• Biomedical Science Letters
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• v.29 no.3
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• pp.190-200
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• 2023

Skeletal muscle, essential for metabolism, thermoregulation, and immunity, undergoes myogenic differentiation that results in myotube formation. Trans-anethole (TA), the major constituent in essential oil produced by anise, star anise, and fennel, whose function in skeletal muscle has not yet been elucidated. Therefore, we investigated whether TA influenced muscle differentiation in mouse C2C12 myoblasts. Cells were induced to differentiate using a differentiation medium with or without TA (50 or 200 mg/mL) daily for 5 days. We measured myotube length and diameter after differentiation days 1, 3, and 5 and analyzed the expression of myogenic markers (myoblast determination protein 1, myogenin, myocyte enhancer factor 2, muscle creatine kinase, and myosin heavy chain) and atrophy-related genes (atrogin-1 and muscle ring finger-1 [MuRF-1]) using quantitative real-time PCR. Additionally, we observed the expression of total protein kinase B (Akt) and phosphorylated Akt (p-Akt) using western blotting. Our data showed that TA significantly induced the formation of smaller and thinner myotubes and reduced the myogenic factor expression. Furthermore, the atrogin-1 and MuRF-1 expression markedly increased by TA. Consistent with these findings, TA significantly decreased the expression of total Akt and p-Akt. Taken together, these results indicate that TA inhibits myogenic differentiation of C2C12 cells via reduction of both total Akt and p-Akt. Our findings may provide valuable insights into the impact of PAA on individuals at risk of muscle atrophy.

• Animal Bioscience
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• v.36 no.12
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• pp.1775-1784
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• 2023

Objective: The aim of this study was to reveal the role and regulatory mechanism of miR-188-5p in the proliferation and differentiation of goat muscle satellite cells. Methods: Goat skeletal muscle satellite cells isolated in the pre-laboratory were used as the test material. First, the expression of miR-188-5p in goat muscle tissues at different developmental stages was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). In addition, miR-188-5p was transfected into goat skeletal muscle satellite cells by constructing mimics and inhibitors of miR-188-5p, respectively. The changes of differentiation marker gene expression were detected by qPCR method. Results: It was highly expressed in adult goat latissimus dorsi and leg muscles, goat fetal skeletal muscle, and at the differentiation stage of muscle satellite cells. Overexpression and interference of miR-188-5p showed that miR-188-5p inhibited the proliferation and promoted the differentiation of goat muscle satellite cells. Target gene prediction and dual luciferase assays showed that miR-188-5p could target the 3'untranslated region of the calcium/calmodulin dependent protein kinase II beta (CAMK2B) gene and inhibit luciferase activity. Further functional studies revealed that CAMK2B promoted the proliferation and inhibited the differentiation of goat muscle satellite cells, whereas si-CAMK2B restored the function of miR-188-5p inhibitor. Conclusion: These results suggest that miR-188-5p inhibits the proliferation and promotes the differentiation of goat muscle satellite cells by targeting CAMK2B. This study will provide a theoretical reference for future studies on the molecular mechanisms of skeletal muscle development in goats.

• Animal Bioscience
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• v.34 no.6
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• pp.1078-1087
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• 2021

Objective: Skeletal muscle satellite cells (SMSCs) are significant for the growth, regeneration, and maintenance of skeletal muscle after birth. However, currently, few studies have been performed on the isolation, culture and inducing differentiation of goose muscle satellite cells. Previous studies have shown that C1q and tumor necrosis factor-related protein 3 (CTRP3) participated in the process of muscle growth and development, but its role in the goose skeletal muscle development is not yet clear. This study aimed to isolate, culture, and identify the goose SMSCs in vitro. Additionally, to explore the function of CTRP3 in goose SMSCs. Methods: Goose SMSCs were isolated using 0.25% trypsin from leg muscle (LM) of 15 to 20 day fertilized goose eggs. Cell differentiation was induced by transferring the cells to differentiation medium with 2% horse serum and 1% penicillin streptomycin. Immunofluorescence staining of Desmin and Pax7 was used to identify goose SMSCs. Quantitative realtime polymerase chain reaction and western blot were applied to explore developmental expression profile of CTRP3 in LM and the regulation of CTRP3 on myosin heavy chains (MyHC), myogenin (MyoG) expression and Notch signaling pathway related genes expression. Results: The goose SMSCs were successfully isolated and cultured. The expression of Pax7 and Desmin were observed in the isolated cells. The expression of CTRP3 decreased significantly during leg muscle development. Overexpression of CTRP3 could enhance the expression of two myogenic differentiation marker genes, MyHC and MyoG. But knockdown of CTRP3 suppressed their expression. Furthermore, CTRP3 could repress the mRNA level of Notch signaling pathway-related genes, notch receptor 1, notch receptor 2 and hairy/enhancer-of-split related with YRPW motif 1, which previously showed a negative regulation in myoblast differentiation. Conclusion: These findings provide a useful cell model for the future research on goose muscle development and suggest that CTRP3 may play an essential role in skeletal muscle growth of goose.

• Journal of Life Science
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• v.22 no.4
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• pp.447-455
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• 2012

Akt plays an important role in a variety of cellular physiologies such as growth, proliferation, and differentiation. In skeletal muscle, Akt has been implicated in regulating regeneration, hypertrophy, and atrophy. In this study, the role of Akt has been examined during skeletal muscle differentiation. Culturing C2C12 myoblasts under low serum (1% horse serum) and high density converted cell morphology from a round shape to an elongated and multi-nucleated shape. Morphological changes were initiated from day 2 of differentiation. In addition, the expression of both myogenin G and myogenin D was elevated from day 2 of differentiation. Skeletal muscle differentiation was abolished by silencing Akt1 or Akt2, but was significantly enhanced by the over-expression of either Akt1 or Akt2. The activation of Akt was observed from day 2 of differentiation and disappeared after day 7. The expression of kruppel-like factor 4 was observed from day 6 of differentiation. Moreover, this expression was blocked in cells silencing either Akt1 or Akt2. In addition, the promoter activity of kruppel-like factor 4 was significantly reduced in cells silencing Akt1 or Akt2. These results suggest that Akt regulates skeletal muscle differentiation through the regulation of kruppel-like factor 4 expression.

• 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.

• BMB Reports
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• v.52 no.1
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• pp.64-69
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• 2019

The loss of skeletal muscle, called sarcopenia, is an inevitable event during the aging process, and significantly impacts quality of life. Autophagy is known to reduce muscle atrophy caused by dysfunctional organelles, even though the molecular mechanism remains unclear. Here, we have discuss the current understanding of exercise-induced autophagy activation in skeletal muscle regeneration and remodeling, leading to sarcopenia intervention. With aging, dysregulation of autophagy flux inhibits lysosomal storage processes involved in muscle biogenesis. AMPK-ULK1 and the $FoxO/PGC-1{\alpha}$ signaling pathways play a critical role in the induction of autophagy machinery in skeletal muscle, thus these pathways could be targets for therapeutics development. Autophagy has been also shown to be a critical regulator of stem cell fate, which determines satellite cell differentiation into muscle fiber, thereby increasing muscle mass. This review aims to provide a comprehensive understanding of the physiological role of autophagy in skeletal muscle aging and sarcopenia.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.9
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• pp.1244-1249
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• 2002

Myostatin (growth differentiation factor (GDF)-8), is one member of the transforming growth factor $\beta$ superfamily. Investigations of GDF-8 null mice and double-muscled cattle revealed that GDF-8 has a profound influence upon skeletal muscle growth. Therefore, the GDF-8 effect upon the productive performance of pigs is worth exploring. In the present study, the nucleotide sequences and expression levels of GDF-8 genes in European pigs (Landrace and Duroc) and Asian pigs (Taoyuan and Small-ear) were evaluated. Based upon their genetic background these breeds possess significantly distinct growth rate and muscle productionphenotypes. Our sequence data showed that the nucleotide sequences of European and Asian pigs were 100% similar. Postnatal expression of GDF-8 gene in skeletal muscles, from birth to 12 mo of age, among different breeds was measured. GDF-8 expression levels in the longissimus muscle of neonatal European breed littermates were the highest, however it declined significantly (p<0.05) at 1 and 3 mo, and then increased gradually at 6 to 12 mo. The Asian breeds, however, GDF-8 expression level increased markedly at 3 mo and maintained a constant level thereafter. The results indicate that rather than polymorphism within the GDF-8 functional sequence between European and Asia breeds, it was relative to the gene regulation in postnatal muscle growth.

• BMB Reports
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• v.55 no.2
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• pp.104-109
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• 2022

Skeletal myogenesis is essential to keep muscle mass and integrity, and impaired myogenesis is closely related to the etiology of muscle wasting. Recently, miR-141-3p has been shown to be induced under various conditions associated with muscle wasting, such as aging, oxidative stress, and mitochondrial dysfunction. However, the functional significance and mechanism of miR-141-3p in myogenic differentiation have not been explored to date. In this study, we investigated the roles of miR-141-3p on CFL2 expression, proliferation, and myogenic differentiation in C2C12 myoblasts. MiR-141-3p appeared to target the 3'UTR of CFL2 directly and suppressed the expression of CFL2, an essential factor for actin filament (F-actin) dynamics. Transfection of miR-141-3p mimic in myoblasts increased F-actin formation and augmented nuclear Yes-associated protein (YAP), a key component of mechanotransduction. Furthermore, miR-141-3p mimic increased myoblast proliferation and promoted cell cycle progression throughout the S and G2/M phases. Consequently, miR-141-3p mimic led to significant suppressions of myogenic factors expression, such as MyoD, MyoG, and MyHC, and hindered the myogenic differentiation of myoblasts. Thus, this study reveals the crucial role of miR-141-3p in myogenic differentiation via CFL2-YAP-mediated mechanotransduction and provides implications of miRNA-mediated myogenic regulation in skeletal muscle homeostasis.

• 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.

• Biomedical Science Letters
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• v.12 no.3
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• pp.201-208
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• 2006

PI3-kinase activity through p85, the regulatory subunit of class IA PI3-kinase, is indispensable for the growth, differentiation, and survival of skeletal muscle cells, but little is known about the function of other regulatory subunits such as p55 and p50. We examined the subcellular localization and the expression of the regulatory subunits of class IA PI3-kinase in L6 myoblasts. Both p55 and p50 as well as p85 were expressed in L6 myoblasts. Whereas p85 was localized at both cytosolic and nuclear tractions, p55 and p50 were localized at only the nuclear traction. During the differentiation of L6 myoblasts, the protein concentrations of both p55 and p50 were decreased but that of p85 was not significantly changed. Menadione-induced oxidative stress induced the translocation of p85 from cytosol to nucleus and the increase of p55 expression. These results suggest that the regulatory subunits of class IA PI3-kinase play an important role in L6 myoblasts.