• Journal of Biomedical Engineering Research
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• v.38 no.6
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• pp.308-312
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• 2017

Skeletal muscle function plays a very important role in quality of life. However, skeletal muscle causes functional decline under aging or some diseases. Exercise and muscle training are good solutions to delay sarcopenia, but there are limitations to those who are uncomfortable in exercise. For this reason, alternative interventions for muscle sarcopenia are required, and many studies proved the increase of skeletal muscle mass by electrical stimulation. In conventional studies, however, mouse skeletal muscle cells have been mostly used in experiments to identify electrical stimulation conditions while human derived cells have not been frequently utilized in these studies. Stimulation used for rehabilitation has been uniformly treated without the consideration of aging. In addition, many studies have been used with conventional petri dish usually requiring many numbers of cells, which is not appropriate for rare. Moreover, they are not usually condition uniformity of electrical field. In this study, we have developed an electrical stimulation device which consumes a small amount of cells and can form a uniform electrical field. With the system, we analyzed the skeletal muscle differentiation and Myotube thickness depending on the electrical stimulation condition.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.9
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• pp.1288-1302
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• 2011

The aim of this study was to analyze the proteome expression of bovine satellite cells from longissimus dorsi (LD), deep pectoral (DP) and semitendinosus (ST) muscle depots during in vitro myogenic differentiation. Proteomic profiling by twodimensional gel electrophoresis and mass spectrometry of differentiating satellite cells revealed a total of 38 proteins that were differentially regulated among the three depots. Among differentially regulated proteins, metabolic proteins like lactate dehydrogenase (LDH), malate dehydrogenase (MDH) were found to be up regulated in ST, while alpha-enolase (NNE) in LD and DP depot satellite cells were down regulated. Also, our analysis found that there was a prominent up regulation of cytoskeletal proteins like actin, actincapping protein and transgelin along with chaperone proteins like heat shock protein beta 1 (HSPB 1) and T-complex protein 1 (TCP-1). Among other up regulated proteins, LIM domain containing protein, annexin 2 and Rho GDP-dissociation inhibitor 1 (Rho GDI) are observed, which were already proven to be involved in the myogeneis. More interestingly, satellite cells from ST depot were found to have a higher myotube formation rate than the cells from the other two depots. Taken together, our results demonstrated that, proteins involved in glucose metabolism, cytoskeletal modeling and protein folding plays a key role in the myogenic differentiation of bovine satellite cells.

• BMB Reports
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• v.40 no.6
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• pp.921-927
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• 2007

UCP2 and UCP3 are members of the uncoupling protein family, which may play roles in energy homeostasis. In order to determine the regulation of the predominant expression of UCP3 in skeletal muscle, the effects of differentiation and myogenic regulatory factors on the promoter activities of the mouse UCP2 and UCP3 genes were studied. Reporter plasmids, containing approximately 3 kb of the 5'-upstream region of the mouse UCP2 and UCP3 genes, were transfected into C2C12 myoblasts, which were then induced to differentiate. Differentiation positively induced the reporter expression about 20-fold via the UCP3 promoter, but by only 2-fold via the UCP2 promoter. C2C12 myoblasts were cotransfected with expression vectors for myogenin and/or MyoD as well as reporter constructs. The simultaneous expression of myogenin and MyoD caused an additional 20-fold increase in the reporter expression via the UCP3 promoter, but only a weak effect via the UCP2 promoter. In L6 myoblasts, only MyoD activated the UCP3 promoter, but in 3T3-L1 cells neither factor activated the UCP3 promoter, indicating that additional cofactors are required, which are present only in C2C12 myoblasts. The expression of UCP2 and UCP3 is differentially regulated during muscle differentiation due to the different responsiveness of their promoter regions to myogenin and MyoD.

• 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.

• BMB Reports
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• v.56 no.8
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• pp.445-450
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• 2023

The development of atherosclerotic cardiovascular disease is associated with the phenotypic switching of vascular smooth muscle cells (SMCs) from a contractile to a synthetic state, leading to cell migration and proliferation. Platelet-derived growth factor-BB (PDGF-BB) modulates this de-differentiation by initiating a number of biological processes. In this study, we show that gene expression of hyaluronic acid (HA) and proteoglycan link protein 1 (HAPLN1) was upregulated during differentiation of human aortic SMCs (HASMCs) into a contractile state, but downregulated upon during PDGF-BB-induced dedifferentiation. This is the first study showing that the treatment of HASMCs with full-length recombinant human HAPLN1 (rhHAPLN1) significantly reversed PDGF-BB-induced decrease in the protein levels of contractile markers (SM22α, α-SMA, calponin, and SM-MHC), and inhibited the proliferation and migration of HASMCs induced by PDGF-BB. Furthermore, our results show that rhHAPLN1 significantly inhibited the phosphorylation of FAK, AKT, STAT3, p38 MAPK and Raf mediated by the binding of PDGF-BB to PDGFRβ. Together, these results indicated that rhHAPLN1 can suppress the PDGF-BB-stimulated phenotypic switching and subsequent de-differentiation of HASMCs, highlighting its potential as a novel therapeutic target for atherosclerosis and other vascular diseases.

• Journal of Life Science
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• v.21 no.2
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• pp.183-193
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• 2011

Migration and differentiation of mesenchymal stem cells are crucial for tissue regeneration in response to injury. Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates a variety of biological processes, including proliferation, survival, differentiation and motility. In the present study, we determined the role of S1P in migration and differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs). S1P stimulated migration of BMSCs in a dose- and time-dependent manner, and pre-incubation of the cells with pertussis toxin completely abrogated S1P-induced migration, suggesting involvement of Gi-coupled receptors in S1P-induced cell migration. S1P elicited elevation of intracellular concentration of $Ca^{2+}$ ($[Ca^{2+}]_i$) and pretreatment with VPC23019, an antagonist of $S1P_1/S1P_3$, blocked S1P-induced migration and increase of $[Ca^{2+}]_i$. Small interfering RNA-mediated knockdown of endogenous $S1P_1$ attenuated S1P-induced migration of BMSCs. Furthermore, S1P treatment induced expression of $\alpha$-smooth muscle actin ($\alpha$-SMA), a smooth muscle marker, and pretreatment with VPC23019 abrogated S1P-induced $\alpha$-SMA expression. S1P induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), and pretreatment of cells with SB202190, an inhibitor of p38 MAPK, or adenoviral overexpression of a dominant-negative mutant of the p38 MAPK blocked S1P-induced cell migration and $\alpha$-SMA expression. Taken together, these results suggest that S1P stimulates migration and smooth muscle differentiation of BMSCs through an $S1P_1$-p38 MAPK-dependent mechanism.

• Animal Bioscience
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• v.34 no.10
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• pp.1590-1599
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• 2021

Objective: This study investigates the expression patterns of toll-like receptors (TLRs) and intracellular mediators in horse muscle cells after exercise, and the relationship between TLRS expression in stressed horse muscle cells and immune cell migration toward them. Methods: The expression patterns of the TLRs (TLR2, TLR4, and TLR8) and downstream signaling pathway-related genes (myeloid differentiation primary response 88 [MYD88]; activating transcription factor 3 [ATF3]) are examined in horse tissues, and horse peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs) and muscles in response to exercise, using the quantitative reverse transcription-polymerase chain reaction (qPCR). Expressions of chemokine receptor genes, i.e., C-X-C motif chemokine receptor 2 (CXCR2) and C-C motif chemokine receptor 5 (CCR5), are studied in PBMCs and PMNs. A horse muscle cell line is developed by transfecting SV-T antigen into fetal muscle cells, followed by examination of muscle-specific genes. Horse muscle cells are treated with stressors, i.e., cortisol, hydrogen peroxide (H₂O₂), and heat, to mimic stress conditions in vitro, and the expression of TLR4 and TLR8 are examined in stressed muscle cells, in addition to migration activity of PBMCs toward stressed muscle cells. Results: The qPCR revealed that TLR4 message was expressed in cerebrum, cerebellum, thymus, lung, liver, kidney, and muscle, whereas TLR8 expressed in thymus, lung, and kidney, while TLR2 expressed in thymus, lung, and kidney. Expressions of TLRs, i.e., TLR4 and TLR8, and mediators, i.e., MYD88 and ATF3, were upregulated in muscle, PBMCs and PMNs in response to exercise. Expressions of CXCR2 and CCR5 were also upregulated in PBMCs and PMNs after exercise. In the muscle cell line, TLR4 and TLR8 expressions were upregulated when cells were treated with stressors such as cortisol, H₂O₂, and heat. Migration of PBMCs toward stressed muscle cells was increased by exercise and oxidative stresses, and combinations of these. Treatment with methylsulfonylmethane (MSM), an antioxidant on stressed muscle cells, reduced migration of PBMCs toward stressed muscle cells. Conclusion: In this study, we have successfully cultured horse skeletal muscle cells, isolated horse PBMCs, and established an in vitro system for studying stress-related gene expressions and function. Expression of TLR4, TLR8, CXCR2, and CCR5 in horse muscle cells was higher in response to stressors such as cortisol, H₂O₂, and heat, or combinations of these. In addition, migration of PBMCs toward muscle cells was increased when muscle cells were under stress, but inhibition of reactive oxygen species by MSM modulated migratory activity of PBMCs to stressed muscle cells. Further study is necessary to investigate the biological function(s) of the TLR gene family in horse muscle cells.

• The Journal of Internal Korean Medicine
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• v.28 no.2
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• pp.333-345
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• 2007

Determination and differentiation of cells in the skeletal muscle lineage is positively regulated by cell-cell contact. differentiation proteins proposed to mediate this effect include both classical MyoD and MEF members : potential interactions between the promyogenic activities of these classes of protein, however, are unknown. We show here that MyoD and MEF, two promyogenic family members that determine to each other in a cis fashion, form ineraction with MyoD- and MEF. These proteins contain myosin heavy chains and are enriched at sites of cell-cell contact between myoblasts, Therefore, In differentiation of MyoD MEF from CST (Chungsimyeonjatang) interact dependently, suggesting that the interactions occur in a cis fashio : consistent with this conclusion, MyoD-mediated differentiation is required for myoblast to occur by CST. Inhibition in myoblasts of a MyoD by STP in its ability to associate with MEF interferes with differentiation as assessed by morphological and transcription level, suggesting that this interaction is functionally important in myogenesis. Also, some of the differentiation-mediated proteins that are required for myogenesis seem to be based on interdependent activities of promyogenic classical SMAD-subfamilly.

• Animal cells and systems
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• v.14 no.3
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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.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.3
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• pp.439-448
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• 2018

Objective: Myogenic satellite cells were isolated from semitendinosus muscle of prepubertal Korean black goat to observe the differential effect of linolenic and retinoic acid in thepresence of thiazolidinediones (TZD) and also to observe the production insulin sensitive preadipocyte. Methods: Cells were characterized for their stemness with cluster of differentiation 34 (CD34), CD13, CD106, CD44, Vimentin surface markers using flow cytometry. Cells characterized themselves as possessing significant (p<0.05) levels of CD13, CD34, CD106, Vimentin revealing their stemness potential. Goat myogenic satellite cells also exhibited CD44, indicating that they possessed a % of stemness factors of adipose lineage apart from their inherent stemness of paxillin factors 3/7. Results: Cells during proliferation stayed absolutely and firmly within the myogenic fate without any external cues and continued to show a significant (p<0.05) fusion index % to express myogenic differentiation, myosin heavy chain, and smooth muscle actin in 2% horse serum. However, confluent myogenic satellite cells were the ones easily turning into adipogenic lineage. Intriguingly, upregulation in adipose specific genetic markers such as peroxisome proliferation-activated receptor ${\gamma}$, adiponectin, lipoprotein lipase, and CCAAT/enhancer binding protein ${\alpha}$ were observed and confirmed in all given treatments. However, the amount of adipogenesis was found to be statistically significant (p<0.01) with linolenic acid as compared to retinoic acid in combination with TZD's. Conclusion: Retinoic acid was found to produce smaller preadipocytes which have been assumed to have insulin sensitization and hence retinoic acid could be used as a potential agent to sensitize tissues to insulin in combination with TZD's to treat diabetic conditions in humans and animals in future.