• Proceedings of the Korean Vacuum Society Conference
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• 2015.08a
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• pp.158-158
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• 2015

Myoblast are myogenic precursors that proliferate, activate, and differentiate on muscle injury to sustain the regenerative capacity of skeletal muscle; The neuronal isoform of nitric oxide synthase (nNOS, termed also NOS-I) is expressed in normal adult skeletal muscle, suggesting important functions for Nitric oxide (NO) in muscle biology1,2,3. However, the expression and subcellular localization of NO in muscle development and myoblast differentiation are largely unknown. In this study, we examined effects of the nitric oxide generated by a microwave plasma torch, on proliferation/differentiation of rat myoblastic L6 cells. Experimental data pertaining to nitric oxide production are presented in terms of the oxygen input in units of cubic centimetres per minute. The various levels of nitric oxide are observed depending on the flow rate of nitrogen gas, the ratio of oxygen gas, and the microwave power4. In order to evaluate the potential of nitric oxide as an activator of cell differentiation, we applied nitric oxide generated from the microwave plasma torch to L6 skeletal muscles. Differentiation of L6 cells into myotubes was significantly enhanced the differentiation after nitric oxide treatment. Nitric oxide treatment also increase the expression of myogenesis marker proteins and mRNA level, such as myogenin and myosin heavy chain (MHC), as well as cyclic guanosine monophosphate (cGMP), However during the myotube differentiation we found that NO activate oxidative stress signaling erks expression. Therefore, these results establish a role of NO and cGMP in regulating myoblast differentiation and elucidate their mechanism of action, providing a direct link with oxidative stress signalling, which is a key player in myogenesis. Based on these findings, nitric oxide generated by plasma can be used as a possible activator of cell differentiation and tissue regeneration.

• 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.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.4
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• pp.479-486
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• 2016

A previous genome-wide association study (GWAS) exposed histone deacetylase 2 (HDAC2) as a possible candidate gene for breast muscle weight in chickens. The present research has examined the possible role of HDAC2 in skeletal muscle development in chickens. Gene expression was measured by quantitative polymerase chain reaction in breast and thigh muscles during both embryonic (four ages) and post-hatch (five ages) development and in cultures of primary myoblasts during both proliferation and differentiation. The expression of HDAC2 increased significantly across embryonic days (ED) in breast (ED 14, 16, 18, and 21) and thigh (ED 14 and 18, and ED 14 and 21) muscles suggesting that it possibly plays a role in myoblast hyperplasia in both breast and thigh muscles. Transcript abundance of HDAC2 identified significantly higher in fast growing muscle than slow growing in chickens at d 90 of age. Expression of HDAC2 during myoblast proliferation in vitro declined between 24 h and 48 h when expression of the marker gene paired box 7 (PAX7) increased and cell numbers increased throughout 72 h of culture. During induced differentiation of myoblasts to myotubes, the abundance of HDAC2 and the marker gene myogenic differentiation 1 (MYOD1), both increased significantly. Taken together, it is suggested that HDAC2 is most likely involved in a suppressive fashion in myoblast proliferation and may play a positive role in myoblast differentiation. The present results confirm the suggestion that HDAC2 is a functional gene for pre-hatch and post-hatch (fast growing muscle) development of chicken skeletal muscle.

• The Korean Journal of Zoology
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• v.31 no.3
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• pp.207-217
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• 1988

A myotrophic protein that seemed to he eseentiai for the hision of chick embryonic myoblasts in culture was isolated from chick embryo extrad and was found to be identical or at least similar to the iron-transporting protein, transferrin. Embryo extract seemed to contain, in addition to this myotrophic protein, a heat stable protein that inhibits the fusion of myoblasts. Iron seemed to he necessary for myoblasts to fuse and it was supposed that the role of the myotrophic protein m myoblast fusion is to supply iron to the cell. The numher of the myotrophic protein receptors on myoblast surface membrane decreased immediately after the start of myoblast fusion, supposedly due to the decreased need of iron after the fusion once commenced. It was estimated that endocytosis of myotrophic protein took about 10 minutes and one recycling about 2 hours. The accumulation of iron in myoblasts continued linearly with cultre time and endocytosis of the myotrophic protein occured at a constant rate.

• Journal of Life Science
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• v.31 no.2
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• pp.137-143
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• 2021

Muscle atrophy refers to a decrease in muscle cells due to damage to muscle fibers. It is reported that muscle atrophy is caused by heart disease, diabetes, and other chronic diseases related to aging. The purpose of this study is to reveal the inhibitory effects of seaweed extracts, which are widely consumed in Korea, and alginic acid on muscle cell damage in muscle atrophy and regeneration models. We found that seaweed extracts (U) and alginic acid (A) attenuated TNF-α-induced muscle atrophy in differentiated C2C12 myoblast cells and inhibited muscle atrophy markers such as MuRF1 and MAFbx. In addition, U and A also regulated ubiquitination marker FoxO1 protein. To confirm the muscle regeneration effect in animal tissue, cardiotoxin (CTX) was used for the regeneration model. Six hours after CTX injection, gastrocnemius muscle volume was increased compared to control. Otherwise, the muscle volume of the U and A treatment groups was not changed. U and A also upregulated regeneration markers MyHC and PGC-1α in a CTX mouse model. These results indicate that seaweed extracts and alginic acid, a seaweed component, are applicable to senile sarcopenia by inhibiting muscle loss and promoting muscle regeneration.

• 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.

• The Korean Journal of Zoology
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• v.35 no.3
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• pp.322-331
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• 1992

To investigate the svnthyesis of muscle proteins during differentiation of chicken myoblast, cvtosolic and membrane fractions were used for both sodium dodecvl sulfate polvcrylamide gel eBectrophoresis and two-dimensional gel electrophoresis. An extensive cell fusion was observed in 4 day culture. In the protein pattern of the cvtosolic fraction from SDS-PAGE. several protein bands including 250 kDa and 46 kDa showed remarkable changes during culture. the protein of 46 kDa was the most prominent one ann its optical density was the highest in 5 day culture (OD = 1.30). In the membrane fraction, band of 19.8 kDa showed the highest absorbance with 0.93 OD at 12 hr after initial plating and decreased gradually thereafter to 0.23 in 5 nay culture. From the results of two-dimensional gel electrophoresis of cytosolic fraction, the 46 kDa spot was observed as ko separated forms from culture 2 nary culture, and the sixte of this spot was the largest in 5 nay culture. In the pattern of membrane protein, the extensive appearance of newiv synthesized Proteins was found in a naut culture, but no Prominent spot was observed throughout culture. From the results of the present clay, we found that, during myoblast differentiation, the most prominent proteins were bands of 46 kDa and 19.8 kDa in cvtosolic and membrane fraction, respectively, and the appearance of new proteins was initiated at 48 hr after initial plating, and the 46 kDa protein was predominant in the cytoplasm of late culture in which extensive cell fusion was observed.

• Journal of Ginseng Research
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• v.42 no.1
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• pp.116-121
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• 2018

Background: Black ginseng (BG) has greatly enhanced pharmacological activities relative to white or red ginseng. However, the effect and molecular mechanism of BG on muscle growth has not yet been examined. In this study, we investigated whether BG could regulate myoblast differentiation and myotube hypertrophy. Methods: BG-treated C2C12 myoblasts were differentiated, followed by immunoblotting for myogenic regulators, immunostaining for a muscle marker, myosin heavy chain or immunoprecipitation analysis for myogenic transcription factors. Results: BG treatment of C2C12 cells resulted in the activation of Akt, thereby enhancing hetero-dimerization of MyoD and E proteins, which in turn promoted muscle-specific gene expression and myoblast differentiation. BG-treated myoblasts formed larger multinucleated myotubes with increased diameter and thickness, accompanied by enhanced Akt/mTOR/p70S6K activation. Furthermore, the BG treatment of human rhabdomyosarcoma cells restored myogenic differentiation. Conclusion: BG enhances myoblast differentiation and myotube hypertrophy by activating Akt/mTOR/p70S6k axis. Thus, our study demonstrates that BG has promising potential to treat or prevent muscle loss related to aging or other pathological conditions, such as diabetes.

• The Korean Journal of Zoology
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• v.33 no.1
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• pp.108-118
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• 1990

Fibronectin is a glycoprotein found in the extracellular matrix as well as in the serum, and has been known to exert pronouned effed on the myoblast fusion. Our previous studies have suggested that the decrease of fibronectin levels during myogenesis is due to the decreased availability of the receptor for the 28 kDa fragrnent of fibronetin. In the fusion-blocked myoblasts by EGTA, the levels of fibronetin and binding of 28 kDa fragment decreased but far less than the control level. In contrast, the levels of fibronetin and binding of 28 kDa fragment decreased to the control level in the myoblast released from the fusion block. On this account, we suggest that the decrease of fibronetin levels during myoblast fusion is closely associated with the loss or alteration of the receptor for 28 kDa fragment. Mild trypsin treatment decreased the binding of the 28 kDa fragment to the myoblasts significandy. Similarly, the presence of gangliosides in the binding media decreased the binding of the 28 kDa fragment in a dose-dependent manner. Furthermore, gel overlay of 125 I-28 kDa fragment on the SDS-PAGE of the myoblast homogenates revealed that the 28 kDa fragment bound to a 43 kDa protein and to gangliosides as well. These results suggest that myoblast fusion is correlated with decrease of the receptor for the 28 kDa fragment and that the receptor might be a glycoprotein that contains glyco-conjugate found in gangliosides.