• BMB Reports
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• v.51 no.7
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• pp.350-355
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• 2018

HnRNPK is a multifunctional protein that participates in chromatin remodeling, transcription, RNA splicing, mRNA stability and translation. Here, we uncovered the function of hnRNPK in regulating the proliferation and differentiation of myoblasts. hnRNPK was mutated in the C2C12 myoblast cell line using the CRISPR/Cas9 system. A decreased proliferation rate was observed in hnRNPK-mutated cells, suggesting an impaired proliferation phenotype. Furthermore, increased G2/M phase, decreased S phase and increased sub-G1 phase cells were detected in the hnRNPK-mutated cell lines. The expression analysis of key cell cycle regulators indicated mRNA of Cyclin A2 was significantly increased in the mutant myoblasts compared to the control cells, while Cyclin B1, Cdc25b and Cdc25c were decreased sharply. In addition to the myoblast proliferation defect, the mutant cells exhibited defect in myotube formation. The myotube formation marker, myosin heavy chain (MHC), was decreased sharply in hnRNPK-mutated cells compared to control myoblasts during differentiation. The deficiency in hnRNPK also resulted in the repression of Myog expression, a key myogenic regulator during differentiation. Together, our data demonstrate that hnRNPK is required for myoblast proliferation and differentiation and may be an essential regulator of myoblast function.

• The Korean Journal of Zoology
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• v.36 no.3
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• pp.433-438
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• 1993

In the previous paper (Choi et al., 1992), we found that a large but transient elevation in intracellular cGMP levels occur concomitant with the myoblast fusion. To establish the physiological significance of the elevation of cGMP levels, the change in guanylate cyclase activity dudng myoblast fusion and the correlation hetween various chemicals that may affect guanylate cyclase adivity and myoblast fusion were examined. Sodium nitroprusside, a nitric oxide-forming compound, induced a precocious fusion and increased guanylate cyclase activity compared to the control. Furthermore, L-NG-monomethyl arginine, specific inhibitor of L-arginine: nitric oxide synthase, inhibited the cell fusion in a dose-dependent manner, without affecting biochemical differentiation. On the basis of our present findings, we propose that the onset of myoblast fusion is somehow correlated with the rise in cellular cGMP levels that is regulated by the activation or inhibItIon of soluble guanylate cyclase, via as yet undefined mechanism but possibly through L-arginine: nitric oxide pathway.

• The Korean Journal of Zoology
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• v.32 no.1
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• pp.40-47
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• 1989

Alteration of intracellular calcium ion Concentration by adding of either calcium ionophore A23187 or EGTA in culture medium at 24 hr after cell plating resulted in remarkable changes in the progression of differentiation of chick embryo myoblast. When separated myoblast proteins using two-dimensional gel electrophoresis, synthesis patterns of several proteins changed upon the addition of either A23187 or EGTA. Treatment of A23187 and calciumactivated neutral protease at 24 hr after initial plating caused an increase in the rate of fusion compared to control culture. However, EGTA inhibited the myoblast fusion to a marked degree. A23187 treated at 24hr also increased the activity of protein kinase C during the fusionprogressed period. It seems that intracellular calcium ion plays an important role in the myoblast differentiation in vitro together with the protein kinase C and calcium-activated neutral protease.

• The Korea Journal of Herbology
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• v.35 no.3
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• pp.9-16
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• 2020

Objectives : Sarcopenia is a disease that leads to a decrease in skeletal muscle, and the importance of prevention and treatment thereof is increasing in an aging society. However, there is a definite limitation of exercise therapy for sarcopenia, and thus, there is an urgent need for a pharmacologic research to the treatment of sarcopenia. Methods : 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay was performed to confirm the antioxidant efficacy of water extract of Lacca Sinica Exsiccata (WELSE). To determine the effect of WELSE on myoblast activity, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed. To confirm the effect of WELSE on the differentiation of myoblast into myotubes, protein expression levels of myosin heavy chain 3 (Myh3) and paired box 3/7 (pax3/7) were confirmed by immunoblot analysis. In addition, immunofluorescence microscopy was performed to confirm the effect on myotube formation of WELSE. Results : It was confirmed that WELSE had high antioxidant activity and showed no cytotoxicity to myoblast up to 200 ㎍/㎖ concentration. Myoblast was treated with WELSE at a concentration of 100 ㎍/㎖ and differentiated for 5 days. The expression of Myh3, which forms myotubes, was promoted and the morphology of myotubes was changed and Increasing the thickness. Conclusions : In this paper, we confirmed the excellent antioxidant efficacy of WELSE and positive effects on muscle differentiation and myotube formation. These results suggest valuable as a material for pharmaceutical research on the prevention and treatment of sarcopenia.

• The Korean Journal of Zoology
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• v.27 no.2
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• pp.73-84
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• 1984

In order to investigate the effect of neurotransmitter on myoblast differentiation in vitro, the effects of dopamine and epinephrine on myoblast fusion and on the intracellular cAMP level in cultured myoblasts were examined. Dopamine $(3\\times10^{-5}M)$ and epinephrine $(3\\times10^{-5}M)$, when added at 34 hr after cell plating, markedly inhibited myoblast fusion, and dopamine was more potent than epinephrine. Both dopamine and epinephrine had no effect on intracellular cAMP level. At the same time, exogeneous dbcAMP, $PGE_1$, and aspirin were used to examine whether cAMP is involved in myoblast differentiation. dbcAMP $(1\\times10^{-4}M)$ inhibited myoblast fusion, whereas $PGE_1 (3\\times10^{-6}M)$ had no inhibitory effect, rather enhancing myoblast fusion. Aspirin, an inhibitor of PG synthetase, was shown to inhibit myoblast fusion. Possible mechanism by which dopamine or epinephrine at a specific concentration used inhibits myoblast fusion is discussed.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.8
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• pp.1183-1189
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• 2017

Objective: Owing to the public availability of complete genome sequences, including avian species, massive bioinformatics analyses may be conducted for computational gene prediction and the identification of gene regulatory networks through various informatics tools. However, to evaluate the biofunctional activity of a predicted target gene, in vivo and in vitro functional genomic analyses should be a prerequisite. Methods: Due to a lack of quail genomic sequence information, we first identified the partial genomic structure and sequences of the quail SH3 domain containing ring finger 2 (SH3RF2) gene. Subsequently, SH3RF2 was knocked out using clustered regularly interspaced short palindromic repeat/Cas9 technology and single cell-derived SH3RF2 mutant sublines were established to study the biofunctional activity of SH3RF2 in quail myoblast (QM7) cells during muscle differentiation. Results: Through a T7 endonuclease I assay and genotyping analysis, we established an SH3RF2 knockout (KO) QM7#4 subline with 61 and 155 nucleotide deletion mutations in SH3RF2. After the induction of myotube differentiation, the expression profiles were analyzed and compared between regular QM7 and SH3RF2 KO QM7#4 cells by global RNA sequencing and bioinformatics analysis. Conclusion: We did not detect any statistically significant role of SH3RF2 during myotube differentiation in QM7 myoblast cells. However, additional experiments are necessary to examine the biofunctional activity of SH3RF2 in cell proliferation and muscle growth.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.7
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• pp.1029-1036
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• 2017

Objective: In the livestock industry, the regulatory mechanisms of muscle proliferation and differentiation can be applied to improve traits such as growth and meat production. We investigated the regulatory pathway of MyoD and its role in muscle differentiation in quail myoblast cells. Methods: The MyoD gene was mutated by the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology and single cell-derived MyoD mutant sublines were identified to investigate the global regulatory mechanism responsible for muscle differentiation. Results: The mutation efficiency was 73.3% in the mixed population, and from this population we were able to establish two QM7 MyoD knockout subline (MyoD KO QM7#4) through single cell pick-up and expansion. In the undifferentiated condition, paired box 7 expression in MyoD KO QM7#4 cells was not significantly different from regular QM7 (rQM7) cells. During differentiation, however, myotube formation was dramatically repressed in MyoD KO QM7#4 cells. Moreover, myogenic differentiation-specific transcripts and proteins were not expressed in MyoD KO QM7#4 cells even after an extended differentiation period. These results indicate that MyoD is critical for muscle differentiation. Furthermore, we analyzed the global regulatory interactions by RNA sequencing during muscle differentiation. Conclusion: With CRISPR/Cas9-mediated genomic editing, single cell-derived sublines with a specific knockout gene can be adapted to various aspects of basic research as well as in functional genomics studies.

• Animal cells and systems
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• v.1 no.1
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• pp.63-69
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• 1997

We have previously reported that NF-kB is involved in the regulation of nitric oxide synthase gene expression during differentiation of chick embryonic myoblasts. However, how NF-kB is timely activated during myogenesis remains elusive. One of the most prominent events in myogenesis is myoblast membrane fusion, which is accompanied with massive cytoskeletal reorganization. Here we show that the activity of NF-kB markedly increases in L6 rat myogenic cells that have just initiated morphological changes by treating nocodazole, a microtubule-disrupting agent. Furthermore, the induction of NF-kB activation was closely correlated with the myoblast fusion. In addition, a variety of agents that disrupt microtubules stimulated the myoblast fusion as well as the induction of NF-kB activation. In contrast, taxol, a microtubule-stabilizing agent, suppressed the induction of NF-kB activation and inhibited spontaneous differentiation of L6 cells as well. In addition, we found that the NF-KB in the cells consists of p50/p65 heterodimers. These results support the idea that reorganization of microtubule at early stages of differentiation plays a role as a signal for NF-KB activation during myogenesis.

• Animal cells and systems
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• v.1 no.4
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• pp.641-646
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• 1997

Lysophosphatidic acid (LPA; 1-acyl-glycerol-3-phosphate) has been known as an intercellular phospholipid messenger with a wide range of biological activities. In this study, the effect of LPA on both the proliferation and differentiation of rat E63 myoblasts has been investigated. In the serum-free Insulin-Transferrin-Selenium (ITS) media, the proliferation of E63 cells was largely restricted. Addition of LPA into the ITS media strongly promoted the cell proliferation and resulted in two to four fold increase of cell number. Furthermore, it appeared to increase the percent fusion in a dose-dependent manner up to 15 ug/ml. The synthesis of myosin heavy chain (MHC) was increased by LPA as well. These results indicate that LPA is able to promote both cell proliferation and differentiation in rat E63 myoblasts. Suramin, known to have uncoupling activity on growth factor-receptor interaction, was tested for antagonistic activity in myoblast proliferation and differentiation. Myoblasts grown in the ITS medium containing LPA were able to proliferate well even in the presence high concentration of suramin whereas myoblast differentiation was completely blocked by 30 ug/ml of suramin. The inhibitory effect of suramin on the myoblast differentiation was completely reversible by removing the suramin. This result indicates that the intracellular signaling pathway of LPA leading to cell proliferation might be distinct from that leading to cell differentiation on E63 myoblasts. Also, the antagonistic effect of suramin suggests that the differentiation activity elicited by LPA might be mediated by a specific G protein-coupled receptor.

• Journal of Physiology & Pathology in Korean Medicine
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• v.28 no.4
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• pp.411-416
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• 2014

Simple formulas (單方) of muscle section in Donguibogam (東醫寶鑑) have long been prescribed for strengthening muscle and/or prevention of age-related muscle loss. However, biological activity and mechanisms by which they influence myoblast differentiation have not been studied. Therefore, in this study, we evaluated the effects of 14 simple formulas on myoblast differentiation in C2C12 myoblast cells under non-cytotoxic ($0.5mg/m{\ell}$) conditions. C2C12 cells were treated with water extracts of simple formulas for 72 h, and RT-PCR was performed to determine the gene expression levels of myogenic regulatory factors (MRFs), including myoD, myogenin, MRF4, myf5, and insulin like growth factor-1 (IGF-1). Treatment with Colocasiae Rhizoma (CR), Pini Semen (PS), and Sesami Semen (SS) resulted in a significant increase in expression of myogenin in C2C12 cells. Treatment with Allii Macrostemi Bulbus (AM), Colocasiae Rhizoma (CR), and Pini Semen (PS) also resulted in increased expression of MRF4 in C2C12 cells. In addition, enhanced expression of IGF-1 was observed in treatment with Eucommiae cortex (EC), Dioscoreae Rhizoma (DR), Colocasiae Rhizoma (CR), Pini Semen (PS), and Sesami Semen (SS) in C2C12 cells. These results indicate that simple formulas of muscle section in Donguibogam could potentially enhance myoblast differentiation at least in part via increasing expression of myogenin, and/or MRF4 and/or IGF-1.