamina expressed in normal mouse and human. Laminin alpha2 chain (LAMA2), however, is absent from muscles of some congenital muscular dystrophy patients and the dystrophia muscularis (dy/dy) mouse model. LAMA2 restoration was investigated following cell transplantation in vivo in dy/dy mouse. Allogeneic primary muscle cell cultures expressing the beta-galactosidase transgene under control of a muscular promoter, or histocompatible primary muscle cell cultures, were transplanted into dy/dy mouse muscles. FK506 immunosuppression was used in noncompatible models. All transplanted animals expressed LAMA2 in these immunologically-controlled models, and the degrees of LAMA2 restoration were shown to depend on the age of the animal at transplantation, on muscle pretreatment, and on duration time after transplantation in some cases. LAMA2 did not always colocalize with new or hybrid muscle fibers formed by the fusion of donor myoblasts. LAMA2 deposition around muscle fibers was often segmental and seemed to radiate from the center to the periphery of the injection site. Allogeneic conditionally immortalized pure myogenic cells expressing the beta-galactosidase transgene were characterized in vitro and in vivo. When injected into FK506-immunosuppressed dy/dy mice, these cells formed new or hybrid muscle fibers but essentially did not express LAMA2 in vivo. These data show that partial LAMA2 restoration is achieved in LAMA2-deficient dy/dy mouse by primary muscle cell culture transplantation. However, not all myoblasts, or myoblasts alone, or the muscle fibers they form are capable of LAMA2 secretion and deposition in vivo.
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Sun, Yujia, Tianqi Zhao, Yaoyao Ma, Xinyi Wu, Yongjiang Mao, Zhangping Yang, and Hong Chen. "New Insight into Muscle-Type Cofilin (CFL2) as an Essential Mediator in Promoting Myogenic Differentiation in Cattle." Bioengineering 9, no. 12 (November 25, 2022): 729. http://dx.doi.org/10.3390/bioengineering9120729.
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Meat quality and meat composition are not separated from the influences of animal genetic improvement systems; the growth and development of skeletal muscle are the primary factors in agricultural meat production and meat quality. Though the muscle-type cofilin (CFL2) gene has a crucial influence on skeletal muscle fibers and other related functions, the epigenetic modification mechanism of the CFL2 gene regulating meat quality remains elusive. After exploring the spatiotemporal expression data of CFL2 gene in a group of samples from fetal bovine, calf, and adult cattle, we found that the level of CFL2 gene in muscle tissues increased obviously with cattle age, whereas DNA methylation levels of CFL2 gene in muscle tissues decreased significantly along with cattle age by BSP and COBRA, although DNA methylation levels and mRNA expression levels basically showed an opposite trend. In cell experiments, we found that bta-miR-183 could suppress primary bovine myoblast differentiation by negatively regulated CFL2. In addition, we packaged recombinant adenovirus vectors for CFL2 gene knockout and overexpression and found that the CFL2 gene could promote the differentiation of primary bovine myoblasts by regulating marker genes MYOD, MYOG and MYH3. Therefore, CFL2 is an essential mediator for promoting myogenic differentiation by regulating myogenic marker genes in cattle myoblasts.
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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.
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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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Saha, Madhurima, Skylar A. Rizzo, Manashwi Ramanathan, Rylie M. Hightower, Katherine E. Santostefano, Naohiro Terada, Richard S. Finkel, et al. "Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy." Human Molecular Genetics 28, no. 14 (April 2, 2019): 2365–77. http://dx.doi.org/10.1093/hmg/ddz064.
Full textAbstract:
Abstract MEGF10 myopathy is a rare inherited muscle disease that is named after the causative gene, MEGF10. The classic phenotype, early onset myopathy, areflexia, respiratory distress and dysphagia, is severe and immediately life-threatening. There are no disease-modifying therapies. We performed a small molecule screen and follow-up studies to seek a novel therapy. A primary in vitro drug screen assessed cellular proliferation patterns in Megf10-deficient myoblasts. Secondary evaluations were performed on primary screen hits using myoblasts derived from Megf10−/− mice, induced pluripotent stem cell-derived myoblasts from MEGF10 myopathy patients, mutant Drosophila that are deficient in the homologue of MEGF10 (Drpr) and megf10 mutant zebrafish. The screen yielded two promising candidates that are both selective serotonin reuptake inhibitors (SSRIs), sertraline and escitalopram. In depth follow-up analyses demonstrated that sertraline was highly effective in alleviating abnormalities across multiple models of the disease including mouse myoblast, human myoblast, Drosophila and zebrafish models. Sertraline also restored deficiencies of Notch1 in disease models. We conclude that SSRIs show promise as potential therapeutic compounds for MEGF10 myopathy, especially sertraline. The mechanism of action may involve the Notch pathway.
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Ross, J. J., M. J. Duxson, and A. J. Harris. "Neural determination of muscle fibre numbers in embryonic rat lumbrical muscles." Development 100, no. 3 (July 1, 1987): 395–409. http://dx.doi.org/10.1242/dev.100.3.395.
Full textAbstract:
The generation and development of muscle cells in the IVth hindlimb lumbrical muscle of the rat was studied following total or partial denervation. Denervation was carried out by injection of beta-bungarotoxin (beta-BTX), a neurotoxin which binds to and destroys peripheral nerves. Primary myotubes were generated in denervated muscles and reached their normal stable number on embryonic day 17 (E17). This number was not maintained and denervated muscles examined on E19 or E21 contained many degenerating primary myotubes. Embryos injected with beta-bungarotoxin (beta-BTX) on E12 or E13 suffered a partial loss of motoneurones, resulting in a reduced number of axons in the L4 ventral root (the IVth lumbrical muscle is supplied by axons in L4, L5 and L6 ventral roots) and reduced numbers of nerve terminals in the intrinsic muscles of the hindfoot. Twitch tension measurements showed that all myotubes in partly innervated muscles examined on E21 contracted in response to nerve stimulation. Primary myotubes were formed and maintained at normal numbers in muscles with innervation reduced throughout development, but a diminished number of secondary myotubes formed by E21. The latter was correlated with a reduction in number of mononucleate cells within the muscles. If beta-BTX was injected on E18 to denervate muscles after primary myotube formation was complete, E21 embryo muscles contained degenerating primary myotubes. After injection to denervate muscles on E19, the day secondary myotubes begin to form, E21 muscles possessed normal numbers of primary myotubes. In both cases, secondary myotube formation had stopped about 1 day after the injection and the number of mononucleate cells was greatly reduced, indicating that cessation of secondary myotube generation was most probably due to exhaustion of the supply of competent myoblasts. We conclude that nerve terminals regulate the number of secondary myotubes by stimulating mitosis in a nerve-dependent population of myoblasts and that activation of these myoblasts requires the physical presence of nerve terminals as well as activation of contraction in primary myotubes.
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Skuk, Daniel, Nicolas Caron, Marlyne Goulet, Brigitte Roy, Francisco Espinosa, and Jacques P. Tremblay. "Dynamics of the Early Immune Cellular Reactions after Myogenic Cell Transplantation." Cell Transplantation 11, no. 7 (October 2002): 671–81. http://dx.doi.org/10.3727/000000002783985378.
Full textAbstract:
The role of immune cells in the early donor cell death/survival following myoblast transplantation is confusing, one of the reasons being the lack of data about the immune reactions following cell transplantation. We used outbred mice as hosts for transplantation of primary cultured muscle cells and T-antigen-immortalized myoblasts. The host muscles were analyzed 1 h to 7 days after cell injection. No net loss of the donor primary cultured cell population was observed in this period. The immune cellular reaction in this case was: 1) a brief (<48 h) neutrophil invasion; 2) macrophage infiltration from days 1 to 7; 3) a specific response involving CTL and few NK cells (days 6 and 7), preceded by a low CD4+ cell infiltration starting at day 3. In contrast, donor-immortalized myoblasts completely disappeared during the 7-day follow-up. In this case, an intense infiltration of CTL and macrophages, with moderate CD4+ infiltration and lower amounts of NK cells, was observed starting at day 2. The nonspecific immune response at days 0 and 1 was similar for both types of donor cells. The present observations set a basis to interpret the role of immune cells on the early death/survival of donor cells following myoblast transplantation.
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Aguiari, Paola, Yan-Yun Liu, Astgik Petrosyan, Sheue-Yann Cheng, Gregory A. Brent, Laura Perin, and Anna Milanesi. "Persistent COUP-TFII Expression Underlies the Myopathy and Impaired Muscle Regeneration Observed in Resistance to Thyroid Hormone-Alpha." Journal of the Endocrine Society 5, Supplement_1 (May 1, 2021): A814. http://dx.doi.org/10.1210/jendso/bvab048.1658.
Full textAbstract:
Abstract Myopathic changes, including muscular dystrophy and weakness, are commonly described in hypothyroid and hyperthyroid patients. Thyroid hormone signaling, via activation of thyroid nuclear receptor alpha (THRA), plays an essential role in maintaining muscle mass, function, and regeneration. A mouse model of resistance to thyroid hormone carrying a frameshift mutation in the THRA gene (THRA-PV) is associated with accelerated skeletal muscle loss with aging and impaired regeneration after injury(1,2). We previously demonstrated that the expression of nuclear orphan receptor chicken ovalbumin upstream promoter-factor II (COUP-TFII, or Nr2f2) persists during myogenic differentiation in THRA-PV myoblasts and skeletal muscle of aged THRA- PV mice. COUP-TFII is known to regulate myogenesis negatively and has a role in Duchenne-like Muscular Dystrophies(3). COUP-TFII physically and functionally interacts with THRA in primary myoblasts isolated from WT and THRA-PV mice, as demonstrated via co-immunoprecipitation and chromatin-immunoprecipitation. We observed that satellite cells from THRA-PV mice display impaired myoblast proliferation and in vitro myogenic differentiation compared to WT cells. However, the silencing of COUP-TFII expression using siRNA probes restores in vitro myogenic potential of THRA-PV myoblasts and shifts the mRNA expression profile closer to WT myoblasts, with a higher proliferation of myoblasts and a higher number of fully differentiated myotubes after 5 days of myogenic induction. Moreover, RNAseq analysis on myoblasts from THRA-PV mice after COUP-TFII knockdown shows that COUP-TFII silencing reverses the transcriptomic profile of THRA-PV myoblasts and results in reactivation of pathways involved in muscle function and extracellular matrix remodeling/deposition. These findings indicate that the persistent COUP-TFII expression in THRA-PV mice is responsible for the abnormal muscle phenotype. In conclusion, COUP-TFII and THRA cooperate during murine post-natal myogenesis, and COUP-TFII is critical for the accelerated skeletal muscle loss with aging and impaired muscle regeneration after injury in THRA-PV mice. These studies can help increase our knowledge of the mechanisms involved in thyroid hormone signaling during skeletal muscle regeneration, ultimately increasing the possibility of designing more specific treatments for patients with thyroid hormone-induced myopathies. References: 1. Milanesi, A., et al., Endocrinology 2016; 2. Kaneshige, M. et al., Proc Natl Acad Sci U S 2001; 3. Lee HJ, et al, Sci Rep. 2017.
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Fornaro, Mara, Aaron C. Hinken, Saul Needle, Erding Hu, Anne-Ulrike Trendelenburg, Angelika Mayer, Antonia Rosenstiel, et al. "Mechano-growth factor peptide, the COOH terminus of unprocessed insulin-like growth factor 1, has no apparent effect on myoblasts or primary muscle stem cells." American Journal of Physiology-Endocrinology and Metabolism 306, no. 2 (January 15, 2014): E150—E156. http://dx.doi.org/10.1152/ajpendo.00408.2013.
Full textAbstract:
A splice form of IGF-1, IGF-1Eb, is upregulated after exercise or injury. Physiological responses have been ascribed to the 24-amino acid COOH-terminal peptide that is cleaved from the NH3-terminal 70-amino acid mature IGF-1 protein. This COOH-terminal peptide was termed “mechano-growth factor” (MGF). Activities claimed for the MGF peptide included enhancing muscle satellite cell proliferation and delaying myoblast fusion. As such, MGF could represent a promising strategy to improve muscle regeneration. Thus, at our two pharmaceutical companies, we attempted to reproduce the claimed effect of MGF peptides on human and mouse muscle myoblast proliferation and differentiation in vitro. Concentrations of peptide up to 500 ng/ml failed to increase the proliferation of C2C12 cells or primary human skeletal muscle myoblasts. In contrast, all cell types exhibited a proliferative response to mature IGF-1 or full-length IGF-1Eb. MGF also failed to inhibit the differentiation of myoblasts into myotubes. To address whether the response to MGF was lost in these tissue culture lines, we measured proliferation and differentiation of primary mouse skeletal muscle stem cells exposed to MGF. This, too, failed to demonstrate a significant effect. Finally, we tested whether MGF could alter a separate documented in vitro effect of the peptide, activation of p-ERK, but not p-Akt, in cardiac myocytes. Although a robust response to IGF-1 was observed, there were no demonstrated activating responses from the native or a stabilized MGF peptide. These results call in to question whether there is a physiological role for MGF.
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Shintani-Ishida, Kaori, Riko Tsurumi, and Hiroshi Ikegaya. "Decrease in the expression of muscle-specific miRNAs, miR-133a and miR-1, in myoblasts with replicative senescence." PLOS ONE 18, no. 1 (January 17, 2023): e0280527. http://dx.doi.org/10.1371/journal.pone.0280527.
Full textAbstract:
Muscles that are injured or atrophied by aging undergo myogenic regeneration. Although myoblasts play a pivotal role in myogenic regeneration, their function is impaired with aging. MicroRNAs (miRNAs) are also involved in myogenic regeneration. MiRNA (miR)-1 and miR-133a are muscle-specific miRNAs that control the proliferation and differentiation of myoblasts. In this study, we determined whether miR-1 and miR-133a expression in myoblasts is altered with cellular senescence and involved in senescence-impaired myogenic differentiation. C2C12 murine skeletal myoblasts were converted to a replicative senescent state by culturing to a high passage number. Although miR-1 and miR-133a expression was largely induced during myogenic differentiation, expression was suppressed in cells at high passage numbers (passage 10 and/or passage 20). Although the senescent myoblasts exhibited a deterioration of myogenic differentiation, transfection of miR-1 or miR-133a into myoblasts ameliorated cell fusion. Treatment with the glutaminase 1 inhibitor, BPTES, removed senescent cells from C2C12 myoblasts with a high passage number, whereas myotube formation and miR-133a expression was increased. In addition, primary cultured myoblasts prepared from aged C57BL/6J male mice (20 months old) exhibited a decrease in miR-1 and miR-133a levels compared with younger mice (3 months old). The results suggest that replicative senescence suppresses muscle-specific miRNA expression in myoblasts, which contributes to the senescence-related dysfunction of myogenic regeneration.
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Nikovits, William, Gordon M. Cann, Ruijin Huang, Bodo Christ, and Frank E. Stockdale. "Patterning of fast and slow fibers within embryonic muscles is established independently of signals from the surrounding mesenchyme." Development 128, no. 13 (July 1, 2001): 2537–44. http://dx.doi.org/10.1242/dev.128.13.2537.
Full textAbstract:
During embryonic development, and before functional innervation, a highly stereotypic pattern of slow- and fast-contracting primary muscle fibers is established within individual muscles of the limbs, from distinct populations of myoblasts. A difference between the fiber-type pattern found within chicken and quail pectoral muscles was exploited to investigate the contributions of somite-derived myogenic precursors and lateral plate-derived mesenchymal stroma to the establishment of muscle fiber-type patterns. Chimeric chicken/quail embryos were constructed by reciprocal transplantation of somites or lateral plate mesoderm at stages prior to muscle formation. Muscle fibers derived from quail myogenic precursors that had migrated into chicken stroma showed a quail pattern of mixed fast- and slow-contracting muscle fibers. Conversely, chicken myogenic precursors that had migrated into quail stroma showed a chicken pattern of nearly exclusive fast muscle fiber formation. These results demonstrate in vivo an intrinsic commitment to fiber-type on the part of the myoblast, independent of extrinsic signals it receives from the mesenchymal stroma in which it differentiates.
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Tadros, L. B., P. M. Taylor, and M. J. Rennie. "Characteristics of glutamine transport in primary tissue culture of rat skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 265, no. 1 (July 1, 1993): E135—E144. http://dx.doi.org/10.1152/ajpendo.1993.265.1.e135.
Full textAbstract:
Glutamine transport was studied in preconfluent monolayered, mononucleated myoblasts (4 days old) and in fused, multinucleated, differentiated myotubes (10 days old), both prepared from neonatal rat skeletal muscle. The initial (60 s) rate of 50 microM glutamine uptake in myoblasts and myotubes was stereospecific, saturable, and largely (80%) Na+ dependent. At glutamine concentrations of 0.01–1 mM, Na(+)-dependent uptake showed saturation kinetics: in myoblasts, the Michaelis constant (Km) was 197 +/- 38 microM, maximum velocity (Vmax) was 1,165 +/- 60 pmol.min-1.mg protein-1; in myotubes, Km was 174 +/- 51 microM and Vmax was 1,435 +/- 47 pmol.min-1.mg protein-1. The Na(+)-dependent glutamine uptake was Li+ tolerant in both myoblasts and myotubes. The Na(+)-dependent uptake of 50 microM L-[3H]glutamine was investigated in the presence of various amino acids at 0.01–10 mM. Histidine and asparagine competitively inhibited glutamine uptake, but inhibition by serine was noncompetitive; glutamate, arginine, leucine, and 2-aminobicyclo(2,2,1)heptane-2-carboxylate (BCH) had no significant inhibitory effects; 2-(methyl-amino)isobutyrate (MeAIB) caused a small but significant inhibition. In parallel with a stimulation of glucose transport, addition of insulin stimulated Na(+)-dependent glutamine uptake within 1 h by a maximum of 27% in myoblasts and 42% in myotubes (half-maximal stimulation at 0.3 nM insulin). Glucagon had no effect. Kinetic analysis revealed that the insulin-stimulated increase in glutamine transport was due to a Vmax effect, which was cycloheximide inhibitable. The insulin-stimulated increase was Li+ tolerant and not inhibited by MeAIB or cysteine at 1 mM. The results indicate that the predominant glutamine transporter of neonatal rat skeletal muscle cells in primary tissue culture in System Nm. System Nm also appears to be the major insulin-sensitive glutamine transport component in skeletal muscle. Primary muscle culture appears to be a useful preparation for studying glutamine transport and its regulation.
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Jing, Jiongjie, Yong Pu, Jeremy Gingrich, and Almudena Veiga-Lopez. "Gestational Exposure to Bisphenol A and Bisphenol S Leads to Fetal Skeletal Muscle Hypertrophy Independent of Sex." Toxicological Sciences 172, no. 2 (September 10, 2019): 292–302. http://dx.doi.org/10.1093/toxsci/kfz198.
Full textAbstract:
Abstract Gestational exposure to bisphenol A (BPA) can lead to offspring insulin resistance. However, despite the role that the skeletal muscle plays in glucose homeostasis, it remains unknown whether gestational exposure to BPA, or its analog bisphenol S (BPS), impairs skeletal muscle development. We hypothesized that gestational exposure to BPA or BPS will impair fetal muscle development and lead to muscle-specific insulin resistance. To test this, pregnant sheep (n = 7–8/group) were exposed to BPA or BPS from gestational day (GD) 30 to 100. At GD120, fetal skeletal muscle was harvested to evaluate fiber size, fiber type, and gene and protein expression related to myogenesis, fiber size, fiber type, and inflammation. Fetal primary myoblasts were isolated to evaluate proliferation and differentiation. In fetal skeletal muscle, myofibers were larger in BPA and BPS groups in both females and males. BPA females had higher MYH1 (reflective of type-IIX fast glycolytic fibers), whereas BPS females had higher MYH2 and MYH7, and higher myogenic regulatory factors (Myf5, MyoG, MyoD, and MRF4) mRNA expression. No differences were observed in males. Myoblast proliferation was not altered in gestationally BPA- or BPS-exposed myoblasts, but upon differentiation, area and diameter of myotubes were larger independent of sex. Females had larger myofibers and myotubes than males in all treatment groups. In conclusion, gestational exposure to BPA or BPS does not result in insulin resistance in fetal myoblasts but leads to fetal fiber hypertrophy in skeletal muscle independent of sex and alters fiber type distribution in a sex-specific manner.
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Porrello, Alessandro, Maria Antonietta Cerone, Sabrina Coen, Aymone Gurtner, Giulia Fontemaggi, Letizia Cimino, Giulia Piaggio, Ada Sacchi, and Silvia Soddu. "P53 Regulates Myogenesis by Triggering the Differentiation Activity of Prb." Journal of Cell Biology 151, no. 6 (December 11, 2000): 1295–304. http://dx.doi.org/10.1083/jcb.151.6.1295.
Full textAbstract:
The p53 oncosuppressor protein regulates cell cycle checkpoints and apoptosis, but increasing evidence also indicates its involvement in differentiation and development. We had previously demonstrated that in the presence of differentiation-promoting stimuli, p53-defective myoblasts exit from the cell cycle but do not differentiate into myocytes and myotubes. To identify the pathways through which p53 contributes to skeletal muscle differentiation, we have analyzed the expression of a series of genes regulated during myogenesis in parental and dominant–negative p53 (dnp53)-expressing C2C12 myoblasts. We found that in dnp53-expressing C2C12 cells, as well as in p53−/− primary myoblasts, pRb is hypophosphorylated and proliferation stops. However, these cells do not upregulate pRb and have reduced MyoD activity. The transduction of exogenous TP53 or Rb genes in p53-defective myoblasts rescues MyoD activity and differentiation potential. Additionally, in vivo studies on the Rb promoter demonstrate that p53 regulates the Rb gene expression at transcriptional level through a p53-binding site. Therefore, here we show that p53 regulates myoblast differentiation by means of pRb without affecting its cell cycle–related functions.
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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.
Full textAbstract:
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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Gower, H. J., S. E. Moore, G. Dickson, V. L. Elsom, R. Nayak, and F. S. Walsh. "Cloning and characterization of a myoblast cell surface antigen defined by 24.1D5 monoclonal antibody." Development 105, no. 4 (April 1, 1989): 723–31. http://dx.doi.org/10.1242/dev.105.4.723.
Full textAbstract:
Monoclonal antibody 24.1D5 reacts specifically with an epitope expressed on the cell surface of mononucleate myoblasts in primary cultures of human skeletal muscle cells, but not with either multinucleate myotubes or fibroblasts. Polypeptides of 60 and 100 X 10(3) Mr were identified by immunoblotting with the McAb. Human muscle cDNAs encoding the 24.1D5 epitope were used to study further the structure and expression of 24.1D5 during skeletal muscle development. Two mRNA species of 3.0 and 2.5 kb were identified in primary cultures of human skeletal muscle and in mouse muscle cell lines. The levels of both transcripts decreased during myotube formation in vitro and were similarly decreased during myogenesis in the mouse embryo. 24.1D5 mRNAs were expressed by multipotential cells and myoblast derivatives of the mouse embryonic cell line C3H10T1/2, suggesting that 24.1D5 is expressed at an early stage during skeletal muscle development.
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Wilson, Magdalene O., Kathleen T. Scougall, Jarupa Ratanamart, Elizabeth A. McIntyre, and James A. M. Shaw. "Tetracycline-regulated secretion of human (pro)insulin following plasmid-mediated transfection of human muscle." Journal of Molecular Endocrinology 34, no. 2 (April 2005): 391–403. http://dx.doi.org/10.1677/jme.1.01646.
Full textAbstract:
Long-term secretion of insulin by host muscle following transduction with an insulin gene construct offers the potential of gene therapy for diabetes without immunosuppression. Clinical implementation will be dependent on proof of principle in human tissue and a system for safely regulating basal insulin levels. Liposomal co-transfection with a tetracycline-responsive wild type human preproinsulin (pTRE-hppI1) or mutant construct (pTRE-hppI4), in which PC2 and PC3 cleavage sites were altered to form tetrabasic consensus sites for furin, together with pTet-off (coding for a transactivating protein) was evaluated in the C2C12 mouse myoblast cell line and human myoblasts following establishment in primary culture. In the absence of tetracycline, (pro)insulin secretion in C2C12 and human myoblasts transfected with tetracycline-responsive hppI1 and hppI4 constructs was comparable to that following transfection with equivalent constructs under the control of a constitutively active cytomegaloviral promoter. Percentage processing to mature insulin was <5% in C2C12 and human myoblasts transfected with pTet-off/pTRE-hppI1 but >90% in C2C12 cells and 45–60% in human myoblasts on transfection with pTet-off/pTRE-hppI4. Incremental dose-responsive suppression of proinsulin secretion was demonstrated in C2C12 and human myoblasts expressing pTet-off/pTRE-hppI1 following incubation with tetracycline (0–100 μg/ml) for up to 72 h. Reversibility was confirmed following tetracycline withdrawal. Dose-responsive tetracycline-inducible repression of mature insulin secretion was confirmed in C2C12 cells following transfection with pTet-off/pTRE-hppI4. Regulation of human proinsulin biosynthesis and secretion has been attained in vivo following plasmid-mediated gene transfer to rat skeletal muscle and oral tetracycline administration. In conclusion, processing to mature insulin has been confirmed following plasmid-mediated gene transfer to human muscle in addition to in vitro- and in vivo-regulated human proinsulin secretion employing the safe and well-tolerated antibiotic, tetracycline.
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Akpulat, Ugur. "Morphological examination is more effective than cell viability assays in the characterization of myotube atrophy." Annals of Medical Research 30, no. 11 (2023): 1. http://dx.doi.org/10.5455/annalsmedres.2023.07.170.
Full textAbstract:
Skeletal muscle atrophy is a significant health problem associated particularly with aging, cancer, and metabolic diseases, leading to a decline in quality of life and responses to treatment. During skeletal muscle atrophy, the balance between protein synthesis and degradation in the tissue is disrupted in favor of protein degradation. This condition results in a reduction in muscle mass and loss of strength and function in the muscles. In order to elucidate the molecular mechanisms causing skeletal muscle atrophy, prevent the processes leading to atrophy, and test the effectiveness of emerging therapeutics, in vitro models that mimic atrophic conditions are commonly used. In these models, typically utilizing primary myoblasts obtained from tissues or myoblast cell lines, either cell viability assays or myotube morphology analyses are employed to determine the degree of atrophy occurring during differentiation. Here, the commonly used MTT cell viability assay and myotube diameter analysis were mutually evaluated to determine which one better characterizes myotube atrophy. While myotube diameter measurement can grade the extent of atrophy in myotubes, the MTT assay, although unable to directly assess the degree of myotube atrophy, demonstrated that the decrease in cell viability indicates the cause of myoblast atrophy.