• Title, Summary, Keyword: Muscle Differentiation

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Muscle differentiation induced up-regulation of calcium-related gene expression in quail myoblasts

  • Park, Jeong-Woong;Lee, Jeong Hyo;Kim, Seo Woo;Han, Ji Seon;Kang, Kyung Soo;Kim, Sung-Jo;Park, Tae Sub
    • Asian-Australasian Journal of Animal Sciences
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    • v.31 no.9
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    • pp.1507-1515
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    • 2018
  • Objective: In the poultry industry, the most important economic traits are meat quality and carcass yield. Thus, many studies were conducted to investigate the regulatory pathways during muscle differentiation. To gain insight of muscle differentiation mechanism during growth period, we identified and validated calcium-related genes which were highly expressed during muscle differentiation through mRNA sequencing analysis. Methods: We conducted next-generation-sequencing (NGS) analysis of mRNA from undifferentiated QM7 cells and differentiated QM7 cells (day 1 to day 3 of differentiation periods). Subsequently, we obtained calcium related genes related to muscle differentiation process and examined the expression patterns by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Results: Through RNA sequencing analysis, we found that the transcription levels of six genes (troponin C1, slow skeletal and cardiac type [TNNC1], myosin light chain 1 [MYL1], MYL3, phospholamban [PLN], caveolin 3 [CAV3], and calsequestrin 2 [CASQ2]) particularly related to calcium regulation were gradually increased according to days of myotube differentiation. Subsequently, we validated the expression patterns of calcium-related genes in quail myoblasts. These results indicated that TNNC1, MYL1, MYL3, PLN, CAV3, CASQ2 responded to differentiation and growth performance in quail muscle. Conclusion: These results indicated that calcium regulation might play a critical role in muscle differentiation. Thus, these findings suggest that further studies would be warranted to investigate the role of calcium ion in muscle differentiation and could provide a useful biomarker for muscle differentiation and growth.

Influence of co-culturing muscle satellite cells with preadipocytes on the differentiation of adipocytes and muscle cells isolated from Korean native cattle

  • Choi, Chang Weon
    • Korean Journal of Agricultural Science
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    • v.45 no.4
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    • pp.715-723
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    • 2018
  • The present study was done to investigate the effect of co-culturing muscle satellite cells (MSCs) and intramuscular preadipocytes (IPs) on the differentiation of adipocytes and muscle cells isolated from Korean native cattle. MSCs and IPs were single-cultured in 10% fetal bovine serum/Dulbecco's modified Eagles medium (FBS/DMEM) for 48 h followed by culturing in 5% FBS/DMEM as the growth media. Then, the growth media was replaced by differentiation media composed of 2% FBS/DMEM without any additives for the single- or co-culture of muscle cells and intramuscular adipocytes to induce the differentiation of both cell types. Cell differentiation was measured by morphological investigation and cytosolic enzyme analysis of glycerol-3-phosphate dehydrogenase (GPDH) for the adipocytes and creatine kinase (CK) for the muscle cells. In the morphological test, the presence of muscle cells did not stimulate adipocyte differentiation showing more differentiation of the adipocytes in the single-culture compared to the co-culture condition. However, the differentiation of muscle cells was promoted by adipocytes in the co-culture. The results of the enzymatic analysis were highly associated with the morphological results with a statistically higher GPDH activity (p < 0.05) appearing in the single-culture than in the co-culture, whereas the opposite was true for the CK activity of the muscle cells (p < 0.05). By manipulating in vivo the milieu using a co-culture, we could detect the difference in the rate of cell differentiation and suggest that a co-culture system is a more reliable and precise technique compared to a single-culture. Further studies on various co-culture trials including supplementation of differentiating substances, gene expression analysis, etc. should be done to obtain practical and fundamental data.

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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    • 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.

Investigation of the effect of Terminalia chebula fruit extract and its active ingredient, gallic aicd on muscle differentiation (가자(訶子) 추출물과 그 유효성분 갈산이 근분화에 미치는 영향)

  • Cheon, Seonghye;Lee, Hyo Seong;Han, Hyo Sang;Kim, Kee Kwang
    • The Korea Journal of Herbology
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    • v.34 no.2
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    • pp.59-66
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    • 2019
  • Objectives : Decrease in muscle mass and loss of muscle function due to aging are associated with various diseases. As interest in healthy aging increases, efforts to prevent and treat muscle hypoxia as an illness are increasing. Considering the physical limitations, a pharmacologic approach to the treatment of myopenia is needed. Methods : Terminalia chebula Rets has a wide range of pharmacological effects and is used as a medicinal product in traditional medicine. However, the drug effect on the treatment of muscle disorders has not been revealed. The purpose of this study was to evaluate the value of water extract of Terminalia chebula (WETC) as a therapeutic agent to relieve symptoms of muscle hypoxia. Results : WETC showed strong radical scavenging ability. In addition, WETC increased cell activity of myoblast, and we observed that WETC induces myoblast differentiation by immunoblot analysis using differentiation protein markers as well as cell morphology of myoblast. Based on these results, we examined the effect of chebulic acid, chebulagic acid, gallic acid, geraniin, and punicalagin on cell activity and differentiation of myoblasts. Gallic acid significantly increased cell activity of myoblast, and it was found to be an effective substance which not only induces myoblast differentiation but also promotes proliferation. Conclusions : We suggest that the WETC with antioxidant effect and its indicator gallic acid on cell activity, proliferation and differentiation of myoblast can be studied and developed as a food and medicine for prevention and treatment of various muscle diseases.

Differentiation of mouse embryonic stem cell into smooth muscle cells by DBcAMP and retinoic acid (DBcAMP와 retinoic acid를 이용한 마우스 배아줄기의 평활근세포 분화)

  • Park, Sung-Soo;Kang, Ju-Won
    • Korean Journal of Veterinary Service
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    • v.31 no.4
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    • pp.449-456
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    • 2008
  • The differentiation of mouse embryonic stem(ES) cell into smooth muscle cells(SMC) may play a major role in cardiovascular development and under pathophysiological conditions. Therefore, in the present study, we have examined the differentiation of ES cells and its related gene expression. SMC differentiation was indicated by cellular morphology and time-dependent induction of dibutyryl adenosine 3,5-cyclic monophosphate(DBcAMP)and retinoic acid(RA) on smooth muscle ${\alpha}$-actin($SM{\alpha}A$), smooth muscle myosin heavy chain(SMMHC) gene expression. The control was undifferentiated ES cells(protein expressions represent 50-60kDaOct-4). The results of this study show that morphology of embryoid body and confirmation of $SM{\alpha}A$ expression by immunocytochemistry. Moreover, SMMHC and desmin expression was significantly increased by time dependent manner(5, 7, 15 days), in contrast to $SM{\alpha}A$ expression was slightly decreased on 15days. In conclusion, DBcAMP and RA stimulate mouse ES cells differentiation into SMC and enhanced $SM{\alpha}A$, SMMHC and desmin expression.

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
    • Proceedings of the Korean Vacuum Society Conference
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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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Leukotriene B4 Regulates Proliferation and Differentiation of Cultured Rat Myoblasts via the BLT1 Pathway

  • Sun, Ru;Ba, Xueqing;Cui, Lingling;Xue, Yan;Zeng, Xianlu
    • Molecules and Cells
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    • v.27 no.4
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    • pp.403-408
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    • 2009
  • Skeletal muscle regeneration is a highly orchestrated process initiated by activation of adult muscle satellite cells. Upon muscle injury, the inflammatory process is always accompanied by muscle regeneration. Leukotriene $B_4$ is one of the essential inflammatory mediators. We isolated and cultured primary satellite cells. RT-PCR showed that myoblasts expressed mRNA for $LTB_4$ receptors BLT1 and BLT2, and $LTB_4$ promoted myoblast proliferation and fusion. Quantitative real-time PCR and immunoblotting showed that $LTB_4$ treatment expedited the expression process of differentiation markers MyoD and M-cadherin. U-75302, a specific BLT1 inhibitor, but not LY2552833, a specific BLT2 inhibitor, blocked proliferation and differentiation of myoblasts induced by $LTB_4$, which implies the involvement of the BLT1 pathway. Overall, the data suggest that $LTB_4$ contributes to muscle regeneration by accelerating proliferation and differentiation of satellite cells.

β-catenin protein utilized by Tumour necrosis factor-α in porcine preadipocytes to suppress differentiation

  • Luo, Xiao;Li, Hui-Xia;Liu, Rong-Xin;Wu, Zong-Song;Yang, Ying-Juan;Yang, Gong-She
    • BMB Reports
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    • v.42 no.6
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    • pp.338-343
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    • 2009
  • The Wnt/$\beta$-catenin signaling pathway alters adipocyte differentiation by inhibiting adipogenic gene expression. $\beta$-catenin plays a central role in the Wnt/$\beta$-catenin signaling pathway. In this study, we revealed that tumour necrosis factor-$\alpha$ (TNF-$\alpha$), a potential negative regulator of adipocyte differentiation, inhibits porcine adipogenesis through activation of the Wnt/$\beta$-catenin signaling pathway. Under the optimal concentration of TNF-$\alpha$, the intracellular $\beta$-catenin protein was stabilized. Thus, the intracellular lipid accumulation of porcine preadipocyte was suppressed and the expression of important adipocyte marker genes, including peroxisome proliferator-activated receptor-$\gamma$ (PPAR$\gamma$) and CCAAT/enhancer binding protein-$\alpha$ (C/EBP$\alpha$), were inhibited. However, a loss of $\beta$-catenin in porcine preadipocytes enhanced the adipogenic differentiation and attenuated TNF-$\alpha$ induced anti-adipogenesis. Taken together, this study indicated that TNF-$\alpha$ inhibits adipogenesis through stabilization of $\beta$-catenin protein in porcine preadipocytes.

Transcriptional Profiling of Differentially Expressed Genes in Porcine Satellite Cell

  • Jeong, Jin Young;Kim, Jang Mi;Rajesh, Ramanna Valmiki;Suresh, Sekar;Jang, Gul Won;Lee, Kyung-Tai;Kim, Tae Hun;Park, Mina;Jeong, Hak Jae;Kim, Kyung Woon;Cho, Yong Min;Lee, Hyun-Jeong
    • Reproductive and Developmental Biology
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    • v.37 no.4
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    • pp.233-245
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    • 2013
  • Muscle satellite cell (SC) is responsible for postnatal muscle growth, repair, and regeneration. Satellite cell is an important source of multi-potent stem cell process and differentiation into adipogenic, myogenic, and osteoblastogenic. The objective of this study was to identify alter of transcriptome during differentiation in porcine satellite cell and to elevated transcriptome at different stages of postnatal development to gain insight into the differences in differentiated PSC. We used RNA-seq technique to investigate the transcriptomes during differentiation in pig muscle. Sequence reads were obtained from Illumina HiSeq2000. Differentially expressed genes (DEG) were detected by EdgeR. Gene ontology (GO) terms are powerful tool for unification among representation genes or products. In study of GO biological terms, functional annotation clustering involved in cell cycle, apoptosis, extracellular matrix, phosphorylation, proteolysis, and cell signaling in differences stage. Taken together, these results would be contributed to a better understanding of muscle biology and processes underlying differentiation. Our results suggest that the source of DEGs could be better understanding of the mechanism of muscle differentiation and transdifferentiation.

Research article Black ginseng activates Akt signaling, thereby enhancing myoblast differentiation and myotube growth

  • Lee, Soo-Yeon;Go, Ga-Yeon;Vuong, Tuan Anh;Kim, Jee Won;Lee, Sullim;Jo, Ayoung;An, Jun Min;Kim, Su-Nam;Seo, Dong-Wan;Kim, Jin-Seok;Kim, Yong Kee;Kang, Jong-Sun;Lee, Sang-Jin;Bae, Gyu-Un
    • 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.