• 제목/요약/키워드: Myogenic differentiation

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Myotube differentiation in clustered regularly interspaced short palindromic repeat/Cas9-mediated MyoD knockout quail myoblast cells

  • Kim, Si Won;Lee, Jeong Hyo;Park, Byung-Chul;Park, Tae Sub
    • Asian-Australasian Journal of Animal Sciences
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    • 제30권7호
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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.

NF-kB Activation by Disruption of Microtubule Array during Myogenesis of L6 Cells

  • Sangmyung Rhee;Lee, Kun-Ho;Hyockman Kwon
    • Animal cells and systems
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    • 제1권1호
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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.

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Role of Exogenous Nitric Oxide Generated through Microwave Plasma Activate the Oxidative Signaling Components in Differentiation of Myoblast cells into Myotube

  • Kumar, Naresh;Shaw, Priyanka;Attri, Pankaj;Uhm, Han Sup;Choi, Eun Ha
    • 한국진공학회:학술대회논문집
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    • 한국진공학회 2015년도 제49회 하계 정기학술대회 초록집
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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.

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Effect of Deer Antler Extract on Muscle Differentiation and 5-Aminoimidazole-4-Carboxamide Ribonucleoside (AICAR)-Induced Muscle Atrophy in C2C12 Cells

  • Jo, Kyungae;Jang, Woo Young;Yun, Beom Sik;Kim, Jin Soo;Lee, Hyun-Sun;Chang, Yeok Boo;Suh, Hyung Joo
    • 한국축산식품학회지
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    • 제41권4호
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    • pp.623-635
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    • 2021
  • The effect of deer antler extract on muscle differentiation and muscle atrophy were evaluated to minimize muscle loss following aging. Various deer antler extracts (HWE, hot water extract of deer antler; FE, HWE of fermented deer antler; ET, enzyme-assisted extract of deer antler; UE, extract prepared by ultrasonication of deer antler) were evaluated for their effect on muscle differentiation and inhibition of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced muscle atrophy in C2C12 cells. Morphological changes according to the effect of antler extracts on muscle differentiation were confirmed by Jenner-Giemsa staining. In addition, the expression levels of genes related to muscle differentiation and atrophy were confirmed through qRT-PCR. In the presence of antler extracts, the length and thickness of myotubes and myogenin differentiation 1 (MyoD1) and myogenic factor 5 (Myf5) gene expression were increased compared to those in the control group (CON). Gene expression of AMP-activated protein kinase (AMPK), MyoD1, and myogenin, along with the muscle atrophy factors muscle RING finger-1 (MuRF-1) and forkhead box O3a (FoxO3a) upon addition of deer antler extracts to muscle-atrophied C2C12 cells was determined by qRT-PCR after treatment with AICAR. The expression of MuRF-1 and FoxO3a decreased in the groups treated with antler extracts compared to that in the group treated with AICAR alone. In addition, gene expression of MyoD1 and myogenin in the muscle atrophy cell model was significantly increased compared that into the CON. Therefore, our findings indicate that antler extract can increase the expression of MyoD1, Myf5 and myogenin, inhibit muscle atrophy, and promote muscle differentiation.

Effects of alpha-linolenic acid and essential amino acids on the proliferation and differentiation of C2C12 myoblasts

  • Zhou, Dongjie;Li, Xiao-Han;Lee, Song‑Hee;Heo, Geun;Cui, Xiang-Shun
    • 한국동물생명공학회지
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    • 제37권1호
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    • pp.17-26
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    • 2022
  • Alpha-linolenic acid is an important polyunsaturated fatty acid that exhibits anticancer, anti-inflammatory, and antioxidative effects. In this study, we investigated the protective effects of alpha-linolenic acid on the cell proliferation and differentiation of C2C12 cells under essential amino acid-deficient conditions. Different concentrations of alpha-linolenic acid and essential amino acids were added to the growth and differentiation media. The concentrations of 10 µM of alpha-linolenic acid and 2% essential amino acid were chosen for subsequent experiments. Supplementation with alpha-linolenic acid and essential amino acids improved the proliferation and differentiation of C2C12 cells and significantly increased the mRNA levels of catalase, superoxide dismutase, B-cell lymphoma-2, and beclin-1 as well as the protein levels of PPARγ coactivator-1α compared to those in the controls. Moreover, supplementation with alpha-linolenic acid and essential amino acids reduced the levels of phosphorylated H2A.X variant histone, Bcl-2-associated X, p53, and light chain 3 during C2C12 cell proliferation, and increased the expression levels of myogenic factors 4 (myogenin) and 5 during C2C12 cell differentiation. Overall, we determined that alpha-linolenic acid and essential amino acids maintained the cell proliferation and differentiation of C2C12 cells via their anti-oxidative, anti-apoptotic, and anti-autophagic effects.

Myogenic Differentiation of p53- and Rb-deficient Immortalized and Transformed Bovine Fibroblasts in Response to MyoD

  • Jin, Xun;Lee, Joong-Seub;Kwak, Sungwook;Jung, Ji-Eun;Kim, Tae-Kyung;Xuo, Chenxiong;Hong, Zhongshan;Li, Zhehu;Kim, Sun-Myoung;Whang, Kwang Youn;Hong, Ki-Chang;You, Seungkwon;Choi, Yun-Jaie;Kim, Hyunggee
    • Molecules and Cells
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    • 제21권2호
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    • pp.206-212
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    • 2006
  • We have established in culture a spontaneously immortalized bovine embryonic fibroblast (BEF) cell line that has lost p53 and $p16^{INK4a}$ functions. MyoD is a muscle-specific regulator capable of inducing myogenesis in a number of cell types. When the BEF cells were transduced with MyoD they differentiated efficiently to desmin-positive myofibers in the presence of 2% horse serum and 1.7 nM insulin. The myogenic differentiation of this cell line was more rapid and obvious than that of C2C12 cells, as judged by morphological changes and expression of various muscle regulatory factors. To confirm that lack of the p53 and $p16^{INK4a}$ pathway does not prevent MyoD-mediated myogenesis, we established a cell line transformed with SV40LT (BEFV) and introduced MyoD into it. In the presence of 2% horse serum and 1.7 nM insulin, the MyoD-transduced BEFV cells differentiated like the MyoD-transduced BEFS cells, and displayed a similar pattern of expression of muscle regulatory proteins. Taken together, our results indicate that MyoD overexpression overcomes the defect in muscle differentiation associated with immortalization and cell transformation caused by the loss of p53 and Rb functions.

The multifunctional RNA-binding protein hnRNPK is critical for the proliferation and differentiation of myoblasts

  • Xu, Yongjie;Li, Rui;Zhang, Kaili;Wu, Wei;Wang, Suying;Zhang, Pengpeng;Xu, Haixia
    • BMB Reports
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    • 제51권7호
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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.

Phosphoinositide 3-kinase regulates myogenin expression at both the transcriptional and post-transcriptional level during myogenesis

  • Woo, Joo-Hong;Kim, Min-Jeong;Kim, Hye-Sun
    • Animal cells and systems
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    • 제14권3호
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    • pp.147-154
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    • 2010
  • It is well-established that phosphoinositide 3-kinase (PI3-kinase) regulates myogenesis by inducing transcription of myogenin, a key muscle regulatory factor, at the initiation of myoblast differentiation. In this study, we investigated the role of PI3-kinase in cells that have committed to differentiation. PI3-kinase activity increases during myogenesis, and this increase is sustained during the myogenic process; however, its function after the induction of differentiation has not been investigated. We show that LY294002, a PI3-kinase inhibitor, blocked myoblast fusion even after myogenin expression initially increased. In contrast to the inhibitory effects of LY294002 on myogenin mRNA levels during the initiation of differentiation, LY294002 blocked the accumulation of myogenin protein without affecting its mRNA level after differentiation was induced. Treatment with cycloheximide, a translation inhibitor, or actinomycin D, a transcription inhibitor, indicated that the stability of myogenin protein is lower than that of its mRNA. LY294002 inhibited the activities of several important translation factors, including eukaryotic elongation factor-2(eEF2), by altering their phosphorylation status. In addition, LY294002 blocked the incorporation of [$^{35}S$]methionine into newly synthesized proteins. Since myogenin has a relatively short half-life, LY294002-mediated inhibition of post-transcriptional processes resulted in a rapid depletion of myogenin protein. In summary, these results suggest that PI3-kinase plays an important role in regulating the expression of myogenin through post-transcriptional mechanisms after differentiation has been induced.

돼지 고기의 아나볼릭 스테로이드가 Myogenic Satellite Cell의 증식과 분화에 미치는 영향 (Effects of Anabolic Steroids of Pork on Proliferation and Differentiation of Myogenic Satellite Cell)

  • 이동목;이기호;전용필;전태훈;최인호
    • 한국축산식품학회지
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    • 제30권5호
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    • pp.842-850
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    • 2010
  • 성호르몬은 근육의 발달(아나볼릭 효과)에 관여하는 것으로 잘 알려져 왔으며 인체의 치료제로 넓게 사용되고 있다. 돼지고기에는 주로 성선(정소와 난소)에서 생산되어 고기에 축적된 다양한 아나볼릭 효과를 나타내는 성호르몬이 많이 존재하는 것으로 알려져 있다. 따라서 본 연구는 돼지고기에 존재하는 아나볼릭 스테로이드를 추출하여 근육세포의 증식과 분화에 미치는 영향을 파악하고자 실시하였다. 본 연구에서 돼지의 각 조직으로부터 아나볼릭 스테로이드를 추출하는 3가지 방법(M1, M2, M3)을 개발하였다. 3가지 모든 추출방법에서 아나볼릭 스테로이드가 추출되었으며 특히 M3에서 조직 1g당 가장 많은 양을 획득할 수 있었다. M3를 이용하여 아나볼릭 스테로이드를 추출했을 때 돼지고기의 근육과 지방에서는 스테로이드 호르몬 양이 수컷에서 가장 높았고, 혈청에서는 nandrolone 과 testosterone의 양은 수컷에서 가장 높았으며, estrone와 $17{\beta}$-estradiol의 양은 비슷하였다. 근육세포 증식은 돼지 아나볼릭 스테로이드의 농도 의존적으로 촉진되었으며, M3에서 가장 빠르게 증식되었다. 근육세포의 분화는 추출된 아나볼릭 스테로이드 처리구가 대조구보다 더 효과적으로 분화가 잘 되는 것을 세포 모양과 유전자(AR, myoD, desmin, myogenin)를 통하여 확인할 수 있었다. 특히 M3에서 추출된 아나볼릭 스테로이드에서 myogenin과 AR 유전자가 유의적으로 높게 발현되었다(p<0.001). 전체적으로 돼지의 아나볼릭 스테로이드는 근육세포의 성장과 분화를 촉진하였다. 따라서 돼지고기에 있는 아나볼릭 스테로이드의 섭취를 통해 인체 근육의 성장과 발달에 긍정적 효과를 나타낼 수 있을 것으로 사료된다.

Proteomic Analysis of Bovine Longissimus Muscle Satellite Cells during Adipogenic Differentiation

  • Rajesh, Ramanna Valmiki;Park, Mi-Rim;Heo, Kang-Nyeong;Yoon, Du-Hak;Kim, Tae-Hun;Lee, Hyun-Jeong
    • Asian-Australasian Journal of Animal Sciences
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    • 제24권5호
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    • pp.685-695
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    • 2011
  • Satellite cells are skeletal muscle progenitor/stem cells that reside between the basal lamina and plasma membranes of skeletal fibers in vivo. These cells can give rise to both myogenic and adipogenic cells. Given the possible role for differentiation of satellite cells into adipocytes in marbling and in some pathological disorders like sarcopenia, knowledge of the proteins involved in such process remains obscure. Using two-dimensional polyacrylamide gel electrophoresis coupled with mass spectrometry, we investigated the proteins that are differentially expressed during adipogenic differentiation of satellite cells from bovine longissimus muscle. Our proteome mapping strategy to identify the differentially expressed intracellular proteins during adipogenic differentiation revealed a total of 25 different proteins. The proteins up-regulated during adipogenic differentiation of satellite cells like Cathepsin H precursor, Retinal dehydrogenase 1, Enoyl-CoA hydratase, Ubiquinol-cytochrome-c reductase, T-complex protein 1 subunit beta and ATP synthase D chain were found to be associated with lipid metabolism. The down-regulated proteins like LIM protein, annexin proteins, cofilin-1, Rho GDP-dissociation inhibitor 1 and septin-2, identified in the present study were found to be associated with myogenesis. These results clearly demonstrate that the adipogenic conversion of muscle satellite cells is associated with the up-regulated and down-regulated proteins involved in adipogenesis and myogenesis respectively.