WorldWideScience

Sample records for c2c12 myoblast differentiation

  1. β‐Taxilin participates in differentiation of C2C12 myoblasts into myotubes

    Energy Technology Data Exchange (ETDEWEB)

    Sakane, Hiroshi; Makiyama, Tomohiko; Nogami, Satoru; Horii, Yukimi [Department of Molecular and Cell Biology, Graduate school of Medicine, Dokkyo Medical University, 880 Kitakobayashi, Mibu-town, Tochigi 321-0293 (Japan); Akasaki, Kenji [Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima 729-0292 (Japan); Shirataki, Hiromichi, E-mail: hiro-sh@dokkyomed.ac.jp [Department of Molecular and Cell Biology, Graduate school of Medicine, Dokkyo Medical University, 880 Kitakobayashi, Mibu-town, Tochigi 321-0293 (Japan)

    2016-07-15

    Myogenesis is required for the development of skeletal muscle. Accumulating evidence indicates that the expression of several genes are upregulated during myogenesis and these genes play pivotal roles in myogenesis. However, the molecular mechanism underlying myogenesis is not fully understood. In this study, we found that β-taxilin, which is specifically expressed in the skeletal muscle and heart tissues, was progressively expressed during differentiation of C2C12 myoblasts into myotubes, prompting us to investigate the role of β-taxilin in myogenesis. In C2C12 cells, knockdown of β-taxilin impaired the fusion of myoblasts into myotubes, and decreased the diameter of myotubes. We also found that β-taxilin interacted with dysbindin, a coiled-coil-containing protein. Knockdown of dysbindin conversely promoted the fusion of myoblasts into myotubes and increased the diameter of myotubes in C2C12 cells. Furthermore, knockdown of dysbindin attenuated the inhibitory effect of β-taxilin depletion on myotube formation of C2C12 cells. These results demonstrate that β-taxilin participates in myogenesis through suppressing the function of dysbindin to inhibit the differentiation of C2C12 myoblasts into myotubes. - Highlights: • β‐Taxilin is progressively expressed during differentiation of C2C12 cell. • Knockdown of β-taxilin impaired C2C12 myotube formation. • β‐Taxilin interacted with dysbindin. • Knockdown of dysbindin promoted C2C12 myotube formation. • The function of β-taxilin in C2C12 myotube formation depends on dysbindin.

  2. Folic acid is necessary for proliferation and differentiation of C2C12 myoblasts.

    Science.gov (United States)

    Hwang, Seong Y; Kang, Yong J; Sung, Bokyung; Jang, Jung Y; Hwang, Na L; Oh, Hye J; Ahn, Yu R; Kim, Hong J; Shin, Jin H; Yoo, Mi-Ae; Kim, Cheol M; Chung, Hae Y; Kim, Nam D

    2017-05-04

    Folic acid, a water soluble B vitamin, plays an important role in cellular metabolic activities, such as functioning as a cofactor in one-carbon metabolism for DNA and RNA synthesis as well as nucleotide and amino acid biosynthesis in the body. A lack of dietary folic acid can lead to folic acid deficiency and result in several health problems, including macrocytic anemia, elevated plasma homocysteine, cardiovascular disease, birth defects, carcinogenesis, muscle weakness, and walking difficulty. However, the effect of folic acid deficiency on skeletal muscle development and its molecular mechanisms are unknown. We, therefore, investigated the effect of folic acid deficiency on myogenesis in skeletal muscle cells and found that folic acid deficiency induced proliferation inhibition and cell cycle breaking as well as cellular senescence in C2C12 myoblasts, implying that folic acid deficiency influences skeletal muscle development. Folic acid deficiency also inhibited differentiation of C2C12 myoblasts and induced deregulation of the cell cycle exit and many cell cycle regulatory genes. It inhibited expression of muscle-specific marker MyHC as well as myogenic regulatory factor (myogenin). Moreover, immunocytochemistry and Western blot analyses revealed that DNA damage was more increased in folic acid-deficient medium-treated differentiating C2C12 cells. Furthermore, we found that folic acid resupplementation reverses the effect on the cell cycle and senescence in folic acid-deficient C2C12 myoblasts but does not reverse the differentiation of C2C12 cells. Altogether, the study results suggest that folic acid is necessary for normal development of skeletal muscle cells. © 2017 Wiley Periodicals, Inc.

  3. Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Malone, Caroline M.P.; Domaschenz, Renae; Amagase, Yoko [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom); Dunham, Ian [EMBL-European Bioinformatics Institute (EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD (United Kingdom); Murai, Kasumi [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom); Jones, Philip H., E-mail: phj20@cam.ac.uk [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom)

    2011-07-01

    Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, but F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.

  4. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven, E-mail: hvwang@mail.ncku.edu.tw

    2014-09-26

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro.

  5. Docosahexaenoyl ethanolamide improves glucose uptake and alters endocannabinoid system gene expression in proliferating and differentiating C2C12 myoblasts

    Directory of Open Access Journals (Sweden)

    Jeffrey eKim

    2014-03-01

    Full Text Available Skeletal muscle is a major storage site for glycogen and a focus for understanding insulin resistance and type-2-diabetes. New evidence indicates that overactivation of the peripheral endocannabinoid system (ECS in skeletal muscle diminishes insulin sensitivity. Specific n-6 and n-3 polyunsaturated fatty acids (PUFA are precursors for the biosynthesis of ligands that bind to and activate the cannabinoid receptors. The function of the ECS and action of PUFA in skeletal muscle glucose uptake was investigated in proliferating and differentiated C2C12 myoblasts treated with either 25µM of arachidonate (AA or docosahexaenoate (DHA, 25µM of EC [anandamide (AEA, 2-arachidonoylglycerol (2-AG, docosahexaenoylethanolamide (DHEA], 1µM of CB1 antagonist NESS0327, and CB2 antagonist AM630. Compared to the BSA vehicle control cell cultures in both proliferating and differentiated myoblasts those treated with DHEA, the EC derived from the n-3 PUFA DHA, had higher 24 h glucose uptake, while AEA and 2-AG, the EC derived from the n-6 PUFA AA, had lower basal glucose uptake. Adenylyl cyclase mRNA was higher in myoblasts treated with DHA in both proliferating and differentiated states while those treated with AEA or 2-AG were lower compared to the control cell cultures. Western blot and qPCR analysis showed higher expression of the cannabinoid receptors in differentiated myoblasts treated with DHA while the opposite was observed with AA. These findings indicate a compensatory effect of DHA and DHEA compared to AA-derived ligands on the ECS and associated ECS gene expression and higher glucose uptake in myoblasts.Key Words: endocannabinoid system •C2C12 myoblasts cannabinoid receptors glucose uptake gene expression DHEA • polyunsaturated fatty acids

  6. Glycogenome expression dynamics during mouse C2C12 myoblast differentiation suggests a sequential reorganization of membrane glycoconjugates

    Directory of Open Access Journals (Sweden)

    Dupuy Fabrice

    2009-10-01

    Full Text Available Abstract Background Several global transcriptomic and proteomic approaches have been applied in order to obtain new molecular insights on skeletal myogenesis, but none has generated any specific data on glycogenome expression, and thus on the role of glycan structures in this process, despite the involvement of glycoconjugates in various biological events including differentiation and development. In the present study, a quantitative real-time RT-PCR technology was used to profile the dynamic expression of 375 glycogenes during the differentiation of C2C12 myoblasts into myotubes. Results Of the 276 genes expressed, 95 exhibited altered mRNA expression when C2C12 cells differentiated and 37 displayed more than 4-fold up- or down-regulations. Principal Component Analysis and Hierarchical Component Analysis of the expression dynamics identified three groups of coordinately and sequentially regulated genes. The first group included 12 down-regulated genes, the second group four genes with an expression peak at 24 h of differentiation, and the last 21 up-regulated genes. These genes mainly encode cell adhesion molecules and key enzymes involved in the biosynthesis of glycosaminoglycans and glycolipids (neolactoseries, lactoseries and ganglioseries, providing a clearer indication of how the plasma membrane and extracellular matrix may be modified prior to cell fusion. In particular, an increase in the quantity of ganglioside GM3 at the cell surface of myoblasts is suggestive of its potential role during the initial steps of myogenic differentiation. Conclusion For the first time, these results provide a broad description of the expression dynamics of glycogenes during C2C12 differentiation. Among the 37 highly deregulated glycogenes, 29 had never been associated with myogenesis. Their biological functions suggest new roles for glycans in skeletal myogenesis.

  7. Mechanical stimuli on C2C12 myoblasts affect myoblast differentiation, focal adhesion kinase phosphorylation and galectin-1 expression

    DEFF Research Database (Denmark)

    Grossi, Alberto Blak; Lametsch, Rene; Karlsson, Anders H

    2011-01-01

    Mechanical forces are crucial in the regulation of cell morphology and function. At the cellular level, these forces influence myoblast differentiation and fusion. In this study we applied mechanical stimuli to embryonic muscle cells using magnetic microbeads, a method shown to apply stress...... by mechanical stimulation including Galectin-1, Annexin III, and RhoGDI. In this study we demonstrate how the combination of this method of mechanical stimuli and proteomic analysis can be a powerful tool to detect proteins that are potentially interacting in biochemical pathways or complex cellular mechanisms...... during the process of myoblast differentiation. We determined an increase in expression and changes in cellular localization of Galectin-1, in mechanically stimulated myoblasts. A potential involvement of Galectin-1 in myoblast differentiation is presented....

  8. MiRNA-199a-3p Regulates C2C12 Myoblast Differentiation through IGF-1/AKT/mTOR Signal Pathway

    Directory of Open Access Journals (Sweden)

    Long Jia

    2013-12-01

    Full Text Available MicroRNAs constitute a class of ~22-nucleotide non-coding RNAs. They modulate gene expression by associating with the 3' untranslated regions (3' UTRs of messenger RNAs (mRNAs. Although multiple miRNAs are known to be regulated during myoblast differentiation, their individual roles in muscle development are still not fully understood. In this study, we showed that miR-199a-3p was highly expressed in skeletal muscle and was induced during C2C12 myoblasts differentiation. We also identified and confirmed several genes of the IGF-1/AKT/mTOR signal pathway, including IGF-1, mTOR, and RPS6KA6, as important cellular targets of miR-199a-3p in myoblasts. Overexpression of miR-199a-3p partially blocked C2C12 myoblast differentiation and the activation of AKT/mTOR signal pathway, while interference of miR-199a-3p by antisense oligonucleotides promoted C2C12 differentiation and myotube hypertrophy. Thus, our studies have established miR-199a-3p as a potential regulator of myogenesis through the suppression of IGF-1/AKT/mTOR signal pathway.

  9. MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Zhiyuan; Sun, Xiaorui; Xu, Dequan; Xiong, Yuanzhu; Zuo, Bo, E-mail: zuobo@mail.hzau.edu.cn

    2015-11-27

    Myogenesis is a complex process including myoblast proliferation, differentiation and myotube formation and is controlled by myogenic regulatory factors (MRFs), MyoD, MyoG, Myf5 and Myf6 (also known as MRF4). MicroRNA is a kind of ∼22 nt-long non-coding small RNAs, and act as key transcriptional or post-transcriptional regulators of gene expression. Identification of miRNAs involved in the regulation of muscle genes could improve our understanding of myogenesis process. In this study, we investigated the regulation of Myf6 gene by miRNAs. We showed that miR-374b specifically bound to the 3'untranslated region (UTR) of Myf6 and down-regulated the expression of Myf6 gene at both mRNA and protein level. Furthermore, miR-374b is ubiquitously expressed in the tissues of adult C57BL6 mouse, and the mRNA abundance increases first and then decreases during C2C12 myoblasts differentiation. Over-expression of miR-374b impaired C2C12 cell differentiation, while inhibiting miR-374b expression by 2′-O-methyl antisense oligonucleotides promoted C2C12 cell differentiation. Taken together, our findings identified miR-374b directly targets Myf6 and negatively regulates myogenesis. - Highlights: • MiR-374b directly targets 3′UTR of Myf6. • MiR-374b negatively regulates Myf6 in C2C12 cells. • MiR-374b abundance significiently changes during C2C12 cells differentiation. • MiR-374b negatively regulates C2C12 cells differentiation.

  10. Retinoid acid-induced microRNA-27b-3p impairs C2C12 myoblast proliferation and differentiation by suppressing α-dystrobrevin

    Energy Technology Data Exchange (ETDEWEB)

    Li, Nan; Tang, Yi; Liu, Bo; Cong, Wei; Liu, Chao, E-mail: liuchao_19760711@yahoo.com; Xiao, Jing, E-mail: xiaoj@dmu.edu.cn

    2017-01-15

    We previously reported that excess retinoic acid (RA) resulted in hypoplastic and derangement of myofilaments in embryonic tongue by inhibiting myogenic proliferation and differentiation through CamKIID pathway. Our further studies revealed that the expression of a series of miRNAs was altered by RA administration in embryonic tongue as well as in C2C12 cells. Thus, if excess RA impairs myogenic proliferation and differentiation through miRNAs is taken into account. In present study, miR-27b-3p was found up-regulated in RA-treated C2C12 cells as in embryonic tongue, and predicted to target the 3′UTR of α-dystrobrevin (DTNA). Luciferase reporter assays confirmed the direct interaction between miR-27b-3p and the 3′UTR of DTNA. MiR-27b-3p mimics recapitulated the RA repression on DTNA expression, C2C12 proliferation and differentiation, while the miR-27b-3p inhibitor circumvented these defects resulting from excess RA. As expected, the effects of siDTNA on C2C12 were coincided with those by RA treatment or miR-27b-3p mimics. Therefore, these findings indicated that excess RA inhibited the myoblast proliferation and differentiation by up-regulating miR-27b-3p to target DTNA, which implied a new mechanism in myogenic hypoplasia. - Highlights: • A mechanism that RA results in tongue deformity by disrupting the myogenesis. • A non-muscle specific miR mediating the RA suppression on tongue myogenesis. • A target gene of non-muscle specific miR involved in RA induced tongue deformity.

  11. DRAGON, a GPI-anchored membrane protein, inhibits BMP signaling in C2C12 myoblasts.

    Science.gov (United States)

    Kanomata, Kazuhiro; Kokabu, Shoichiro; Nojima, Junya; Fukuda, Toru; Katagiri, Takenobu

    2009-06-01

    Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation of myoblasts via binding to cell surface receptors. Repulsive guidance molecules (RGMs) have been identified as BMP co-receptors. We report here that DRAGON/RGMb, a member of the RGM family, suppressed BMP signaling in C2C12 myoblasts via a novel mechanism. All RGMs were expressed in C2C12 cells that were differentiated into myocytes and osteoblastic cells, but RGMc was not detected in immature cells. In C2C12 cells, only DRAGON suppressed ALP and Id1 promoter activities induced by BMP-4 or by constitutively activated BMP type I receptors. This inhibition by DRAGON was dependent on the secretory form of the von Willbrand factor type D domain. DRAGON even suppressed BMP signaling induced by constitutively activated Smad1. Over-expression of neogenin did not alter the inhibitory capacity of DRAGON. Taken together, these findings indicate that DRAGON may be an inhibitor of BMP signaling in C2C12 myoblasts. We also suggest that a novel molecule(s) expressed on the cell membrane may mediate the signal transduction of DRAGON in order to suppress BMP signaling in C2C12 myoblasts.

  12. The Mouse C2C12 Myoblast Cell Surface N-Linked Glycoproteome

    Science.gov (United States)

    Gundry, Rebekah L.; Raginski, Kimberly; Tarasova, Yelena; Tchernyshyov, Irina; Bausch-Fluck, Damaris; Elliott, Steven T.; Boheler, Kenneth R.; Van Eyk, Jennifer E.; Wollscheid, Bernd

    2009-01-01

    Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and β-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes. PMID:19656770

  13. Nanoparticle-mediated intracellular lipid accumulation during C2C12 cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Tsukahara, Tamotsu, E-mail: ttamotsu@shinshu-u.ac.jp [Department of Integrative Physiology and Bio-System Control, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Haniu, Hisao, E-mail: hhaniu@shinshu-u.ac.jp [Institute of Carbon Science and Technology, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan)

    2011-03-25

    Research highlights: {yields} HTT2800 has a significant effect on intracellular lipid accumulation. {yields} HTT2800 reduced muscle-specific genes and led to the emergence of adipocyte-related genes. {yields} HT2800 converts the differentiation pathway of C2C12 myoblasts to that of adipoblast-like cells. -- Abstract: In this report, we sought to elucidate whether multiwall carbon nanotubes are involved in the modulation of the proliferation and differentiation of the skeletal muscle cell line C2C12. Skeletal muscle is a major mass peripheral tissue that accounts for 40% of total body weight and 50% of energy consumption. We focused on the differentiation pathway of myoblasts after exposure to a vapor-grown carbon fiber, HTT2800, which is one of the most highly purified carbon nanotubes. This treatment leads in parallel to the expression of a typical adipose differentiation program. We found that HTT2800 stimulated intracellular lipid accumulation in C2C12 cells. We have also shown by quantified PCR analysis that the expression of adipose-related genes was markedly upregulated during HTT2800 exposure. Taken together, these results suggest that HTT2800 specifically converts the differentiation pathway of C2C12 myoblasts to that of adipoblast-like cells.

  14. Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes.

    Science.gov (United States)

    Schöneich, Christian; Dremina, Elena; Galeva, Nadezhda; Sharov, Victor

    2014-01-01

    Muscle cell apoptosis accompanies normal muscle development and regeneration, as well as degenerative diseases and aging. C2C12 murine myoblast cells represent a common model to study muscle differentiation. Though it was already shown that myogenic differentiation of C2C12 cells is accompanied by enhanced apoptosis in a fraction of cells, either the cell population sensitive to apoptosis or regulatory mechanisms for the apoptotic response are unclear so far. In the current study we characterize apoptotic phenotypes of different types of C2C12 cells at all stages of differentiation, and report here that myotubes of differentiated C2C12 cells with low levels of anti-apoptotic Bcl-2 expression are particularly vulnerable to apoptosis even though they are displaying low levels of pro-apoptotic proteins Bax, Bak and Bad. In contrast, reserve cells exhibit higher levels of Bcl-2 and high resistance to apoptosis. The transfection of proliferating myoblasts with Bcl-2 prior to differentiation did not protect against spontaneous apoptosis accompanying differentiation of C2C12 cells but led to Bcl-2 overexpression in myotubes and to significant protection from apoptotic cell loss caused by exposure to hydrogen peroxide. Overall, our data advocate for a Bcl-2-dependent mechanism of apoptosis in differentiated muscle cells. However, downstream processes for spontaneous and hydrogen peroxide induced apoptosis are not completely similar. Apoptosis in differentiating myoblasts and myotubes is regulated not through interaction of Bcl-2 with pro-apoptotic Bcl-2 family proteins such as Bax, Bak, and Bad.

  15. BAMBI Promotes C2C12 Myogenic Differentiation by Enhancing Wnt/β-Catenin Signaling

    Directory of Open Access Journals (Sweden)

    Qiangling Zhang

    2015-08-01

    Full Text Available Bone morphogenic protein and activin membrane-bound inhibitor (BAMBI is regarded as an essential regulator of cell proliferation and differentiation that represses transforming growth factor-β and enhances Wnt/β-catenin signaling in various cell types. However, its role in skeletal muscle remains largely unknown. In the current study, we found that the expression level of BAMBI peaked in the early differentiation phase of the C2C12 rodent myoblast cell line. Knockdown of BAMBI via siRNA inhibited C2C12 differentiation, indicated by repressed MyoD, MyoG, and MyHC expression as well as reductions in the differentiation and fusion indices. BAMBI knockdown reduced the activity of Wnt/β-catenin signaling, as characterized by the decreased nuclear translocation of β-catenin and the lowered transcription of Axin2, which is a well-documented target gene of the Wnt/β-catenin signaling pathway. Furthermore, treatment with LiCl, an activator of Wnt/β-catenin signaling, rescued the reduction in C2C12 differentiation caused by BAMBI siRNA. Taken together, our data suggest that BAMBI is required for normal C2C12 differentiation, and that its role in myogenesis is mediated by the Wnt/β-catenin pathway.

  16. Geigerin-induced cytotoxicity in a murine myoblast cell line (C2C12

    Directory of Open Access Journals (Sweden)

    Christo J. Botha

    2017-01-01

    Full Text Available Geigeria poisoning in sheep, locally known as ‘vermeersiekte’, is an economically important plant poisoning in southern Africa. The toxic principles contained by the toxic plants are believed to be several sesquiterpene lactones, such as geigerin, vermeeric acid and vermeerin, which cause striated muscle lesions in small stock. Because of ethical issues surrounding the use of live animals in toxicity studies, there is currently a dire need to establish an in vitro model that can be used to replace traditional animal experimentation. The objective of this study was to determine the cytotoxicity of geigerin in a murine myoblast cell line (C2C12 using methyl-thiazol-tetrazolium (MTT and lactate dehydrogenase (LDH assays, annexin V and propidium iodide (PI flow cytometry and transmission electron microscopy (TEM. Mouse myoblasts were exposed to 2.0 mM, 2.5 mM and 5.0 mM geigerin for 24, 48 and 72 h. A concentration-dependent cytotoxic response was observed. Apoptosis was detected by means of annexin V flow cytometry during the first 24 h and apoptotic bodies were also visible on TEM. According to the LDH and PI flow cytometry results, myoblast cell membranes were not injured. We concluded that the murine myoblast cell line (C2C12 is a suitable model for future studies planned to evaluate the cytotoxicity of other and combinations of sesquiterpene lactones, with and without metabolic activation, implicated in ‘vermeersiekte’ and to elucidate the subcellular effects of these myotoxins on cultured myoblasts.

  17. Uniaxial cyclic strain of human adipose–derived mesenchymal stem cells and C2C12 myoblasts in coculture

    Directory of Open Access Journals (Sweden)

    James M Dugan

    2014-03-01

    Full Text Available Tissue engineering skeletal muscle in vitro is of great importance for the production of tissue-like constructs for treating tissue loss due to traumatic injury or surgery. However, it is essential to find new sources of cells for muscle engineering as efficient in vitro expansion and culture of primary myoblasts are problematic. Mesenchymal stem cells may be a promising source of myogenic progenitor cells and may be harvested in large numbers from adipose tissue. As skeletal muscle is a mechanically dynamic tissue, we have investigated the effect of cyclic mechanical strain on the myogenic differentiation of a coculture system of murine C2C12 myoblasts and human adipose–derived mesenchymal stem cells. Fusion of mesenchymal stem cells with nascent myotubes and expression of human sarcomeric proteins was observed, indicating the potential for myogenic differentiation of human mesenchymal stem cells. Cyclic mechanical strain did not affect the fusion of mesenchymal stem cells, but maturation of myotubes was perturbed.

  18. The mouse C2C12 myoblast cell surface N-linked glycoproteome: identification, glycosite occupancy, and membrane orientation.

    Science.gov (United States)

    Gundry, Rebekah L; Raginski, Kimberly; Tarasova, Yelena; Tchernyshyov, Irina; Bausch-Fluck, Damaris; Elliott, Steven T; Boheler, Kenneth R; Van Eyk, Jennifer E; Wollscheid, Bernd

    2009-11-01

    Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and beta-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes.

  19. Cyclic stretch facilitates myogenesis in C2C12 myoblasts and rescues thiazolidinedione-inhibited myotube formation

    Directory of Open Access Journals (Sweden)

    Ya-Ju eChang

    2016-03-01

    Full Text Available Thiazolidinedione (TZD, a specific peroxisome proliferator-activated receptor r (PPARr agonist, was developed to control blood glucose in diabetes patients. However, several side effects were reported that increased the risk of heart failure. We used C2C12 myoblasts to investigate the role of PPARs and their transcriptional activity during myotube formation. The role of mechanical stretch during myogenesis was also explored by applying cyclic stretch to the differentiating C2C12 myoblasts with 10% strain deformation at 1 Hz. The myogenesis medium (MM, composed of Dulbecco’s modified Eagle’s medium with 2% horse serum, facilitated myotube formation with increased myosin heavy chain and a-smooth muscle actin (a-SMA protein expression. The PPAR protein and PPAR response element (PPRE promoter activity decreased during MM induction. Cyclic stretch further facilitated the myogenesis in MM with increased a-SMA and decreased PPARr protein expression and inhibited PPRE promoter activity. Adding a PPARr agonist (TZD to the MM stopped the myogenesis and restored the PPRE promoter activity, whereas a PPARr antagonist (GW9662 significantly increased the myotube number and length. During the myogenesis induction, application of cyclic stretch rescued the inhibitory effects of TZD. These results provide novel perspectives for mechanical stretch to interplay and rescue the dysfunction of myogenesis with the involvement of PPARr and its target drugs.

  20. Graphene oxide increases the viability of C2C12 myoblasts microencapsulated in alginate.

    Science.gov (United States)

    Ciriza, J; Saenz del Burgo, L; Virumbrales-Muñoz, M; Ochoa, I; Fernandez, L J; Orive, G; Hernandez, R M; Pedraz, J L

    2015-09-30

    Cell microencapsulation represents a great promise for long-term drug delivery, but still several challenges need to be overcome before its translation into the clinic, such as the long term cell survival inside the capsules. On this regard, graphene oxide has shown to promote proliferation of different cell types either in two or three dimensions. Therefore, we planned to combine graphene oxide with the cell microencapsulation technology. We first studied the effect of this material on the stability of the capsules and next we analyzed the biocompatibility of this chemical compound with erythropoietin secreting C2C12 myoblasts within the microcapsule matrix. We produced 160 μm-diameter alginate microcapsules with increasing concentrations of graphene oxide and did not find modifications on the physicochemical parameters of traditional alginate microcapsules. Moreover, we observed that the viability of encapsulated cells within alginate microcapsules containing specific graphene oxide concentrations was enhanced. These results provide a relevant step for the future clinical application of graphene oxide on cell microencapsulation. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. The Mouse C2C12 Myoblast Cell Surface N-Linked Glycoproteome

    OpenAIRE

    Gundry, Rebekah L.; Raginski, Kimberly; Tarasova, Yelena; Tchernyshyov, Irina; Bausch-Fluck, Damaris; Elliott, Steven T.; Boheler, Kenneth R.; Van Eyk, Jennifer E.; Wollscheid, Bernd

    2009-01-01

    Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing ...

  2. Stimulating effect of graphene oxide on myogenesis of C2C12 myoblasts on RGD peptide-decorated PLGA nanofiber matrices.

    Science.gov (United States)

    Shin, Yong Cheol; Lee, Jong Ho; Kim, Min Jeong; Hong, Suck Won; Kim, Bongju; Hyun, Jung Keun; Choi, Yu Suk; Park, Jong-Chul; Han, Dong-Wook

    2015-01-01

    In the field of biomedical engineering, many studies have focused on the possible applications of graphene and related nanomaterials due to their potential for use as scaffolds, coating materials and delivery carriers. On the other hand, electrospun nanofiber matrices composed of diverse biocompatible polymers have attracted tremendous attention for tissue engineering and regenerative medicine. However, their combination is intriguing and still challenging. In the present study, we fabricated nanofiber matrices composed of M13 bacteriophage with RGD peptide displayed on its surface (RGD-M13 phage) and poly(lactic-co-glycolic acid, PLGA) and characterized their physicochemical properties. In addition, the effect of graphene oxide (GO) on the cellular behaviors of C2C12 myoblasts, which were cultured on PLGA decorated with RGD-M13 phage (RGD/PLGA) nanofiber matrices, was investigated. Our results revealed that the RGD/PLGA nanofiber matrices have suitable physicochemical properties as a tissue engineering scaffold and the growth of C2C12 myoblasts were significantly enhanced on the matrices. Moreover, the myogenic differentiation of C2C12 myoblasts was substantially stimulated when they were cultured on the RGD/PLGA matrices in the presence of GO. In conclusion, these findings propose that the combination of RGD/PLGA nanofiber matrices and GO can be used as a promising strategy for skeletal tissue engineering and regeneration.

  3. Propolis Ethanol Extract Stimulates Cytokine and Chemokine Production through NF-κB Activation in C2C12 Myoblasts

    Directory of Open Access Journals (Sweden)

    Kohei Washio

    2015-01-01

    Full Text Available Myoblast activation is a triggering event for muscle remodeling. We assessed the stimulatory effects of propolis, a beehive product, on myoblasts. After an 8 h treatment with 100 μg/mL of Brazilian propolis ethanol extract, expression of various chemokines, including CCL-2 and CCL-5, and cytokines, such as IL-6, increased. This propolis-induced cytokine production appears to depend on NF-κB activation, because the IKK inhibitor BMS-345541 repressed mRNA levels of CCL-2 by ~66%, CCL-5 by ~81%, and IL-6 by ~69% after propolis treatment. Supernatant from propolis-conditioned C2C12 cells upregulated RAW264 macrophage migration. The supernatant also stimulated RAW264 cells to produce angiogenic factors, including VEGF-A and MMP-12. Brazilian green propolis therefore causes myoblasts to secrete cytokines and chemokines, which might contribute to tissue remodeling of skeletal muscle.

  4. Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Eom, Young Woo [Cell Therapy and Tissue Engineering Center, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Biomedical Research Institute, Lifeliver Co., Ltd., Suwon (Korea, Republic of); Lee, Jong Eun; Yang, Mal Sook; Jang, In Keun; Kim, Hyo Eun; Lee, Doo Hoon; Kim, Young Jin [Biomedical Research Institute, Lifeliver Co., Ltd., Suwon (Korea, Republic of); Park, Won Jin [Dr. Park' s Aesthetic Clinic, Seoul (Korea, Republic of); Kong, Jee Hyun; Shim, Kwang Yong [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Lee, Jong In, E-mail: oncochem@yonsei.ac.kr [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Kim, Hyun Soo, E-mail: khsmd@unitel.co.kr [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of)

    2011-04-29

    Highlights: {yields} hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. {yields} Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. {yields} hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.

  5. Fusion and differentiation of murine C2C12 skeletal muscle cells that express Trichinella spiralis p43 protein.

    Science.gov (United States)

    Jasmer, Douglas P; Kwak, Dongmi

    2006-02-01

    The ability of a 43 kDa stichocyte protein from Trichinella spiralis (Tsp43) to interfere with mammalian skeletal muscle gene expression was investigated. A MYC-tagged Tsp43 construct was expressed as a recombinant protein in C2C12 myoblasts. Transfection with low amounts of expression plasmid was required for successful expression of the protein. This construct had apparent toxic effects on transfected myoblasts and ectopic green fluorescent protein expression was suppressed in myoblasts co-transfected with the Tsp43 construct. These effects may result from similarities of Tsp43 to DNase II. Use of the general DNase inhibitor aurintricarboxylic acid (ATA) enhanced expression of MYC-Tsp43 in transfected muscle cells. Myoblasts transfected with Tsp43 did not fuse well when cultured under differentiation conditions without ATA. In contrast, transfected myoblasts transiently cultured with ATA underwent fusion and differentiation. Under short-term differentiation conditions without ATA, unfused myoblasts nevertheless expressed both MYC-Tsp43 and myosin heavy chain. Collectively, the results support that Tsp43 has a role in the T. spiralis life cycle that is distinct from repressing muscle gene expression during the muscle phase of infection. While the function of Tsp43 as a DNase is under debate, the effects of ATA on transfected muscle cells were consistent with this possibility.

  6. Graphene oxide-stimulated myogenic differentiation of C2C12 cells on PLGA/RGD peptide nanofiber matrices

    Science.gov (United States)

    Shin, Y. C.; Lee, J. H.; Kim, M. J.; Hong, S. W.; Oh, J.-W.; Kim, C.-S.; Kim, B.; Hyun, J. K.; Kim, Y.-J.; Han, D.-W.

    2015-07-01

    During the last decade, much attention has been paid to graphene-based nanomaterials because they are considered as potential candidates for biomedical applications such as scaffolds for tissue engineering and substrates for the differentiation of stem cells. Until now, electrospun matrices composed of various biodegradable copolymers have been extensively developed for tissue engineering and regeneration; however, their use in combination with graphene oxide (GO) is novel and challenging. In this study, nanofiber matrices composed of poly(lactic-co-glycolic acid, PLGA) and M13 phage with RGD peptide displayed on its surface (RGD peptide-M13 phage) were prepared as extracellular matrix (ECM)-mimicking substrates. RGD peptide is a tripeptide (Arg-Gly-Asp) found on ECM proteins that promotes various cellular behaviors. The physicochemical properties of PLGA and RGD peptide-M13 phage (PLGA/RGD peptide) nanofiber matrices were characterized by atomic force microscopy, Fourier-transform infrared spectroscopy and thermogravimetric analysis. In addition, the growth of C2C12 mouse myoblasts on the PLGA/RGD peptide matrices was examined by measuring the metabolic activity. Moreover, the differentiation of C2C12 mouse myoblasts on the matrices when treated with GO was evaluated. The cellular behaviors, including growth and differentiation of C2C12 mouse myoblasts, were substantially enhanced on the PLGA/RGD peptide nanofiber matrices when treated with GO. Overall, these findings suggest that the PLGA/RGD peptide nanofiber matrices can be used in combination with GO as a novel strategy for skeletal tissue regeneration.

  7. Fatty acids increase glucose uptake and metabolism in C2C12 myoblasts stably transfected with human lipoprotein lipase.

    Science.gov (United States)

    Capell, Warren H; Schlaepfer, Isabel R; Wolfe, Pamela; Watson, Peter A; Bessesen, Daniel H; Pagliassotti, Michael J; Eckel, Robert H

    2010-10-01

    Cellular effects of FFA might differ from those of lipoprotein triglyceride (TG)-derived fatty acids (TGFA). The aim of the current study was to examine the relationship between lipoprotein lipase (LPL) expression, TGFA, or FFA availability and glucose metabolism in the absence of insulin in C2C12 myoblasts. Control myoblasts or myoblasts stably transfected with human lipoprotein lipase (C2/LPL; 15-fold greater LPL activity) were incubated for 12 h in fetal bovine serum-free medium in the absence or presence of Intralipid-20. Intracellular retention of labeled medium glucose was assessed in a subset of experiments. In the presence of Intralipid, medium glucose disappearance was increased in C2/LPL cells but not in control cells. In both cell types, glucose label retention in cellular TG was increased in the presence of Intralipid; incubation with albumin-bound oleate produced similar results. In the presence of Intralipid, the LPL hydrolytic inhibitor tetrahydrolipstatin blocked excess glucose retention in cellular TG but did not significantly decrease glucose disappearance in C2/LPL cells. Changes in glucose transport or hexokinase II did not explain the altered glucose disappearance in C2/LPL cells. Our results suggest that LPL overexpression in these cells leads to chronic metabolic adaptations that alter glucose uptake and retention.

  8. Conessine Interferes with Oxidative Stress-Induced C2C12 Myoblast Cell Death through Inhibition of Autophagic Flux.

    Directory of Open Access Journals (Sweden)

    Hyunju Kim

    Full Text Available Conessine, a steroidal alkaloid isolated from Holarrhena floribunda, has anti-malarial activity and interacts with the histamine H3 receptor. However, the cellular effects of conessine are poorly understood. Accordingly, we evaluated the involvement of conessine in the regulation of autophagy. We searched natural compounds that modulate autophagy, and conessine was identified as an inhibitor of autophagic flux. Conessine treatment induced the formation of autophagosomes, and p62, an autophagic adapter, accumulated in the autophagosomes. Reactive oxygen species such as hydrogen peroxide (H2O2 result in muscle cell death by inducing excessive autophagic flux. Treatment with conessine inhibited H2O2-induced autophagic flux in C2C12 myoblast cells and also interfered with cell death. Our results indicate that conessine has the potential effect to inhibit muscle cell death by interfering with autophagic flux.

  9. Conessine Interferes with Oxidative Stress-Induced C2C12 Myoblast Cell Death through Inhibition of Autophagic Flux.

    Science.gov (United States)

    Kim, Hyunju; Lee, Kang Il; Jang, Minsu; Namkoong, Sim; Park, Rackhyun; Ju, Hyunwoo; Choi, Inho; Oh, Won Keun; Park, Junsoo

    2016-01-01

    Conessine, a steroidal alkaloid isolated from Holarrhena floribunda, has anti-malarial activity and interacts with the histamine H3 receptor. However, the cellular effects of conessine are poorly understood. Accordingly, we evaluated the involvement of conessine in the regulation of autophagy. We searched natural compounds that modulate autophagy, and conessine was identified as an inhibitor of autophagic flux. Conessine treatment induced the formation of autophagosomes, and p62, an autophagic adapter, accumulated in the autophagosomes. Reactive oxygen species such as hydrogen peroxide (H2O2) result in muscle cell death by inducing excessive autophagic flux. Treatment with conessine inhibited H2O2-induced autophagic flux in C2C12 myoblast cells and also interfered with cell death. Our results indicate that conessine has the potential effect to inhibit muscle cell death by interfering with autophagic flux.

  10. Extremely Low-Frequency Electromagnetic Fields Affect Myogenic Processes in C2C12 Myoblasts: Role of Gap-Junction-Mediated Intercellular Communication

    Directory of Open Access Journals (Sweden)

    Caterina Morabito

    2017-01-01

    Full Text Available Extremely low-frequency electromagnetic fields (ELF-EMFs can interact with biological systems. Although they are successfully used as therapeutic agents in physiatrics and rehabilitative practice, they might represent environmental pollutants and pose a risk to human health. Due to the lack of evidence of their mechanism of action, the effects of ELF-EMFs on differentiation processes in skeletal muscle were investigated. C2C12 myoblasts were exposed to ELF-EMFs generated by a solenoid. The effects of ELF-EMFs on cell viability and on growth and differentiation rates were studied using colorimetric and vital dye assays, cytomorphology, and molecular analysis of MyoD and myogenin expression, respectively. The establishment of functional gap junctions was investigated analyzing connexin 43 expression levels and measuring cell permeability, using microinjection/dye-transfer assays. The ELF-EMFs did not affect C2C12 myoblast viability or proliferation rate. Conversely, at ELF-EMF intensity in the mT range, the myogenic process was accelerated, through increased expression of MyoD, myogenin, and connexin 43. The increase in gap-junction function suggests promoting cell fusion and myotube differentiation. These data provide the first evidence of the mechanism through which ELF-EMFs may provide therapeutic benefits and can resolve, at least in part, some conditions of muscle dysfunction.

  11. Extremely Low-Frequency Electromagnetic Fields Affect Myogenic Processes in C2C12 Myoblasts: Role of Gap-Junction-Mediated Intercellular Communication.

    Science.gov (United States)

    Morabito, Caterina; Steimberg, Nathalie; Rovetta, Francesca; Boniotti, Jennifer; Guarnieri, Simone; Mazzoleni, Giovanna; Mariggiò, Maria A

    2017-01-01

    Extremely low-frequency electromagnetic fields (ELF-EMFs) can interact with biological systems. Although they are successfully used as therapeutic agents in physiatrics and rehabilitative practice, they might represent environmental pollutants and pose a risk to human health. Due to the lack of evidence of their mechanism of action, the effects of ELF-EMFs on differentiation processes in skeletal muscle were investigated. C2C12 myoblasts were exposed to ELF-EMFs generated by a solenoid. The effects of ELF-EMFs on cell viability and on growth and differentiation rates were studied using colorimetric and vital dye assays, cytomorphology, and molecular analysis of MyoD and myogenin expression, respectively. The establishment of functional gap junctions was investigated analyzing connexin 43 expression levels and measuring cell permeability, using microinjection/dye-transfer assays. The ELF-EMFs did not affect C2C12 myoblast viability or proliferation rate. Conversely, at ELF-EMF intensity in the mT range, the myogenic process was accelerated, through increased expression of MyoD, myogenin, and connexin 43. The increase in gap-junction function suggests promoting cell fusion and myotube differentiation. These data provide the first evidence of the mechanism through which ELF-EMFs may provide therapeutic benefits and can resolve, at least in part, some conditions of muscle dysfunction.

  12. A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kodaka, Manami [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Yang, Zeyu [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Ultrasound, Shengjing Hospital of China Medical University, Shenyang (China); Nakagawa, Kentaro; Maruyama, Junichi [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Xu, Xiaoyin [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Breast Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou (China); Sarkar, Aradhan; Ichimura, Ayana [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Nasu, Yusuke [Department of Breast Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou (China); Ozawa, Takeaki [Department of Chemistry, School of Science, The University of Tokyo, Tokyo (Japan); Iwasa, Hiroaki [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Ishigami-Yuasa, Mari [Chemical Biology Screening Center, Tokyo Medical and Dental University, Tokyo (Japan); Ito, Shigeru [Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo (Japan); Kagechika, Hiroyuki [Chemical Biology Screening Center, Tokyo Medical and Dental University, Tokyo (Japan); Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo (Japan); and others

    2015-08-15

    The development of the efficient screening system of detecting compounds that promote myogenesis and prevent muscle atrophy is important. Mouse C2C12 cells are widely used to evaluate myogenesis but the procedures of the assay are not simple and the quantification is not easy. We established C2C12 cells expressing the N-terminal green fluorescence protein (GFP) and the C-terminal GFP (GFP1–10 and GFP11 cells). GFP1–10 and GFP11 cells do not exhibit GFP signals until they are fused. The signal intensity correlates with the expression of myogenic markers and myofusion. Myogenesis-promoting reagents, such as insulin-like growth factor-1 (IGF1) and β-guanidinopropionic acid (GPA), enhance the signals, whereas the poly-caspase inhibitor, z-VAD-FMK, suppresses it. GFP signals are observed when myotubes formed by GFP1–10 cells are fused with single nuclear GFP11 cells, and enhanced by IGF1, GPA, and IBS008738, a recently-reported myogenesis-promoting reagent. Fusion between myotubes formed by GFP1–10 and GFP11 cells is associated with the appearance of GFP signals. IGF1 and GPA augment these signals, whereas NSC23766, Rac inhibitor, decreases them. The conditioned medium of cancer cells suppresses GFP signals during myogenesis and reduces the width of GFP-positive myotubes after differentiation. Thus the novel split GFP-based assay will provide the useful method for the study of myogenesis, myofusion, and atrophy. - Highlights: • C2C12 cells expressing split GFP proteins show GFP signals when mix-cultured. • The GFP signals correlate with myogenesis and myofusion. • The GFP signals attenuate under the condition that muscle atrophy is induced.

  13. Sodium arsenite represses the expression of myogenin in C2C12 mouse myoblast cells through histone modifications and altered expression of Ezh2, Glp, and Igf-1

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Gia-Ming [Environmental Toxicology Graduate Program, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States); Present address: The University of Chicago, Section of Hematology/Oncology, 900 E. 57th Street, Room 7134, Chicago, IL 60637 (United States); Bain, Lisa J., E-mail: lbain@clemson.edu [Environmental Toxicology Graduate Program, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States); Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States)

    2012-05-01

    Arsenic is a toxicant commonly found in water systems and chronic exposure can result in adverse developmental effects including increased neonatal death, stillbirths, and miscarriages, low birth weight, and altered locomotor activity. Previous studies indicate that 20 nM sodium arsenite exposure to C2C12 mouse myocyte cells delayed myoblast differentiation due to reduced myogenin expression, the transcription factor that differentiates myoblasts into myotubes. In this study, several mechanisms by which arsenic could alter myogenin expression were examined. Exposing differentiating C2C12 cells to 20 nM arsenic increased H3K9 dimethylation (H3K9me2) and H3K9 trimethylation (H3K9me3) by 3-fold near the transcription start site of myogenin, which is indicative of increased repressive marks, and reduced H3K9 acetylation (H3K9Ac) by 0.5-fold, indicative of reduced permissive marks. Protein expression of Glp or Ehmt1, a H3-K9 methyltransferase, was also increased by 1.6-fold in arsenic-exposed cells. In addition to the altered histone remodeling status on the myogenin promoter, protein and mRNA levels of Igf-1, a myogenic growth factor, were significantly repressed by arsenic exposure. Moreover, a 2-fold induction of Ezh2 expression, and an increased recruitment of Ezh2 (3.3-fold) and Dnmt3a (∼ 2-fold) to the myogenin promoter at the transcription start site (− 40 to + 42), were detected in the arsenic-treated cells. Together, we conclude that the repressed myogenin expression in arsenic-exposed C2C12 cells was likely due to a combination of reduced expression of Igf-1, enhanced nuclear expression and promoter recruitment of Ezh2, and altered histone remodeling status on myogenin promoter (− 40 to + 42). -- Highlights: ► Igf-1 expression is decreased in C2C12 cells after 20 nM arsenite exposure. ► Arsenic exposure alters histone remodeling on the myogenin promoter. ► Glp expression, a H3–K9 methyltransferase, was increased in arsenic-exposed cells. ► Ezh2

  14. 4,4'-Dichlorodiphenyltrichloroethane (DDT) and 4,4'-dichlorodiphenyldichloroethylene (DDE) inhibit myogenesis in C2C12 myoblasts.

    Science.gov (United States)

    Kim, Jonggun; Park, Min Young; Kim, Yoo; Yoon, Kyong Sup; Clark, John Marshall; Park, Yeonhwa; Whang, Kwang-Youn

    2017-12-01

    Most countries have banned the use of 4,4'-dichlorodiphenyltrichloroethane (DDT). However, owing to its extremely high lipophilic characteristics, DDT and its metabolite 4,4'-dichlorodiphenyldichloroethylene (DDE) are ubiquitous in the environment and in many types of food. The positive correlation between exposure to insecticides, including DDT and DDE, and weight gain, resulting in impaired energy metabolism in offspring following perinatal DDT and DDE exposure, was previously reported. Therefore the influence of DDT and DDE on myogenesis using C2C12 myoblasts was investigated in this study. DDT and DDE decreased myotube formation dose- and time-dependently. Among myogenic regulatory factors, DDT and DDE mainly decreased MyoD1 and Myf5 expression. DDT and DDE treatment also altered Myostatin expression, phosphorylation of protein kinase B, p70 ribosomal protein S6 kinase, forkhead box O protein 3 and mammalian target of rapamycin, resulting in attenuation of myotube formation. These results may have significant implications for understanding the effects of developmental exposure of DDT and DDE on myogenesis and development of obesity and type 2 diabetes later in life. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  15. Hybrid Alginate-Protein-Coated Graphene Oxide Microcapsules Enhance the Functionality of Erythropoietin Secreting C2C12 Myoblasts.

    Science.gov (United States)

    Saenz Del Burgo, Laura; Ciriza, Jesús; Acarregui, Argia; Gurruchaga, Haritz; Blanco, Francisco Javier; Orive, Gorka; Hernández, Rosa María; Pedraz, Jose Luis

    2017-03-06

    The beneficial effect of combining alginate hydrogel with graphene oxide (GO) on microencapsulated C2C12-myoblast viability has recently been described. However, the commercially available GO lacks homogeneity in size, this parameter being of high relevance for the cell fate in two-dimensional studies. In three-dimensional applications the capacity of this material for binding different kinds of proteins can result in the reduction of de novo released protein that can effectively reach the vicinity of the microcapsules. Undoubtedly, this could be an important hurdle in its clinical use when combined with alginate-PLL microcapsules. Here, we demonstrate that the homogenization of GO nanoparticles is not a mandatory preparation step in order to get the best of this material upon cell microencapsulation. In fact, when the superficial area of these particles is increased, higher amounts of the therapeutic protein erythropoietin (EPO) are adsorbed on their surface. On the other hand, we have been able to improve even more the favorable effects of this graphene derivative on microencapsulated cell viability by forming a protein biocorona. These proteins block the potential binding sites of EPO and, therefore, enhance the amount of therapeutic drug that is released. Finally, we prove that these hybrid alginate-protein-coated GO-microcapsules are functional in vivo.

  16. Internalization and fate of silica nanoparticles in C2C12 skeletal muscle cells: evidence of a beneficial effect on myoblast fusion.

    Science.gov (United States)

    Poussard, Sylvie; Decossas, Marion; Le Bihan, Olivier; Mornet, Stéphane; Naudin, Grégoire; Lambert, Olivier

    2015-01-01

    The use of silica nanoparticles for their cellular uptake capability opens up new fields in biomedical research. Among the toxicological effects associated with their internalization, silica nanoparticles induce apoptosis that has been recently reported as a biochemical cue required for muscle regeneration. To assess whether silica nanoparticles could affect muscle regeneration, we used the C2C12 muscle cell line to study the uptake of fluorescently labeled NPs and their cellular trafficking over a long period. Using inhibitors of endocytosis, we determined that the NP uptake was an energy-dependent process mainly involving macropinocytosis and clathrin-mediated pathway. NPs were eventually clustered in lysosomal structures. Myoblasts containing NPs were capable of differentiation into myotubes, and after 7 days, electron microscopy revealed that the NPs remained primarily within lysosomes. The presence of NPs stimulated the formation of myotubes in a dose-dependent manner. NP internalization induced an increase of apoptotic myoblasts required for myoblast fusion. At noncytotoxic doses, the NP uptake by skeletal muscle cells did not prevent their differentiation into myotubes but, instead, enhanced the cell fusion.

  17. Static magnetic fields inhibit proliferation and disperse subcellular localization of gamma complex protein3 in cultured C2C12 myoblast cells.

    Science.gov (United States)

    Kim, SeungChan; Im, Wooseok

    2010-05-01

    Magnetic fields may delay the rate of cell cycle progression, and there are reports that magnetic fields induce neurite outgrowth in cultured neuronal cells. To demonstrate whether magnetic field also effects on myoblast cells in cell growth, C2C12 cell lines were cultured and 2000G static magnetic field was applied. After 48 h of incubation, both the WST-1 assay (0.01 magnetic fields inhibit the proliferation of cultured C2C12 cells. Immunocytochemistry for alpha and tubulin gamma complex protein (TUBA and GCP3) was made and applying a static magnetic field-dispersed tubulin GCP3 formation, a intracellular apparatus for tubulin structuring in cell division. This protein expression was not altered by western blot. This study indicates that applying a static magnetic field alters the subcellular localizing of GCP3, and may delay the cell growth in cultured C2C12 myoblast cells.

  18. Enigma homolog 1 promotes myogenic gene expression and differentiation of C2C12 cells.

    Science.gov (United States)

    Ito, Jumpei; Takita, Masatoshi; Takimoto, Koichi; Maturana, Andrés D

    2013-06-07

    The Enigma homolog (ENH) gene generates several splicing variants. The initially identified ENH1 possesses one PDZ and three LIM domains, whereas ENH2~4 lack the latter domains. The splicing switch from ENH1 to LIM-less ENHs occurs during development/maturation of skeletal and heart muscles. We examined for the roles of ENH splicing variants in muscle differentiation using C2C12 cells. Cells stably expressing ENH1 exhibited significantly higher MyoD and myogenin mRNA levels before differentiation and after 5 days in low serum-differentiating medium than mock-transfected cells. ENH1-stable transformants also retained the ability to exhibit elongated morphology with well-extended actin fibers following differentiation. In contrast, cells stably expressing ENH3 or ENH4 did not show myotube-like morphology or reorganization of actin fibers following culture in the differentiating medium. Transient overexpression of ENH1 using adenovirus supported the increased expression of muscle marker mRNAs and the formation of well-organized stress fibers, whereas ENH4 overexpression prevented these morphological changes. Furthermore, specific suppression of ENH1 expression by RNAi caused a significant reduction in MyoD mRNA level and blocked the morphological changes. These results suggest that ENH1 with multiple protein-protein interaction modules is essential for differentiation of striated muscles, whereas ectopic expression of LIM-less ENH disrupts normal muscle differentiation. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Catalytic activity of nuclear PLC-beta(1) is required for its signalling function during C2C12 differentiation.

    Science.gov (United States)

    Ramazzotti, Giulia; Faenza, Irene; Gaboardi, Gian Carlo; Piazzi, Manuela; Bavelloni, Alberto; Fiume, Roberta; Manzoli, Lucia; Martelli, Alberto M; Cocco, Lucio

    2008-11-01

    Here we report that PLC-beta(1) catalytic activity plays a role in the increase of cyclin D3 levels and induces the differentiation of C2C12 skeletal muscle cells. PLC-beta(1) mutational analysis revealed the importance of His(331) and His(378) for the catalysis. The expression of PLC-beta(1) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that PLC-beta(1) activates cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLC-beta(1) is a crucial regulator of the mouse cyclin D3 gene. We show that after insulin treatment cyclin D3 mRNA levels are lower in cells overexpressing the PLC-beta(1) catalytically inactive form in comparison to wild type cells. We describe a novel signalling pathway elicited by PLC-beta(1) that modulates AP-1 activity. Gel mobility shift assay and supershift performed with specific antibodies indicate that the c-jun binding site is located in a cyclin D3 promoter region specifically regulated by PLC-beta(1) and that c-Jun binding activity is significantly increased by insulin and PLC-beta(1) overexpression. Mutation of AP-1 site decreased the basal cyclin D3 promoter activity and eliminated its induction by insulin and PLC-beta(1). These results hint at the fact that PLC-beta(1) catalytic activity signals a c-jun/AP-1 target gene, i.e. cyclin D3, during myogenic differentiation.

  20. Response of C2C12 Myoblasts to Hypoxia: The Relative Roles of Glucose and Oxygen in Adaptive Cellular Metabolism

    Directory of Open Access Journals (Sweden)

    Wei Li

    2013-01-01

    Full Text Available Background. Oxygen and glucose are two important nutrients for mammalian cell function. In this study, the effect of glucose and oxygen concentrations on C2C12 cellular metabolism was characterized with an emphasis on detecting whether cells show oxygen conformance (OC in response to hypoxia. Methods. After C2C12 cells being cultured in the levels of glucose at 0.6 mM (LG, 5.6 mM (MG, or 23.3 mM(HG under normoxic or hypoxic (1% oxygen condition, cellular oxygen consumption, glucose consumption, lactate production, and metabolic status were determined. Short-term oxygen consumption was measured with a novel oxygen biosensor technique. Longer-term measurements were performed with standard glucose, lactate, and cell metabolism assays. Results. It was found that oxygen depletion in normoxia is dependent on the glucose concentration in the medium. Cellular glucose uptake and lactate production increased significantly in hypoxia than those in normoxia. In hypoxia the cellular response to the level of glucose was different to that in normoxia. The metabolic activities decreased while glucose concentration increased in normoxia, while in hypoxia, metabolic activity was reduced in LG and MG, but unchanged in HG condition. The OC phenomenon was not observed in the present study. Conclusions. Our findings suggested that a combination of low oxygen and low glucose damages the viability of C2C12 cells more seriously than low oxygen alone. In addition, when there is sufficient glucose, C2C12 cells will respond to hypoxia by upregulating anaerobic respiration, as shown by lactate production.

  1. Fibroblast Growth Factor 21 Promotes C2C12 Cells Myogenic Differentiation by Enhancing Cell Cycle Exit

    Directory of Open Access Journals (Sweden)

    Xinyi Liu

    2017-01-01

    Full Text Available Fibroblast growth factor 21 (FGF21, a secretion protein, functions as a pivotal regulator of energy metabolism and is being considered as a therapeutic candidate in metabolic syndromes. However, the roles of FGF21 in myogenic differentiation and cell cycle remain obscure. In this study, we investigated the function of FGF21 in myogenesis and cell cycle exit using C2C12 cell line. Our data showed that the expression of myogenic genes as well as cell cycle exit genes was increased after FGF21 overexpression, and FGF21 overexpression induces cell cycle arrest. Moreover, cell cycle genes were decreased in FGF21 overexpression cells while they were increased in FGF21 knockdown cells. Further, FGF21/P53/p21/Cyclin-CDK has been suggested as the key pathway for cell cycle exit mediated by FGF21 in C2C12 cells. Also, we deduce that FGF21 promotes the initiation of myogenic differentiation mainly through enhancing cell cycle exit of C2C12 cells. Taken together, our results demonstrated that FGF21 promotes cell cycle exit and enhances myogenic differentiation of C2C12 cells. This study provided new evidence that FGF21 promotes myogenic differentiation, which could be useful for better understanding the roles of FGF21 in myogenesis.

  2. α-linolenic acid reduces TNF-induced apoptosis in C2C12 myoblasts by regulating expression of apoptotic proteins

    Directory of Open Access Journals (Sweden)

    Felicia Carotenuto

    2016-11-01

    Full Text Available Impaired regeneration and consequent muscle wasting is a major feature of muscle degenerative diseases. Nutritional interventions as adjuvant strategy for preventing such conditions are recently gaining increasing attention. Ingestion of n3-polyunsaturated fatty acids has been suggested to have a positive impact on muscle diseases. We recently demonstrated that the dietary n3-fatty acid, alpha-linolenic acid (ALA, exerts potent beneficial effects in preserving skeletal muscle regeneration in models of muscle dystrophy. To better elucidate the underlying mechanism we investigate here on the expression level of the anti- and pro-apototic proteins, as well as caspase-3 activity, in C2C12 myoblasts challenged with pathological levels of TNF. The results demonstrated that ALA protective effect on C2C12 myoblasts was associated to an increased Bcl-2/Bax ratio. Indeed, the effect of ALA was directed to rescue Bcl-2 expression and decrease Bax expression both affected in an opposite way by TNF treatment. This effect was associated with a decrease in caspase-3 activity by ALA. TNF is a major pro-inflammatory cytokine that is expressed in damaged skeletal muscle, therefore, counteract inflammatory signals in the muscle microenvironment represents a critical strategy to ameliorate skeletal muscle pathologies

  3. Ethanol extract of Prunus mume fruit attenuates hydrogen peroxide-induced oxidative stress and apoptosis involving Nrf2/HO-1 activation in C2C12 myoblasts

    Directory of Open Access Journals (Sweden)

    Ji Sook Kang

    Full Text Available ABSTRACT The fruit of the Prunus mume (Siebold Siebold & Zucc., Rosaceae (Korean name: Maesil has long been used as a health food or valuable medicinal material in traditional herb medicine in Southeast Asian countries. In this study, we determined the potential therapeutic efficacy of the ethanol extract of P. mume fruits (EEPM against H2O2-induced oxidative stress and apoptosis in the murine skeletal muscle myoblast cell line C2C12, and sought to understand the associated molecular mechanisms. The results indicated that exposure of C2C12 cells to H2O2 caused a reduction in cell viability by increasing the generation of intracellular reactive oxygen species and by disrupting mitochondrial membrane permeability, leading to DNA damage and apoptosis. However, pretreatment of the cells with EEPM before H2O2 exposure effectively attenuated these changes, suggesting that EEPM prevented H2O2-induced mitochondria-dependent apoptosis. Furthermore, the increased ex-pression and phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2 and up-regulation of heme oxygenase-1 (HO-1, a phase II antioxidant enzyme, were detected in EEPM-treated C2C12 cells. We also found that zinc protoporphyrin IX, an HO-1 inhibitor, attenuated the protective effects of EEPM against H2O2-induced reactive oxygen species accumulation and cytotoxicity. Therefore, these results indicate that the activation of the Nrf2/HO-1 pathway might be involved in the protection of EEPM against H2O2-induced cellular oxidative damage. In conclusion, these results show that EEPM contributes to the prevention of oxidative damage and could be used as a nutritional agent for oxidative stress-related diseases.

  4. ANKRD1 modulates inflammatory responses in C2C12 myoblasts through feedback inhibition of NF-κB signaling activity

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xin-Hua [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Bauman, William A. [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Cardozo, Christopher, E-mail: chris.cardozo@va.gov [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States)

    2015-08-14

    Transcription factors of the nuclear factor-kappa B (NF-κB) family play a pivotal role in inflammation, immunity and cell survival responses. Recent studies revealed that NF-κB also regulates the processes of muscle atrophy. NF-κB activity is regulated by various factors, including ankyrin repeat domain 2 (AnkrD2), which belongs to the muscle ankyrin repeat protein family. Another member of this family, AnkrD1 is also a transcriptional effector. The expression levels of AnkrD1 are highly upregulated in denervated skeletal muscle, suggesting an involvement of AnkrD1 in NF-κB mediated cellular responses to paralysis. However, the molecular mechanism underlying the interactive role of AnkrD1 in NF-κB mediated cellular responses is not well understood. In the current study, we examined the effect of AnkrD1 on NF-κB activity and determined the interactions between AnkrD1 expression and NF-κB signaling induced by TNFα in differentiating C2C12 myoblasts. TNFα upregulated AnkrD1 mRNA and protein levels. AnkrD1-siRNA significantly increased TNFα-induced transcriptional activation of NF-κB, whereas overexpression of AnkrD1 inhibited TNFα-induced NF-κB activity. Co-immunoprecipitation studies demonstrated that AnkrD1 was able to bind p50 subunit of NF-κB and vice versa. Finally, CHIP assays revealed that AnkrD1 bound chromatin at a NF-κB binding site in the AnrkD2 promoter and required NF-κB to do so. These results provide evidence of signaling integration between AnkrD1 and NF-κB pathways, and suggest a novel anti-inflammatory role of AnkrD1 through feedback inhibition of NF-κB transcriptional activity by which AnkrD1 modulates the balance between physiological and pathological inflammatory responses in skeletal muscle. - Highlights: • AnkrD1 is upregulated by TNFα and represses NF-κB-induced transcriptional activity. • AnkrD1 binds to p50 subunit of NF-κB and is recruited to NF-κB bound to chromatin. • AnkrD1 mediates a feed-back inhibitory loop

  5. Overexpression of Striated Muscle Activator of Rho Signaling (STARS) Increases C2C12 Skeletal Muscle Cell Differentiation.

    Science.gov (United States)

    Wallace, Marita A; Della Gatta, Paul A; Ahmad Mir, Bilal; Kowalski, Greg M; Kloehn, Joachim; McConville, Malcom J; Russell, Aaron P; Lamon, Séverine

    2016-01-01

    Skeletal muscle growth and regeneration depend on the activation of satellite cells, which leads to myocyte proliferation, differentiation and fusion with existing muscle fibers. Skeletal muscle cell proliferation and differentiation are tightly coordinated by a continuum of molecular signaling pathways. The striated muscle activator of Rho signaling (STARS) is an actin binding protein that regulates the transcription of genes involved in muscle cell growth, structure and function via the stimulation of actin polymerization and activation of serum-response factor (SRF) signaling. STARS mediates cell proliferation in smooth and cardiac muscle models; however, whether STARS overexpression enhances cell proliferation and differentiation has not been investigated in skeletal muscle cells. We demonstrate for the first time that STARS overexpression enhances differentiation but not proliferation in C2C12 mouse skeletal muscle cells. Increased differentiation was associated with an increase in the gene levels of the myogenic differentiation markers Ckm, Ckmt2 and Myh4, the differentiation factor Igf2 and the myogenic regulatory factors (MRFs) Myf5 and Myf6. Exposing C2C12 cells to CCG-1423, a pharmacological inhibitor of SRF preventing the nuclear translocation of its co-factor MRTF-A, had no effect on myotube differentiation rate, suggesting that STARS regulates differentiation via a MRTF-A independent mechanism. These findings position STARS as an important regulator of skeletal muscle growth and regeneration.

  6. α-Actinin involvement in Z-disk assembly during skeletal muscle C2C12 cells in vitro differentiation.

    Science.gov (United States)

    Salucci, S; Baldassarri, V; Falcieri, E; Burattini, S

    2015-01-01

    α-Actinin is involved in the assembly and maintenance of muscle fibers. α-Actinin is required to cross-link actin filaments and to connect the actin cytoskeleton to the cell membrane and it is necessary for the attachment of actin filaments to Z-disks in skeletal muscle fibers and to dense bodies in smooth muscle ones. In addition to its mechanical role, sarcomeric α-actinin interacts with proteins involved in a variety of signaling and metabolic pathways. The aim of this work is to monitor Z-disk formation, in order to clear up the role of sarcomeric α-actinin in undifferentiated stage, after 4 days of differentiation (intermediate differentiation stage) and after 7 days of differentiation (fully differentiated stage). For this purpose, C2C12 murine skeletal muscle cells, grown in vitro, were analyzed at three time points of differentiation. Confocal laser scanner microscopy and transmission electron microscopy have been utilized for α-actinin immunolocalization. Both techniques reveal that in undifferentiated cells labeling appears uniformly distributed in the cytoplasm with punctate α-actinin Z-bodies. Moreover, we found that when differentiation is induced, α-actinin links at first membrane-associated proteins, then it aligns longitudinally across the cytoplasm and finally binds actin, giving rise to Z-disks. These findings evidence α-actinin involvement in sarcomeric development, suggesting for this protein an important role in stabilizing the muscle contractile apparatus. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. C2C12 myotubes inhibit the proliferation and differentiation of 3T3-L1 preadipocytes by reducing the expression of glucocorticoid receptor gene

    Energy Technology Data Exchange (ETDEWEB)

    Chu, Weiwei; Wei, Wei; Yu, Shigang; Han, Haiyin; Shi, Xiaoli [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Sun, Wenxing [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); College of Public Health, Nantong University, Nantong 226019 (China); Gao, Ying [College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095 (China); Zhang, Lifan [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Chen, Jie, E-mail: jiechen@njau.edu.cn [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China)

    2016-03-25

    Obesity is a well-established risk factor to health for its relationship with insulin resistance, diabetes and metabolic syndrome. Myocyte-adipocyte crosstalk model plays a significant role in studying the interaction of muscle and adipose development. Previous related studies mainly focus on the effects of adipocytes on the myocytes activity, however, the influence of myotubes on the preadipocytes development remains unclear. The present study was carried out to settle this issue. Firstly, the co-culture experiment showed that the proliferation, cell cycle, and differentiation of 3T3-L1 preadipocytes were arrested, and the apoptosis was induced, by differentiated C2C12 myotubes. Next, the sensitivity of 3T3-L1 preadipocytes to glucocorticoids (GCs), which was well known as cell proliferation, differentiation, apoptosis factor, was decreased after co-cultured with C2C12 myotubes. What's more, our results showed that C2C12 myotubes suppressed the mRNA and protein expression of glucocorticoid receptor (GR) in 3T3-L1 preadipocytes, indicating the potential mechanism of GCs sensitivity reduction. Taken together, we conclude that C2C12 myotubes inhibited 3T3-L1 preadipocytes proliferation and differentiation by reducing the expression of GR. These data suggest that decreasing GR by administration of myokines may be a promising therapy for treating patients with obesity or diabetes. - Highlights: • C2C12 myotubes inhibited proliferation and differentiation of 3T3-L1 preadipocytes. • C2C12 myotubes arrested cell cycle of 3T3-L1 preadipocytes. • C2C12 myotubes induced apoptosis of 3T3-L1 preadipocytes. • C2C12 inhibit 3T3-L1 cells by reducing the expression of glucocorticoid receptor gene.

  8. Expressional studies of the aldehyde oxidase (AOX1) gene during myogenic differentiation in C2C12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kamli, Majid Rasool; Kim, Jihoe; Pokharel, Smritee; Jan, Arif Tasleem [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Lee, Eun Ju [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Bovine Genome Resources Bank, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Choi, Inho, E-mail: inhochoi@ynu.ac.kr [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Bovine Genome Resources Bank, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of)

    2014-08-08

    Highlights: • AOX1 contributes to the formation of myotube. • Silencing of AOX1 reduces myotube formation. • AOX1 regulates MyoG gene expression. • AOX1 contributes to myogenesis via H{sub 2}O{sub 2}. - Abstract: Aldehyde oxidases (AOXs), which catalyze the hydroxylation of heterocycles and oxidation of a wide variety of aldehydic compounds, have been present throughout evolution from bacteria to humans. While humans have only a single functional aldehyde oxidase (AOX1) gene, rodents are endowed with four AOXs; AOX1 and three aldehyde oxidase homologs (AOH1, AOH2 and AOH3). In continuation of our previous study conducted to identify genes differentially expressed during myogenesis using a microarray approach, we investigated AOX1 with respect to its role in myogenesis to conceptualize how it is regulated in C2C12 cells. The results obtained were validated by silencing of the AOX1 gene. Analysis of their fusion index revealed that formation of myotubes showed a marked reduction of up to 40% in AOX1{sub kd} cells. Expression of myogenin (MYOG), one of the marker genes used to study myogenesis, was also found to be reduced in AOX1{sub kd} cells. AOX1 is an enzyme of pharmacological and toxicological importance that metabolizes numerous xenobiotics to their respective carboxylic acids. Hydrogen peroxide (H{sub 2}O{sub 2}) produced as a by-product in this reaction is considered to be involved as a part of the signaling mechanism during differentiation. An observed reduction in the level of H{sub 2}O{sub 2} among AOX1{sub kd} cells confirmed production of H{sub 2}O{sub 2} in the reaction catalyzed by AOX1. Taken together, these findings suggest that AOX1 acts as a contributor to the process of myogenesis by influencing the level of H{sub 2}O{sub 2}.

  9. Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation and cell differentiation

    DEFF Research Database (Denmark)

    Grossi, Alberto; Lawson, Moira Ann

    have shown that m-calpain is necessary for myoblast fusion leading to the formation of muscle fibers and that inhibition of this enzyme restricts myotube formation. Whether there is a link between stretchor load induced signaling and m-calpain expression and activation is not known. Using a magnetic...... documented and has been shown to affect transcription of specific gene sequences, protein synthesis, the immune system and increase in Ca2+ influx. The past 10 years has seen a dramatic increase in the understanding of how proteolytic enzymes such as calpains can affect the growth of muscle. In vivo studies...... bead stimulation assay and a C2C12 mouse myoblast cell population, we have found that mechanical stimulation via laminin receptors leads to an increase in m-calpain expression, an enzyme found to be required for muscle cell fusion. After a short period of stimulation, m-calpain relocates into focal...

  10. Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells.

    Science.gov (United States)

    Gallea, S; Lallemand, F; Atfi, A; Rawadi, G; Ramez, V; Spinella-Jaegle, S; Kawai, S; Faucheu, C; Huet, L; Baron, R; Roman-Roman, S

    2001-05-01

    Bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta (TGF-beta) superfamily, is able to induce osteoblastic differentiation of C2C12 cells. Both Smad and mitogen-activated protein kinase (MAPK) pathways are essential components of the TGF-beta superfamily signaling machinery. Although Smads have been demonstrated to participate in the BMP-2-induced osteoblastic differentiation of C2C12 cells, the role of MAPK has not been addressed. This report shows that BMP-2 activates ERK and p38, but not JNK, in C2C12 cells. Pretreatment of cells with the p38 inhibitor, SB203580, dramatically reduced BMP-2-induced expression of the osteoblast markers alkaline phosphatase (ALP) and osteocalcin (OC). Nevertheless, overexpression of MKK3, a protein kinase that phosphorylates and activates p38, failed to induce ALP or OC expression in the absence of BMP-2, indicating that p38 activation is necessary but not sufficient for the acquisition of the osteoblast phenotype by these cells. Although ALP induction was increased slightly in the presence of PD-98059, a selective inhibitor of the ERK cascade, this compound significantly inhibited both steady-state and BMP-2-induced OC RNA levels. Our results indicate that p38 and ERK cascades play a crucial role in the osteoblast differentiation of C2C12 cells mediated by BMP-2.

  11. A fractionation method to identify qauntitative changes in protein expression mediated by IGF-1 on the proteome of murine C2C12 myoblasts

    Directory of Open Access Journals (Sweden)

    Friedmann Theodore

    2009-08-01

    Full Text Available Abstract Although much is known about signal transduction downstream of insulin-like growth factor-1 (IGF-1, relatively little is known about the global changes in protein expression induced by this hormone. In this study, the acute effects of IGF-1 on the proteome of murine C2C12 cells were examined. Cells were treated with IGF-1 for up to 24 hours, lysed, and fractionated into cytosolic, nuclear, and insoluble portions. Proteins from the cytosolic fraction were further separated using a new batch ion-exchange chromatography method to reduce sample complexity, followed by two-dimensional (2D electrophoresis, and identification of selected proteins by mass spectrometry. PDQuest software was utilized to identify and catalogue temporal changes in protein expression during IGF-1 stimulation. In response to IGF-1 stimulation, expression of 23 proteins increased at least three-fold and expression of 17 proteins decreased at least three-fold compared with control un-stimulated C2C12 cells. Changes in expression of selected proteins from each group, including Rho-GDI, cofillin, RAD50, enolase, IκB kinase b (IκBKb and Hsp70 were confirmed by Western blotting. Additionally, the position of 136 'landmark' proteins whose expression levels and physicochemical properties did not change appreciably or consistently during IGF-1 treatment were mapped and identified. This characterization of large-scale changes in protein expression in response to growth factor stimulation of C2C12 cells will further help to establish a comprehensive understanding of the networks and pathways involved in the action of IGF-1.

  12. Photobiomodulation Protects and Promotes Differentiation of C2C12 Myoblast Cells Exposed to Snake Venom

    National Research Council Canada - National Science Library

    Silva, Luciana Miato Gonçalves; Silva, Camila Aparecida Alves da; Silva, Aline da; Vieira, Rodolfo Paula; Mesquita-Ferrari, Raquel Agnelli; Cogo, José Carlos; Zamuner, Stella Regina

    2016-01-01

    .... Several studies have shown the effectiveness of low level laser (LLL) therapy in reducing local myonecrosis induced by Bothropic venoms, however the mechanism involved in this effect is unknown...

  13. Dopamine-Incorporated Dual Bioactive Electroactive Shape Memory Polyurethane Elastomers with Physiological Shape Recovery Temperature, High Stretchability, and Enhanced C2C12 Myogenic Differentiation.

    Science.gov (United States)

    Zhao, Xin; Dong, Ruonan; Guo, Baolin; Ma, Peter X

    2017-09-06

    Soft tissue engineering needs elastic biomaterials not only mimicking the elasticity of soft tissue but also possessing multiple bioactivity to promote cell adhesion, proliferation, and differentiation, which still remain ongoing challenges. Herein, we synthesized a series of dopamine-incorporated dual bioactive electroactive shape memory polyurethane elastomers by combining the properties of elastomeric poly(citric acid-co-polycaprolactone) (CA-PCL) polyurethane elastomer, bioactive dopamine (DA), and electroactive aniline hexamer (AH). The chemical structures, electroactivity, conductivity, thermal properties, hydrophilicity and hydration ability, mechanical properties, and degradability of the polyurethane elastomers were systematically characterized. The elastomers showed excellent shape fixity ratio and shape recovery ability under physiological conditions. The elastomers' elongation and stress were tailored by the AH content, whereas the hydrophilicity and hydration ability of the elastomers were adjusted by the content of DA and AH, as well as the doping state of AH. The viability and proliferation results of C2C12 cells seeded on the elastomers showed their excellent cytocompatibility. Additionally, by analyzing the protein and gene level, the promotion effect on myogenic differentiation of C2C12 cells by these elastomers compared to that by control groups (PCL80 000, CA-PCL elastomer, and CA-PCL elastomer with the DA segment) was demonstrated. Furthermore, the results from subcutaneous implantation confirmed the elastomers' mild host response in vivo. These results represent that these dopamine-incorporated dual bioactive electroactive shape memory polyurethane elastomers are promising candidates for soft tissue regeneration that is sensitive to electrical signals.

  14. C2C12 co-culture on a fibroblast substratum enables sustained survival of contractile, highly differentiated myotubes with peripheral nuclei and adult fast myosin expression.

    Science.gov (United States)

    Cooper, S T; Maxwell, A L; Kizana, E; Ghoddusi, M; Hardeman, E C; Alexander, I E; Allen, D G; North, K N

    2004-07-01

    We describe a simple culture method for obtaining highly differentiated clonal C2C12 myotubes using a feeder layer of confluent fibroblasts, and document the expression of contractile protein expression and aspects of myofibre morphology using this system. Traditional culture methods using collagen- or laminin-coated tissue-culture plastic typically results in a cyclic pattern of detachment and reformation of myotubes, rarely producing myotubes of a mature adult phenotype. C2C12 co-culture on a fibroblast substratum facilitates the sustained culture of contractile myotubes, resulting in a mature sarcomeric register with evidence for peripherally migrating nuclei. Immunoblot analysis demonstrates that desmin, tropomyosin, sarcomeric actin, alpha-actinin-2 and slow myosin are detected throughout myogenic differentiation, whereas adult fast myosin heavy chain isoforms, members of the dystrophin-associated complex, and alpha-actinin-3 are not expressed at significant levels until >6 days of differentiation, coincident with the onset of contractile activity. Electrical stimulation of mature myotubes reveals typical and reproducible calcium transients, demonstrating functional maturation with respect to calcium handling proteins. Immunocytochemical staining demonstrates a well-defined sarcomeric register throughout the majority of myotubes (70-80%) and a striated staining pattern is observed for desmin, indicating alignment of the intermediate filament network with the sarcomeric register. We report that culture volume affects the fusion index and rate of sarcomeric development in developing myotubes and propose that a fibroblast feeder layer provides an elastic substratum to support contractile activity and likely secretes growth factors and extracellular matrix proteins that assist myotube development. Copyright 2004 Wiley-Liss, Inc.

  15. Application of direct HPTLC-MALDI for the qualitative and quantitative profiling of neutral and acidic glycosphingolipids: the case of NEU3 overexpressing C2C12 murine myoblasts.

    Science.gov (United States)

    Torretta, Enrica; Vasso, Michele; Fania, Chiara; Capitanio, Daniele; Bergante, Sonia; Piccoli, Marco; Tettamanti, Guido; Anastasia, Luigi; Gelfi, Cecilia

    2014-05-01

    Glycosphingolipids (GSLs) are a class of ubiquitous lipids characterized by a wide structural repertoire and a variety of functional implications. Importantly, altered levels have been correlated with different diseases, suggesting their crucial role in health. Conventional methods for the characterization and quantification are based on high-performance TLC (HPTLC) separation and comparison with the migration distance of standard samples or on MS. We set up and herein report the application of an ImagePrep method for glycosphingolipids qualitative and quantitative profiling through direct HPTLC-MALDI with particular application to wild-type and NEU3 sialidase-overexpressing C2C12 myoblasts. Lipids were analyzed by HPTLC, coupled with MALDI-TOF, and the resulting GSLs profiles were compared to the [³H]sphingolipids HPTLC patterns obtained after metabolic radiolabeling. GSLs detection by HPTLC-MALDI was optimized by testing different methods for matrix delivery and by performing quantitative analyses using serial dilutions of GSLs standards. Through this approach an accurate analysis of each variant of neutral and acidic GSLs, including the detection of different fatty-acid chain variants for each GSL, was provided and these results demonstrated that HPTLC-MALDI is an easy and high-throughput analytical method for GSLs profiling, suggesting its use for an early detection of markers in different diseases, including cancer and heart ischemia. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. miR-145a-5p Promotes Myoblast Differentiation

    Directory of Open Access Journals (Sweden)

    Jingjing Du

    2016-01-01

    Full Text Available MicroRNAs are a class of 18–22-nucleotide noncoding RNAs that posttranscriptionally regulate gene expression and have been shown to play an important role during myoblast differentiation. In this study, we found that the expression of miR-145a-5p was gradually increased during C2C12 myoblast differentiation, and miR-145a-5p inhibitors or mimics significantly suppressed or promoted the relative expression of specific myogenesis related marker genes. Moreover, overexpression or inhibition of miR-145a-5p enhanced or repressed the expression of some special genes involved in the endogenous Wnt signaling pathway during C2C12 myoblast differentiation, including Wnt5a, LRP5, Axin2, and β-catenin. These results indicated that miR-145a-5p might be considered as a new myogenic differentiation-associated microRNA that can promote C2C12 myoblast differentiation by enhancing genes related to myoblasts differentiation.

  17. N-methyl-D-aspartate (NMDA) impairs myogenesis in C2C12 cells.

    Science.gov (United States)

    Auh, Q-SChick; Park, Kyung-Ran; Lee, Myeong-Ok; Hwang, Mi-Jin; Kang, Soo-Kyung; Hong, Jung-Pyo; Yun, Hyung-Mun; Kim, Eun-Cheol

    2017-09-01

    N-methyl-d-aspartate (NMDA) is expressed in sensory neurons and plays important roles in peripheral pain mechanisms. The aim of this study was to examine the effects and molecular mechanisms of NMDA on C2C12 myoblast proliferation and differentiation. Cytotoxicity and differentiation were examined by the MTT assay, reverse transcription-polymerase chain reaction, and immunofluorescence. NMDA had no cytotoxicity (10-500 μM) and inhibited myoblastic differentiation of C2C12 cells, as assessed by F-actin immunofluorescence and levels of mRNAs encoding myogenic markers such as myogenin and myosin heavy-chain 2. It inhibited phosphorylation of mammalian target of rapamycin (mTOR) by inactivating mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38. It induced reactive oxygen species production. Furthermore, NMDA-suppressed expression of F-actin was reversed by adding the antioxidant N-acetylcysteine. Collectively, these results indicate that NMDA impairs myogenesis or myogenic differentiation in C2C12 cells through the mTOR/MAPK signaling pathways and may lead to skeletal muscle degeneration. Muscle Nerve 56: 510-518, 2017. © 2016 Wiley Periodicals, Inc.

  18. Regulation of myoblast differentiation by metabolic perturbations induced by metformin.

    Directory of Open Access Journals (Sweden)

    Theodora Pavlidou

    Full Text Available The metabolic perturbation caused by calorie restriction enhances muscle repair by playing a critical role in regulating satellite cell availability and activity in the muscles of young and old mice. To clarify the underlying mechanisms we asked whether myoblast replication and differentiation are affected by metformin, a calorie restriction-mimicking drug. C2C12, a mouse myoblast cell line, readily differentiate in vitro and fuse to form myotubes. However, when incubated with metformin, C2C12 slow their replication and do not differentiate. Interestingly, lower doses of metformin promote myogenic differentiation. We observe that metformin treatment modulates the expression of cyclins and cyclin inhibitors thereby inducing a cell cycle perturbation that causes a delay in the G2/M transition. The effect of metformin treatment is reversible since after drug withdrawal, myoblasts can re-enter the cell cycle and/or differentiate, depending on culture conditions. Myoblasts cultured under metformin treatment fail to up-regulate MyoD and p21cip1, a key step in cell cycle exit and terminal differentiation. Although the details of the molecular mechanisms underlying the effect of the drug on myoblasts still need to be clarified, we propose that metformin negatively affects myogenic differentiation by inhibiting irreversible exit from the cell cycle through reduction of MyoD and p21cip1 levels.

  19. JAZF1 promotes proliferation of C2C12 cells, but retards their myogenic differentiation through transcriptional repression of MEF2C and MRF4—Implications for the role of Jazf1 variants in oncogenesis and type 2 diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Yuasa, Katsutoshi; Aoki, Natsumi; Hijikata, Takao, E-mail: hijikata@musashino-u.ac.jp

    2015-08-15

    Single-nucleotide polymorphisms associated with type 2 diabetes (T2D) have been identified in Jazf1, which is also involved in the oncogenesis of endometrial stromal tumors. To understand how Jazf1 variants confer a risk of tumorigenesis and T2D, we explored the functional roles of JAZF1 and searched for JAZF1 target genes in myogenic C2C12 cells. Consistent with an increase of Jazf1 transcripts during myoblast proliferation and their decrease during myogenic differentiation in regenerating skeletal muscle, JAZF1 overexpression promoted cell proliferation, whereas it retarded myogenic differentiation. Examination of myogenic genes revealed that JAZF1 overexpression transcriptionally repressed MEF2C and MRF4 and their downstream genes. AMP deaminase1 (AMPD1) was identified as a candidate for JAZF1 target by gene array analysis. However, promoter assays of Ampd1 demonstrated that mutation of the putative binding site for the TR4/JAZF1 complex did not alleviate the repressive effects of JAZF1 on promoter activity. Instead, JAZF1-mediated repression of Ampd1 occurred through the MEF2-binding site and E-box within the Ampd1 proximal regulatory elements. Consistently, MEF2C and MRF4 expression enhanced Ampd1 promoter activity. AMPD1 overexpression and JAZF1 downregulation impaired AMPK phosphorylation, while JAZF1 overexpression also reduced it. Collectively, these results suggest that aberrant JAZF1 expression contributes to the oncogenesis and T2D pathogenesis. - Highlights: • JAZF1 promotes cell cycle progression and proliferation of myoblasts. • JAZF1 retards myogenic differentiation and hypertrophy of myotubes. • JAZF1 transcriptionally represses Mef2C and Mrf4 expression. • JAZF1 has an impact on the phosphorylation of AMPK.

  20. Methylcobalamin promotes proliferation and migration and inhibits apoptosis of C2C12 cells via the Erk1/2 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Okamoto, Michio [Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Tanaka, Hiroyuki, E-mail: tanahiro-osk@umin.ac.jp [Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Okada, Kiyoshi [Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kuroda, Yusuke [Department of Orthopaedic Surgery, Kansai Rosai Hospital, 3-1-69 Inabaso, Amagasaki, Hyogo 660-8511 (Japan); Nishimoto, Shunsuke; Murase, Tsuyoshi; Yoshikawa, Hideki [Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2014-01-17

    Highlights: •Methylcobalamin activated the Erk1/2 signaling pathway in C2C12 cells. •Methylcobalamin promoted the proliferation and migration in C2C12 cells. •C2C12 cell apoptosis during differentiation was inhibited by methylcobalamin. -- Abstract: Methylcobalamin (MeCbl) is a vitamin B12 analog that has some positive effects on peripheral nervous disorders. Although some previous studies revealed the effects of MeCbl on neurons, its effect on the muscle, which is the final target of motoneuron axons, remains to be elucidated. This study aimed to determine the effect of MeCbl on the muscle. We found that MeCbl promoted the proliferation and migration of C2C12 myoblasts in vitro and that these effects are mediated by the Erk1/2 signaling pathway without affecting the activity of the Akt signaling pathway. We also demonstrated that MeCbl inhibits C2C12 cell apoptosis during differentiation. Our results suggest that MeCbl has beneficial effects on the muscle in vitro. MeCbl administration may provide a novel therapeutic approach for muscle injury or degenerating muscle after denervation.

  1. The roles of supernatant of macrophage treated by excretory-secretory products from muscle larvae of Trichinella spiralis on the differentiation of C2C12 myoblasts

    Science.gov (United States)

    The excretory-secretory products (ESPs) released by the muscle-larvae (ML) stage of Trichinella spiralis have been suggested to be involved in nurse cell formation. However, the molecular mechanisms by which ML-ESPs modulate nurse cell formation remain unclear. Macrophages exert either beneficial or...

  2. Ethanol decreases agrin-induced acetylcholine receptor clustering in C2C12 myotube culture.

    Science.gov (United States)

    Owen, David B; Chamberlain, Kevin T; Shishido, Sonia; Grow, Wade A

    2010-03-01

    We investigated the effect of ethanol on skeletal muscle development using C2C12 cell culture. The ethanol concentrations of 10mM, 25mM, and 100mM, were tested because plasma samples of alcohol-dependent individuals fall within this range. We assessed two specific events in skeletal muscle development, the fusion of myoblasts to form myotubes and the acetylcholine receptor (AChR) clustering associated with neuromuscular synapse formation. We report that ethanol does not effect myotube formation or the viability of myoblasts or myotubes in C2C12 cell culture. However, ethanol does effect AChR clustering on C2C12 myotubes. As motor neurons approach skeletal muscle during development, agrin is released by motor neurons and induces AChR clustering on muscle fibers. In our experiments, agrin was applied to cell cultures during the period when myoblasts fuse to form myotubes. In cell cultures exposed to ethanol during myotube formation, agrin-induced AChR clustering was decreased compared to untreated cultures. In cell cultures exposed to ethanol during myoblast proliferation, with ethanol removed during myotube formation, agrin-induced AChR clustering was unaffected. We conclude that exposure to a physiologically relevant concentration of ethanol during the specific period of myotube formation decreases agrin-induced AChR clustering. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  3. Developmental Changes is Expression of Beta-Adrenergic Receptors in Cultures of C2C12 Skeletal Muscle Cells

    Science.gov (United States)

    Young, Ronald B.; Bridge, K. Y.; Vaughn, J. R.

    2000-01-01

    beta-Adrenergic receptor (bAR) agonists have been reported to modulate growth in several mammalian and avian species, and bAR agonists presumably exert their physiological action on skeletal muscle cells through this receptor. Because of the importance of bAR regulation on muscle protein metabolism in muscle cells, the objectives of this study were to determine the developmental expression pattern of the bAR population in C2C12 skeletal muscle cells, and to analyze changes in both the quantity and isoform expression of the major muscle protein, myosin. The number of bAR in mononucleated C2C12 cells was approximately 8,000 bAR per cell, which is comparable with the population reported in several other nonmuscle cell types. However, the bar population increased after myoblast fusion to greater than 50,000 bAR per muscle cell equivalent. The reasons for this apparent over-expression of bAR in C2C12 cells is not known. The quantity of myosin also increased after C2C12 myoblast fusion, but the quantity of myosin was less than that reported in primary muscle cell cultures. Finally, at least five different isoforms of myosin heavy chain could be resolved in C2C12 cells, and three of these exhibited either increased or decreased developmental regulation relative to the others. Thus, C2C12 myoblasts undergo developmental regulation of bAR population and myosin heavy chain isoform expression.

  4. CD36 is required for myoblast fusion during myogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Park, Seung-Yoon [Department of Biochemistry, College of Medicine, Dongguk University and Medical Institute of Dongguk University, Gyeongju 780-714 (Korea, Republic of); Yun, Youngeun [Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Kim, In-San, E-mail: iskim@knu.ac.kr [Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Biomedical Research Institute, Korea Institute Science and Technology, Seoul (Korea, Republic of)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer CD36 expression was induced during myogenic differentiation. Black-Right-Pointing-Pointer CD36 expression was localized in multinucleated myotubes. Black-Right-Pointing-Pointer The expression of myogenic markers is attenuated in CD36 knockdown C2C12 cells. Black-Right-Pointing-Pointer Knockdown of CD36 significantly inhibited myotube formation during differentiation. -- Abstract: Recently, CD36 has been found to be involved in the cytokine-induced fusion of macrophage. Myoblast fusion to form multinucleated myotubes is required for myogenesis and muscle regeneration. Because a search of gene expression database revealed the attenuation of CD36 expression in the muscles of muscular dystrophy patients, the possibility that CD36 could be required for myoblast fusion was investigated. CD36 expression was markedly up-regulated during myoblast differentiation and localized in multinucleated myotubes. Knockdown of endogenous CD36 significantly decreased the expression of myogenic markers as well as myotube formation. These results support the notion that CD36 plays an important role in cell fusion during myogenic differentiation. Our finding will aid the elucidation of the common mechanism governing cell-to-cell fusion in various fusion models.

  5. Crosstalk between MLO-Y4 osteocytes and C2C12 muscle cells is mediated by the Wnt/β-catenin pathway.

    Science.gov (United States)

    Huang, Jian; Romero-Suarez, Sandra; Lara, Nuria; Mo, Chenglin; Kaja, Simon; Brotto, Leticia; Dallas, Sarah L; Johnson, Mark L; Jähn, Katharina; Bonewald, Lynda F; Brotto, Marco

    2017-10-01

    We examined the effects of osteocyte secreted factors on myogenesis and muscle function. MLO-Y4 osteocyte-like cell conditioned media (CM) (10%) increased ex vivo soleus muscle contractile force by ~25%. MLO-Y4 and primary osteocyte CM (1-10%) stimulated myogenic differentiation of C2C12 myoblasts, but 10% osteoblast CMs did not enhance C2C12 cell differentiation. Since WNT3a and WNT1 are secreted by osteocytes, and the expression level of Wnt3a is increased in MLO-Y4 cells by fluid flow shear stress, both were compared, showing WNT3a more potent than WNT1 in inducing myogenesis. Treatment of C2C12 myoblasts with WNT3a at concentrations as low as 0.5ng/mL mirrored the effects of both primary osteocyte and MLO-Y4 CM by inducing nuclear translocation of β-catenin with myogenic differentiation, suggesting that Wnts might be potential factors secreted by osteocytes that signal to muscle cells. Knocking down Wnt3a in MLO-Y4 osteocytes inhibited the effect of CM on C2C12 myogenic differentiation. Sclerostin (100ng/mL) inhibited both the effects of MLO-Y4 CM and WNT3a on C2C12 cell differentiation. RT-PCR array results supported the activation of the Wnt/β-catenin pathway by MLO-Y4 CM and WNT3a. These results were confirmed by qPCR showing up-regulation of myogenic markers and two Wnt/β-catenin downstream genes, Numb and Flh1. We postulated that MLO-Y4 CM/WNT3a could modulate intracellular calcium homeostasis as the trigger mechanism for the enhanced myogenesis and contractile force. MLO-Y4 CM and WNT3a increased caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and the expression of genes directly associated with intracellular Ca2+ signaling and homeostasis. Together, these data show that in vitro and ex vivo, osteocytes can stimulate myogenesis and enhance muscle contractile function and suggest that Wnts could be mediators of bone to muscle signaling, likely via modulation of intracellular Ca2+ signaling and the Wnt

  6. L-carnitine protects C2C12 cells against mitochondrial superoxide overproduction and cell death

    Science.gov (United States)

    Le Borgne, Françoise; Ravaut, Gaétan; Bernard, Arnaud; Demarquoy, Jean

    2017-01-01

    AIM To identify and characterize the protective effect that L-carnitine exerted against an oxidative stress in C2C12 cells. METHODS Myoblastic C2C12 cells were treated with menadione, a vitamin K analog that engenders oxidative stress, and the protective effect of L-carnitine (a nutrient involved in fatty acid metabolism and the control of the oxidative process), was assessed by monitoring various parameters related to the oxidative stress, autophagy and cell death. RESULTS Associated with its physiological function, a muscle cell metabolism is highly dependent on oxygen and may produce reactive oxygen species (ROS), especially under pathological conditions. High levels of ROS are known to induce injuries in cell structure as they interact at many levels in cell function. In C2C12 cells, a treatment with menadione induced a loss of transmembrane mitochondrial potential, an increase in mitochondrial production of ROS; it also induces autophagy and was able to provoke cell death. Pre-treatment of the cells with L-carnitine reduced ROS production, diminished autophagy and protected C2C12 cells against menadione-induced deleterious effects. CONCLUSION In conclusion, L-carnitine limits the oxidative stress in these cells and prevents cell death. PMID:28289521

  7. Three-dimensional co-culture of C2C12/PC12 cells improves skeletal muscle tissue formation and function.

    Science.gov (United States)

    Ostrovidov, Serge; Ahadian, Samad; Ramon-Azcon, Javier; Hosseini, Vahid; Fujie, Toshinori; Parthiban, S Prakash; Shiku, Hitoshi; Matsue, Tomokazu; Kaji, Hirokazu; Ramalingam, Murugan; Bae, Hojae; Khademhosseini, Ali

    2017-02-01

    Engineered muscle tissues demonstrate properties far from native muscle tissue. Therefore, fabrication of muscle tissues with enhanced functionalities is required to enable their use in various applications. To improve the formation of mature muscle tissues with higher functionalities, we co-cultured C2C12 myoblasts and PC12 neural cells. While alignment of the myoblasts was obtained by culturing the cells in micropatterned methacrylated gelatin (GelMA) hydrogels, we studied the effects of the neural cells (PC12) on the formation and maturation of muscle tissues. Myoblasts cultured in the presence of neural cells showed improved differentiation, with enhanced myotube formation. Myotube alignment, length and coverage area were increased. In addition, the mRNA expression of muscle differentiation markers (Myf-5, myogenin, Mefc2, MLP), muscle maturation markers (MHC-IId/x, MHC-IIa, MHC-IIb, MHC-pn, α-actinin, sarcomeric actinin) and the neuromuscular markers (AChE, AChR-ε) were also upregulated. All these observations were amplified after further muscle tissue maturation under electrical stimulation. Our data suggest a synergistic effect on the C2C12 differentiation induced by PC12 cells, which could be useful for creating improved muscle tissue. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  8. Transthyretin is a key regulator of myoblast differentiation.

    Directory of Open Access Journals (Sweden)

    Eun Ju Lee

    Full Text Available Transthyretin (TTR is a known carrier protein for thyroxine (T4 and retinol-binding protein in the blood that is primarily synthesized in the liver and choroid plexus of the brain. Herein, we report that the TTR gene is expressed in skeletal muscle tissue and up-regulated during myotube formation in C2C12 cells. TTR silencing (TTRkd significantly reduced myogenin expression and myotube formation, whereas myogenin silencing (MYOGkd did not have any effect on TTR gene expression. Both TTRkd and MYOGkd led to a decrease in calcium channel related genes including Cav1.1, STIM1 and Orai1. A significant decrease in intracellular T4 uptake during myogenesis was observed in TTRkd cells. Taken together, the results of this study suggest that TTR initiates myoblast differentiation via affecting expression of the genes involved during early stage of myogenesis and the genes related to calcium channel.

  9. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) activates promyogenic signaling pathways, thereby promoting myoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sang-Jin; Go, Ga-Yeon; Yoo, Miran; Kim, Yong Kee [Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Seo, Dong-Wan [College of Pharmacy, Dankook University, Cheonan 330-714 (Korea, Republic of); Kang, Jong-Sun [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon 440-746 (Korea, Republic of); Bae, Gyu-Un, E-mail: gbae@sookmyung.ac.kr [Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of)

    2016-01-29

    Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) regulates postnatal myogenesis by alleviating myostatin activity, but the molecular mechanisms by which it regulates myogenesis are not fully understood. In this study, we investigate molecular mechanisms of PPARβ/δ in myoblast differentiation. C2C12 myoblasts treated with a PPARβ/δ agonist, GW0742 exhibit enhanced myotube formation and muscle-specific gene expression. GW0742 treatment dramatically activates promyogenic kinases, p38MAPK and Akt, in a dose-dependent manner. GW0742-stimulated myoblast differentiation is mediated by p38MAPK and Akt, since it failed to restore myoblast differentiation repressed by inhibition of p38MAPK and Akt. In addition, GW0742 treatment enhances MyoD-reporter activities. Consistently, overexpression of PPARβ/δ enhances myoblast differentiation accompanied by elevated activation of p38MAPK and Akt. Collectively, these results suggest that PPARβ/δ enhances myoblast differentiation through activation of promyogenic signaling pathways. - Highlights: • A PPARβ/δ agonist, GW0742 promotes myoblast differentiation. • GW0742 activates both p38MAPK and Akt activation in myogenic differentiation. • GW0742 enhances MyoD activity for myogenic differentiation. • Overexpression of PPARβ/δ enhances myoblast differentiation via activating promyogenic signaling pathways. • This is the first finding for agonistic mechanism of PPARβ/δ in myogenesis.

  10. NANOG restores the impaired myogenic differentiation potential of skeletal myoblasts after multiple population doublings

    Directory of Open Access Journals (Sweden)

    Aref Shahini

    2018-01-01

    Full Text Available Adult skeletal muscle regeneration relies on the activity of satellite cells residing in the skeletal muscle niche. However, systemic and intrinsic factors decrease the myogenic differentiation potential of satellite cells thereby impairing muscle regeneration. Here we present data showing that late passage C2C12 myoblasts exhibited significantly impaired myogenic differentiation potential that was accompanied by impaired expression of myogenic regulatory factors (Myf5, MyoD, Myogenin, and MRF4 and members of myocyte enhancer factor 2 family. Notably, ectopic expression of NANOG preserved the morphology and restored the myogenic differentiation capacity of late passage myoblasts, possibly by restoring the expression level of these myogenic factors. Muscle regeneration was effective in 2D cultures and in 3D skeletal microtissues mimicking the skeletal muscle niche. The presence of NANOG was required for at least 15 days to reverse the impaired differentiation potential of myoblasts. However, it was critical to remove NANOG during the process of maturation, as it inhibited myotube formation. Finally, myoblasts that were primed by NANOG maintained their differentiation capacity for 20 days after NANOG withdrawal, suggesting potential epigenetic changes. In conclusion, these results shed light on the potential of NANOG to restore the myogenic differentiation potential of myoblasts, which is impaired after multiple rounds of cellular division, and to reverse the loss of muscle regeneration.

  11. Protein kinase D2 is an essential regulator of murine myoblast differentiation.

    Directory of Open Access Journals (Sweden)

    Alexander Kleger

    Full Text Available Muscle differentiation is a highly conserved process that occurs through the activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to generate new myofibers. A defined pattern of myogenic transcription factors is orchestrated during this process and is regulated via distinct signaling cascades involving various intracellular signaling pathways, including members of the protein kinase C (PKC family. The protein kinase D (PKD isoenzymes PKD1, -2, and -3, are prominent downstream targets of PKCs and phospholipase D in various biological systems including mouse and could hence play a role in muscle differentiation. In the present study, we used a mouse myoblast cell line (C2C12 as an in vitro model to investigate the role of PKDs, in particular PKD2, in muscle stem cell differentiation. We show that C2C12 cells express all PKD isoforms with PKD2 being highly expressed. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated during the initiation of mouse myoblast differentiation. Selective inhibition of PKCs or PKDs by pharmacological inhibitors blocked myotube formation. Depletion of PKD2 by shRNAs resulted in a marked inhibition of myoblast cell fusion. PKD2-depleted cells exhibit impaired regulation of muscle development-associated genes while the proliferative capacity remains unaltered. Vice versa forced expression of PKD2 increases myoblast differentiation. These findings were confirmed in primary mouse satellite cells where myotube fusion was also decreased upon inhibition of PKDs. Active PKD2 induced transcriptional activation of myocyte enhancer factor 2D and repression of Pax3 transcriptional activity. In conclusion, we identify PKDs, in particular PKD2, as a major mediator of muscle cell differentiation in vitro and thereby as a potential novel target for the modulation of muscle regeneration.

  12. Stimulated myoblast differentiation on graphene oxide-impregnated PLGA-collagen hybrid fibre matrices.

    Science.gov (United States)

    Shin, Yong Cheol; Lee, Jong Ho; Jin, Linhua; Kim, Min Jeong; Kim, Yong-Joo; Hyun, Jung Keun; Jung, Tae-Gon; Hong, Suck Won; Han, Dong-Wook

    2015-03-12

    Electrospinning is a simple and effective method for fabricating micro- and nanofiber matrices. Electrospun fibre matrices have numerous advantages for use as tissue engineering scaffolds, such as high surface area-to-volume ratio, mass production capability and structural similarity to the natural extracellular matrix (ECM). Therefore, electrospun matrices, which are composed of biocompatible polymers and various biomaterials, have been developed as biomimetic scaffolds for the tissue engineering applications. In particular, graphene oxide (GO) has recently been considered as a novel biomaterial for skeletal muscle regeneration because it can promote the growth and differentiation of myoblasts. Therefore, the aim of the present study was to fabricate the hybrid fibre matrices that stimulate myoblasts differentiation for skeletal muscle regeneration. Hybrid fibre matrices composed of poly(lactic-co-glycolic acid, PLGA) and collagen (Col) impregnated with GO (GO-PLGA-Col) were successfully fabricated using an electrospinning process. Our results indicated that the GO-PLGA-Col hybrid matrices were comprised of randomly-oriented continuous fibres with a three-dimensional non-woven porous structure. Compositional analysis showed that GO was dispersed uniformly throughout the GO-PLGA-Col matrices. In addition, the hydrophilicity of the fabricated matrices was significantly increased by blending with a small amount of Col and GO. The attachment and proliferation of the C2C12 skeletal myoblasts were significantly enhanced on the GO-PLGA-Col hybrid matrices. Furthermore, the GO-PLGA-Col matrices stimulated the myogenic differentiation of C2C12 skeletal myoblasts, which was enhanced further under the culture conditions of the differentiation media. Taking our findings into consideration, it is suggested that the GO-PLGA-Col hybrid fibre matrices can be exploited as potential biomimetic scaffolds for skeletal tissue engineering and regeneration because these GO

  13. Leptin impairs myogenesis in C2C12 cells through JAK/STAT and MEK signaling pathways.

    Science.gov (United States)

    Pijet, Maja; Pijet, Barbara; Litwiniuk, Anna; Pajak, Beata; Gajkowska, Barbara; Orzechowski, Arkadiusz

    2013-02-01

    Reduced lean body mass in genetically obese (ob/ob) or anorectic/cachectic subjects prompted us to verify the hypothesis whether leptin, white adipose tissue cytokine, might be a negative organizer of myogenesis. Recombinant leptin (100 ng/mL) stimulated mitogenesis together with the raise in T(202/)Y(204)P-ERK1/2 protein expression. Concomitantly, it impaired cell viability and muscle fiber formation from C2C12 mouse myoblasts. Detailed acute and chronic studies with the use of metabolic inhibitors revealed that both JAK/STAT3 and MEK/MAPK but not PI3-K/AKT/GSK-3β signaling pathways were activated by leptin, and that STAT3 (Y(705)P-STAT3) and MEK (T(202/)Y(204)P-ERK1/2) mediate these effects. In contrary, insulin evoked PI3-K-dependent phosphorylation of AKT (S(473)) and GSK-3β (S(9)) and insulin surpassed leptin-dependent inhibition of myogenic differentiation in PI3-K-dependent manner. GSK-3β seems to play dual role in muscle development. Insulin-dependent effect on GSK-3β (S(9)P-GSK-3β) led to accelerated myotube construction. In contrary, leptin through MEK-dependent manner caused GSK-3β phosphorylation (Y(216)P-GSK-3β) with resultant drop in myoblast fusion. Summing up, partially opposite effects of insulin and leptin on skeletal muscle growth emphasize the importance of interplay between these cytokines. They determine how muscle mass is gained or lost. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. LKB1 destabilizes microtubules in myoblasts and contributes to myoblast differentiation.

    Directory of Open Access Journals (Sweden)

    Isma Mian

    Full Text Available BACKGROUND: Skeletal muscle myoblast differentiation and fusion into multinucleate myotubes is associated with dramatic cytoskeletal changes. We find that microtubules in differentiated myotubes are highly stabilized, but premature microtubule stabilization blocks differentiation. Factors responsible for microtubule destabilization in myoblasts have not been identified. FINDINGS: We find that a transient decrease in microtubule stabilization early during myoblast differentiation precedes the ultimate microtubule stabilization seen in differentiated myotubes. We report a role for the serine-threonine kinase LKB1 in both microtubule destabilization and myoblast differentiation. LKB1 overexpression reduced microtubule elongation in a Nocodazole washout assay, and LKB1 RNAi increased it, showing LKB1 destabilizes microtubule assembly in myoblasts. LKB1 levels and activity increased during myoblast differentiation, along with activation of the known LKB1 substrates AMP-activated protein kinase (AMPK and microtubule affinity regulating kinases (MARKs. LKB1 overexpression accelerated differentiation, whereas RNAi impaired it. CONCLUSIONS: Reduced microtubule stability precedes myoblast differentiation and the associated ultimate microtubule stabilization seen in myotubes. LKB1 plays a positive role in microtubule destabilization in myoblasts and in myoblast differentiation. This work suggests a model by which LKB1-induced microtubule destabilization facilitates the cytoskeletal changes required for differentiation. Transient destabilization of microtubules might be a useful strategy for enhancing and/or synchronizing myoblast differentiation.

  15. Unveiling novel genes upregulated by both rhBMP2 and rhBMP7 during early osteoblastic transdifferentiation of C2C12 cells

    Directory of Open Access Journals (Sweden)

    Sogayar Mari C

    2011-09-01

    Full Text Available Abstract Findings We set out to analyse the gene expression profile of pre-osteoblastic C2C12 cells during osteodifferentiation induced by both rhBMP2 and rhBMP7 using DNA microarrays. Induced and repressed genes were intercepted, resulting in 1,318 induced genes and 704 repressed genes by both rhBMP2 and rhBMP7. We selected and validated, by RT-qPCR, 24 genes which were upregulated by rhBMP2 and rhBMP7; of these, 13 are related to transcription (Runx2, Dlx1, Dlx2, Dlx5, Id1, Id2, Id3, Fkhr1, Osx, Hoxc8, Glis1, Glis3 and Cfdp1, four are associated with cell signalling pathways (Lrp6, Dvl1, Ecsit and PKCδ and seven are associated with the extracellular matrix (Ltbp2, Grn, Postn, Plod1, BMP1, Htra1 and IGFBP-rP10. The novel identified genes include: Hoxc8, Glis1, Glis3, Ecsit, PKCδ, LrP6, Dvl1, Grn, BMP1, Ltbp2, Plod1, Htra1 and IGFBP-rP10. Background BMPs (bone morphogenetic proteins are members of the TGFβ (transforming growth factor-β super-family of proteins, which regulate growth and differentiation of different cell types in various tissues, and play a critical role in the differentiation of mesenchymal cells into osteoblasts. In particular, rhBMP2 and rhBMP7 promote osteoinduction in vitro and in vivo, and both proteins are therapeutically applied in orthopaedics and dentistry. Conclusion Using DNA microarrays and RT-qPCR, we identified both previously known and novel genes which are upregulated by rhBMP2 and rhBMP7 during the onset of osteoblastic transdifferentiation of pre-myoblastic C2C12 cells. Subsequent studies of these genes in C2C12 and mesenchymal or pre-osteoblastic cells should reveal more details about their role during this type of cellular differentiation induced by BMP2 or BMP7. These studies are relevant to better understanding the molecular mechanisms underlying osteoblastic differentiation and bone repair.

  16. Cytoprotective Effect of Hispidin against Palmitate-Induced Lipotoxicity in C2C12 Myotubes

    Directory of Open Access Journals (Sweden)

    Jun Myoung Park

    2015-03-01

    Full Text Available It is well known that Phellinus linteus, which produces hispidin and its derivatives, possesses antioxidant activities. In this study, we investigated whether hispidin has protective effects on palmitate-induced oxidative stress in C2C12 skeletal muscle cells. Our results showed that palmitate treatment in C2C12 myotubes increased ROS generation and cell death as compared with the control. However, pretreatment of hispidin for 8 h improved the survival of C2C12 myotubes against palmitate-induced oxidative stress via inhibition of intracellular ROS production. Hispidin also inhibited palmitate-induced apoptotic nuclear condensation in C2C12 myotubes. In addition, we found that hispidin can suppress cleavage of caspase-3, expression of Bax, and NF-κB translocation. Therefore, these results suggest that hispidin is capable of protecting C2C12 myotubes against palmitate-induced oxidative stress.

  17. Cytoprotective Role of Nrf2 in Electrical Pulse Stimulated C2C12 Myotube.

    Directory of Open Access Journals (Sweden)

    Masaki Horie

    Full Text Available Regular physical exercise is central to a healthy lifestyle. However, exercise-related muscle contraction can induce reactive oxygen species and reactive nitrogen species (ROS/RNS production in skeletal muscle. The nuclear factor-E2-related factor-2 (Nrf2 transcription factor is a cellular sensor for oxidative stress. Regulation of nuclear Nrf2 signaling regulates antioxidant responses and protects organ structure and function. However, the role of Nrf2 in exercise- or contraction-induced ROS/RNS production in skeletal muscle is not clear. In this study, using differentiated C2C12 cells and electrical pulse stimulation (EPS of muscle contraction, we explored whether Nrf2 plays a role in the skeletal muscle response to muscle contraction-induced ROS/RNS. We found that EPS (40 V, 1 Hz, 2 ms stimulated ROS/RNS accumulation and Nrf2 activation. We also showed that expression of NQO1, HO-1 and GCLM increased after EPS-induced muscle contraction and was remarkably suppressed in cells with Nrf2 knockdown. We also found that the antioxidant N-acetylcysteine (NAC significantly attenuated Nrf2 activation after EPS, whereas the nitric oxide synthetase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME did not. Furthermore, Nrf2 knockdown after EPS markedly decreased ROS/RNS redox potential and cell viability and increased expression of the apoptosis marker Annexin V in C2C12 myotubes. These results indicate that Nrf2 activation and expression of Nrf2 regulated-genes protected muscle against the increased ROS caused by EPS-induced muscle contraction. Thus, our findings suggest that Nrf2 may be a key factor for preservation of muscle function during muscle contraction.

  18. Permethrin alters adipogenesis in 3T3-L1 adipocytes and causes insulin resistance in C2C12 myotubes.

    Science.gov (United States)

    Kim, Jonggun; Park, Yooheon; Yoon, Kyong Sup; Clark, J Marshall; Park, Yeonhwa

    2014-09-01

    Pyrethroids are a class of insecticides structurally derived from the naturally occurring insecticides called pyrethrins. Along with emerging evidence that exposure to insecticides is linked to altered weight gain and glucose homeostasis, exposure to pyrethroids has been linked to altered blood glucose levels in humans. Thus, the purpose of this study was to determine the role of permethrin on lipid and glucose metabolisms. Permethrin was treated to 3T3-L1 adipocytes and C2C12 myoblasts to determine its role in lipid and glucose metabolisms, respectively. Permethrin treatment resulted in increased expression of key markers of adipogenesis and lipogenesis in adipocytes. Permethrin significantly reduced insulin-stimulated glucose uptake in myotubes. This is the first report on the role of permethrin in altered lipid metabolism in adipocytes and impaired glucose homeostasis in myotubes. These results may help elucidate fundamental underlying mechanisms between insecticide exposure, particularly permethrin, and potential risk of developing obesity and its comorbidities. © 2014 Wiley Periodicals, Inc.

  19. Syncytin-1 in differentiating human myoblasts

    DEFF Research Database (Denmark)

    Bjerregard, Bolette; Ziomkiewicz, Iwona; Schulz, Alexander

    2014-01-01

    Myoblasts fuse to form myotubes, which mature into skeletal muscle fibres. Recent studies indicate that an endogenous retroviral fusion gene, syncytin-1, is important for myoblast fusions in man. We have now expanded these data by examining the immunolocalization of syncytin in human myoblasts...... fusions. Thus, syncytin is involved in human myoblast fusions and is localized in areas of contact between fusing cells. Moreover, syncytin and caveolin-3 might interact at the level of the sarcolemma....

  20. Pannexin Channels Mediate the Acquisition of Myogenic Commitment in C2C12 Reserve Cells Promoted by P2 Receptor Activation

    Directory of Open Access Journals (Sweden)

    Manuel Antonio Riquelme

    2015-05-01

    Full Text Available The acquisition of myoblast commitment to the myogenic linage requires rises in intracellular free Ca2+ concentration ([Ca2+]i. Putative cell membrane pathways involved in these [Ca2+]i increments are P2 receptors (P2Rs as well as connexin (Cx and/or pannexin (Panx hemichannels and channels (Cx HChs and Panx Chs, respectively, which are known to permeate Ca2+. Reserve cells (RCs are uncommitted myoblasts obtained from differentiated C2C12 cell cultures, which acquire commitment upon replating. Regarding these cells, we found that extracellular ATP increases the [Ca2+]i via P2Rs. Moreover, ATP increases the plasma membrane permeability to small molecules and a non-selective membrane current, both of which were inhibited by Cx HCh/Panx1Ch blockers. However, RCs exposed to divalent cation-free saline solution, which is known to activate Cx HChs (but not Panx Chs, did not enhance membrane permeability, thus ruling out the possible involvement of Cx HChs. Moreover, ATP-induced membrane permeability was inhibited with blockers of P2Rs that activate Panx Chs. In addition, exogenous ATP induced the expression of myogenic commitment and increased MyoD levels, which was prevented by the inhibition of P2Rs or knockdown of Panx1 Chs. Similarly, increases in MyoD levels induced by ATP released by RCs were inhibited by Panx Ch/Cx HCh blockers. Myogenic commitment acquisition thus requires a feed-forward mechanism mediated by extracellular ATP, P2Rs and Panx Chs.

  1. Pannexin channels mediate the acquisition of myogenic commitment in C2C12 reserve cells promoted by P2 receptor activation

    Science.gov (United States)

    Riquelme, Manuel A.; Cea, Luis A.; Vega, José L.; Puebla, Carlos; Vargas, Aníbal A.; Shoji, Kenji F.; Subiabre, Mario; Sáez, Juan C.

    2015-01-01

    The acquisition of myoblast commitment to the myogenic linage requires rises in intracellular free Ca2+ concentration ([Ca2+]i). Putative cell membrane pathways involved in these [Ca2+]i increments are P2 receptors (P2Rs) as well as connexin (Cx) and/or pannexin (Panx) hemichannels and channels (Cx HChs and Panx Chs), respectively, which are known to permeate Ca2+. Reserve cells (RCs) are uncommitted myoblasts obtained from differentiated C2C12 cell cultures, which acquire commitment upon replating. Regarding these cells, we found that extracellular ATP increases the [Ca2+]i via P2Rs. Moreover, ATP increases the plasma membrane permeability to small molecules and a non-selective membrane current, both of which were inhibited by Cx HCh/Panx1Ch blockers. However, RCs exposed to divalent cation-free saline solution, which is known to activate Cx HChs (but not Panx Chs), did not enhance membrane permeability, thus ruling out the possible involvement of Cx HChs. Moreover, ATP-induced membrane permeability was inhibited with blockers of P2Rs that activate Panx Chs. In addition, exogenous ATP induced the expression of myogenic commitment and increased MyoD levels, which was prevented by the inhibition of P2Rs or knockdown of Panx1 Chs. Similarly, increases in MyoD levels induced by ATP released by RCs were inhibited by Panx Ch/Cx HCh blockers. Myogenic commitment acquisition thus requires a feed-forward mechanism mediated by extracellular ATP, P2Rs, and Panx Chs. PMID:26000275

  2. Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation

    DEFF Research Database (Denmark)

    Grossi, Alberto; Karlsson, Anders H; Lawson, Moira Ann

    2008-01-01

    Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. During embryonic development, myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Sti...

  3. File list: InP.Myo.10.AllAg.C2C12 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Myo.10.AllAg.C2C12 mm9 Input control Muscle C2C12 SRX1482291,SRX262224,SRX26222...695944 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Myo.10.AllAg.C2C12.bed ...

  4. File list: NoD.Myo.50.AllAg.C2C12 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Myo.50.AllAg.C2C12 mm9 No description Muscle C2C12 SRX683676,DRX000201,SRX68367...020490,DRX020492 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Myo.50.AllAg.C2C12.bed ...

  5. File list: His.Myo.20.AllAg.C2C12 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Myo.20.AllAg.C2C12 mm9 Histone Muscle C2C12 SRX1482269,SRX1482270,SRX1482268,SR...04,SRX143610,SRX115555,SRX103216 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Myo.20.AllAg.C2C12.bed ...

  6. Differentiation-Associated Downregulation of Poly(ADP-Ribose Polymerase-1 Expression in Myoblasts Serves to Increase Their Resistance to Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Gábor Oláh

    Full Text Available Poly(ADP-ribose polymerase 1 (PARP-1, the major isoform of the poly (ADP-ribose polymerase family, is a constitutive nuclear and mitochondrial protein with well-recognized roles in various essential cellular functions such as DNA repair, signal transduction, apoptosis, as well as in a variety of pathophysiological conditions including sepsis, diabetes and cancer. Activation of PARP-1 in response to oxidative stress catalyzes the covalent attachment of the poly (ADP-ribose (PAR groups on itself and other acceptor proteins, utilizing NAD+ as a substrate. Overactivation of PARP-1 depletes intracellular NAD+ influencing mitochondrial electron transport, cellular ATP generation and, if persistent, can result in necrotic cell death. Due to their high metabolic activity, skeletal muscle cells are particularly exposed to constant oxidative stress insults. In this study, we investigated the role of PARP-1 in a well-defined model of murine skeletal muscle differentiation (C2C12 and compare the responses to oxidative stress of undifferentiated myoblasts and differentiated myotubes. We observed a marked reduction of PARP-1 expression as myoblasts differentiated into myotubes. This alteration correlated with an increased resistance to oxidative stress of the myotubes, as measured by MTT and LDH assays. Mitochondrial function, assessed by measuring mitochondrial membrane potential, was preserved under oxidative stress in myotubes compared to myoblasts. Moreover, basal respiration, ATP synthesis, and the maximal respiratory capacity of mitochondria were higher in myotubes than in myoblasts. Inhibition of the catalytic activity of PARP-1 by PJ34 (a phenanthridinone PARP inhibitor exerted greater protective effects in undifferentiated myoblasts than in differentiated myotubes. The above observations in C2C12 cells were also confirmed in a rat-derived skeletal muscle cell line (L6. Forced overexpression of PARP1 in C2C12 myotubes sensitized the cells to oxidant

  7. Regulation of nonmuscle myosin II during 3-methylcholanthrene induced dedifferentiation of C2C12 myotubes

    Energy Technology Data Exchange (ETDEWEB)

    Dey, Sumit K.; Saha, Shekhar; Das, Provas; Das, Mahua R.; Jana, Siddhartha S., E-mail: bcssj@iacs.res.in

    2014-08-01

    3-Methylcholanthrene (3MC) induces tumor formation at the site of injection in the hind leg of mice within 110 days. Recent reports reveal that the transformation of normal muscle cells to atypical cells is one of the causes for tumor formation, however the molecular mechanism behind this process is not well understood. Here, we show in an in vitro study that 3MC induces fragmentation of multinucleate myotubes into viable mononucleates. These mononucleates form colonies when they are seeded into soft agar, indicative of cellular transformation. Immunoblot analysis reveals that phosphorylation of myosin regulatory light chain (RLC{sub 20}) is 5.6±0.5 fold reduced in 3MC treated myotubes in comparison to vehicle treated myotubes during the fragmentation of myotubes. In contrast, levels of myogenic factors such as MyoD, Myogenin and cell cycle regulators such as Cyclin D, Cyclin E1 remain unchanged as assessed by real-time PCR array and reverse transcriptase PCR analysis, respectively. Interestingly, addition of the myosin light chain kinase inhibitor, ML-7, enhances the fragmentation, whereas phosphatase inhibitor perturbs the 3MC induced fragmentation of myotubes. These results suggest that decrease in RLC{sub 20} phosphorylation may be associated with the fragmentation step of dedifferentiation. - Highlights: • 3-Methylcholanthrene induces fragmentation of C2C12-myotubes. • Dedifferentiation can be divided into two steps – fragmentation and proliferation. • Fragmentation is associated with rearrangement of nonmuscle myosin II. • Genes associated with differentiation and proliferation are not altered during fragmentation. • Phosphorylation of myosin regulatory light chain is reduced during fragmentation.

  8. Acute high-caffeine exposure increases autophagic flux and reduces protein synthesis in C2C12 skeletal myotubes

    OpenAIRE

    Hughes, M. A.; Downs, R. M.; Webb, G. W.; Crocker, C. L.; Kinsey, S.T.; Baumgarner, Bradley L.

    2017-01-01

    Caffeine is a highly catabolic dietary stimulant. High caffeine concentrations (1–10 mM) have previously been shown to inhibit protein synthesis and increase protein degradation in various mammalian cell lines. The purpose of this study was to examine the effect of short-term caffeine exposure on cell signaling pathways that regulate protein metabolism in mammalian skeletal muscle cells. Fully differentiated C2C12 skeletal myotubes either received vehicle (DMSO) or 5 mM caffeine for 6 h. Our ...

  9. Biocompatible 3D printed polymers via fused deposition modelling direct C2C12 cellular phenotype in vitro.

    Science.gov (United States)

    Rimington, Rowan P; Capel, Andrew J; Christie, Steven D R; Lewis, Mark P

    2017-08-22

    The capability to 3D print bespoke biologically receptive parts within short time periods has driven the growing prevalence of additive manufacture (AM) technology within biological settings, however limited research concerning cellular interaction with 3D printed polymers has been undertaken. In this work, we used skeletal muscle C2C12 cell line in order to ascertain critical evidence of cellular behaviour in response to multiple bio-receptive candidate polymers; polylactic acid (PLA), acrylonitrile butadiene styrene (ABS), polyethylene terephthalate (PET) and polycarbonate (PC) 3D printed via fused deposition modelling (FDM). The extrusion based nature of FDM elicited polymer specific topographies, within which C2C12 cells exhibited reduced metabolic activity when compared to optimised surfaces of tissue culture plastic, however assay viability readings remained high across polymers outlining viable phenotypes. C2C12 cells exhibited consistently high levels of morphological alignment across polymers, however differential myotube widths and levels of transcriptional myogenin expression appeared to demonstrate response specific thresholds at which varying polymer selection potentiates cellular differentiation, elicits pre-mature early myotube formation and directs subsequent morphological phenotype. Here we observed biocompatible AM polymers manufactured via FDM, which also appear to hold the potential to simultaneously manipulate the desired biological phenotype and enhance the biomimicry of skeletal muscle cells in vitro via AM polymer choice and careful selection of machine processing parameters. When considered in combination with the associated design freedom of AM, this may provide the opportunity to not only enhance the efficiency of creating biomimetic models, but also to precisely control the biological output within such scaffolds.

  10. Expression of Thyroid Stimulating Hormone Receptor mRNA in Mouse C2C12 Skeletal Muscle Cells

    Directory of Open Access Journals (Sweden)

    Jung Hun Ohn

    2013-06-01

    Full Text Available BackgroundWe analyzed whether thyroid stimulating hormone receptor (TSH-R is expressed in a skeletal muscle cell line and if TSH has influence on the differentiation of muscle cells or on the determination of muscle fiber types.MethodsTSH-R gene expression was detected with nested real-time polymerase chain reaction (RT-PCR in C2C12, a mouse skeletal muscle cell line. The effect of TSH on myotube differentiation was assessed by microscopic examination of myotube formation and through the measurement of expression of muscle differentiation markers, i.e., myogenin and myoD, and muscle type-specific genes, i.e., MyHC1, MyHC2a, and MyHC2b, with quantitative RT-PCR before and after incubation of C2C12 myotube with TSH.ResultsTSH-R was expressed in the mouse skeletal muscle cell line. However, treatment with TSH had little effect on the differentiation of muscle cells, although the expression of the muscle differention marker myogenin was significantly increased after TSH treatment. Treatment of TSH did not affect the expression of muscle type-specific genes.ConclusionTSH-R is expressed in a mouse skeletal muscle cell line, but the role of TSH receptor signaling in skeletal muscle needs further investigation.

  11. Mechanical Stimulation of C2C12 Cells Increases m-Calpain Expression and Activity, Focal Adhesion Plaque Degradation and Cell Fusion

    DEFF Research Database (Denmark)

    Grossi, Alberto; Lawson, Moira Ann; Karlsson, Anders H

    to stretch- or load-induced signaling is now beginning to be understood as a factor which affects gene sequences, protein synthesis and an increase in Ca2+ infux in myocytes. Evidence of the involvement of Ca2+ dependent activity in myoblast fusion, cell membrane and cytoskeleton component reorganization due......Abstract Mechanical Stimulation of C2C12 Cells Increases m-calpain Expression and Activity, Focal Adhesion Plaque Degradation and Cell Fusion A. Grossi, A. H. Karlsson, M. A. Lawson; Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark...

  12. Micropatterning topology on soft substrates affects myoblast proliferation and differentiation.

    Science.gov (United States)

    Zatti, Susi; Zoso, Alice; Serena, Elena; Luni, Camilla; Cimetta, Elisa; Elvassore, Nicola

    2012-02-07

    Micropatterning techniques and substrate engineering are becoming useful tools to investigate several aspects of cell-cell interaction biology. In this work, we rationally study how different micropatterning geometries can affect myoblast behavior in the early stage of in vitro myogenesis. Soft hydrogels with physiological elastic modulus (E = 15 kPa) were micropatterned in parallel lanes (100, 300, and 500 μm width) resulting in different local and global myoblast densities. Proliferation and differentiation into multinucleated myotubes were evaluated for murine and human myoblasts. Wider lanes showed a decrease in murine myoblast proliferation: (69 ± 8)% in 100 μm wide lanes compared to (39 ± 7)% in 500 μm lanes. Conversely, fusion index increased in wider lanes: from (46 ± 7)% to (66 ± 7)% for murine myoblasts, and from (15 ± 3)% to (36 ± 2)% for human primary myoblasts, using a patterning width of 100 and 500 μm, respectively. These results are consistent with both computational modeling data and conditioned medium experiments, which demonstrated that wider lanes favor the accumulation of endogenous secreted factors. Interestingly, human primary myoblast proliferation is not affected by patterning width, which may be because the high serum content of their culture medium overrides the effect of secreted factors. These data highlight the role of micropatterning in shaping the cellular niche through secreted factor accumulation, and are of paramount importance in rationally understanding myogenesis in vitro for the correct design of in vitro skeletal muscle models.

  13. Bromopropane compounds inhibit osteogenesis by ERK-dependent Runx2 inhibition in C2C12 cells.

    Science.gov (United States)

    Jeong, Hyung Min; Choi, You Hee; Jeong, Hye Gwang; Jeong, Tae Cheon; Lee, Kwang Youl

    2014-02-01

    Bromopropane (BP) is a halogenated alkan compound used in various industries as chemical intermediates, extraction solvents, and degreasing compounds. Halogenated alkan compounds can damage the nervous system, immune system, and hematopoietic and reproductive functions in animals and humans. However, the effect of BPs on bone formation has not yet been examined. This study examined the effects of BPs on osteoblast differentiation and analyzed the mechanisms involved in C2C12, mesenchymal stem cells. BPs dose dependently reduced the alkaline phosphatase activity, expression levels and promoter activity of bone marker genes. Additionally, 1,2-dibromopropane (1,2-DBP) significantly reduced the levels and transcriptional activity of Runx2 and Osterix, major bone transcription factors, in BMP2 induced C2C12 cells. Furthermore, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) were significantly inhibited by 1,2-DBP. These results demonstrate that BPs inhibit osteoblast differentiation by suppressing Runx2 and Osterix through the ERK/JNK pathway.

  14. Sarcolipin expression is repressed by endoplasmic reticulum stress in C2C12 myotubes.

    Science.gov (United States)

    Takahashi, Nobuhiko; Kimura, Atsushi P; Naito, Sumiyoshi; Yoshida, Mika; Kumano, Osamu; Suzuki, Takeshi; Itaya, Satoshi; Moriya, Mitsuru; Tsuji, Masahiro; Ieko, Masahiro

    2017-11-01

    Sarcolipin is a transmembrane protein expressed in the sarco/endoplasmic reticulum of skeletal and atrial muscles in large animals. Sarcolipin plays crucial roles in heat production through modifying the function of sarco/endoplasmic reticulum Ca2+ ATPase, thereby being involved in thermogenesis and systemic metabolism. In skeletal muscle, endoplasmic reticulum (ER) stress has been implicated in several conditions, such as insulin resistance, muscle diseases, and hypo/hyper-contraction. Here, we investigated the effect of ER stress on sarcolipin expression in skeletal muscle cells, C2C12 myotubes. First, gene expression of sarcolipin was confirmed in the cells during myogenesis. Then, ER stress was induced in C2C12 myotubes by treatment with tunicamycin or thapsigargin. Sarcolipin messenger RNA (mRNA) and protein expression were significantly reduced by ER stress induction. The reduction was independent of inositol-requiring element 1 (IRE1), which is activated by ER stress and has potent endonuclease activity, when evaluated by treatment with an IRE1 inhibitor, 4μ8C. On the other hand, sarcolipin mRNA stability was reduced under the ER stress when evaluated by treatment with actinomycin D. In conclusion, these results show that ER stress represses sarcolipin expression due to changes in mRNA stability in C2C12 myotubes.

  15. The critical role of myostatin in differentiation of sheep myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chenxi [College of Life Science and Technology, Xinjiang University, Urumqi (China); Xinjiang Laboratory of Animal Biotechnology, Urumqi (China); Li, Wenrong; Zhang, Xuemei; Zhang, Ning; He, Sangang; Huang, Juncheng [Xinjiang Laboratory of Animal Biotechnology, Urumqi (China); Laboratory of Grass-fed Animal Genetics, Breeding and Reproduction of Ministry of Agriculture, Urumqi (China); Animal Biotechnological Research Center, Xinjiang Academy of Animal Science, Urumqi (China); Ge, Yubin [The State Engineering Laboratory of AIDS Vaccine, College of Life Science, Jilin University, Changchun (China); Liu, Mingjun, E-mail: xjlmj2004@yahoo.com.cn [Xinjiang Laboratory of Animal Biotechnology, Urumqi (China); Laboratory of Grass-fed Animal Genetics, Breeding and Reproduction of Ministry of Agriculture, Urumqi (China); Animal Biotechnological Research Center, Xinjiang Academy of Animal Science, Urumqi (China)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Identification of the effective and specific shRNA to knockdown MSTN. Black-Right-Pointing-Pointer Overexpression of MSTN reversibly suppressed myogenic differentiation. Black-Right-Pointing-Pointer shRNA knockdown of endogenous MSTN promoted ovine myoblast differentiation. Black-Right-Pointing-Pointer MSTN inhibits myogenic differentiation through down-regulation of MyoD and Myogenin and up-regulation of Smad3. Black-Right-Pointing-Pointer Provides a promise for the generation of transgenic sheep to improve meat productivity. -- Abstract: Myostatin [MSTN, also known as growth differentiation factor 8 (GDF8)], is an inhibitor of skeletal muscle growth. Blockade of MSTN function has been reported to result in increased muscle mass in mice. However, its role in myoblast differentiation in farm animals has not been determined. In the present study, we sought to determine the role of MSTN in the differentiation of primary sheep myoblasts. We found that ectopic overexpression of MSTN resulted in lower fusion index in sheep myoblasts, which indicated the repression of myoblast differentiation. This phenotypic change was reversed by shRNA knockdown of the ectopically expressed MSTN in the cells. In contrast, shRNA knockdown of the endogenous MSTN resulted in induction of myogenic differentiation. Additional studies revealed that the induction of differentiation by knocking down the ectopically or endogenously expressed MSTN was accompanied by up-regulation of MyoD and myogenin, and down-regulation of Smad3. Our results demonstrate that MSTN plays critical role in myoblast differentiation in sheep, analogous to that in mice. This study also suggests that shRNA knockdown of MSTN could be a potentially promising approach to improve sheep muscle growth, so as to increase meat productivity.

  16. File list: Pol.Myo.50.AllAg.C2C12 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Myo.50.AllAg.C2C12 mm9 RNA polymerase Muscle C2C12 SRX142527,SRX101682,SRX10168...3,SRX142518,SRX062103,SRX062102,SRX1176761,SRX1176745,SRX1176759,SRX1176747 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Myo.50.AllAg.C2C12.bed ...

  17. File list: ALL.Myo.20.AllAg.C2C12 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Myo.20.AllAg.C2C12 mm9 All antigens Muscle C2C12 SRX1482269,SRX262223,SRX262224...,SRX683673,DRX000355,SRX103216,SRX373248,SRX039345,SRX695944,DRX000353 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Myo.20.AllAg.C2C12.bed ...

  18. File list: ALL.Myo.50.AllAg.C2C12 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Myo.50.AllAg.C2C12 mm9 All antigens Muscle C2C12 SRX1482269,SRX262223,SRX262224...,SRX115557,SRX115559,SRX062124,DRX020490,DRX020492,SRX022849,SRX022851 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Myo.50.AllAg.C2C12.bed ...

  19. File list: Pol.Myo.05.AllAg.C2C12 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Myo.05.AllAg.C2C12 mm9 RNA polymerase Muscle C2C12 SRX142527,SRX142518,SRX10168...3,SRX101682,SRX062102,SRX062103,SRX1176745,SRX1176761,SRX1176747,SRX1176759 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Myo.05.AllAg.C2C12.bed ...

  20. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Itoigawa, Yoshiaki [Tohoku University School of Medicine, Sendai (Japan); Juntendo University School of Medicine, Tokyo (Japan); Kishimoto, Koshi N., E-mail: kishimoto@med.tohoku.ac.jp [Tohoku University School of Medicine, Sendai (Japan); Okuno, Hiroshi; Sano, Hirotaka [Tohoku University School of Medicine, Sendai (Japan); Kaneko, Kazuo [Juntendo University School of Medicine, Tokyo (Japan); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan)

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  1. Infectious prions accumulate to high levels in non proliferative C2C12 myotubes.

    Directory of Open Access Journals (Sweden)

    Allen Herbst

    Full Text Available Prion diseases are driven by the strain-specific, template-dependent transconformation of the normal cellular prion protein (PrP(C into a disease specific isoform PrP(Sc. Cell culture models of prion infection generally use replicating cells resulting in lower levels of prion accumulation compared to animals. Using non-replicating cells allows the accumulation of higher levels of PrP(Sc and, thus, greater amounts of infectivity. Here, we infect non-proliferating muscle fiber myotube cultures prepared from differentiated myoblasts. We demonstrate that prion-infected myotubes generate substantial amounts of PrP(Sc and that the level of infectivity produced in these post-mitotic cells, 10(5.5 L.D.50/mg of total protein, approaches that observed in vivo. Exposure of the myotubes to different mouse-adapted agents demonstrates strain-specific replication of infectious agents. Mouse-derived myotubes could not be infected with hamster prions suggesting that the species barrier effect is intact. We suggest that non-proliferating myotubes will be a valuable model system for generating infectious prions and for screening compounds for anti-prion activity.

  2. Characterization and Functional Analysis of Extracellular Vesicles and Muscle-Abundant miRNAs (miR-1, miR-133a, and miR-206 in C2C12 Myocytes and mdx Mice.

    Directory of Open Access Journals (Sweden)

    Yasunari Matsuzaka

    Full Text Available Duchenne muscular dystrophy (DMD is a progressive neuromuscular disorder. Here, we show that the CD63 antigen, which is located on the surface of extracellular vesicles (EVs, is associated with increased levels of muscle-abundant miRNAs, namely myomiRs miR-1, miR-133a, and miR-206, in the sera of DMD patients and mdx mice. Furthermore, the release of EVs from the murine myoblast C2C12 cell line was found to be modulated by intracellular ceramide levels in a Ca2+-dependent manner. Next, to investigate the effects of EVs on cell survival, C2C12 myoblasts and myotubes were cultured with EVs from the sera of mdx mice or C2C12 cells overexpressing myomiRs in presence of cellular stresses. Both the exposure of C2C12 myoblasts and myotubes to EVs from the serum of mdx mice, and the overexpression of miR-133a in C2C12 cells in presence of cellular stress resulted in a significant decrease in cell death. Finally, to assess whether miRNAs regulate skeletal muscle regeneration in vivo, we intraperitoneally injected GW4869 (an inhibitor of exosome secretion into mdx mice for 5 and 10 days. Levels of miRNAs and creatine kinase in the serum of GW4869-treated mdx mice were significantly downregulated compared with those of controls. The tibialis anterior muscles of the GW4869-treated mdx mice showed a robust decrease in Evans blue dye uptake. Collectively, these results indicate that EVs and myomiRs might protect the skeletal muscle of mdx mice from degeneration.

  3. Genome-wide examination of myoblast cell cycle withdrawal duringdifferentiation

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Xun; Collier, John Michael; Hlaing, Myint; Zhang, Leanne; Delshad, Elizabeth H.; Bristow, James; Bernstein, Harold S.

    2002-12-02

    Skeletal and cardiac myocytes cease division within weeks of birth. Although skeletal muscle retains limited capacity for regeneration through recruitment of satellite cells, resident populations of adult myocardial stem cells have not been identified. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that C2C12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. C2C12 cells were differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis. BrdU incorporation decreased dramatically in confluent cultures 48 hr after addition of horse serum, as cells started to form myotubes. This finding was preceded by up-regulation of MyoD, followed by myogenin, and activation of Bcl-2. Cyclin D1 was expressed in proliferating cultures and became undetectable in cultures containing 40 percent fused myotubes, as levels of p21(WAF1/Cip1) increased and alpha-actin became detectable. Because C2C12 myoblasts withdraw from the cell cycle during myocyte differentiation following a course that recapitulates this process in vivo, we performed a genome-wide screen to identify other gene products involved in this process. Using microarrays containing approximately 10,000 minimally redundant mouse sequences that map to the UniGene database of the National Center for Biotechnology Information, we compared gene expression profiles between proliferating, differentiating, and differentiated C2C12 cells and verified candidate genes demonstrating differential expression by RT-PCR. Cluster analysis of differentially expressed genes revealed groups of gene products involved in cell cycle withdrawal, muscle differentiation, and apoptosis. In addition, we identified several genes, including DDAH2 and Ly

  4. Melatonin protects against uric acid-induced mitochondrial dysfunction, oxidative stress, and triglyceride accumulation in C2C12myotubes.

    Science.gov (United States)

    Maarman, Gerald J; Andrew, Brittany M; Blackhurst, Dee M; Ojuka, Edward O

    2017-04-01

    Excess uric acid has been shown to induce oxidative stress, triglyceride accumulation, and mitochondrial dysfunction in the liver and is an independent predictor of type-2 diabetes. Skeletal muscle plays a dominant role in type 2 diabetes and presents a large surface area to plasma uric acid. However, the effects of uric acid on skeletal muscle are underinvestigated. Our aim was therefore to characterize the effects of excessive uric acid on oxidative stress, triglyceride content, and mitochondrial function in skeletal muscle C 2 C 12 myotubes and assess how these are modulated by the antioxidant molecule melatonin. Differentiated C 2 C 12 myotubes were exposed to 750 µM uric acid or uric acid + 10 nM melatonin for 72 h. Compared with control, uric acid increased triglyceride content by ~237%, oxidative stress by 32%, and antioxidant capacity by 135%. Uric acid also reduced endogenous ROUTINE respiration, complex II-linked oxidative phosphorylation, and electron transfer system capacities. Melatonin counteracted the effects of uric acid without further altering antioxidant capacity. Our data demonstrate that excess uric acid has adverse effects on skeletal muscle similar to those previously reported in hepatocytes and suggest that melatonin at a low physiological concentration of 10 nM may be a possible therapy against some adverse effects of excess uric acid. NEW & NOTEWORTHY Few studies have investigated the effects of uric acid on skeletal muscle. This study shows that hyperuricemia induces mitochondrial dysfunction and triglyceride accumulation in skeletal muscle. The findings may explain why hyperuricemia is an independent predictor of diabetes. Copyright © 2017 the American Physiological Society.

  5. The effect of eicosapentaenoic and docosahexaenoic acid on protein synthesis and breakdown in murine C2C12 myotubes

    Energy Technology Data Exchange (ETDEWEB)

    Kamolrat, Torkamol [Musculoskeletal Research Programme, Institute of Medical Sciences, University of Aberdeen, AB25 2ZD (United Kingdom); Gray, Stuart R., E-mail: s.r.gray@abdn.ac.uk [Musculoskeletal Research Programme, Institute of Medical Sciences, University of Aberdeen, AB25 2ZD (United Kingdom)

    2013-03-22

    Highlights: ► EPA can enhance protein synthesis and retard protein breakdown in muscle cells. ► These effects were concurrent with increases in p70s6k and FOXO3a phosphorylation. ► EPA may be a useful tool in the treatment of muscle wasting conditions. -- Abstract: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been found to stimulate protein synthesis with little information regarding their effects on protein breakdown. Furthermore whether there are distinct effects of EPA and DHA remains to be established. The aim of the current study was to determine the distinct effects of EPA and DHA on protein synthesis, protein breakdown and signalling pathways in C2C12 myotubes. Fully differentiated C2C12 cells were incubated for 24 h with 0.1% ethanol (control), 50 μM EPA or 50 μM DHA prior to experimentation. After serum (4 h) and amino acid (1 h) starvation cells were stimulated with 2 mM L-leucine and protein synthesis measured using {sup 3}H-labelled phenylalanine. Protein breakdown was measured using {sup 3}H-labelled phenylalanine and signalling pathways (Akt, mTOR, p70S6k, 4EBP1, rps6 and FOXO3a) via Western blots. Data revealed that after incubation with EPA protein synthesis was 25% greater (P < 0.05) compared to control cells, with no effect of DHA. Protein breakdown was 22% (P < 0.05) lower, compared to control cells, after incubation with EPA, with no effect of DHA. Analysis of signalling pathways revealed that both EPA and DHA incubation increased (P < 0.05) p70s6k phosphorylation, EPA increased (P < 0.05) FOXO3a phosphorylation, with no alteration in other signalling proteins. The current study has demonstrated distinct effects of EPA and DHA on protein metabolism with EPA showing a greater ability to result in skeletal muscle protein accretion.

  6. Pregnancy-associated Plasma Protein-A Regulates Myoblast Proliferation and Differentiation through an Insulin-like Growth Factor-dependent Mechanism*

    OpenAIRE

    Kumar, Ashok; Mohan, Subburaman; Newton, Jacqueline; Rehage, Mark; Tran, Kiet; Baylink, David J.; Qin, Xuezhong

    2005-01-01

    Pregnancy-associated plasma protein-A (PAPP-A), a member of the metalloproteinase superfamily, is an important regulator of mammalian growth and development. However, the role of PAPP-A and its mechanism of action in various cellular processes remain unknown. In this study, we have investigated the role of PAPP-A in skeletal myogenesis using C2C12 myoblasts. Recombinant PAPP-A was purified from the conditioned medium of HT1080 cells overexpressing PAPP-A. Treatment of C2C12 myoblasts with PAP...

  7. Conessine Interferes with Oxidative Stress-Induced C2C12 Myoblast Cell Death through Inhibition of Autophagic Flux

    OpenAIRE

    Hyunju Kim; Kang Il Lee; Minsu Jang; Sim Namkoong; Rackhyun Park; Hyunwoo Ju; Inho Choi; Won Keun Oh; Junsoo Park

    2016-01-01

    Conessine, a steroidal alkaloid isolated from Holarrhena floribunda, has anti-malarial activity and interacts with the histamine H3 receptor. However, the cellular effects of conessine are poorly understood. Accordingly, we evaluated the involvement of conessine in the regulation of autophagy. We searched natural compounds that modulate autophagy, and conessine was identified as an inhibitor of autophagic flux. Conessine treatment induced the formation of autophagosomes, and p62, an autophagi...

  8. Acute high-caffeine exposure increases autophagic flux and reduces protein synthesis in C2C12 skeletal myotubes.

    Science.gov (United States)

    Hughes, M A; Downs, R M; Webb, G W; Crocker, C L; Kinsey, S T; Baumgarner, Bradley L

    2017-04-01

    Caffeine is a highly catabolic dietary stimulant. High caffeine concentrations (1-10 mM) have previously been shown to inhibit protein synthesis and increase protein degradation in various mammalian cell lines. The purpose of this study was to examine the effect of short-term caffeine exposure on cell signaling pathways that regulate protein metabolism in mammalian skeletal muscle cells. Fully differentiated C2C12 skeletal myotubes either received vehicle (DMSO) or 5 mM caffeine for 6 h. Our analysis revealed that caffeine promoted a 40% increase in autolysosome formation and a 25% increase in autophagic flux. In contrast, caffeine treatment did not significantly increase the expression of the skeletal muscle specific ubiquitin ligases MAFbx and MuRF1 or 20S proteasome activity. Caffeine treatment significantly reduced mTORC1 signaling, total protein synthesis and myotube diameter in a CaMKKβ/AMPK-dependent manner. Further, caffeine promoted a CaMKII-dependent increase in myostatin mRNA expression that did not significantly contribute to the caffeine-dependent reduction in protein synthesis. Our results indicate that short-term caffeine exposure significantly reduced skeletal myotube diameter by increasing autophagic flux and promoting a CaMKKβ/AMPK-dependent reduction in protein synthesis.

  9. Potentiated Osteoinductivity via Cotransfection with BMP-2 and VEGF Genes in Microencapsulated C2C12 Cells

    Directory of Open Access Journals (Sweden)

    Yang Shen

    2015-01-01

    Full Text Available Microcapsules with entrapped cells hold great promise for repairing bone defects. Unfortunately, the osteoinductivity of microcapsules has been restricted by many factors, among which the deficiency of functional proteins is a significant priority. We potentiated the osteoinductivity of microencapsulated cells via cotransfection with BMP-2 and VEGF genes. Various tissue-derived mesenchymal stem cells and cell lines were compared for BMP-2 and VEGF cotransfection. Ethidium bromide (EB/Calcein AM staining revealed that all of the cell categories could survive for 4 weeks after microencapsulation. An ELISA assay indicated that all microencapsulated BMP-2 or VEGF transfected cells could secrete gene products constitutively for 1 month. Particularly, the recombinant microencapsulated C2C12 cells released the most desirable level of BMP-2 and VEGF. Further experiments demonstrated that microencapsulated BMP-2 and VEGF cotransfected C2C12 cells generated both BMP-2 and VEGF for 4 weeks. Additionally, the cotransfection of BMP-2 and VEGF in microencapsulated C2C12 cells showed a stronger osteogenic induction against BMSCs than individual BMP-2-transfected microencapsulated C2C12 cells. These results demonstrated that the cotransfection of BMP-2 and VEGF into microencapsulated C2C12 cells is of potent utility for the potentiation of bone regeneration, which would provide a promising clinical strategy for cellular therapy in bone defects.

  10. Multiple AMPK activators inhibit l-carnitine uptake in C2C12 skeletal muscle myotubes.

    Science.gov (United States)

    Shaw, Andy; Jeromson, Stewart; Watterson, Kenneth R; Pediani, John D; Gallagher, Iain J; Whalley, Tim; Dreczkowski, Gillian; Brooks, Naomi; Galloway, Stuart D; Hamilton, D Lee

    2017-06-01

    Mutations in the gene that encodes the principal l-carnitine transporter, OCTN2, can lead to a reduced intracellular l-carnitine pool and the disease Primary Carnitine Deficiency. l-Carnitine supplementation is used therapeutically to increase intracellular l-carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake, we hypothesized that AMPK-activating compounds and insulin would increase l-carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level, and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase l-carnitine uptake at 100 nM. However, l-carnitine uptake was modestly increased at a dose of 150 nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10 mM, 5 mM, 1 mM, 0.5 mM), A23187 (10 μM)], inhibit mitochondrial function [sodium azide (75 μM), rotenone (1 μM), berberine (100 μM), DNP (500 μM)], or directly activate AMPK [AICAR (250 μM)] were assessed for their ability to regulate l-carnitine uptake. All compounds tested significantly inhibited l-carnitine uptake. Inhibition by caffeine was not dantrolene (10 μM) sensitive despite dantrolene inhibiting caffeine-mediated calcium release. Saturation curve analysis suggested that caffeine did not competitively inhibit l-carnitine transport. To assess the potential role of AMPK in this process, we assessed the ability of the AMPK inhibitor Compound C (10 μM) to rescue the effect of caffeine. Compound C offered a partial rescue of l-carnitine uptake with 0.5 mM caffeine, suggesting that AMPK may play a role in the inhibitory effects of caffeine. However, caffeine likely inhibits l-carnitine uptake by alternative mechanisms independently of calcium release. PKA activation or direct interference with transporter function may play a role. Copyright © 2017 the American Physiological Society.

  11. Downregulation of lipin-1 induces insulin resistance by increasing intracellular ceramide accumulation in C2C12 myotubes.

    Science.gov (United States)

    Huang, Shujuan; Huang, Suling; Wang, Xi; Zhang, Qingli; Liu, Jia; Leng, Ying

    2017-01-01

    Dysregulation of lipid metabolism in skeletal muscle is involved in the development of insulin resistance. Mutations in lipin-1, a key lipid metabolism regulator leads to significant systemic insulin resistance in fld mice. However, the function of lipin-1 on lipid metabolism and insulin sensitivity in skeletal muscle is still unclear. Herein we demonstrated that downregulation of lipin-1 in C2C12 myotubes by siRNA transfection suppressed insulin action, characterized by reduced insulin stimulated Akt phosphorylation and glucose uptake. Correspondingly, decreased lipin-1 expression was observed in palmitate-induced insulin resistance in C2C12 myotubes, suggested that lipin-1 might play a role in the etiology of insulin resistance in skeletal muscle. The insulin resistance induced by lipin-1 downregulation was related to the disturbance of lipid homeostasis. Lipin-1 silencing reduced intracellular DAG and TAG levels, but elevated ceramide accumulation in C2C12 myotubes. Moreover, the impaired insulin stimulated Akt phosphorylation and glucose uptake caused by lipin-1 silencing could be blocked by the pretreatment with SPT inhibitor myriocin, ceramide synthase inhibitor FB1, or PP2A inhibitor okadaic acid, suggested that the increased ceramide accumulation might be responsible for the development of insulin resistance induced by lipin-1 silencing in C2C12 myotubes. Meanwhile, decreased lipin-1 expression also impaired mitochondrial function in C2C12 myotubes. Therefore, our study suggests that lipin-1 plays an important role in lipid metabolism and downregulation of lipin-1 induces insulin resistance by increasing intracellular ceramide accumulation in C2C12 myotubes. These results offer a molecular insight into the role of lipin-1 in the development of insulin resistance in skeletal muscle.

  12. Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System.

    Science.gov (United States)

    Chaturvedi, Vishal; Dye, Danielle E; Kinnear, Beverley F; van Kuppevelt, Toin H; Grounds, Miranda D; Coombe, Deirdre R

    2015-01-01

    Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.

  13. Efeitos do ultra-som terapêutico contínuo sobre a proliferação e viabilidade de células musculares C2C12 Effects of continuous therapeutic ultrasound on proliferation and viability of C2C12 muscle cells

    Directory of Open Access Journals (Sweden)

    Paola Pelegrineli Artilheiro

    2010-06-01

    Full Text Available O ultra-som terapêutico (US é um recurso bioestimulante utilizado para propiciar reparo muscular de melhor qualidade e menor duração, mas o potencial terapêutico do US contínuo não está totalmente estabelecido. O objetivo deste trabalho foi avaliar o efeito do US contínuo sobre a proliferação e viabilidade de células musculares precursoras (mioblastos C2C12. Mioblastos C2C12 foram cultivados em meio de cultura contendo 10% de soro fetal bovino e irradiados com US contínuo nas freqüências de 1 e 3 MHz nas intensidades de 0,2 e 0,5 W/cm2, durante 2 e 5 minutos. A viabilidade e proliferação celular foram avaliadas após 24, 48 e 72 h de incubação. Grupos não-irradiados serviram como controle. Foram realizados experimentos independentes em cada condição acima, e os dados obtidos submetidos à análise estatística. Os resultados mostram que não houve diferença estatisticamente significativa na proliferação e viabilidade celular entre os mioblastos tratados com US e as culturas controles após os diferentes períodos de incubação, em todos os parâmetros avaliados. Conclui-se que o US contínuo, nos parâmetros avaliados, não foi capaz de alterar a proliferação e viabilidade dos mioblastos.Therapeutic ultrasound (US is a biophysical stimulation resource widely used in order to promote better, faster muscle repair, but the effectiveness of continuous US in treating injuries is not fully established. The aim of the present in vitro study was to assess the effects of continuous ultrasound on viability and proliferation of skeletal muscle precursor cells (C2C12 myoblasts. C2C12 myoblasts were cultured in a medium containing 10% foetal bovine serum and irradiated with continuous ultrasound at 1 and 3 MHz frequencies, at intensities of 0.2 and 0.5 W/cm² for 2 and 5 minutes. Cell viability and proliferation were assessed after different incubation periods (24, 48 and 72 h. Non-irradiated groups served as control and data were

  14. Developmental regulation of expression of the alpha 1 and alpha 2 subunits mRNAs of the voltage-dependent calcium channel in a differentiating myogenic cell line.

    Science.gov (United States)

    Varadi, G; Orlowski, J; Schwartz, A

    1989-07-03

    The voltage-dependent calcium channel (VDCC) in skeletal muscle probably plays a key role in transducing membrane charge movement to the calcium release channel. We report here that the expression of VDCC alpha 1 and alpha 2 mRNAs is developmentally regulated in differentiating C2C12 myogenic cells. The alpha 1 mRNA is not detectable in the myoblast form of C2C12 cells while its expression is induced 20-fold in differentiated myotubes. In contrast, the alpha 2 mRNA is weakly expressed in myoblasts but is also induced upon myogenic differentiation.

  15. Screening with a novel cell-based assay for TAZ activators identifies a compound that enhances myogenesis in C2C12 cells and facilitates muscle repair in a muscle injury model.

    Science.gov (United States)

    Yang, Zeyu; Nakagawa, Kentaro; Sarkar, Aradhan; Maruyama, Junichi; Iwasa, Hiroaki; Bao, Yijun; Ishigami-Yuasa, Mari; Ito, Shigeru; Kagechika, Hiroyuki; Hata, Shoji; Nishina, Hiroshi; Abe, Shinya; Kitagawa, Masanobu; Hata, Yutaka

    2014-05-01

    The transcriptional coactivator with a PDZ-binding motif (TAZ) cooperates with various transcriptional factors and plays various roles. Immortalized human mammalian epithelial MCF10A cells form spheres when TAZ is overexpressed and activated. We developed a cell-based assay using sphere formation by TAZ-expressing MCF10A cells as a readout to screen 18,458 chemical compounds for TAZ activators. Fifty compounds were obtained, and 47 were confirmed to activate the TAZ-dependent TEAD-responsive reporter activity in HEK293 cells. We used the derived subset of compounds as a TAZ activator candidate minilibrary and searched for compounds that promote myogenesis in mouse C2C12 myoblast cells. In this study, we focused on one compound, IBS008738. IBS008738 stabilizes TAZ, increases the unphosphorylated TAZ level, enhances the association of MyoD with the myogenin promoter, upregulates MyoD-dependent gene transcription, and competes with myostatin in C2C12 cells. TAZ knockdown verifies that the effect of IBS008738 depends on endogenous TAZ in C2C12 cells. IBS008738 facilitates muscle repair in cardiotoxin-induced muscle injury and prevents dexamethasone-induced muscle atrophy. Thus, this cell-based assay is useful to identify TAZ activators with a variety of cellular outputs. Our findings also support the idea that TAZ is a potential therapeutic target for muscle atrophy.

  16. ZnO nanoparticles augment ALT, AST, ALP and LDH expressions in C2C12 cells

    OpenAIRE

    Pandurangan, Muthuraman; Kim, Doo Hwan

    2015-01-01

    The present study aimed to investigate the effect of ZnO nanoparticles on alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) enzyme expressions in C2C12 cells. ZnO nanoparticles are widely used in the several cosmetic lotions and other biomedical products. Several studies report on ZnO nanoparticle mediated cytotoxicity. However, there are no reports on the effect of ZnO nanoparticles on ALT, AST, ALP and LDH enzyme expressions...

  17. Activated Integrin-Linked Kinase Negatively Regulates Muscle Cell Enhancement Factor 2C in C2C12 Cells

    Directory of Open Access Journals (Sweden)

    Zhenguo Dong

    2015-01-01

    Full Text Available Our previous study reported that muscle cell enhancement factor 2C (MEF2C was fully activated after inhibition of the phosphorylation activity of integrin-linked kinase (ILK in the skeletal muscle cells of goats. It enhanced the binding of promoter or enhancer of transcription factor related to proliferation of muscle cells and then regulated the expression of these genes. In the present investigation, we explored whether ILK activation depended on PI3K to regulate the phosphorylation and transcriptional activity of MEF2C during C2C12 cell proliferation. We inhibited PI3K activity in C2C12 with LY294002 and then found that ILK phosphorylation levels and MEF2C phosphorylation were decreased and that MCK mRNA expression was suppressed significantly. After inhibiting ILK phosphorylation activity with Cpd22 and ILK-shRNA, we found MEF2C phosphorylation activity and MCK mRNA expression were increased extremely significantly. In the presence of Cpd22, PI3K activity inhibition increased MEF2C phosphorylation and MCK mRNA expression indistinctively. We conclude that ILK negatively and independently of PI3K regulated MEF2C phosphorylation activity and MCK mRNA expression in C2C12 cells. The results provide new ideas for the study of classical signaling pathway of PI3K-ILK-related proteins and transcription factors.

  18. Specific knockdown of delta-sarcoglycan gene in C2C12 in vitro causes post-translational loss of other sarcoglycans without mechanical stress.

    Science.gov (United States)

    Honda, Michiyo; Hosoda, Mari; Kanzawa, Nobuyuki; Tsuchiya, Takahide; Toyo-oka, Teruhiko

    2009-03-01

    The precise role of delta-sarcoglycan (SG) that is constitutively expressed in skeletal muscle cells and may serve for maintaining the sarcolemmal integrity has not been identified. The delta-SG protein is at first among SG complex. To specifically identify the role in C(2)C(12) cells during the myogenesis, we screened several RNA interference (RNAi) candidates at first, and knocked down both levels of the mRNA and protein, employing adenovirus-mediated RNAi. We found no morphological alteration at both myoblast and myotube stages by suppression of delta-SG. The specific knockdown of delta-SG accompanied a concomitant decrease of alpha-, beta-, and gamma-SGs preserving normal levels of each transcript. As for the localization, alpha-, beta-, and gamma-SGs were weakly stained on the cell membrane in delta-SG knockdown cells, whereas each SG in control cell was localized both on the cell membrane and myoplasm abundantly. This enhanced post-translational loss would represent similitude of the progression of cardiomuscular diseases in vitro. Different from cardiac muscle cells, skeletal muscle cell culture without muscle contraction may imply that mechanical stress per se is not primarily involved in the progression of limb-girdle muscular dystrophy. Furthermore, we have observed translocation of calpain-2 to cell membrane in delta-SG knockdown cells, suggesting that Ca(2+)-sensitive proteases, calpains closely take part in post-translational proteolysis.

  19. Insulin sensitizing effects of oligomannuronate-chromium (III complexes in C2C12 skeletal muscle cells.

    Directory of Open Access Journals (Sweden)

    Cui Hao

    Full Text Available BACKGROUND: It was known that the insulin resistance in skeletal muscle is a major pathogenic factor in diabetes mellitus. Therefore prevention of metabolic disorder caused by insulin resistance and improvement of insulin sensitivity are very important for the therapy of type 2 diabetes. In the present study, we investigated the ability of marine oligosaccharides oligomannuronate and its chromium (III complexes from brown alga to enhance insulin sensitivity in C2C12 skeletal muscle cells. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that oligomannuronate, especially its chromium (III complexes, enhanced insulin-stimulated glucose uptake and increased the mRNA expression of glucose transporter 4 (GLUT4 and insulin receptor (IR after their internalization into C2C12 skeletal muscle cells. Additionally, oligosaccharides treatment also significantly enhanced the phosphorylation of proteins involved in both AMP activated protein kinase (AMPK/acetyl-CoA carboxylase (ACC and phosphoinositide 3-kinase (PI3K/protein kinase B (Akt signaling pathways in C2C12 cells, indicating that the oligosaccharides activated both the insulin signal pathway and AMPK pathways as their mode of action. Moreover, oligosaccharides distributed to the mitochondria after internalization into C2C12 cells and increased the expression of transcriptional regulator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α, carnitine palmitoyl transferase-1 (CPT-1, and phosphorylated acetyl-CoA carboxylase (p-ACC, which suggested that the actions of these oligosaccharides might be associated with mitochondria through increasing energy expenditure. All of these effects of marine oligosaccharides were comparable to that of the established anti-diabetic drug, metformin. In addition, the treatment with oligosaccharides showed less toxicity than that of metformin. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that oligomannuonate and its chromium (III complexes improved

  20. Bone morphogenetic protein-2 functions as a negative regulator in the differentiation of myoblasts, but not as an inducer for the formations of cartilage and bone in mouse embryonic tongue

    Directory of Open Access Journals (Sweden)

    Suzuki Erika

    2011-07-01

    Full Text Available Abstract Background In vitro studies using the myogenic cell line C2C12 demonstrate that bone morphogenetic protein-2 (BMP-2 converts the developmental pathway of C2C12 from a myogenic cell lineage to an osteoblastic cell lineage. Further, in vivo studies using null mutation mice demonstrate that BMPs inhibit the specification of the developmental fate of myogenic progenitor cells. However, the roles of BMPs in the phases of differentiation and maturation in skeletal muscles have yet to be determined. The present study attempts to define the function of BMP-2 in the final stage of differentiation of mouse tongue myoblast. Results Recombinant BMP-2 inhibited the expressions of markers for the differentiation of skeletal muscle cells, such as myogenin, muscle creatine kinase (MCK, and fast myosin heavy chain (fMyHC, whereas BMP-2 siRNA stimulated such markers. Neither the recombinant BMP-2 nor BMP-2 siRNA altered the expressions of markers for the formation of cartilage and bone, such as osteocalcin, alkaline phosphatase (ALP, collagen II, and collagen X. Further, no formation of cartilage and bone was observed in the recombinant BMP-2-treated tongues based on Alizarin red and Alcian blue stainings. Neither recombinant BMP-2 nor BMP-2 siRNA affected the expression of inhibitor of DNA binding/differentiation 1 (Id1. The ratios of chondrogenic and osteogenic markers relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a house keeping gene were approximately 1000-fold lower than those of myogenic markers in the cultured tongue. Conclusions BMP-2 functions as a negative regulator for the final differentiation of tongue myoblasts, but not as an inducer for the formation of cartilage and bone in cultured tongue, probably because the genes related to myogenesis are in an activation mode, while the genes related to chondrogenesis and osteogenesis are in a silencing mode.

  1. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Fei; Kishida, Tsunao; Ejima, Akika [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Gojo, Satoshi [Department of Cardiac Support, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mazda, Osam, E-mail: mazda@koto.kpu-m.ac.jp [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan)

    2013-02-08

    Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases.

  2. Metabolic profiling of heat or anoxic stress in mouse C2C12 myotubes using multinuclear magnetic resonance spectroscopy

    DEFF Research Database (Denmark)

    Straadt, Ida K; Young, Jette F; Petersen, Bent O

    2010-01-01

    In the present study, the metabolic effects of heat and anoxic stress in myotubes from the mouse cell line C2C12 were investigated by using a combination of (13)C, (1)H, and (31)P nuclear magnetic resonance (NMR) spectroscopy and enrichment with [(13)C]-glucose. Both the (13)C and the (1)H NMR...... spectra showed reduced levels of the amino acids alanine, glutamate, and aspartate after heat or anoxic stress. The decreases were smallest at 42 degrees C, larger at 45 degrees C, and most pronounced after anoxic conditions. In addition, in both the (1)H and the (31)P NMR spectra, decreases in the high...

  3. Focal adhesion kinase activation is necessary for stretch-induced alignment and enhanced differentiation of myogenic precursor cells

    DEFF Research Database (Denmark)

    Andersen, Jens Isak; Pennisi, Cristian Pablo; Fink, Trine

    2017-01-01

    ), they display uniform alignment and an enhanced rate of differentiation. In this work, we explored the role of FAK activation by using C2C12 myoblasts that were grown on flexible culture plates and exposed to UCTS during the early differentiation phase. After 24 h, the cells oriented perpendicularly...

  4. Promotion of Glucose Uptake in C2C12 Myotubes by Cereal Flavone Tricin and Its Underlying Molecular Mechanism.

    Science.gov (United States)

    Kim, Sohyun; Go, Gwang-Woong; Imm, Jee-Young

    2017-05-17

    The effect of tricin, a methylated flavone widely distributed in cereals, on glucose uptake and the underlying molecular mechanism was investigated using C2C12 myotubes. Tricin significantly increased glucose uptake in C2C12 myotubes, regardless of the absence (1.4-fold at 20 μM) or presence (1.6-fold at 20 μM) of insulin. The GLUT4 expression on the plasma membrane was increased 1.6-fold after tricin treatment (20 μM) in the absence of insulin. Tricin treatment significantly activated the insulin-dependent cell signaling pathway, including the activation of insulin receptor substrate-1 (IRS1), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and AKT substrate of 160 kDa (AS160). The oral administration of tricin (64 and 160 mg kg -1 of body weight day -1 ) also significantly lowered blood glucose levels in glucose-loaded C57BL/6 mice (p < 0.05). These results suggest that tricin has great potential to be used as a functional agent for glycemic control.

  5. Cell-Adhesive Matrices Composed of RGD Peptide-Displaying M13 Bacteriophage/Poly(lactic-co-glycolic acid) Nanofibers Beneficial to Myoblast Differentiation.

    Science.gov (United States)

    Shin, Yong Cheol; Lee, Jong Ho; Jin, Linhua; Kim, Min Jeong; Kim, Chuntae; Hong, Suck Won; Oh, Jin Woo; Han, Dong-Wook

    2015-10-01

    Recently, there has been considerable effort to develop suitable scaffolds for tissue engineering applications. Cell adhesion is a prerequisite for cells to survive. In nature, the extracellular matrix (ECM) plays this role. Therefore, an ideal scaffold should be structurally similar to the natural ECM and have biocompatibility and biodegradability. In addition, the scaffold should have biofunctionality, which provides the potent ability to enhance the cellular behaviors, such as adhesion, proliferation and differentiation. This study concentrates on fabricating cell-adhesive matrices composed of RGD peptide-displaying M13 bacteriophage (RGD-M13 phage) and poly(lactic-co-glycolic acid, PLGA) nanofibers. Long rod-shaped M13 bacteriophages are non-toxic and can express many desired proteins on their surface. A genetically engineered M13 phage was constructed to display RGD peptides on its surface. PLGA is a biodegradable polymer with excellent biocompatibility and suitable physicochemical property for adhesive matrices. In this study, RGD-M13 phage/PLGA hybrid nanofiber matrices were fabricated by electrospinning. The physicochemical properties of these matrices were characterized by scanning electron microscopy, atomic force microscopy, Raman spectroscopy, and contact angle measurement. In addition, the cellular behaviors, such as the initial attachment, proliferation and differentiation, were analyzed by a CCK-8 assay and immunofluorescence staining to evaluate the potential application of these matrices to tissue engineering scaffolds. The RGD-M13 phage/PLGA nanofiber matrices could enhance the cellular behaviors and promote the differentiation of C2C12 myoblasts. These results suggest that the RGD-M13 phage/PLGA nanofiber matrices are beneficial to myoblast differentiation and can serve as effective tissue engineering scaffolds.

  6. FSHD myoblasts fail to downregulate intermediate filament protein vimentin during myogenic differentiation.

    Directory of Open Access Journals (Sweden)

    Lipinski M.

    2011-10-01

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is an autosomal dominant hereditary neuromuscular disorder. The clinical features of FSHD include weakness of the facial and shoulder girdle muscles followed by wasting of skeletal muscles of the pelvic girdle and lower extremities. Although FSHD myoblasts grown in vitro can be induced to differentiate into myotubes by serum starvation, the resulting FSHD myotubes have been shown previously to be morphologically abnormal. Aim. In order to find the cause of morphological anomalies of FSHD myotubes we compared in vitro myogenic differentiation of normal and FSHD myoblasts at the protein level. Methods. We induced myogenic differentiation of normal and FSHD myoblasts by serum starvation. We then compared protein extracts from proliferating myoblasts and differentiated myotubes using SDS-PAGE followed by mass spectrometry identification of differentially expressed proteins. Results. We demonstrated that the expression of vimentin was elevated at the protein and mRNA levels in FSHD myotubes as compared to normal myotubes. Conclusions. We demonstrate for the first time that in contrast to normal myoblasts, FSHD myoblasts fail to downregulate vimentin after induction of in vitro myogenic differentiation. We suggest that vimentin could be an easily detectable marker of FSHD myotubes

  7. Dehydroepiandrosterone activates AMP kinase and regulates GLUT4 and PGC-1α expression in C2C12 myotubes

    Energy Technology Data Exchange (ETDEWEB)

    Yokokawa, Takumi [Laboratory of Sports and Exercise Medicine, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto (Japan); Sato, Koji [Graduate School of Sport & Health Science, Ritsumeikan University, Shiga (Japan); Iwanaka, Nobumasa [The Graduate School of Science and Engineering, Ritsumeikan University, Shiga (Japan); Honda, Hiroki [Graduate School of Sport & Health Science, Ritsumeikan University, Shiga (Japan); Higashida, Kazuhiko [Faculty of Sport Science, Waseda University, Saitama (Japan); Iemitsu, Motoyuki [Graduate School of Sport & Health Science, Ritsumeikan University, Shiga (Japan); Hayashi, Tatsuya [Laboratory of Sports and Exercise Medicine, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto (Japan); Hashimoto, Takeshi, E-mail: thashimo@fc.ritsumei.ac.jp [Graduate School of Sport & Health Science, Ritsumeikan University, Shiga (Japan)

    2015-07-17

    Exercise and caloric restriction (CR) have been reported to have anti-ageing, anti-obesity, and health-promoting effects. Both interventions increase the level of dehydroepiandrosterone (DHEA) in muscle and blood, suggesting that DHEA might partially mediate these effects. In addition, it is thought that either 5′-adenosine monophosphate-activated protein kinase (AMPK) or peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mediates the beneficial effects of exercise and CR. However, the effects of DHEA on AMPK activity and PGC-1α expression remain unclear. Therefore, we explored whether DHEA in myotubes acts as an activator of AMPK and increases PGC-1α. DHEA exposure increased glucose uptake but not the phosphorylation levels of Akt and PKCζ/λ in C2C12 myotubes. In contrast, the phosphorylation levels of AMPK were elevated by DHEA exposure. Finally, we found that DHEA induced the expression of the genes PGC-1α and GLUT4. Our current results might reveal a previously unrecognized physiological role of DHEA; the activation of AMPK and the induction of PGC-1α by DHEA might mediate its anti-obesity and health-promoting effects in living organisms. - Highlights: • We assessed whether dehydroepiandrosterone (DHEA) activates AMPK and PGC-1α. • DHEA exposure increased glucose uptake in C2C12 myotubes. • The phosphorylation levels of AMPK were elevated by DHEA exposure. • DHEA induced the expression of the genes PGC-1α and GLUT4. • AMPK might mediate the anti-obesity and health-promoting effects of DHEA.

  8. New gene targets of PGC-1α and ERRα co-regulation in C2C12 myotubes.

    Science.gov (United States)

    Nsiah-Sefaa, Abena; Brown, Erin L; Russell, Aaron P; Foletta, Victoria C

    2014-12-01

    As a transcriptional coactivator, PGC-1α contributes to the regulation of a broad range of metabolic processes in skeletal muscle health and disease; however, there is limited information about the genes it transcriptionally regulates. To identify new potential gene targets of PGC-1α regulation, mouse C2C12 myotubes were screened by microarray analysis following PGC-1α overexpression. Genes with an mRNA expression of 2.5-fold or more (P genes were singled out if they had no previous connection to PGC-1α regulation or characterization in skeletal muscle, or were unannotated with no known function. Following confirmation of their regulation by PGC-1α using qPCR analysis, eight genes were focused on for further investigation (Akr1b10, Rmnd1, 1110008P14Rik, 1700021F05Rik, Mtfp1, Mrm1, Oxnad1 and Cluh). Bioinformatics indicated a number of the genes were linked to a range of metabolic-related functions including fatty acid oxidation, oxido-reductase activity, and mitochondrial remodeling and transport. Treating C2C12 myotubes for 6 h with AICAR, a known activator of AMP kinase and inducer of Pgc-1α gene expression, increased the mRNA levels of both Pgc-1α (P genes to contain either a consensus or near consensus response elements for the estrogen-related receptor α (ERRα), a key transcription factor-binding partner of PGC-1α in skeletal muscle. Furthermore, knockdown of endogenous ERRα levels partially or completely blocked the induction of gene expression of all genes by PGC-1α, while each gene was significantly upregulated in the presence of a constitutively active form of ERRα (P regulation of these genes by PGC-1α and its signaling pathway in skeletal muscle.

  9. Pyropia yezoensis peptide PYP1‑5 protects against dexamethasone‑induced muscle atrophy through the downregulation of atrogin1/MAFbx and MuRF1 in mouse C2C12 myotubes.

    Science.gov (United States)

    Lee, Min-Kyeong; Kim, Young-Min; Kim, In-Hye; Choi, Youn-Hee; Nam, Taek-Jeong

    2017-06-01

    Skeletal muscle atrophy refers to the decline in muscle mass and strength that occurs under various conditions, including aging, starvation, cancer and other cachectic diseases. Muscle atrophy caused by aging, known as sarcopenia, primarily occurs after 50 years of age. Muscle atrophy‑related genes, including atrogin1/muscle atrophy F‑box (MAFbx) and muscle RING finger 1 (MuRF1), are expressed early in the muscle atrophy process, and their expression precedes the loss of muscle mass. The present study investigated the potential anti‑atrophic effects of the Pyropia yezoensis peptide PYP1‑5. The MTS assay did not detect cytotoxic effects of PYP1‑5 on C2C12 mouse myoblast cells. Subsequently, the anti‑atrophic effects of PYP1‑5 on skeletal muscle cells was examined by treating C2C12 myotubes with 100 µM dexamethasone (DEX) and/or 500 ng/ml PYP1‑5 for 24 h. Compared with the control, myotube diameter was reduced in DEX‑treated cells, whereas PYP1‑5 treatment protected against DEX‑induced muscle atrophy. MAFbx and MuRF1 protein and mRNA expression levels were detected by western blot analysis and reverse transcription‑quantitative polymerase chain reaction, respectively. The results demonstrated that PYP1‑5 significantly reduced the expression of atrogin1/MAFbx and MuRF1. Therefore, data from the present study suggest that PYP1‑5 inhibits the expression of atrogin1/MAFbx and MuRF1 in C2C12 cells, and these characteristics may be of value in the development of anti‑atrophy functional foods.

  10. Pyropia yezoensis peptide PYP1-5 protects against dexamethasone-induced muscle atrophy through the downregulation of atrogin1/MAFbx and MuRF1 in mouse C2C12 myotubes

    Science.gov (United States)

    Lee, Min-Kyeong; Kim, Young-Min; Kim, In-Hye; Choi, Youn-Hee; Nam, Taek-Jeong

    2017-01-01

    Skeletal muscle atrophy refers to the decline in muscle mass and strength that occurs under various conditions, including aging, starvation, cancer and other cachectic diseases. Muscle atrophy caused by aging, known as sarcopenia, primarily occurs after 50 years of age. Muscle atrophy-related genes, including atrogin1/muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1), are expressed early in the muscle atrophy process, and their expression precedes the loss of muscle mass. The present study investigated the potential anti-atrophic effects of the Pyropia yezoensis peptide PYP1-5. The MTS assay did not detect cytotoxic effects of PYP1-5 on C2C12 mouse myoblast cells. Subsequently, the anti-atrophic effects of PYP1-5 on skeletal muscle cells was examined by treating C2C12 myotubes with 100 µM dexamethasone (DEX) and/or 500 ng/ml PYP1-5 for 24 h. Compared with the control, myotube diameter was reduced in DEX-treated cells, whereas PYP1-5 treatment protected against DEX-induced muscle atrophy. MAFbx and MuRF1 protein and mRNA expression levels were detected by western blot analysis and reverse transcription-quantitative polymerase chain reaction, respectively. The results demonstrated that PYP1-5 significantly reduced the expression of atrogin1/MAFbx and MuRF1. Therefore, data from the present study suggest that PYP1-5 inhibits the expression of atrogin1/MAFbx and MuRF1 in C2C12 cells, and these characteristics may be of value in the development of anti-atrophy functional foods. PMID:28393223

  11. Isolation of human foetal myoblasts and its application for microencapsulation.

    Science.gov (United States)

    Li, Anna Aihua; Bourgeois, Jacqueline; Potter, Murray; Chang, Patricia L

    2008-01-01

    Foetal cells secrete more growth factors, generate less immune response, grow and proliferate better than adult cells. These characteristics make them desirable for recombinant modification and use in microencapsulated cellular gene therapeutics. We have established a system in vitro to obtain a pure population of primary human foetal myoblasts under several rounds of selection with non-collagen coated plates and identified by desmin staining. These primary myoblasts presented good proliferation ability and better differentiation characteristics in monolayer and after microencapsulation compared to murine myoblast C2C12 cells based on creatine phosphokinase (CPK), major histocompatibility complex (MHC) and multi-nucleated myotubule determination. The lifespan of primary myoblasts was 70 population doublings before entering into senescent state, with a population time of 18-24 hrs. Hence, we have developed a protocol for isolating human foetal primary myoblasts with excellent differentiation potential and robust growth and longevity. They should be useful for cell-based therapy in human clinical applications with microencapsulation technology.

  12. Isolation of human foetal myoblasts and its application for microencapsulation

    Science.gov (United States)

    Li, Anna Aihua; Bourgeois, Jacqueline; Potter, Murray; Chang, Patricia L

    2008-01-01

    Abstract Foetal cells secrete more growth factors, generate less immune response, grow and proliferate better than adult cells. These characteristics make them desirable for recombinant modification and use in microencapsulated cellular gene therapeutics. We have established a system in vitro to obtain a pure population of primary human foetal myoblasts under several rounds of selection with non-collagen coated plates and identified by desmin staining. These primary myoblasts presented good proliferation ability and better differentiation characteristics in monolayer and after microencapsulation compared to murine myoblast C2C12 cells based on creatine phosphokinase (CPK), major histocompatibility complex (MHC) and multi-nucleated myotubule determination. The lifespan of primary myoblasts was 70 population doublings before entering into senescent state, with a population time of 18–24 hrs. Hence, we have developed a protocol for isolating human foetal primary myoblasts with excellent differentiation potential and robust growth and longevity. They should be useful for cell-based therapy in human clinical applications with microencapsulation technology. PMID:18366454

  13. Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75NTR and α9β1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model.

    Science.gov (United States)

    Ettinger, Keren; Lecht, Shimon; Arien-Zakay, Hadar; Cohen, Gadi; Aga-Mizrachi, Shlomit; Yanay, Nurit; Saragovi, H Uri; Nedev, Hinyu; Marcinkiewicz, Cezary; Nevo, Yoram; Lazarovici, Philip

    2012-12-01

    The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75(NTR), α9β1 integrin, and its regulator ADAM12, but not trkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and βNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9β1 integrin; and (c) p75(NTR) antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4 days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60-70%, 85-90% and 60-80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of βNGF, added either 1h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and βNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75(NTR) and α9β1 integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9β1 in NGF-induced signaling in skeletal muscle model, resembling the role of trkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets. Copyright © 2012 Elsevier Inc. All

  14. Isolation, Culturing, and Differentiation of Primary Myoblasts from Skeletal Muscle of Adult Mice.

    Science.gov (United States)

    Hindi, Lubna; McMillan, Joseph D; Afroze, Dil; Hindi, Sajedah M; Kumar, Ashok

    2017-05-05

    Myogenesis is a multi-step process that leads to the formation of skeletal muscle during embryonic development and repair of injured myofibers. In this process, myoblasts are the main effector cell type which fuse with each other or to injured myofibers leading to the formation of new myofibers or regeneration of skeletal muscle in adults. Many steps of myogenesis can be recapitulated through in vitro differentiation of myoblasts into myotubes. Most laboratories use immortalized myogenic cells lines that also differentiate into myotubes. Although these cell lines have been found quite useful to delineating the regulatory mechanisms of myogenesis, they often show a great degree of variability depending on the origin of the cells and culture conditions. Primary myoblasts have been suggested as the most physiologically relevant model for studying myogenesis in vitro. However, due to their low abundance in adult skeletal muscle, isolation of primary myoblasts is technically challenging. In this article, we describe an improved protocol for the isolation of primary myoblasts from adult skeletal muscle of mice. We also describe methods for their culturing and differentiation into myotubes.

  15. APPL1 promotes glucose uptake in response to mechanical stretch via the PKCζ-non-muscle myosin IIa pathway in C2C12 myotubes.

    Science.gov (United States)

    Saito, Tsugumichi; Okada, Shuichi; Shimoda, Yoko; Tagaya, Yuko; Osaki, Aya; Yamada, Eijiro; Shibusawa, Ryo; Nakajima, Yasuyo; Ozawa, Atsushi; Satoh, Tetsurou; Mori, Masatomo; Yamada, Masanobu

    2016-11-01

    Expression of adaptor protein, phosphotyrosine interaction, pleckstrin homology domain, and leucine zipper containing 1 (APPL1) promoted glucose transporter 4 (GLUT4) translocation and glucose uptake in adipose and muscle tissues in response to stimulation with insulin, adiponectin, or exercise. In response to mechanical stretch, knockdown of APPL1 in C2C12 myotubes suppressed glucose uptake. APPL1-induced increased glucose uptake was mediated by protein kinase C (PKC) ζ but not AKT, AMPK, or calmodulin-dependent protein kinase. In myotubes overexpressing APPL1, PKCζ was phosphorylated and translocated to the plasma membrane (PM) in response to mechanical stretch. Phosphorylated PKCζ co-immunoprecipitated with protein phosphatase 2A (PP2A) under basal conditions, but dissociated upon myotube stretching. Moreover, stretch-induced phosphorylated PKCζ co-immunoprecipitated with non-muscle myosin IIa. Blebbistatin, an inhibitor of myosin II ATPase activity, suppressed APPL1-mediated stretch-induced glucose uptake and PKCζ translocation. Taken together these data demonstrate that in response to mechanical stretch, APPL1 enhances glucose uptake by modulating the activation and localization of PKCζ, as well as its functional interaction with both PP2A and myosin IIa. These findings support a new function for non-muscle myosin IIa in differentiated myotubes. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Novel lncRNAs in myogenesis: a miR-31 overlapping transcript controls myoblast differentiation.

    KAUST Repository

    Ballarino, Monica

    2014-12-15

    Transcriptome analysis allowed the identification of new long noncoding RNAs differentially expressed during murine myoblast differentiation. These transcripts were classified on the basis of their expression under proliferating versus differentiated conditions, muscle-restricted activation, and subcellular localization. Several species displayed preferential expression in dystrophic (mdx) versus wild-type muscles, indicating their possible link with regenerative processes. One of the identified transcripts, lnc-31, even if originating from the same nuclear precursor of miR-31, is produced by a pathway mutually exclusive. We show that lnc-31 and its human homologue hsa-lnc-31 are expressed in proliferating myoblasts, where they counteract differentiation. In line with this, both species are more abundant in mdx muscles and in human Duchenne muscular dystrophy (DMD) myoblasts, than in their normal counterparts. Altogether, these data suggest a crucial role for lnc-31 in controlling the differentiation commitment of precursor myoblasts and indicate that its function is maintained in evolution despite the poor sequence conservation with the human counterpart.

  17. Stimulating effect of graphene oxide on myogenesis of C2C12 myoblasts on RGD peptide-decorated PLGA nanofiber matrices

    National Research Council Canada - National Science Library

    Shin, Yong Cheol; Lee, Jong Ho; Kim, Min Jeong; Hong, Suck Won; Kim, Bongju; Hyun, Jung Keun; Choi, Yu Suk; Park, Jong-Chul; Han, Dong-Wook

    2015-01-01

    .... However, their combination is intriguing and still challenging. In the present study, we fabricated nanofiber matrices composed of M13 bacteriophage with RGD peptide displayed on its surface (RGD-M13 phage) and poly...

  18. Black ginseng activates Akt signaling, thereby enhancing myoblast differentiation and myotube growth

    Directory of Open Access Journals (Sweden)

    Soo-Yeon Lee

    2018-01-01

    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.

  19. Oxygen Modulates the Glutathione Peroxidase Activity during the L6 Myoblast Early Differentiation Process

    Directory of Open Access Journals (Sweden)

    Magdalena Hidalgo

    2014-01-01

    Full Text Available Aim: This work aims to study the regulation of the glutathione peroxidase and catalase activities in myoblasts from the L6 line exposed to 21%, 5% and 1% O2 during the cell differentiation. Material and Methods: Rat L6 myoblasts were grown in 1%, 5% or 21% O2 in the presence or absence of N-acetyl cysteine. The cell proliferation was evaluated by determining the doubling time and kinetics of cultures by counting cells. The cell differentiation was analyzed by determining the myogenic fusion index using antibodies against the myosin heavy chain. The glutathione peroxidase and catalase activities were assayed. The p110-PI3K/Thr308-Akt pathway was studied using western blotting. The oxidative status of the cells was carried out by determining TBARS. Results: 5% O2 improves the glutathione peroxidase activity, p110-PI3K/Thr308-Akt pathway and differentiation while 1% O2 alters all these parameters compared to 21% O2. NAC (0.5 mM can prevent the deleterious effects of hypoxia (1% O2 on the L6 myoblast proliferation and enhances the myoblast differentiation when exposed to 21% O2. TBARS are reduced in 5% O2 compared to both 21% and 1% O2. Conclusion: The glutathione peroxidase activity and p110-PI3K/Thr308-Akt are both modulated in the same way by oxygen.

  20. Characterization of heterokaryons between skeletal myoblasts and somatic cells formed by fusion with HVJ (Sendai virus); effects on myogenic differentiation.

    Science.gov (United States)

    Hirayama, E; Udaka, Y; Kawai, T; Kim, J

    2001-02-01

    In skeletal myogenic differentiation, myoblasts fuse with myogenic cells spontaneously, but do not fuse with non-myogenic cells either in vivo or in vitro, suggesting that the fusion of myoblasts with non-myogenic cells is unsuitable for differentiation. To understand the inevitability of the fusion among myoblasts, we prepared heterokaryons in crosses between quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and rodent non-myogenic cells, such as tumor cells, fibroblasts, or neurogenic cells by HVJ (Sendai virus) and examined how myogenic differentiation was influenced in the prepared heterokaryons, focusing on myogenin expression and myofibril formation as markers of differentiation. When presumptive QM-RSV cells were fused with non-myogenic cells by HVJ and induced to differentiate, both myogenin expression and myofibril formation were suppressed. When myotubes of QM-RSV cells that had already expressed myogenin and formed myofibrils were fused with non-myogenic cells, both myogenin and myofibrils disappeared. Especially, fibrous structures of myofibrils were significantly lost and dots or aggregations of F-actin were formed within 24 hr after formation of heterokaryons. However, the fusion of presumptive or differentiated QM-RSV cells with rodent myoblasts did not disturb myogenin expression or myofibril formation. These results suggest that mutual fusion of myoblasts is indispensable for normal myogenic differentiation irrespective of the species, and that some factors inhibiting myogenic differentiation exist in the cytoplasm of non-myogenic cells, but not in myoblasts.

  1. Effect of alkyl glycerophosphate on the activation of peroxisome proliferator-activated receptor gamma and glucose uptake in C2C12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Tsukahara, Tamotsu, E-mail: ttamotsu@shinshu-u.ac.jp [Department of Integrative Physiology and Bio-System Control, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Haniu, Hisao [Department of Orthopedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Matsuda, Yoshikazu [Clinical Pharmacology Educational Center, Nihon Pharmaceutical University, Ina-machi, Saitama 362-0806 (Japan)

    2013-04-12

    Highlights: •Alkyl-LPA specifically interacts with PPARγ. •Alkyl-LPA treatments induces lipid accumulation in C2C12 cells. •Alkyl-LPA enhanced glucose uptake in C2C12 cells. •Alkyl-LPA-treated C2C12 cells express increased amounts of GLUT4 mRNA. •Alkyl-LPA is a novel therapeutic agent that can be used for the treatment of obesity and diabetes. -- Abstract: Studies on the effects of lipids on skeletal muscle cells rarely examine the effects of lysophospholipids. Through our recent studies, we identified select forms of phospholipids, such as alkyl-LPA, as ligands for the intracellular receptor peroxisome proliferator-activated receptor gamma (PPARγ). PPARγ is a nuclear hormone receptor implicated in many human diseases, including diabetes and obesity. We previously showed that alkyl-LPA is a specific agonist of PPARγ. However, the mechanism by which the alkyl-LPA–PPARγ axis affects skeletal muscle cells is poorly defined. Our objective in the present study was to determine whether alkyl-LPA and PPARγ activation promotes glucose uptake in skeletal muscle cells. Our findings indicate that PPARγ1 mRNA is more abundant than PPARγ2 mRNA in C2C12 cells. We showed that alkyl-LPA (3 μM) significantly activated PPARγ and increased intracellular glucose levels in skeletal muscle cells. We also showed that incubation of C2C12 cells with alkyl-LPA led to lipid accumulation in the cells. These findings suggest that alkyl-LPA activates PPARγ and stimulates glucose uptake in the absence of insulin in C2C12 cells. This may contribute to the plasma glucose-lowering effect in the treatment of insulin resistance.

  2. DHA Inhibits Protein Degradation More Efficiently than EPA by Regulating the PPARγ/NFκB Pathway in C2C12 Myotubes

    Directory of Open Access Journals (Sweden)

    Yue Wang

    2013-01-01

    Full Text Available This study was conducted to evaluate the mechanism by which n-3 PUFA regulated the protein degradation in C2C12 myotubes. Compared with the BSA control, EPA at concentrations from 400 to 600 µM decreased total protein degradation (P0.05. Interestingly, EPA and DHA both still decreased the total protein degradation, although PPARγ knockdown attenuated the suppressive effects of EPA and DHA on the total protein degradation (P<0.01. These results revealed that DHA inhibits protein degradation more efficiently than EPA by regulating the PPARγ/NF-κB pathway in C2C12 myotubes.

  3. Selective inhibition of cathepsin B with cell-permeable CA074Me negatively affects L6 rat myoblast differentiation.

    Science.gov (United States)

    Jane, Derek T; Morvay, Leslie C; Allen, Francis; Sloane, Bonnie F; Dufresne, Michael J

    2002-01-01

    Active cathepsin B, in concert with other cellular proteases, has been implicated in the catabolic restructuring associated with myotube formation during skeletal myoblast cell differentiation (i.e., myogenesis). We have examined this role in differentiating myoblasts using the cell-permeable, cathepsin B selective inhibitor CA074Me. Cathepsin B activity levels in differentiating L6 rat myoblasts treated with CA074Me were significantly lower than levels in control myoblasts. Inhibition of cathepsin B activity by CA074Me occurred at each stage of differentiation and was dose related. Myotube size and number and induced levels of fusion-related creatine phosphokinase activity and myosin heavy-chain protein were reduced from 30 to 50% in CA074Me-treated myoblasts. These reductions were also dose related. In contrast, CA074Me did not affect levels of myogenin, an early marker of myogenesis, or levels of cathepsin L type and myokinase activities, two nonspecific enzymes. The negative effects associated with CA074Me were reversed when the drug was removed. Collectively, these data suggest that active cathepsin B plays a role in myoblast-myoblast fusion and consequently may be necessary for the complete expression of those genes associated with the fusion process.

  4. Heterogeneous activation of a slow myosin gene in proliferating myoblasts and differentiated single myofibers.

    Science.gov (United States)

    Wang, Jing-Hua; Wang, Qiao-Jing; Wang, Chao; Reinholt, Brad; Grant, Alan L; Gerrard, David E; Kuang, Shihuan

    2015-06-01

    Each skeletal muscle contains a fixed ratio of fast and slow myofibers that are distributed in a stereotyped pattern to achieve a specific motor function. How myofibers are specified during development and regeneration is poorly understood. Here we address this question using transgenic reporter mice that indelibly mark the myofiber lineages based on activation of fast or slow myosin. Lineage tracing indicates that during development all muscles have activated the fast myosin gene Myl1, but not the slow myosin gene Myh7, which is activated in all slow but a subset of fast myofibers. Similarly, most nascent myofibers do not activate Myh7 during fast muscle regeneration, but the ratio and pattern of fast and slow myofibers are restored at the completion of regeneration. At the single myofiber level, most mature fast myofibers are heterogeneous in nuclear composition, manifested by mosaic activation of Myh7. Strikingly, Myh7 is activated in a subpopulation of proliferating myoblasts that co-express the myogenic progenitor marker Pax7. When induced to differentiate, the Myh7-activated myoblasts differentiate more readily than the non-activated myoblasts, and have a higher tendency, but not restricted, to become slow myotubes. Together, our data reveal significant nuclear heterogeneity within a single myofiber, and challenge the conventional view that myosin genes are only expressed after myogenic differentiation. These results provide novel insights into the regulation of muscle fiber type specification. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Amino acids and insulin act additively to regulate components of the ubiquitin-proteasome pathway in C2C12 myotubes

    Directory of Open Access Journals (Sweden)

    Lomax Michael A

    2007-03-01

    Full Text Available Abstract Background The ubiquitin-proteasome system is the predominant pathway for myofibrillar proteolysis but a previous study in C2C12 myotubes only observed alterations in lysosome-dependent proteolysis in response to complete starvation of amino acids or leucine from the media. Here, we determined the interaction between insulin and amino acids in the regulation of myotube proteolysis Results Incubation of C2C12 myotubes with 0.2 × physiological amino acids concentration (0.2 × PC AA, relative to 1.0 × PC AA, significantly increased total proteolysis and the expression of 14-kDa E2 ubiquitin conjugating enzyme (p Conclusion In a C2C12 myotube model of myofibrillar protein turnover, amino acid limitation increases proteolysis in a ubiquitin-proteasome-dependent manner. Increasing amino acids or leucine alone, act additively with insulin to down regulate proteolysis and expression of components of ubiquitin-proteasome pathway. The effects of amino acids on proteolysis but not insulin and leucine, are blocked by inhibition of the mTOR signalling pathway.

  6. Plasma membrane characterization, by scanning electron microscopy, of multipotent myoblasts-derived populations sorted using dielectrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Muratore, Massimo, E-mail: M.Muratore@ed.ac.uk [Institute of Integrated Micro and Nano System, School of Engineering, The University of Edinburgh, Edinburgh EH9 3JF (United Kingdom); Mitchell, Steve [Institute of Molecular Plant Science, School of Biological Science, The University of Edinburgh, Edinburgh EH9 3JF (United Kingdom); Waterfall, Martin [Institute of Immunology and Infection Research, School of Biological Science, The University of Edinburgh, Edinburgh EH9 3JT (United Kingdom)

    2013-09-06

    Highlights: •Dielectrophoretic separation/sorting of multipotent cells. •Plasma membrane microvilli structure of C2C12 and fibroblasts by SEM microscopy. •Cell cycle determination by Ki-67 in DEP-sorted cells. •Plasma membrane differences responsible for changes in membrane capacitance. -- Abstract: Multipotent progenitor cells have shown promise for use in biomedical applications and regenerative medicine. The implementation of such cells for clinical application requires a synchronized, phenotypically and/or genotypically, homogenous cell population. Here we have demonstrated the implementation of a biological tag-free dielectrophoretic device used for discrimination of multipotent myoblastic C2C12 model. The multipotent capabilities in differentiation, for these cells, diminishes with higher passage number, so for cultures above 70 passages only a small percentage of cells is able to differentiate into terminal myotubes. In this work we demonstrated that we could recover, above 96% purity, specific cell types from a mixed population of cells at high passage number without any biological tag using dielectrophoresis. The purity of the samples was confirmed by cytometric analysis using the cell specific marker embryonic myosin. To further investigate the dielectric properties of the cell plasma membrane we co-culture C2C12 with similar size, when in suspension, GFP-positive fibroblast as feeder layer. The level of separation between the cell types was above 98% purity which was confirmed by flow cytometry. These levels of separation are assumed to account for cell size and for the plasma membrane morphological differences between C2C12 and fibroblast unrelated to the stages of the cell cycle which was assessed by immunofluorescence staining. Plasma membrane conformational differences were further confirmed by scanning electron microscopy.

  7. α-Syntrophin stabilizes catalase to reduce endogenous reactive oxygen species levels during myoblast differentiation.

    Science.gov (United States)

    Moon, Jae Yun; Choi, Su Jin; Heo, Cheol Ho; Kim, Hwan Myung; Kim, Hye Sun

    2017-07-01

    α-Syntrophin is a component of the dystrophin-glycoprotein complex that interacts with various intracellular signaling proteins in muscle cells. The α-syntrophin knock-down C2 cell line (SNKD), established by infecting lentivirus particles with α-syntrophin shRNA, is characterized by a defect in terminal differentiation and increase in cell death. Since myoblast differentiation is accompanied by intensive mitochondrial biogenesis, the generation of intracellular reactive oxygen species (ROS) is also increased during myogenesis. Two-photon microscopy imaging showed that excessive intracellular ROS accumulated during the differentiation of SNKD cells as compared with control cells. The formation of 4-hydroxynonenal adduct, a byproduct of lipid peroxidation during oxidative stress, significantly increased in differentiated SNKD myotubes and was dramatically reduced by epigallocatechin-3-gallate, a well-known ROS scavenger. Among antioxidant enzymes, catalase was significantly decreased during differentiation of SNKD cells without changes at the mRNA level. Of interest was the finding that the degradation of catalase was rescued by MG132, a proteasome inhibitor, in the SNKD cells. This study demonstrates a novel function of α-syntrophin. This protein plays an important role in the regulation of oxidative stress from endogenously generated ROS during myoblast differentiation by modulating the protein stability of catalase. © 2017 Federation of European Biochemical Societies.

  8. Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts

    DEFF Research Database (Denmark)

    Le Bihan, Marie-Catherine; Barrio, Inigo; Mortensen, Tenna Pavia

    2015-01-01

    Muscle stem cells, or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although they have been extensively studied, the key regulatory steps and the ......Muscle stem cells, or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although they have been extensively studied, the key regulatory steps...... and the complex temporal protein dynamics accompanying the differentiation of primary human muscle cells remain poorly understood. Here, we demonstrate the advantages of applying a MS-based quantitative approach, stable isotope labeling by amino acids in cell culture (SILAC), for studying human myogenesis...... in vitro and characterize the fine-tuned changes in protein expression underlying the dramatic phenotypic conversion of primary mononucleated human muscle cells during in vitro differentiation to form multinucleated myotubes. Using an exclusively optimized triple encoding SILAC procedure, we generated...

  9. Nilotinib impairs skeletal myogenesis by increasing myoblast proliferation

    Directory of Open Access Journals (Sweden)

    Osvaldo Contreras

    2018-02-01

    Full Text Available Abstract Background Tyrosine kinase inhibitors (TKIs are effective therapies with demonstrated antineoplastic activity. Nilotinib is a second-generation FDA-approved TKI designed to overcome Imatinib resistance and intolerance in patients with chronic myelogenous leukemia (CML. Interestingly, TKIs have also been shown to be an efficient treatment for several non-malignant disorders such fibrotic diseases, including those affecting skeletal muscles. Methods We investigated the role of Nilotinib on skeletal myogenesis using the well-established C2C12 myoblast cell line. We evaluated the impact of Nilotinib during the time course of skeletal myogenesis. We compared the effect of Nilotinib with the well-known p38 MAPK inhibitor SB203580. MEK1/2 UO126 and PI3K/AKT LY294002 inhibitors were used to identify the signaling pathways involved in Nilotinib-related effects on myoblast. Adult primary myoblasts were also used to corroborate the inhibition of myoblasts fusion and myotube-nuclei positioning by Nilotinib. Results We found that Nilotinib inhibited myogenic differentiation, reducing the number of myogenin-positive myoblasts and decreasing myogenin and MyoD expression. Furthermore, Nilotinib-mediated anti-myogenic effects impair myotube formation, myosin heavy chain expression, and compromise myotube-nuclei positioning. In addition, we found that p38 MAPK is a new off-target protein of Nilotinib, which causes inhibition of p38 phosphorylation in a similar manner as the well-characterized p38 inhibitor SB203580. Nilotinib induces the activation of ERK1/2 and AKT on myoblasts but not in myotubes. We also found that Nilotinib stimulates myoblast proliferation, a process dependent on ERK1/2 and AKT activation. Conclusions Our findings suggest that Nilotinib may have important negative effects on muscle homeostasis, inhibiting myogenic differentiation but stimulating myoblasts proliferation. Additionally, we found that Nilotinib stimulates the activation

  10. Carbon-based hierarchical scaffolds for myoblast differentiation: Synergy between nano-functionalization and alignment.

    Science.gov (United States)

    Patel, Akhil; Mukundan, Shilpaa; Wang, Wenhu; Karumuri, Anil; Sant, Vinayak; Mukhopadhyay, Sharmila M; Sant, Shilpa

    2016-03-01

    While several scaffolds have been proposed for skeletal muscle regeneration, multiscale hierarchical scaffolds with the complexity of extracellular matrix (ECM) haven't been engineered successfully. By precise control over nano- and microscale features, comprehensive understanding of the effect of multiple factors on skeletal muscle regeneration can be derived. In this study, we engineered carbon-based scaffolds with hierarchical nano- and microscale architecture with controlled physico-chemical properties. More specifically, we built multiscale hierarchy by growing carbon nanotube (CNT) carpets on two types of scaffolds, namely, interconnected microporous carbon foams and aligned carbon fiber mats. Nanostructured CNT carpets offered fine control over nano-roughness and wettability facilitating myoblast adhesion, growth and differentiation into myocytes. However, microporous foam architecture failed to promote their fusion into multinucleated myotubes. On the other hand, aligned fibrous architecture stimulated formation of multinucleated myotubes. Most importantly, nanostructured CNT carpets interfaced with microscale aligned fibrous architecture significantly enhanced myocyte fusion into multinucleated mature myotubes highlighting synergy between nanoscale surface features and micro-/macroscale aligned fibrous architecture in the process of myogenesis. Due to limited regenerative potential of skeletal muscle, strategies stimulating regeneration of functional muscles are important. These strategies are aimed at promoting differentiation of progenitor cells (myoblasts) into multinucleated myotubes, a key initial step in functional muscle regeneration. Recent tissue engineering approaches utilize various scaffolds ranging from decellularized matrices to aligned biomaterial scaffolds. Although, majority of them have focused on nano- or microscale organization, a systematic approach to build the multiscale hierarchy into these scaffolds is lacking. Here, we engineered

  11. Oxidative stress-induced metabolic changes in mouse C2C12 myotubes studied with high-resolution 13C, 1H, and 31P NMR spectroscopy

    DEFF Research Database (Denmark)

    Straadt, Ida K; Young, Jette F; Petersen, Bent O

    2010-01-01

    In this study, stress in relation to slaughter was investigated in a model system by the use of (13)C, (1)H, and (31)P nuclear magnetic resonance (NMR) spectroscopy for elucidating changes in the metabolites in C2C12 myotubes exposed to H(2)O(2)-induced stress. Oxidative stress resulted in lower...... levels of several metabolites, mainly amino acids; however, higher levels of alanine were apparent in the (13)C spectra after incubation with [(13)C(1)]glucose. In the (13)C spectra [(13)C(3)]lactate tended to increase after exposure to increasing concentrations of H(2)O(2); conversely, a tendency...

  12. Bit-1 is an essential regulator of myogenic differentiation.

    Science.gov (United States)

    Griffiths, Genevieve S; Doe, Jinger; Jijiwa, Mayumi; Van Ry, Pam; Cruz, Vivian; de la Vega, Michelle; Ramos, Joe W; Burkin, Dean J; Matter, Michelle L

    2015-05-01

    Muscle differentiation requires a complex signaling cascade that leads to the production of multinucleated myofibers. Genes regulating the intrinsic mitochondrial apoptotic pathway also function in controlling cell differentiation. How such signaling pathways are regulated during differentiation is not fully understood. Bit-1 (also known as PTRH2) mutations in humans cause infantile-onset multisystem disease with muscle weakness. We demonstrate here that Bit-1 controls skeletal myogenesis through a caspase-mediated signaling pathway. Bit-1-null mice exhibit a myopathy with hypotrophic myofibers. Bit-1-null myoblasts prematurely express muscle-specific proteins. Similarly, knockdown of Bit-1 expression in C2C12 myoblasts promotes early differentiation, whereas overexpression delays differentiation. In wild-type mice, Bit-1 levels increase during differentiation. Bit-1-null myoblasts exhibited increased levels of caspase 9 and caspase 3 without increased apoptosis. Bit-1 re-expression partially rescued differentiation. In Bit-1-null muscle, Bcl-2 levels are reduced, suggesting that Bcl-2-mediated inhibition of caspase 9 and caspase 3 is decreased. Bcl-2 re-expression rescued Bit-1-mediated early differentiation in Bit-1-null myoblasts and C2C12 cells with knockdown of Bit-1 expression. These results support an unanticipated yet essential role for Bit-1 in controlling myogenesis through regulation of Bcl-2. © 2015. Published by The Company of Biologists Ltd.

  13. Development of gas chromatography-flame ionization detection system with a single column and liquid nitrogen-free for measuring atmospheric C2-C12 hydrocarbons.

    Science.gov (United States)

    Liu, Chengtang; Mu, Yujing; Zhang, Chenglong; Zhang, Zhibo; Zhang, Yuanyuan; Liu, Junfeng; Sheng, Jiujiang; Quan, Jiannong

    2016-01-04

    A liquid nitrogen-free GC-FID system equipped with a single column has been developed for measuring atmospheric C2-C12 hydrocarbons. The system is consisted of a cooling unit, a sampling unit and a separation unit. The cooling unit is used to meet the temperature needs of the sampling unit and the separation unit. The sampling unit includes a dehydration tube and an enrichment tube. No breakthrough of the hydrocarbons was detected when the temperature of the enrichment tube was kept at -90 °C and sampling volume was 400 mL. The separation unit is a small round oven attached on the cooling column. A single capillary column (OV-1, 30 m × 0.32 mm I.D.) was used to separate the hydrocarbons. An optimal program temperature (-60 ∼ 170 °C) of the oven was achieved to efficiently separate C2-C12 hydrocarbons. There were good linear correlations (R(2)=0.993-0.999) between the signals of the hydrocarbons and the enrichment amount of hydrocarbons, and the relative standard deviation (RSD) was less than 5%, and the method detection limits (MDLs) for the hydrocarbons were in the range of 0.02-0.10 ppbv for sampling volume of 400 mL. Field measurements were also conducted and more than 50 hydrocarbons from C2 to C12 were detected in Beijing city. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C2C12 myotubes

    Directory of Open Access Journals (Sweden)

    Tisdale Michael J

    2008-01-01

    Full Text Available Abstract Background Patients with advanced cancer suffer from cachexia, which is characterised by a marked weight loss, and is invariably associated with the presence of tumoral and humoral factors which are mainly responsible for the depletion of fat stores and muscular tissue. Methods In this work, we used cytotoxicity and enzymatic assays and morphological analysis to examine the effects of a proteolysis-inducing factor (PIF-like molecule purified from ascitic fluid of Walker tumour-bearing rats (WF, which has been suggested to be responsible for muscle atrophy, on cultured C2C12 muscle cells. Results WF decreased the viability of C2C12 myotubes, especially at concentrations of 20–25 μg.mL-1. There was an increase in the content of the pro-oxidant malondialdehyde, and a decrease in antioxidant enzyme activity. Myotubes protein synthesis decreased and protein degradation increased together with an enhanced in the chymotrypsin-like enzyme activity, a measure of functional proteasome activity, after treatment with WF. Morphological alterations such as cell retraction and the presence of numerous cells in suspension were observed, particularly at high WF concentrations. Conclusion These results indicate that WF has similar effects to those of proteolysis-inducing factor, but is less potent than the latter. Further studies are required to determine the precise role of WF in this experimental model.

  15. Trichocyalides A and B, new inhibitors of alkaline phosphatase activity in bone morphogenetic protein-stimulated myoblasts, produced by Trichoderma sp. FKI-5513.

    Science.gov (United States)

    Fukuda, Takashi; Uchida, Ryuji; Ohte, Satoshi; Inoue, Hiroyo; Yamazaki, Hiroyuki; Matsuda, Daisuke; Nonaka, Kenichi; Masuma, Rokurou; Katagiri, Takenobu; Tomoda, Hiroshi

    2012-11-01

    Two new butenolides, designated trichocyalides A and B, were isolated along with the known compound harzianolide, from the culture broth of Trichoderma sp. FKI-5513 by solvent extraction, ODS column chromatography and HPLC. Their structures were elucidated by several spectral analyses, showing that they have the common skeleton of butenofuranone. Trichocyalides A and B inhibited alkaline phosphatase (ALP) activity, a typical marker enzyme of osteoblastic differentiation (IC(50): 83.0 and 187 μM, respectively), in bone morphogenetic protein (BMP)-stimulated C2C12 myoblasts mutant cells, which stably express BMP receptor activity, whereas harzianolide showed no inhibitory activity against ALP even at 500 μM.

  16. Mitochondrial hyperfusion during oxidative stress is coupled to a dysregulation in calcium handling within a C2C12 cell model.

    Directory of Open Access Journals (Sweden)

    Calum J Redpath

    Full Text Available Atrial Fibrillation is the most common sustained cardiac arrhythmia worldwide harming millions of people every year. Atrial Fibrillation (AF abruptly induces rapid conduction between atrial myocytes which is associated with oxidative stress and abnormal calcium handling. Unfortunately this new equilibrium promotes perpetuation of the arrhythmia. Recently, in addition to being the major source of oxidative stress within cells, mitochondria have been observed to fuse, forming mitochondrial networks and attach to intracellular calcium stores in response to cellular stress. We sought to identify a potential role for rapid stimulation, oxidative stress and mitochondrial hyperfusion in acute changes to myocyte calcium handling. In addition we hoped to link altered calcium handling to increased sarcoplasmic reticulum (SR-mitochondrial contacts, the so-called mitochondrial associated membrane (MAM. We selected the C2C12 murine myotube model as it has previously been successfully used to investigate mitochondrial dynamics and has a myofibrillar system similar to atrial myocytes. We observed that rapid stimulation of C2C12 cells resulted in mitochondrial hyperfusion and increased mitochondrial colocalisation with calcium stores. Inhibition of mitochondrial fission by transfection of mutant DRP1K38E resulted in similar effects on mitochondrial fusion, SR colocalisation and altered calcium handling. Interestingly the effects of 'forced fusion' were reversed by co-incubation with the reducing agent N-Acetyl cysteine (NAC. Subsequently we demonstrated that oxidative stress resulted in similar reversible increases in mitochondrial fusion, SR-colocalisation and altered calcium handling. Finally, we believe we have identified that myocyte calcium handling is reliant on baseline levels of reactive oxygen species as co-incubation with NAC both reversed and retarded myocyte response to caffeine induced calcium release and re-uptake. Based on these results we

  17. Amygdalin isolated from Semen Persicae (Tao Ren) extracts induces the expression of follistatin in HepG2 and C2C12 cell lines.

    Science.gov (United States)

    Yang, Chuanbin; Li, Xuechen; Rong, Jianhui

    2014-01-01

    The Chinese medicine formulation ISF-1 (also known as Bu-Yang-Huan-Wu-Tang) for post-stroke rehabilitation could increase the expression of growth-regulating protein follistatin by approximately 4-fold. This study aims to identify the active compounds of ISF-1 for the induction of follistatin expression. Active compounds in ISF-1 responsible for induction of follistatin were identified by a bioactivity-guided fractionation procedure involving liquid-liquid extraction, HPLC separation and RT-PCR detection. The aqueous extracts of seven ISF-1 ingredients including Semen Persicae (Tao Ren) and the S. Persicae-derived fractions were assayed for the induction of follistatin mRNA expression in human hepatocarcinoma HepG2 cells by RT-PCR. The concentrations of isolated compounds were proportionally normalized to the reported IC50 concentration (5.8 mg/mL) of the formulation ISF-1 in HepG2. The active fractions were characterized by reverse-phase HPLC on a C18 column and identified by mass spectrometry. Three ingredients of ISF-1, namely S. Persicae (Tao Ren), Pheretima (Di Long), and Flos Carthami (Hong Hua), induced the expression of follistatin mRNA. Among these, the ingredient S. Persicae were the most active, and amygdalin from S. Persicae extract was identified as a novel follistatin inducer. Amygdalin stimulated the growth of skeletal muscle cell line C2C12 cells in a concentration-dependent manner. Amygdalin isolated from S. Persicae extract in ISF-1 through a bioactivity-guided fractionation procedure induced the expression of follistatin in HepG2 and C2C12 cell lines.

  18. Effect of Low Power Laser Irradiation on the Ability of Cell Growth and Myogenic Differentiation of Myoblasts Cultured In Vitro

    Directory of Open Access Journals (Sweden)

    Cui-Ping Zhang

    2014-01-01

    Full Text Available As a therapeutic modality, low power laser irradiation (LPLI has been used clinically in the treatment of skeletal muscle injuries and other myopathic conditions, but the cellular and molecular mechanisms attributed to this therapy were still unclear. Myoblasts are a type of myogenic stem cells quiescence in mature skeletal muscle fibers and are considered as the source cells during the regenerating process. The purpose of this paper was to investigate the effects of LPLI on the proliferation and myogenic differentiation of the cultured myoblasts and to find out the major candidates responsible for LPLI-induced muscle regeneration in vivo. In this study, primary rat myoblasts were exposed to helium-neon (He-Ne laser. Cell proliferation, differentiation, and the cellular responses to LPLI were monitored by using morphological observation and molecular biological methods. It was found that LPLI at a certain fluence could increase the cell growth potential for myoblasts and further induce more cells entering into S phase of the mitotic cycle as indicated by high levels of bromodeoxyuridine (BrdU incorporation, while at the same time inhibiting their in vitro differentiation and decreasing the expression of myogenic regulatory genes to a certain extent. Taken together, these results provide experimental evidence for the clinical applications of LPLI in regenerating skeletal muscle.

  19. Inhibition of in vitro myogenic differentiation by cellular transcription factor E2F1

    DEFF Research Database (Denmark)

    Wang, J; Helin, K; Jin, P

    1995-01-01

    Terminal differentiation of cultured myocytes requires withdrawal of the cells from the cell cycle. Constitutive overexpression of several oncogenes in myoblasts can inhibit in vitro myogenesis. Here we studied the role of the cellular transcription factor E2F1 on myogenic differentiation. E2F1...... expression is irreversibly down-regulated during differentiation of C2C12 myocytes. Furthermore, deregulated E2F1 expression in C2C12 cells prevented myogenic differentiation. This inhibition of myogenesis was associated with the repression of myogenin expression and an elevated cyclin D1 expression....... Moreover, E2F1-overexpressing myocytes failed to exit the cell cycle under differentiation conditions. These results are consistent with the notion that E2F1 can function as an oncogene and further suggest that E2F1 down-regulation is required for myogenic differentiation....

  20. Metabolic responses to BRL37344 and clenbuterol in soleus muscle and C2C12 cells via different atypical pharmacologies and β2-adrenoceptor mechanisms

    Science.gov (United States)

    Ngala, R A; O'Dowd, J; Wang, S J; Agarwal, A; Stocker, C; Cawthorne, M A; Arch, J R S

    2008-01-01

    Background and purpose: Picomolar concentrations of the β3-adrenoceptor agonist BRL37344 stimulate 2-deoxyglucose uptake in soleus muscle via undefined receptors. Higher concentrations alter uptake, apparently via β2-adrenoceptors. Effects of BRL37344 and β2-adrenoceptor agonists are compared. Experimental approach: Mouse soleus muscles were incubated with 2-deoxy[1-14C]-glucose, [1-14C]-palmitate or [2-14C]-pyruvate, and BRL37344, β2-adrenoceptor agonists and selective β-adrenoceptor antagonists. Formation of 2-deoxy[1-14C]-glucose-6-phosphate or 14CO2 was measured. 2-Deoxy[1-14C]-glucose uptake and β-adrenoceptor mRNA were measured in C2C12 cells. Key results: 10 pM BRL37344, 10 pM clenbuterol and 100 pM salbutamol stimulated 2-deoxyglucose uptake in soleus muscle by 33–54%. The effect of BRL37344 was prevented by 1 μM atenolol but not by 300 nM CGP20712A or IC3118551, or 1 μM SR59230A; that of clenbuterol was prevented by ICI118551 but not atenolol. 10 nM BRL37344 st4mulated 2-deoxyglucose uptake, whereas 100 nM clenbuterol and salbutamol inhibited uptake. These effects were blocked by ICI118551. Similar results were obtained in C2C12 cells, in which only β2-adrenoceptor mRNA could be detected by RT-PCR. 10 nM BRL37344 and 10 pM clenbuterol stimulated muscle palmitate oxidation. In the presence of palmitate, BRL37344 no longer stimulated 2-deoxyglucose uptake and the effect of clenbuterol was not significant. Conclusions and implications: Stimulation of glucose uptake by 10 pM BRL37344 and clenbuterol involves different atypical pharmacologies. Nanomolar concentrations of BRL37344 and clenbuterol, probably acting via β2-adrenoceptors, have opposite effects on glucose uptake. The agonists preferentially stimulate fat rather than carbohydrate oxidation, but stimulation of endogenous fat oxidation cannot explain why 100 nM clenbuterol inhibited 2-deoxyglucose uptake. PMID:18552870

  1. Metabolic responses to BRL37344 and clenbuterol in soleus muscle and C2C12 cells via different atypical pharmacologies and beta2-adrenoceptor mechanisms.

    Science.gov (United States)

    Ngala, R A; O'Dowd, J; Wang, S J; Agarwal, A; Stocker, C; Cawthorne, M A; Arch, J R S

    2008-10-01

    Picomolar concentrations of the beta3-adrenoceptor agonist BRL37344 stimulate 2-deoxyglucose uptake in soleus muscle via undefined receptors. Higher concentrations alter uptake, apparently via beta2-adrenoceptors. Effects of BRL37344 and beta2-adrenoceptor agonists are compared. Mouse soleus muscles were incubated with 2-deoxy[1-(14)C]-glucose, [1-(14)C]-palmitate or [2-(14)C]-pyruvate, and BRL37344, beta2-adrenoceptor agonists and selective beta-adrenoceptor antagonists. Formation of 2-deoxy[1-(14)C]-glucose-6-phosphate or (14)CO2 was measured. 2-Deoxy[1-(14)C]-glucose uptake and beta-adrenoceptor mRNA were measured in C2C12 cells. 10 pM BRL37344, 10 pM clenbuterol and 100 pM salbutamol stimulated 2-deoxyglucose uptake in soleus muscle by 33-54%. The effect of BRL37344 was prevented by 1 microM atenolol but not by 300 nM CGP20712A or IC3118551, or 1 microM SR59230A; that of clenbuterol was prevented by ICI118551 but not atenolol. 10 nM BRL37344 stimulated 2-deoxyglucose uptake, whereas 100 nM clenbuterol and salbutamol inhibited uptake. These effects were blocked by ICI118551. Similar results were obtained in C2C12 cells, in which only beta2-adrenoceptor mRNA could be detected by RT-PCR. 10 nM BRL37344 and 10 pM clenbuterol stimulated muscle palmitate oxidation. In the presence of palmitate, BRL37344 no longer stimulated 2-deoxyglucose uptake and the effect of clenbuterol was not significant. Stimulation of glucose uptake by 10 pM BRL37344 and clenbuterol involves different atypical pharmacologies. Nanomolar concentrations of BRL37344 and clenbuterol, probably acting via beta2-adrenoceptors, have opposite effects on glucose uptake. The agonists preferentially stimulate fat rather than carbohydrate oxidation, but stimulation of endogenous fat oxidation cannot explain why 100 nM clenbuterol inhibited 2-deoxyglucose uptake.

  2. Transient Silencing of a Type IV P-Type ATPase, Atp10c, Results in Decreased Glucose Uptake in C2C12 Myotubes.

    Science.gov (United States)

    Hurst, S E; Minkin, S C; Biggerstaff, J; Dhar, M S

    2012-01-01

    Atp10c is a strong candidate gene for diet-induced obesity and type 2 diabetes. To identify molecular and cellular targets of ATP10C, Atp10c expression was altered in vitro in C2C12 skeletal muscle myotubes by transient transfection with an Atp10c-specific siRNA. Glucose uptake assays revealed that insulin stimulation caused a significant 2.54-fold decrease in 2-deoxyglucose uptake in transfected cells coupled with a significant upregulation of native mitogen-activated protein kinases (MAPKs), p38, and p44/42. Additionally, glucose transporter-1 (GLUT1) was significantly upregulated; no changes in glucose transporter-4 (GLUT4) expression were observed. The involvement of MAPKs was confirmed using the specific inhibitor SB203580, which downregulated the expression of native and phosphorylated MAPK proteins in transfected cells without any changes in insulin-stimulated glucose uptake. Results indicate that Atp10c regulates glucose metabolism, at least in part via the MAPK pathway, and, thus, plays a significant role in the development of insulin resistance and type 2 diabetes.

  3. Transient Silencing of a Type IV P-Type ATPase, Atp10c, Results in Decreased Glucose Uptake in C2C12 Myotubes

    Directory of Open Access Journals (Sweden)

    S. E. Hurst

    2012-01-01

    Full Text Available Atp10c is a strong candidate gene for diet-induced obesity and type 2 diabetes. To identify molecular and cellular targets of ATP10C, Atp10c expression was altered in vitro in C2C12 skeletal muscle myotubes by transient transfection with an Atp10c-specific siRNA. Glucose uptake assays revealed that insulin stimulation caused a significant 2.54-fold decrease in 2-deoxyglucose uptake in transfected cells coupled with a significant upregulation of native mitogen-activated protein kinases (MAPKs, p38, and p44/42. Additionally, glucose transporter-1 (GLUT1 was significantly upregulated; no changes in glucose transporter-4 (GLUT4 expression were observed. The involvement of MAPKs was confirmed using the specific inhibitor SB203580, which downregulated the expression of native and phosphorylated MAPK proteins in transfected cells without any changes in insulin-stimulated glucose uptake. Results indicate that Atp10c regulates glucose metabolism, at least in part via the MAPK pathway, and, thus, plays a significant role in the development of insulin resistance and type 2 diabetes.

  4. Tiam-1, a GEF for Rac1, plays a critical role in metformin-mediated glucose uptake in C2C12 cells.

    Science.gov (United States)

    You, Ga Young; Lee, Jung Ok; Kim, Ji Hae; Kim, Nami; Lee, Soo Kyung; Moon, Ji Wook; Jie, Sha; Lee, Hye Jeong; Kim, Su Jin; Park, Sun Hwa; Kim, Hyeon Soo

    2013-12-01

    Metformin is known to stimulate glucose uptake, but the mechanism for this action is not fully understood. In this study, AMPK activators (AICAR and metformin) increased the expression of T-lymphoma invasion and metastasis-inducing protein-1 (Tiam-1), a Rac1 specific guanine nucleotide exchange factor (GEF), mRNA and protein in skeletal muscle C2C12 cells. Metformin increases the serine-phosphorylation of Tiam-1 by AMPK and induces interaction between Tiam-1 and 14-3-3. Pharmacologic inhibition of AMPK blocks this interaction, indicating that 14-3-3 may be required for induction of Tiam-1 by AMPK. Metformin also increases the phosphorylation of p21-activated kinase 1 (PAK1), a direct downstream target of Rac1, dependent on AMPK. Tiam-1 is down-regulated at high glucose concentrations in cultured cells and in the db/db mouse model of hyperglycemia. Furthermore, Tiam-1 knock-down blocked metformin-induced increase in glucose uptake. These findings suggest that metformin promotes cellular glucose uptake in part through Tiam-1 induction. © 2013. Published by Elsevier Inc. All rights reserved.

  5. Cinnamon extract enhances glucose uptake in 3T3-L1 adipocytes and C2C12 myocytes by inducing LKB1-AMP-activated protein kinase signaling.

    Directory of Open Access Journals (Sweden)

    Yan Shen

    Full Text Available We previously demonstrated that cinnamon extract (CE ameliorates type 1 diabetes induced by streptozotocin in rats through the up-regulation of glucose transporter 4 (GLUT4 translocation in both muscle and adipose tissues. This present study was aimed at clarifying the detailed mechanism(s with which CE increases the glucose uptake in vivo and in cell culture systems using 3T3-L1 adipocytes and C2C12 myotubes in vitro. Specific inhibitors of key enzymes in insulin signaling and AMP-activated protein kinase (AMPK signaling pathways, as well as small interference RNA, were used to examine the role of these kinases in the CE-induced glucose uptake. The results showed that CE stimulated the phosphorylation of AMPK and acetyl-CoA carboxylase. An AMPK inhibitor and LKB1 siRNA blocked the CE-induced glucose uptake. We also found for the first time that insulin suppressed AMPK activation in the adipocyte. To investigate the effect of CE on type 2 diabetes in vivo, we further performed oral glucose tolerance tests and insulin tolerance tests in type 2 diabetes model rats administered with CE. The CE improved glucose tolerance in oral glucose tolerance tests, but not insulin sensitivity in insulin tolerance test. In summary, these results indicate that CE ameliorates type 2 diabetes by inducing GLUT4 translocation via the AMPK signaling pathway. We also found insulin antagonistically regulates the activation of AMPK.

  6. Cinnamon extract enhances glucose uptake in 3T3-L1 adipocytes and C2C12 myocytes by inducing LKB1-AMP-activated protein kinase signaling.

    Science.gov (United States)

    Shen, Yan; Honma, Natsumi; Kobayashi, Katsuya; Jia, Liu Nan; Hosono, Takashi; Shindo, Kazutoshi; Ariga, Toyohiko; Seki, Taiichiro

    2014-01-01

    We previously demonstrated that cinnamon extract (CE) ameliorates type 1 diabetes induced by streptozotocin in rats through the up-regulation of glucose transporter 4 (GLUT4) translocation in both muscle and adipose tissues. This present study was aimed at clarifying the detailed mechanism(s) with which CE increases the glucose uptake in vivo and in cell culture systems using 3T3-L1 adipocytes and C2C12 myotubes in vitro. Specific inhibitors of key enzymes in insulin signaling and AMP-activated protein kinase (AMPK) signaling pathways, as well as small interference RNA, were used to examine the role of these kinases in the CE-induced glucose uptake. The results showed that CE stimulated the phosphorylation of AMPK and acetyl-CoA carboxylase. An AMPK inhibitor and LKB1 siRNA blocked the CE-induced glucose uptake. We also found for the first time that insulin suppressed AMPK activation in the adipocyte. To investigate the effect of CE on type 2 diabetes in vivo, we further performed oral glucose tolerance tests and insulin tolerance tests in type 2 diabetes model rats administered with CE. The CE improved glucose tolerance in oral glucose tolerance tests, but not insulin sensitivity in insulin tolerance test. In summary, these results indicate that CE ameliorates type 2 diabetes by inducing GLUT4 translocation via the AMPK signaling pathway. We also found insulin antagonistically regulates the activation of AMPK.

  7. Effect of testosterone on the regulation of p53 and p66Shc during oxidative stress damage in C2C12 cells.

    Science.gov (United States)

    Pronsato, Lucía; Milanesi, Lorena

    2016-02-01

    Accumulating evidence indicates that apoptosis is activated in the aged skeletal muscle, contributing to sarcopenia. We have previously demonstrated that testosterone protects against hydrogen peroxide (H2O2)-induced apoptosis in C2C12 muscle cells, at different levels: morphological, physiological, biochemical and molecular. In the present study we observed that H2O2 induces the mitochondrial permeability transition pore (mPTP) opening and exerts p53 activation in a time-dependent way, with a maximum response after 1-2h of treatment. Testosterone treatment, prior to H2O2, reduces not only p53 phosphorylation but also p66Shc expression, activation and its mitochondrial localization, at the same time that it prevents the mPTP opening. Furthermore, testosterone diminishes JNK and PKCβI phosphorylation induced by H2O2 and probably contributing thus, to reduce the activation of p66Shc. Thus, the mPTP opening, p53, JNK and PKCβI activation, as well as p66Shc mRNA increase, induced by oxidative stress, were reduced by testosterone pretreatment. The data presented in this work show some of the components upstream of the classical apoptotic pathway, that are activated during oxidative stress and that are points where testosterone exerts its protective action against apoptosis, exposing some of the puzzle pieces of the intricate network that aged skeletal muscle apoptosis represents. Copyright © 2015. Published by Elsevier Inc.

  8. Testosterone exerts antiapoptotic effects against H2O2 in C2C12 skeletal muscle cells through the apoptotic intrinsic pathway.

    Science.gov (United States)

    Pronsato, Lucía; Boland, Ricardo; Milanesi, Lorena

    2012-03-01

    Experimental data indicate that apoptosis is activated in the aged skeletal muscle, contributing to sarcopenia. We have previously demonstrated that testosterone protects against hydrogen peroxide (H(2)O(2))-induced apoptosis in C2C12 muscle cells. Here we identified molecular events involved in the antiapoptotic effect of testosterone. At short times of exposure to H(2)O(2) cells exhibit a defense response but at longer treatment times cells undergo apoptosis. Incubation with testosterone prior to H(2)O(2) induces BAD inactivation, inhibition of poly(ADP-ribose) polymerase cleavage, and a decrease in BAX levels, and impedes the loss of mitochondrial membrane potential, suggesting that the hormone participates in the regulation of the apoptotic intrinsic pathway. Simultaneous treatment with testosterone, H(2)O(2), and the androgen receptor (AR) antagonist, flutamide, reduces the effects of the hormone, pointing to a possible participation of the AR in the antiapoptotic effect. The data presented allow us to begin to elucidate the mechanism by which the hormone prevents apoptosis in skeletal muscle.

  9. Mining the Secretome of C2C12 Muscle Cells: Data Dependent Experimental Approach To Analyze Protein Secretion Using Label-Free Quantification and Peptide Based Analysis.

    Science.gov (United States)

    Grube, Leonie; Dellen, Rafael; Kruse, Fabian; Schwender, Holger; Stühler, Kai; Poschmann, Gereon

    2018-01-24

    Secretome analysis faces several challenges including detection of low abundant proteins and the discrimination of bona fide secreted proteins from false-positive identifications stemming from cell leakage or serum. Here, we developed a two-step secretomics approach and applied it to the analysis of secreted proteins of C2C12 skeletal muscle cells since the skeletal muscle has been identified as an important endocrine organ secreting myokines as signaling molecules. First, we compared culture supernatants with corresponding cell lysates by mass spectrometry-based proteomics and label-free quantification. We identified 672 protein groups as candidate secreted proteins due to their higher abundance in the secretome. On the basis of Brefeldin A mediated blocking of classical secretory processes, we estimated a sensitivity of >80% for the detection of classical secreted proteins for our experimental approach. In the second step, the peptide level information was integrated with UniProt based protein information employing the newly developed bioinformatics tool "Lysate and Secretome Peptide Feature Plotter" (LSPFP) to detect proteolytic protein processing events that might occur during secretion. Concerning the proof of concept, we identified truncations of the cytoplasmic part of the protein Plexin-B2. Our workflow provides an efficient combination of experimental workflow and data analysis to identify putative secreted and proteolytic processed proteins.

  10. Doxycycline Inhibits IL-17-Stimulated MMP-9 Expression by Downregulating ERK1/2 Activation: Implications in Myogenic Differentiation

    Science.gov (United States)

    Obradović, Hristina; Krstić, Jelena; Kukolj, Tamara; Đorđević, Ivana Okić; Jauković, Aleksandra; Jovčić, Gordana

    2016-01-01

    Interleukin 17 (IL-17) is a cytokine with pleiotropic effects associated with several inflammatory diseases. Although elevated levels of IL-17 have been described in inflammatory myopathies, its role in muscle remodeling and regeneration is still unknown. Excessive extracellular matrix degradation in skeletal muscle is an important pathological consequence of many diseases involving muscle wasting. In this study, the role of IL-17 on the expression of matrix metalloproteinase- (MMP-) 9 in myoblast cells was investigated. The expression of MMP-9 after IL-17 treatment was analyzed in mouse myoblasts C2C12 cell line. The increase in MMP-9 production by IL-17 was concomitant with its capacity to inhibit myogenic differentiation of C2C12 cells. Doxycycline (Doxy) treatment protected the myogenic capacity of myoblasts from IL-17 inhibition and, moreover, increased myotubes hypertrophy. Doxy blocked the capacity of IL-17 to stimulate MMP-9 production by regulating IL-17-induced ERK1/2 MAPK activation. Our results imply that MMP-9 mediates IL-17's capacity to inhibit myoblast differentiation during inflammatory diseases and indicate that Doxy can modulate myoblast response to inflammatory induction by IL-17. PMID:28042204

  11. Identification of valid reference genes during the differentiation of human myoblasts

    Directory of Open Access Journals (Sweden)

    Hörmann Karl

    2009-07-01

    Full Text Available Abstract Background Analysis of RNA expression using real-time PCR (qRT-PCR traditionally includes reference genes (RG as an internal control. This practice is being questioned as it becomes increasingly clear that RG may vary considerably under certain experimental conditions. Thus, the validity of a particular RG must be determined for each experimental setting. We used qRT-PCR to measure the levels of six RG, which have been reported in the literature to be invariant. The RG were analyzed in human myoblast cultures under differentiation conditions. We examined the expression by qRT-PCR of mRNA encoding Beta-actin (ACTB, Beta-2-microglobulin (B2M, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, peptidylprolyl isomerase A (PPIA, TATA box binding protein (TBP and ribosomal protein, large, P0 (RPLPO. The mRNA expression of the following genes of interest (GOI were analyzed: skeletal muscle alpha 1 actin (ACTA1, myogenin/myogenic factor 4 (MYOG, embryonic skeletal muscle myosin heavy chain 3 (MYH3 and the activity of creatine phosphokinase (CK. The geNorm, NormFinder and BestKeeper software programs were used to ascertain the most suitable RG to normalize the RNA input. Results Using the geNorm program, RPLPO and TBP were found to be the most stable genes, additionally a suitable normalization factor (NF was calculated. The NormFinder software showed that RPLPO was the most stable, whereas TBP ranked second. BestKeeper program also revealed that RPLPO and TBP as stable genes, but PPIA was the most stable reference gene, whereas GAPDH and ACTB were the worst ranked. Conclusion RNA expression analyses including three independent softwares revealed that RPLPO, TBP as reference genes or NF calculated by geNorm software, are suitable to normalize the mRNA expression in myoblast after culture under differentiation conditions. Significant correlations can be identified between the differentiations markers ACTA1, MYOG, MYH3 and creatine phosphokinase (CK

  12. Rapamycin has a biphasic effect on insulin sensitivity in C2C12 myotubes due to sequential disruption of mTORC1 and mTORC2

    Directory of Open Access Journals (Sweden)

    Lan eYe

    2012-09-01

    Full Text Available Rapamycin, an inhibitor of mTOR complex 1 (mTORC1, improves insulin sensitivity in acute studies in vitro and in vivo by disrupting a negative feedback loop mediated by S6 kinase. We find that rapamycin has a clear biphasic effect on insulin sensitivity in C2C12 myotubes, with enhanced responsiveness during the first hour that declines to almost complete insulin resistance by 24-48 hours. We and others have recently observed that chronic rapamycin treatment induces insulin resistance in rodents, at least in part due to disruption of mTORC2, an mTOR-containing complex that is not acutely sensitive to the drug. Chronic rapamycin treatment may also impair insulin action via the inhibition of mTORC1-dependent mitochondrial biogenesis and activity, which could result in a buildup of lipid intermediates that are known to trigger insulin resistance. We confirmed that rapamycin inhibits expression of PGC-1α, a key mitochondrial transcription factor, and acutely reduces respiration rate in myotubes. However, rapamycin did not stimulate phosphorylation of PKCθ, a central mediator of lipid-induced insulin resistance. Instead, we found dramatic disruption of mTORC2, which coincided with the onset of insulin resistance. Selective inhibition of mTORC1 or mTORC2 by shRNA-mediated knockdown of specific components (Raptor and Rictor, respectively confirmed that mitochondrial effects of rapamycin are mTORC1-dependent, whereas insulin resistance was recapitulated only by knockdown of mTORC2. Thus, mTORC2 disruption, rather than inhibition of mitochondria, causes insulin resistance in rapamycin-treated myotubes, and this system may serve as a useful model to understand the effects of rapamycin on mTOR signaling in vivo.

  13. Extracellular vesicles from a muscle cell line (C2C12 enhance cell survival and neurite outgrowth of a motor neuron cell line (NSC-34

    Directory of Open Access Journals (Sweden)

    Roger D. Madison

    2014-02-01

    Full Text Available Introduction: There is renewed interest in extracellular vesicles over the past decade or 2 after initially being thought of as simple cellular garbage cans to rid cells of unwanted components. Although there has been intense research into the role of extracellular vesicles in the fields of tumour and stem cell biology, the possible role of extracellular vesicles in nerve regeneration is just in its infancy. Background: When a peripheral nerve is damaged, the communication between spinal cord motor neurons and their target muscles is disrupted and the result can be the loss of coordinated muscle movement. Despite state-of-the-art surgical procedures only approximately 10% of adults will recover full function after peripheral nerve repair. To improve upon such results will require a better understanding of the basic mechanisms that influence axon outgrowth and the interplay between the parent motor neuron and the distal end organ of muscle. It has previously been shown that extracellular vesicles are immunologically tolerated, display targeting ligands on their surface, and can be delivered in vivo to selected cell populations. All of these characteristics suggest that extracellular vesicles could play a significant role in nerve regeneration. Methods: We have carried out studies using 2 very well characterized cell lines, the C2C12 muscle cell line and the motor neuron cell line NSC-34 to ask the question: Do extracellular vesicles from muscle influence cell survival and/or neurite outgrowth of motor neurons? Conclusion: Our results show striking effects of extracellular vesicles derived from the muscle cell line on the motor neuron cell line in terms of neurite outgrowth and survival.

  14. Protection of palmitic acid-mediated lipotoxicity by arachidonic acid via channeling of palmitic acid into triglycerides in C2C12

    Science.gov (United States)

    2014-01-01

    Background Excessive saturated fatty acids have been considered to be one of major contributing factors for the dysfunction of skeletal muscle cells as well as pancreatic beta cells, leading to the pathogenesis of type 2 diabetes. Results PA induced cell death in a dose dependent manner up to 1.5 mM, but AA protected substantially lipotoxicity caused by PA at even low concentration of 62 μM, at which monounsaturated fatty acids including palmitoleic acid (POA) and oleic acid (OA) did not protect as much as AA did. Induction of cell death by PA was resulted from mitochondrial membrane potential loss, and AA effectively blocked the progression of apoptosis. Furthermore, AA rescued significantly PA-impaired glucose uptake and -signal transduction of Akt in response to insulin. Based on the observations that polyunsaturated AA generated competently cellular droplets at low concentration within the cytosol of myotubes compared with other monounsaturated fatty acids, and AA-driven lipid droplets were also enhanced in the presence of PA, we hypothesized that incorporation of harmful PA into inert triglyceride (TG) may be responsible for the protective effects of AA against PA-induced lipotoxicity. To address this assumption, C2C12 myotubes were incubated with fluorescent probed-PA analogue 4, 4-difluoro-5, 7-dimethyl-4-boro-3a,4a-diaza-s-indacene-3-hexadecanoic acid (BODIPY FL C16) in the presence of AA and their subsequent lipid profiles were analyzed. The analyses of lipids on thin layer chromatograpy (TLC) showed that fluorescent PA analogue was rapidly channeled into AA-driven TG droplets. Conclusion Taken together, it is proposed that AA diverts PA into inert TG, therefore reducing the availability of harmful PA into intracellular target molecules. PMID:24521082

  15. Protection of palmitic acid-mediated lipotoxicity by arachidonic acid via channeling of palmitic acid into triglycerides in C2C12.

    Science.gov (United States)

    Cheon, Hyae Gyeong; Cho, Young Sik

    2014-02-12

    Excessive saturated fatty acids have been considered to be one of major contributing factors for the dysfunction of skeletal muscle cells as well as pancreatic beta cells, leading to the pathogenesis of type 2 diabetes. PA induced cell death in a dose dependent manner up to 1.5 mM, but AA protected substantially lipotoxicity caused by PA at even low concentration of 62 μM, at which monounsaturated fatty acids including palmitoleic acid (POA) and oleic acid (OA) did not protect as much as AA did. Induction of cell death by PA was resulted from mitochondrial membrane potential loss, and AA effectively blocked the progression of apoptosis. Furthermore, AA rescued significantly PA-impaired glucose uptake and -signal transduction of Akt in response to insulin.Based on the observations that polyunsaturated AA generated competently cellular droplets at low concentration within the cytosol of myotubes compared with other monounsaturated fatty acids, and AA-driven lipid droplets were also enhanced in the presence of PA, we hypothesized that incorporation of harmful PA into inert triglyceride (TG) may be responsible for the protective effects of AA against PA-induced lipotoxicity. To address this assumption, C2C12 myotubes were incubated with fluorescent probed-PA analogue 4, 4-difluoro-5, 7-dimethyl-4-boro-3a,4a-diaza-s-indacene-3-hexadecanoic acid (BODIPY FL C16) in the presence of AA and their subsequent lipid profiles were analyzed. The analyses of lipids on thin layer chromatography (TLC) showed that fluorescent PA analogue was rapidly channeled into AA-driven TG droplets. Taken together, it is proposed that AA diverts PA into inert TG, therefore reducing the availability of harmful PA into intracellular target molecules.

  16. Microbial synthesis of gold nanoparticles using the fungus Penicillium brevicompactum and their cytotoxic effects against mouse mayo blast cancer C 2 C 12 cells.

    Science.gov (United States)

    Mishra, Amrita; Tripathy, Suraj Kumar; Wahab, Rizwan; Jeong, Song-Hoon; Hwang, Inho; Yang, You-Bing; Kim, Young-Soon; Shin, Hyung-Shik; Yun, Soon-Il

    2011-11-01

    Microorganisms, their cell filtrates, and live biomass have been utilized for synthesizing various gold nanoparticles. The shape, size, stability as well as the purity of the bio synthesized nanoparticles become very essential for application purpose. In the present study, gold nanoparticles have been synthesized from the supernatant, live cell filtrate, and biomass of the fungus Penicillium brevicompactum. The fungus has been grown in potato dextrose broth which is also found to synthesize gold nanoparticles. The size of the particles has been investigated by Bio-TEM before purification, following purification and after storing the particles for 3 months under refrigerated condition. Different characterization techniques like X-ray diffraction, Fourier transform infrared spectroscopy, and UV-visible spectroscopy have been used for analysis of the particles. The effect of reaction parameters such as pH and concentration of gold salt have also been monitored to optimize the morphology and dispersity of the synthesized gold nanoparticles. A pH range of 5 to 8 has favored the synthesis process whereas increasing concentration of gold salt (beyond 2 mM) has resulted in the formation of bigger sized and aggregated nanoparticles. Additionally, the cytotoxic nature of prepared nanoparticles has been analyzed using mouse mayo blast cancer C(2)C(12) cells at different time intervals (24, 48, and 72 h) of incubation period. The cells are cultivated in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum with antibiotics (streptopenicillin) at 37°C in a 5% humidified environment of CO(2). The medium has been replenished every other day, and the cells are subcultured after reaching the confluence. The viability of the cells is analyzed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide method.

  17. In vitro assessment of the combined effect of eicosapentaenoic acid, green tea extract and curcumin C3 on protein loss in C2C12 myotubes.

    Science.gov (United States)

    Mirza, Kamran A; Luo, Menghua; Pereira, Suzette; Voss, Anne; Das, Tapas; Tisdale, Michael J

    2016-09-01

    EPA has been clinically shown to reduce muscle wasting during cancer cachexia. This study investigates whether curcumin or green tea extract (GTE) enhances the ability of low doses of eicosapentaenoic acid (EPA) to reduce loss of muscle protein in an in vitro model. A low dose of EPA with minimal anti-cachectic activity was chosen to evaluate any potential synergistic effect with curcumin or GTE. Depression of protein synthesis and increase in degradation was determined in C2C12 myotubes in response to tumour necrosis factor-α (TNF-α) and proteolysis-inducing factor (PIF). EPA (50 μM) or curcumin (10 μg ml(-1)) alone had little effect on protein degradation caused by PIF but the combination produced complete inhibition, as did the combination with GTE (10 μg ml(-1)). In response to TNF-α (25 ng ml(-1))-induced protein degradation, EPA had a small, but not significant effect on protein degradation; however, when curcumin and GTE were combined with EPA, the effect was enhanced. EPA completely attenuated the depression of protein synthesis caused by TNF-α, but not that caused by PIF. The combination of EPA with curcumin produced a significant increase in protein synthesis to both agents. GTE alone or in combination with EPA had no effect on the depression of protein synthesis by TNF-α, but did significantly increase protein synthesis in PIF-treated cells. Both TNF-α and PIF significantly reduced myotube diameter from 17 to 13 μm for TNF-α (23.5%) and 15 μm (11.8%) for PIF However the triple combination of EPA, curcumin and GTE returned diameters to values not significantly different from the control. These results suggest that either curcumin or GTE or the combination could enhance the anti-catabolic effect of EPA on lean body mass.

  18. Extracellular vesicles from a muscle cell line (C2C12) enhance cell survival and neurite outgrowth of a motor neuron cell line (NSC-34).

    Science.gov (United States)

    Madison, Roger D; McGee, Christopher; Rawson, Renee; Robinson, Grant A

    2014-01-01

    There is renewed interest in extracellular vesicles over the past decade or 2 after initially being thought of as simple cellular garbage cans to rid cells of unwanted components. Although there has been intense research into the role of extracellular vesicles in the fields of tumour and stem cell biology, the possible role of extracellular vesicles in nerve regeneration is just in its infancy. When a peripheral nerve is damaged, the communication between spinal cord motor neurons and their target muscles is disrupted and the result can be the loss of coordinated muscle movement. Despite state-of-the-art surgical procedures only approximately 10% of adults will recover full function after peripheral nerve repair. To improve upon such results will require a better understanding of the basic mechanisms that influence axon outgrowth and the interplay between the parent motor neuron and the distal end organ of muscle. It has previously been shown that extracellular vesicles are immunologically tolerated, display targeting ligands on their surface, and can be delivered in vivo to selected cell populations. All of these characteristics suggest that extracellular vesicles could play a significant role in nerve regeneration. We have carried out studies using 2 very well characterized cell lines, the C2C12 muscle cell line and the motor neuron cell line NSC-34 to ask the question: Do extracellular vesicles from muscle influence cell survival and/or neurite outgrowth of motor neurons? Our results show striking effects of extracellular vesicles derived from the muscle cell line on the motor neuron cell line in terms of neurite outgrowth and survival.

  19. Nonionizing radiation as a noninvasive strategy in regenerative medicine: the effect of Ca(2+)-ICR on mouse skeletal muscle cell growth and differentiation.

    Science.gov (United States)

    De Carlo, Flavia; Ledda, Mario; Pozzi, Deleana; Pierimarchi, Pasquale; Zonfrillo, Manuela; Giuliani, Livio; D'Emilia, Enrico; Foletti, Alberto; Scorretti, Riccardo; Grimaldi, Settimio; Lisi, Antonella

    2012-11-01

    Controlling cell differentiation and proliferation with minimal manipulation is one of the most important goals for cell therapy in clinical applications. In this work, we evaluated the hypothesis that the exposure of myoblast cells (C2C12) to nonionizing radiation (tuned at an extremely low-frequency electromagnetic field at calcium-ion cyclotron frequency of 13.75 Hz) may drive their differentiation toward a myogenic phenotype. C2C12 cells exposed to calcium-ion cyclotron resonance (Ca(2+)-ICR) showed a decrease in cellular growth and an increase in the G(0)/G(1) phase. Severe modifications in the shape and morphology and a change in the actin distribution were revealed by the phalloidin fluorescence analysis. A significant upregulation at transcriptional and translational levels of muscle differentiation markers such as myogenin (MYOG), muscle creatine kinase (MCK), and alpha skeletal muscle actin (ASMA) was observed in exposed C2C12 cells. Moreover, the pretreatment with nifedipine (an L-type voltage-gated Ca(2+) channel blocker) led to a reduction of the Ca(2+)-ICR effect. Consequently, it induced a downregulation of the MYOG, MCK, and ASMA mRNA expression affecting adversely the differentiation process. Therefore, our data suggest that Ca(2+)-ICR exposure can upregulate C2C12 differentiation. Although further studies are needed, these results may have important implications in myodegenerative pathology therapies.

  20. SPARC is up-regulated during skeletal muscle regeneration and inhibits myoblast differentiation

    DEFF Research Database (Denmark)

    Petersson, Stine Juhl; Jørgensen, Louise Helskov; Andersen, Ditte C

    2013-01-01

    Skeletal muscle repair is mediated primarily by the muscle stem cell, the satellite cell. Several factors, including extracellular matrix, are known to regulate satellite cell function and regeneration. One factor, the matricellular Secreted Protein Acidic and Rich in Cysteine (SPARC) is highly up......-regulated during skeletal muscle disease, but its function remains elusive. In the present study, we demonstrate a prominent yet transient increase in SPARC mRNA and protein content during skeletal muscle regeneration that correlates with the expression profile of specific muscle factors like MyoD, Myf5, Myf6......, Myogenin, NCAM, CD34, and M-Cadherin, all known to be implicated in satellite cell activation/proliferation following muscle damage. This up regulation was detected in more cell types. Ectopic expression of SPARC in the muscle progenitor cell line C2C12 was performed to mimic the high levels of SPARC seen...

  1. The Tocotrienol-Rich Fraction Is Superior to Tocopherol in Promoting Myogenic Differentiation in the Prevention of Replicative Senescence of Myoblasts.

    Directory of Open Access Journals (Sweden)

    Shy Cian Khor

    Full Text Available Aging results in a loss of muscle mass and strength. Myoblasts play an important role in maintaining muscle mass through regenerative processes, which are impaired during aging. Vitamin E potentially ameliorates age-related phenotypes. Hence, this study aimed to determine the effects of the tocotrienol-rich fraction (TRF and α-tocopherol (ATF in protecting myoblasts from replicative senescence and promoting myogenic differentiation. Primary human myoblasts were cultured into young and senescent stages and were then treated with TRF or ATF for 24 h, followed by an analysis of cell proliferation, senescence biomarkers, cellular morphology and differentiation. Our data showed that replicative senescence impaired the normal regenerative processes of myoblasts, resulting in changes in cellular morphology, cell proliferation, senescence-associated β-galactosidase (SA-β-gal expression, myogenic differentiation and myogenic regulatory factors (MRFs expression. Treatment with both TRF and ATF was beneficial to senescent myoblasts in reclaiming the morphology of young cells, improved cell viability and decreased SA-β-gal expression. However, only TRF treatment increased BrdU incorporation in senescent myoblasts, as well as promoted myogenic differentiation through the modulation of MRFs at the mRNA and protein levels. MYOD1 and MYOG gene expression and myogenin protein expression were modulated in the early phases of myogenic differentiation. In conclusion, the tocotrienol-rich fraction is superior to α-tocopherol in ameliorating replicative senescence-related aberration and promoting differentiation via modulation of MRFs expression, indicating vitamin E potential in modulating replicative senescence of myoblasts.

  2. The Use of Platelet-Rich and Platelet-Poor Plasma to Enhance Differentiation of Skeletal Myoblasts: Implications for the Use of Autologous Blood Products for Muscle Regeneration.

    Science.gov (United States)

    Miroshnychenko, Olga; Chang, Wen-Teh; Dragoo, Jason L

    2017-03-01

    Platelet-rich plasma (PRP) has been used to augment tissue repair and regeneration after musculoskeletal injury. However, there is increasing clinical evidence that PRP does not show a consistent clinical effect. Purpose/Hypothesis: This study aimed to compare the effects of the following non-neutrophil-containing (leukocyte-poor) plasma fractions on human skeletal muscle myoblast (HSMM) differentiation: (1) PRP, (2) modified PRP (Mod-PRP), in which transforming growth factor β1 (TGF-β1) and myostatin (MSTN) were depleted, and (3) platelet-poor plasma (PPP). The hypothesis was that leukocyte-poor PRP would lead to myoblast proliferation (not differentiation), whereas certain modifications of PRP preparations would increase myoblast differentiation, which is necessary for skeletal muscle regeneration. Controlled laboratory study. Blood from 7 human donors was individually processed to simultaneously create leukocyte-poor fractions: PRP, Mod-PRP, PPP, and secondarily spun PRP and Mod-PRP (PRPss and Mod-PRPss, respectively). Mod-PRP was produced by removing TGF-β1 and MSTN from PRP using antibodies attached to sterile beads, while a second-stage centrifugal spin of PRP was performed to remove platelets. The biologics were individually added to cell culture groups. Analysis for induction into myoblast differentiation pathways included Western blot analysis, reverse-transcription polymerase chain reaction, and immunohistochemistry, as well as confocal microscopy to assess polynucleated myotubule formation. HSMMs cultured with PRP showed an increase in proliferation but no evidence of differentiation. Western blot analysis confirmed that MSTN and TGF-β1 could be decreased in Mod-PRP using antibody-coated beads, but this modification mildly improved myoblast differentiation. However, cell culture with PPP, PRPss, and Mod-PRPss led to a decreased proliferation rate but a significant induction of myoblast differentiation verified by increased multinucleated myotubule

  3. A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts

    Directory of Open Access Journals (Sweden)

    Knight James DR

    2012-03-01

    Full Text Available Abstract Background The p38α mitogen-activated protein kinase (MAPK is a critical mediator of myoblast differentiation, and does so in part through the phosphorylation and regulation of several transcription factors and chromatin remodelling proteins. However, whether p38α is involved in processes other than gene regulation during myogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear. Methods To further characterise the involvement of p38α during myoblast differentiation, we developed and applied a simple technique for identifying relevant in vivo kinase substrates and their phosphorylation sites. In addition to identifying substrates for one kinase, the technique can be used in vitro to compare multiple kinases in the same experiment, and we made use of this to study the substrate specificities of the p38α and β isoforms. Results Applying the technique to p38α resulted in the identification of seven in vivo phosphorylation sites on six proteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An in vitro comparison with p38β revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation. Conclusion Our results suggest p38α has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38β and other isoforms cannot compensate for its absence. The substrate-finding approach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and for uncovering the deeper mechanisms of phosphorylation-dependent cell signalling.

  4. Myocyte-derived Tnfsf14 is a survival factor necessary for myoblast differentiation and skeletal muscle regeneration.

    Science.gov (United States)

    Waldemer-Streyer, R J; Chen, J

    2015-12-31

    Adult skeletal muscle tissue has a uniquely robust capacity for regeneration, which gradually declines with aging or is compromised in muscle diseases. The cellular mechanisms regulating adult myogenesis remain incompletely understood. Here we identify the cytokine tumor necrosis factor superfamily member 14 (Tnfsf14) as a positive regulator of myoblast differentiation in culture and muscle regeneration in vivo. We find that Tnfsf14, as well as its cognate receptors herpes virus entry mediator (HVEM) and lymphotoxin β receptor (LTβR), are expressed in both differentiating myocytes and regenerating myofibers. Depletion of Tnfsf14 or either receptor inhibits myoblast differentiation and promotes apoptosis. Our results also suggest that Tnfsf14 regulates myogenesis by supporting cell survival and maintaining a sufficient pool of cells for fusion. In addition, we show that Akt mediates the survival and myogenic function of Tnfsf14. Importantly, local knockdown of Tnfsf14 is found to impair injury-induced muscle regeneration in a mouse model, affirming an important physiological role for Tnfsf14 in myogenesis in vivo. Furthermore, we demonstrate that localized overexpression of Tnfsf14 potently enhances muscle regeneration, and that this regenerative capacity of Tnfsf14 is dependent on Akt signaling. Taken together, our findings reveal a novel regulator of skeletal myogenesis and implicate Tnfsf14 in future therapeutic development.

  5. Culture of skeletal myoblasts from human donors aged over 40 years: dynamics of cell growth and expression of differentiation markers

    Directory of Open Access Journals (Sweden)

    Cherubino Paolo

    2005-05-01

    Full Text Available Abstract Background Local myogenesis, neoangiogenesis and homing of progenitor cells from the bone marrow appear to contribute to repair of the infarcted myocardium. Implantation into heart tissues of autologous skeletal myoblasts has been associated with improved contractile function in animal models and in humans with acute myocardial ischemia. Since heart infarction is most prevalent in individuals of over 40 years of age, we tested whether culture methods available in our laboratory were adequate to obtain sufficient numbers of differentiated skeletal myoblasts from muscle biopsy specimens obtained from patients aged 41 to 91. Methods and results No matter of donor age, differentiated skeletal muscle cells could be produced in vitro in amounts adequate for cellular therapy (≥300 millions. Using desmin as a cytoplasmic marker, about 50% cultured cells were differentiated along myogenic lineages and expressed proteins proper of skeletal muscle (myosin type I and II, actin, actinin, spectrin and dystrophin. Cytogenetic alterations were not detected in cultured muscle cells that had undergone at least 10 population doublings. Molecular methods employed for the screening of persistent viral infections evidenced that HCV failed to replicate in muscle cells cultured from one patient with chronic HCV infection. Conclusion The proposed culture methods appear to hold promise for aged patients not only in the field of cardiovascular medicine, but also in the urologic and orthopedic fields.

  6. DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zirong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China); Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Jin, Guorong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China); Lin, Shuibin [Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Lin, Xiumei [Department of Hematology, Guangzhou First Municipal People' s Hospital, Guangzhou 510180 (China); Gu, Yumei [Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Zhu, Yujuan; Hu, Chengbin; Zhang, Qingjiong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China); Wu, Lizi [Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Shen, Huangxuan, E-mail: shenhx@mail.sysu.edu.cn [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer CDA-II inhibits myogenic differentiation in a dose-dependent manner. Black-Right-Pointing-Pointer CDA-II repressed expression of muscle transcription factors and structural proteins. Black-Right-Pointing-Pointer CDA-II inhibited proliferation and migration of C2C12 myoblasts. -- Abstract: CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiation of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions.

  7. TAK-1/p38/nNFκB signaling inhibits myoblast differentiation by increasing levels of Activin A

    Directory of Open Access Journals (Sweden)

    Trendelenburg Anne

    2012-02-01

    Full Text Available Abstract Background Skeletal-muscle differentiation is required for the regeneration of myofibers after injury. The differentiation capacity of satellite cells is impaired in settings of old age, which is at least one factor in the onset of sarcopenia, the age-related loss of skeletal-muscle mass and major cause of frailty. One important cause of impaired regeneration is increased levels of transforming growth factor (TGF-β accompanied by reduced Notch signaling. Pro-inflammatory cytokines are also upregulated in aging, which led us hypothesize that they might potentially contribute to impaired regeneration in sarcopenia. Thus, in this study, we further analyzed the muscle differentiation-inhibition pathway mediated by pro-inflammatory cytokines in human skeletal muscle cells (HuSKMCs. Methods We studied the modulation of HuSKMC differentiation by the pro-inflammatory cytokines interleukin (IL-1α and tumor necrosis factor (TNF-α The grade of differentiation was determined by either imaging (fusion index or creatine kinase (CK activity, a marker of muscle differentiation. Secretion of TGF-β proteins during differentiation was assessed by using a TGF-β-responsive reporter-gene assay and further identified by means of pharmacological and genetic inhibitors. In addition, signaling events were monitored by western blotting and reverse transcription PCR, both in HuSKMC cultures and in samples from a rat sarcopenia study. Results The pro-inflammatory cytokines IL-1α and TNF-α block differentiation of human myoblasts into myotubes. This anti-differentiation effect requires activation of TGF-β-activated kinase (TAK-1. Using pharmacological and genetic inhibitors, the TAK-1 pathway could be traced to p38 and NFκB. Surprisingly, the anti-differentiation effect of the cytokines required the transcriptional upregulation of Activin A, which in turn acted through its established signaling pathway: ActRII/ALK/SMAD. Inhibition of Activin A signaling was

  8. Stretchable degradable and electroactive shape memory copolymers with tunable recovery temperature enhance myogenic differentiation.

    Science.gov (United States)

    Deng, Zexing; Guo, Yi; Zhao, Xin; Li, Longchao; Dong, Ruonan; Guo, Baolin; Ma, Peter X

    2016-12-01

    Development of flexible degradable electroactive shape memory polymers (ESMPs) with tunable switching temperature (around body temperature) for tissue engineering is still a challenge. Here we designed and synthesized a series of shape memory copolymers with electroactivity, super stretchability and tunable recovery temperature based on poly(ε-caprolactone) (PCL) with different molecular weight and conductive amino capped aniline trimer, and demonstrated their potential to enhance myogenic differentiation from C2C12 myoblast cells. We characterized the copolymers by Fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance ((1)H NMR), cyclic voltammetry (CV), ultraviolet-visible spectroscopy (UV-vis), differential scanning calorimetry (DSC), shape memory test, tensile test and in vitro enzymatic degradation study. The electroactive biodegradable shape memory copolymers showed great elasticity, tunable recovery temperature around 37°C, and good shape memory properties. Furthermore, proliferation and differentiation of C2C12 myoblasts were investigated on electroactive copolymers films, and they greatly enhanced the proliferation, myotube formation and related myogenic differentiation genes expression of C2C12 myoblasts compared to the pure PCL with molecular weight of 80,000. Our study suggests that these electroactive, highly stretchable, biodegradable shape memory polymers with tunable recovery temperature near the body temperature have great potential in skeletal muscle tissue engineering application. Conducting polymers can regulate cell behavior such cell adhesion, proliferation, and differentiation with or without electrical stimulation. Therefore, they have great potential for electrical signal sensitive tissue regeneration. Although conducting biomaterials with degradability have been developed, highly stretchable and electroactive degradable copolymers for soft tissue engineering have been rarely reported. On the other hand, shape

  9. Production of IL-6 by human myoblasts stimulated with Abeta: relevance in the pathogenesis of IBM.

    Science.gov (United States)

    Baron, P; Galimberti, D; Meda, L; Scarpini, E; Conti, G; Cogiamanian, F; Scarlato, G

    2001-11-13

    To determine whether amyloid-beta protein (Abeta) can induce the production of proinflammatory cytokines by cultured normal muscle cells. Sporadic inclusion body myositis (IBM) is characterized by the presence of rimmed vacuoles and fibrillary inclusions of Abeta in muscle fibers, and often inflammatory cells. Endomysial expression of proinflammatory molecules has suggested an ongoing immune process, but the site of sensitization and the mechanisms that trigger an inflammatory reaction is unknown. The authors used Northern blot analysis and specific immunoassays to study the expression and secretion in cell-free supernatants of tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) by purified human myoblasts and C2C12 mouse skeletal muscle cells incubated with Abeta[1-42] or Abeta[25-35] peptides. Nonstimulated muscle cells produced detectable IL-6, whereas secretion of IL-1beta and TNFalpha was absent. Incubation with Abeta peptides increased IL-6 production, whereas TNFalpha and IL-1beta levels remained undetectable. Northern blot analysis of Abeta-stimulated human myoblasts revealed an increase in IL-6 mRNA expression. Cultured muscle cells increase the constitutive production of IL-6 in response to local deposition of Abeta in sporadic IBM. IL-6 could be a CD8(+) proliferation and differentiation agent, an autocrine proteolysis-inducing factor of damaged myotubes, and a proliferation-stimulating agent for satellite cells to replace the destroyed myofibers in IBM.

  10. Functional analysis of SH3 domain containing ring finger 2 during the myogenic differentiation of quail myoblast cells

    Directory of Open Access Journals (Sweden)

    Si Won Kim

    2017-08-01

    Full Text Available Objective Owing to the public availability of complete genome sequences, including avian species, massive bioinformatics analyses may be conducted for computational gene prediction and the identification of gene regulatory networks through various informatics tools. However, to evaluate the biofunctional activity of a predicted target gene, in vivo and in vitro functional genomic analyses should be a prerequisite. Methods Due to a lack of quail genomic sequence information, we first identified the partial genomic structure and sequences of the quail SH3 domain containing ring finger 2 (SH3RF2 gene. Subsequently, SH3RF2 was knocked out using clustered regularly interspaced short palindromic repeat/Cas9 technology and single cell-derived SH3RF2 mutant sublines were established to study the biofunctional activity of SH3RF2 in quail myoblast (QM7 cells during muscle differentiation. Results Through a T7 endonuclease I assay and genotyping analysis, we established an SH3RF2 knockout (KO QM7#4 subline with 61 and 155 nucleotide deletion mutations in SH3RF2. After the induction of myotube differentiation, the expression profiles were analyzed and compared between regular QM7 and SH3RF2 KO QM7#4 cells by global RNA sequencing and bioinformatics analysis. Conclusion We did not detect any statistically significant role of SH3RF2 during myotube differentiation in QM7 myoblast cells. However, additional experiments are necessary to examine the biofunctional activity of SH3RF2 in cell proliferation and muscle growth.

  11. L6E9 Myoblasts Are Deficient of Myostatin and Additional TGF- Members Are Candidates to Developmentally Control Their Fiber Formation

    Directory of Open Access Journals (Sweden)

    Stefania Rossi

    2010-01-01

    Full Text Available This work provides evidence that the robust myoblast differentiation observed in L6E9 cells is causally linked to deficiency of myostatin, which, conversely, has been found to be expressed in C2C12 cells. However, despite the absence of endogenous myostatin, L6E9 myoblasts expressed functional Activin receptors type II (ActRIIs and follistatin as well as the highly related TGF- members Activins and GDF11, suggesting that in this cell line the regulation of fiber size might be under the control of multiple regulators regardless of myostatin. In line with this hypothesis, delivery of a dominant-negative ActRIIb form or the increase of follistatin, as obtained via Trichostatin treatment or stable transfection of a short human follistatin form, enhanced the L6E9 cell differentiation and further increased the size of myotubes, suggesting that L6E9 myoblasts provide a spontaneous myostatin knock-out in vitro model to study TGF- ligands involved in developmental regulation of fiber size.

  12. Cellular Trafficking of Phospholamban and Formation of Functional Sarcoplasmic Reticulum During Myocyte DIfferentiation

    Energy Technology Data Exchange (ETDEWEB)

    Stenoien, David L.; Knyushko, Tatyana V.; Londono, Monica P.; Opresko, Lee; Mayer, M. Uljana; Brady, Scott T.; Squier, Thomas C.; Bigelow, Diana J.

    2007-06-01

    The sarco/endoplasmic reticulum Ca-ATPase (SERCA) family members are transmembrane proteins that play an essential role in regulating intracellular calcium levels. Phospholamban (PLB), a 52 amino acid phosphoprotein, regulates SERCA activity in adult heart and skeletal muscle. Using the C2C12 myocyte cell line, we find endogenous PLB constitutively expressed in both myoblasts and myotubes, whereas SERCA expression coincides with activation of the differentiation program. PLB has a punctuate distribution in myoblasts changing to a reticular distribution in myotubes where it colocalizes with SERCAs. To examine the distribution and dynamics of PLB and SERCA, we expressed fluorescent fusion proteins (GFP, CFP, and YFP) of PLB and SERCA in myoblasts. Coexpressed PLB and SERCA localize to distinct cellular compartments in myoblasts but begin to colocalize as cells differentiate. Fluorescence Recovery After Photobleaching (FRAP) studies show different recovery patterns for each protein in myoblasts confirming their localization to distinct compartments. To extend these studies, we created stable cell lines expressing O6-alkylguanine-DNA alkyltransferase (AGT) fusions with PLB or SERCA to track their localization as myocytes differentiate. These experiments demonstrate that PLB localizes to punctate vesicles in myoblasts and adopts a reticular distribution that coincides with SERCA distribution after differentiation. Colocalization experiments indicate that a subset of PLB in myoblasts colocalizes with endosomes, Golgi, and the plasma membrane however PLB also localizes to other, as yet unidentified vesicles. Our results indicate that differentiation plays a critical role in regulating PLB distribution to ensure its colocalization within the same cellular compartment as SERCA in differentiated cells. The presence and altered distribution of PLB in undifferentiated myoblasts raises the possibility that this protein has additional functions distinct from SERCA regulation.

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

    Directory of Open Access Journals (Sweden)

    Si Won Kim

    2017-07-01

    Full Text Available 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.

  14. Permethrin decreased insulin-stimulated AKT phosphorylation dependent on extracellular signal-regulated kinase-1 (ERK), but not AMP-activated protein kinase α (AMPKα), in C2C12 myotubes.

    Science.gov (United States)

    Sun, Quancai; Peng, Ye; Qi, Weipeng; Kim, Yoo; Clark, John M; Kim, Daeyoung; Park, Yeonhwa

    2017-11-01

    Previously 10 μM permethrin (38.7% cis and 59.4% trans isomers), a pyrethroid insecticide widely used in agriculture and household products for pest control, was reported to reduce insulin-stimulated glucose uptake and phosphorylation of protein kinase B (p-AKT) in C2C12 mouse myotubes. The underlying mechanisms on how permethrin decreases insulin-stimulated AKT phosphorylation, however, are unknown. Thus, the goal of this study was to determine the possible mechanism(s) through which permethrin reduced insulin-stimulated AKT phosphorylation in C2C12 myotubes. Permethrin treatment, at 10 μM, decreased insulin-stimulated membrane glucose transporter type 4 (GLUT4) and AKT phosphorylation, and increased insulin receptor substrate 1 (IRS1) Ser307 phosphorylation in the presence of insulin. The inactivation of AKT by permethrin was independent of AMPKα. ERK inactivation by U0126, however, restored insulin-stimulated AKT phosphorylation, which was decreased by permethrin treatment. These results suggest that permethrin decreased insulin-stimulated AKT phosphorylation via ERK activation, but not by AMPKα inactivation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Inulin Increases Glucose Transport in C2C12 Myotubes and HepG2 Cells via Activation of AMP-Activated Protein Kinase and Phosphatidylinositol 3-Kinase Pathways

    Science.gov (United States)

    Yun, Hee; Lee, Jong Hwa; Park, Chang Eun; Kim, Min-Jung; Min, Byung-Il; Bae, Hyunsu; Choe, Wonchae; Kang, Insug; Kim, Sung-Soo

    2009-01-01

    Abstract Inulin, a naturally occurring, functional food ingredient found in various edible plants, has been reported to exert potential health benefits, including decreased risk of colonic diseases, non–insulin-dependent diabetes, obesity, osteoporosis, and cancer. However, the mechanism of the antidiabetic activity of inulin has not yet been elucidated. In this study, we showed that inulin increased the uptake of glucose in C2C12 myotubes, which was associated with both AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3-K) signaling pathways, but both of these pathways appeared to transmit their signals in an independent manner. Moreover, we found that inulin was able to increase the uptake of glucose in C2C12 myotubes in which insulin resistance was induced by exposing cells to high glucose concentrations. The identical effects of inulin were also observed in HepG2 hepatoma cells. Collectively, we report the antidiabetic activity of inulin and further demonstrate for the first time that such activity is associated with AMPK and PI3-K activation. PMID:19857065

  16. Boehmeria nivea Stimulates Glucose Uptake by Activating Peroxisome Proliferator-Activated Receptor Gamma in C2C12 Cells and Improves Glucose Intolerance in Mice Fed a High-Fat Diet

    Directory of Open Access Journals (Sweden)

    Sung Hee Kim

    2013-01-01

    Full Text Available We examined the antidiabetic property of Boehmeria nivea (L. Gaud. Ethanolic extract of Boehmeria nivea (L. Gaud. (EBN increased the uptake of 2-[N-(nitrobenz-2-oxa-1,3-diazol-4-ylamino]-2-deoxy-d-glucose in C2C12 myotubes. To examine the mechanisms underlying EBN-mediated increase in glucose uptake, we examined the transcriptional activity and expression of peroxisome proliferator-activated receptor gamma (PPAR-γ, a pivotal target for glucose metabolism in C2C12 myotubes. We found that the EBN increased both the transcriptional activity and mRNA expression levels of PPAR-γ. In addition, we measured phosphorylation and expression levels of other targets of glucose metabolism, such as AMP-activated protein kinase (AMPK and protein kinase B (Akt/PKB. We found that EBN did not alter the phosphorylation or expression levels of these proteins in a time- or dose-dependent manner, which suggested that EBN stimulates glucose uptake through a PPAR-γ-dependent mechanism. Further, we investigated the antidiabetic property of EBN using mice fed a high-fat diet (HFD. Administration of 0.5% EBN reduced the HFD-induced increase in body weight, total cholesterol level, and fatty liver and improved the impaired fasting glucose level, blood insulin content, and glucose intolerance. These results suggest that EBN had an antidiabetic effect in cell culture and animal systems and may be useful for preventing diabetes.

  17. Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system

    Science.gov (United States)

    Torgan, C. E.; Burge, S. S.; Collinsworth, A. M.; Truskey, G. A.; Kraus, W. E.

    2000-01-01

    The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p muscle growth and repair during spaceflight.

  18. Characterization of the Methylation Status of and Myogenic Regulator Factors in Cell Myogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Zhe Chao

    2016-07-01

    Full Text Available Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

  19. Mechano-growth factor peptide, the COOH terminus of unprocessed insulin-like growth factor 1, has no apparent effect on myoblasts or primary muscle stem cells.

    Science.gov (United States)

    Fornaro, Mara; Hinken, Aaron C; Needle, Saul; Hu, Erding; Trendelenburg, Anne-Ulrike; Mayer, Angelika; Rosenstiel, Antonia; Chang, Calvin; Meier, Viktor; Billin, Andrew N; Becherer, J David; Brace, Arthur D; Evans, William J; Glass, David J; Russell, Alan J

    2014-01-15

    A splice form of IGF-1, IGF-1Eb, is upregulated after exercise or injury. Physiological responses have been ascribed to the 24-amino acid COOH-terminal peptide that is cleaved from the NH3-terminal 70-amino acid mature IGF-1 protein. This COOH-terminal peptide was termed "mechano-growth factor" (MGF). Activities claimed for the MGF peptide included enhancing muscle satellite cell proliferation and delaying myoblast fusion. As such, MGF could represent a promising strategy to improve muscle regeneration. Thus, at our two pharmaceutical companies, we attempted to reproduce the claimed effect of MGF peptides on human and mouse muscle myoblast proliferation and differentiation in vitro. Concentrations of peptide up to 500 ng/ml failed to increase the proliferation of C2C12 cells or primary human skeletal muscle myoblasts. In contrast, all cell types exhibited a proliferative response to mature IGF-1 or full-length IGF-1Eb. MGF also failed to inhibit the differentiation of myoblasts into myotubes. To address whether the response to MGF was lost in these tissue culture lines, we measured proliferation and differentiation of primary mouse skeletal muscle stem cells exposed to MGF. This, too, failed to demonstrate a significant effect. Finally, we tested whether MGF could alter a separate documented in vitro effect of the peptide, activation of p-ERK, but not p-Akt, in cardiac myocytes. Although a robust response to IGF-1 was observed, there were no demonstrated activating responses from the native or a stabilized MGF peptide. These results call in to question whether there is a physiological role for MGF.

  20. Bone marrow mesenchymal stromal cells stimulate skeletal myoblast proliferation through the paracrine release of VEGF.

    Directory of Open Access Journals (Sweden)

    Chiara Sassoli

    Full Text Available Mesenchymal stromal cells (MSCs are the leading cell candidates in the field of regenerative medicine. These cells have also been successfully used to improve skeletal muscle repair/regeneration; however, the mechanisms responsible for their beneficial effects remain to be clarified. On this basis, in the present study, we evaluated in a co-culture system, the ability of bone-marrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. We found that myoblast proliferation was greatly enhanced in the co-culture as judged by time lapse videomicroscopy, cyclin A expression and EdU incorporation. Moreover, myoblasts immunomagnetically separated from MSCs after co-culture expressed higher mRNA and protein levels of Notch-1, a key determinant of myoblast activation and proliferation, as compared with the single culture. Notch-1 intracellular domain and nuclear localization of Hes-1, a Notch-1 target gene, were also increased in the co-culture. Interestingly, the myoblastic response was mainly dependent on the paracrine release of vascular endothelial growth factor (VEGF by MSCs. Indeed, the addition of MSC-derived conditioned medium (CM to C2C12 cells yielded similar results as those observed in the co-culture and increased the phosphorylation and expression levels of VEGFR. The treatment with the selective pharmacological VEGFR inhibitor, KRN633, resulted in a marked attenuation of the receptor activation and concomitantly inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signaling. In conclusion, this study provides novel evidence for a role of MSCs in stimulating myoblast cell proliferation and suggests that the functional interaction between the two cell types may be exploited for the development of new and more efficient cell-based skeletal muscle repair strategies.

  1. Mouse C2 myoblast cells resist HVJ (Sendai virus)-mediated cell fusion in the proliferating stage but become capable of fusion after differentiation.

    Science.gov (United States)

    Hirayama, E; Nakanishi, M; Honda, N; Kim, J

    1999-05-01

    To investigate the mechanism of myoblast fusion, we attempted to prepare artificial myotubes of mouse C2 myoblast cells using the hemagglutinating virus of Japan (HVJ, Sendai virus). Proliferating C2 cells showed strong resistance to HVJ-mediated cell fusion and remained morphologically unchanged even though massive numbers of virions adsorbed onto their surface. They showed no membrane disruption, which occurs in the early stage of cell fusion induced by HVJ. These observations suggest that proliferating C2 cells are resistant to HVJ-mediated cell fusion. However, upon induction of differentiation, C2 cells gradually became capable of fusion induced by HVJ and then even generated heterokaryons with Ehrlich ascites tumor cells. When differentiated C2 cells that had become fusion-sensitive were treated with HVJ in the presence of EDTA, they did not fuse but degenerated, suggesting that their cell membranes were transiently disrupted by interaction with HVJ. These results suggest that the cell membranes of myoblasts change to a fusion-capable state during the process of differentiation.

  2. Akt2-mediated phosphorylation of Pitx2 controls Ccnd1 mRNA decay during muscle cell differentiation.

    Science.gov (United States)

    Gherzi, R; Trabucchi, M; Ponassi, M; Gallouzi, I-E; Rosenfeld, M G; Briata, P

    2010-06-01

    Paired-like homeodomain 2 (Pitx2), first identified as the gene responsible for the Axenfeld-Rieger syndrome, encodes a protein factor that, controlling cell proliferation in a tissue-specific manner, has a crucial role in morphogenesis. During embryonic development, Pitx2 exerts a role in the expansion of muscle progenitors and is expressed at all stages of myogenic progression. In this study, we show that Pitx2 is phosphorylated by the protein kinase Akt2 and is necessary to ensure proper C2C12 myoblast proliferation and differentiation. Pitx2 associates with a ribonucleoprotein complex that includes the mRNA stabilizing factor HuR and sustains Ccnd1 (also known as Cyclin D1) expression, thereby prolonging its mRNA half-life. When the differentiation program is initiated, phosphorylation by Akt2 impairs the ability of Pitx2 to associate with the Ccnd1 mRNA-stabilizing complex that includes HuR and, as a consequence, Ccnd1 mRNA half-life is shortened. We propose that unphosphorylated Pitx2 is required to favor HuR-mediated Ccnd1 mRNA stabilization, thus sustaining myoblast proliferation. Upon Akt2-phosphorylation, the complex Pitx2/HuR/Ccnd1 mRNA dissociates and Ccnd1 mRNA is destabilized. These events contribute to the switch of C2C12 cells from a proliferating to a differentiating phenotype.

  3. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Chieri [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi, E-mail: tsuyo-i@huhs.ac.jp [Division of Pharmacotherapy, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We investigated the role of S1P signaling for osteoblast differentiation. Black-Right-Pointing-Pointer Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. Black-Right-Pointing-Pointer S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. Black-Right-Pointing-Pointer MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P

  4. Epicatechin elicits MyoD-dependent myoblast differentiation and myogenic conversion of fibroblasts.

    Directory of Open Access Journals (Sweden)

    Sang-Jin Lee

    Full Text Available Prevention of age-associated reduction in muscle mass and function is required to manage a healthy life. Supplemental (--Epicatechin (EC appears to act as a potential regulator for muscle growth and strength. However, its cellular and molecular mechanisms as a potential muscle growth agent have not been studied well. In the current study, we investigated a role of EC in differentiation of muscle progenitors to gain the molecular insight into how EC regulates muscle growth. EC enhanced myogenic differentiation in a dose-dependent manner through stimulation of promyogenic signaling pathways, p38MAPK and Akt. EC treatment elevated MyoD activity by enhancing its heterodimerization with E protein. Consistently, EC also positively regulated myogenic conversion and differentiation of fibroblasts. In conclusion, EC has a potential as a therapeutic or nutraceutical remedy to treat degenerative muscle diseases or age-related muscle weakness.

  5. Mechanical stimuli activation of calpain is required for myoblast differentiation and occurs via an ERK/MAP kinase signaling pathway

    DEFF Research Database (Denmark)

    Grossi, Alberto; Karlsson, Anders H; Lawson, Moira Ann

    tissues. Stimulation due to stretch- or load-induced signaling is now beginning to be understood as a factor which affects various signal transduction pathways, gene sequences and protein synthesis. Evidence of the involvement of mitogen-activated protein kinase (MAPK) cascade activation in myoblast...

  6. Network Analysis for the Identification of Differentially Expressed Hub Genes Using Myogenin Knock-down Muscle Satellite Cells.

    Directory of Open Access Journals (Sweden)

    Adeel Malik

    Full Text Available Muscle, a multinucleate syncytium formed by the fusion of mononuclear myoblasts, arises from quiescent progenitors (satellite cells via activation of muscle-specific transcription factors (MyoD, Myf5, myogenin: MYOG, and MRF4. Subsequent to a decline in Pax7, induction in the expression of MYOG is a hallmark of myoblasts that have entered the differentiation phase following cell cycle withdrawal. It is evident that MYOG function cannot be compensated by any other myogenic regulatory factors (MRFs. Despite a plethora of information available regarding MYOG, the mechanism by which MYOG regulates muscle cell differentiation has not yet been identified. Using an RNA-Seq approach, analysis of MYOG knock-down muscle satellite cells (MSCs have shown that genes associated with cell cycle and division, DNA replication, and phosphate metabolism are differentially expressed. By constructing an interaction network of differentially expressed genes (DEGs using GeneMANIA, cadherin-associated protein (CTNNA2 was identified as the main hub gene in the network with highest node degree. Four functional clusters (modules or communities were identified in the network and the functional enrichment analysis revealed that genes included in these clusters significantly contribute to skeletal muscle development. To confirm this finding, in vitro studies revealed increased expression of CTNNA2 in MSCs on day 12 compared to day 10. Expression of CTNNA2 was decreased in MYOG knock-down cells. However, knocking down CTNNA2, which leads to increased expression of extracellular matrix (ECM genes (type I collagen α1 and type I collagen α2 along with myostatin (MSTN, was not found significantly affecting the expression of MYOG in C2C12 cells. We therefore propose that MYOG exerts its regulatory effects by acting upstream of CTNNA2, which in turn regulates the differentiation of C2C12 cells via interaction with ECM genes. Taken together, these findings highlight a new

  7. ErbB3 binding protein-1 (Ebp1) controls proliferation and myogenic differentiation of muscle stem cells.

    Science.gov (United States)

    Figeac, Nicolas; Serralbo, Olivier; Marcelle, Christophe; Zammit, Peter S

    2014-02-01

    Satellite cells are resident stem cells of skeletal muscle, supplying myoblasts for post-natal muscle growth, hypertrophy and repair. Many regulatory networks control satellite cell function, which includes EGF signalling via the ErbB family of receptors. Here we investigated the role of ErbB3 binding protein-1 (Ebp1) in regulation of myogenic stem cell proliferation and differentiation. Ebp1 is a well-conserved DNA/RNA binding protein that is implicated in cell growth, apoptosis and differentiation in many cell types. Of the two main Ebp1 isoforms, only p48 was expressed in satellite cells and C2C12 myoblasts. Although not present in quiescent satellite cells, p48 was strongly induced during activation, remaining at high levels during proliferation and differentiation. While retroviral-mediated over-expression of Ebp1 had only minor effects, siRNA-mediated Ebp1 knockdown inhibited both proliferation and differentiation of satellite cells and C2C12 myoblasts, with a clear failure of myotube formation. Ebp1-knockdown significantly reduced ErbB3 receptor levels, yet over-expression of ErbB3 in Ebp1 knockdown cells did not rescue differentiation. Ebp1 was also expressed by muscle cells during developmental myogenesis in mouse. Since Ebp1 is well-conserved between mouse and chick, we switched to chick to examine its role in muscle formation. In chick embryo, Ebp1 was expressed in the dermomyotome, and myogenic differentiation of muscle progenitors was inhibited by specific Ebp1 down-regulation using shRNA electroporation. These observations demonstrate a conserved function of Ebp1 in the regulation of embryonic muscle progenitors and adult muscle stem cells, which likely operates independently of ErbB3 signaling. © 2013 Published by Elsevier Inc.

  8. Engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation.

    Science.gov (United States)

    van der Schaft, Daisy W J; van Spreeuwel, Ariane C C; Boonen, Kristel J M; Langelaan, Marloes L P; Bouten, Carlijn V C; Baaijens, Frank P T

    2013-03-19

    Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative (1). The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues (2,3). Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts (4), neonatal muscle derived progenitor cells (5), cells derived from adult muscle tissues from other species such as human (6) or even induced pluripotent stem cells (iPS cells) (7). Cell contractility causes alignment of the cells along the long axis of the construct (8,9) and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent (8). Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while

  9. Mir193b-365 is essential for brown fat differentiation.

    Science.gov (United States)

    Sun, Lei; Xie, Huangming; Mori, Marcelo A; Alexander, Ryan; Yuan, Bingbing; Hattangadi, Shilpa M; Liu, Qingqing; Kahn, C Ronald; Lodish, Harvey F

    2011-07-10

    Mammals have two principal types of fat. White adipose tissue primarily serves to store extra energy as triglycerides, whereas brown adipose tissue is specialized to burn lipids for heat generation and energy expenditure as a defence against cold and obesity. Recent studies have demonstrated that brown adipocytes arise in vivo from a Myf5-positive, myoblastic progenitor by the action of Prdm16 (PR domain containing 16). Here, we identified a brown-fat-enriched miRNA cluster, MiR-193b-365, as a key regulator of brown fat development. Blocking miR-193b and/or miR-365 in primary brown preadipocytes markedly impaired brown adipocyte adipogenesis by enhancing Runx1t1 (runt-related transcription factor 1; translocated to, 1) expression, whereas myogenic markers were significantly induced. Forced expression of Mir193b and/or Mir365 in C2C12 myoblasts blocked the entire programme of myogenesis, and, in adipogenic conditions, miR-193b induced myoblasts to differentiate into brown adipocytes. Mir193b-365 was upregulated by Prdm16 partially through Pparα. Our results demonstrate that Mir193b-365 serves as an essential regulator for brown fat differentiation, in part by repressing myogenesis.

  10. Eburicoic Acid, a Triterpenoid Compound from Antrodia camphorata, Displays Antidiabetic and Antihyperlipidemic Effects in Palmitate-Treated C2C12 Myotubes and in High-Fat Diet-Fed Mice

    Directory of Open Access Journals (Sweden)

    Cheng-Hsiu Lin

    2017-11-01

    Full Text Available This study was designed to investigate the antidiabetic and antihyperlipidemic effects and mechanisms of eburicoic acid (TRR; one component of Antrodia camphorata in vitro and in an animal model for 14 weeks. Expression levels of membrane glucose transporter type 4 (GLUT4; phospho-5′-adenosine monophosphate-activated protein kinase (AMPK/total AMPK; and phospho-Akt/total-Akt in insulin-resistant C2C12 myotube cells were significantly decreased by palmitate; and such decrease was prevented and restored by TRR at different concentrations. A group of control (CON was on low-fat diet over a period of 14 weeks. Diabetic mice; after high-fat-diet (HFD induction for 10 weeks; were randomly divided into six groups and were given once a day oral gavage doses of either TRR (at three dosage levels; fenofibrate (Feno (at 0.25 g/kg body weight; metformin (Metf (at 0.3 g/kg body weight; or vehicle (distilled water (HF group over a period of 4 weeks and still on HFD. Levels of glucose; triglyceride; free fatty acid (FFA; insulin; and leptin in blood were increased in 14-week HFD-fed mice as compared to the CON group; and the increases were prevented by TRR, Feno, or Metf as compared to the HF group. Moreover, HFD-induction displayed a decrease in circulating adiponectin levels, and the decrease was prevented by TRR, Feno, or Metf treatment. The overall effect of TRR is to decrease glucose and triglyceride levels and improved peripheral insulin sensitivity. Eburicoic acid, Feno, and Metf displayed both enhanced expression levels of phospho-AMPK and membrane expression levels of GLUT4 in the skeletal muscle of HFD-fed mice to facilitate glucose uptake with consequent enhanced hepatic expression levels of phospho-AMPK in the liver and phosphorylation of the transcription factor forkhead box protein O1 (FOXO1 but decreased messenger RNA (mRNA of phosphenolpyruvate carboxykinase (PEPCK to inhibit hepatic glucose production; resulting in lowered blood glucose

  11. CRYOPRESERVATION EFFECTS ON RECOMBINANT MYOBLASTS ENCAPSULATED IN ADHESIVE ALGINATE HYDROGELS

    Science.gov (United States)

    Ahmad, Hajira F.; Sambanis, Athanassios

    2013-01-01

    Cell encapsulation in hydrogels is widely used in tissue engineering applications, including encapsulation of islets or other insulin-secreting cells in pancreatic substitutes. Use of adhesive, bio-functionalized hydrogels is receiving increasing attention, as cell-matrix interactions in 3-D can be important for various cell processes. With pancreatic substitutes, studies have indicated benefits of 3-D adhesion on the viability and/or function of insulin-secreting cells. As long-term storage of microencapsulated cells is critical for their clinical translation, cryopreservation of cells in hydrogels is actively being investigated. Previous studies have examined the cryopreservation response of cells encapsulated in non-adhesive hydrogels using conventional freezing and/or vitrification (ice-free cryopreservation), however, none have systematically compared the two cryopreservation methods with cells encapsulated within an adhesive 3-D environment. The latter would be significant, as evidence suggests adhesion influences cellular response to cryopreservation. Thus, the objective of this study was to determine the response to conventional freezing and vitrification of insulin-secreting cells encapsulated in an adhesive biomimetic hydrogel. Recombinant insulin-secreting C2C12 myoblasts were encapsulated in oxidized RGD-alginate and cultured 1 or 4 days post-encapsulation, cryopreserved, and assessed up to 3 days post-warming for metabolic activity and insulin secretion, and one day post-warming for cell morphology. Besides certain transient differences of the vitrified group relative to the Fresh control, both conventional freezing and vitrification maintained metabolism, secretion and morphology of the recombinant C2C12 cells. Thus, due to a simpler procedure and slightly superior results, conventional freezing is recommended over vitrification for the cryopreservation of C2C12 cells in oxidized RGD-modified alginate. PMID:23499987

  12. Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling

    Science.gov (United States)

    Fitzpatrick, Vincent; Fourel, Laure; Destaing, Olivier; Gilde, Flora; Albigès-Rizo, Corinne; Picart, Catherine; Boudou, Thomas

    2017-01-01

    In vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro.

  13. Mesenchymal Stromal Cell Secreted Sphingosine 1-Phosphate (S1P) Exerts a Stimulatory Effect on Skeletal Myoblast Proliferation

    Science.gov (United States)

    Tani, Alessia; Anderloni, Giulia; Pierucci, Federica; Matteini, Francesca; Chellini, Flaminia; Zecchi Orlandini, Sandra; Meacci, Elisabetta

    2014-01-01

    Bone-marrow-derived mesenchymal stromal cells (MSCs) have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration. However, MSC-derived trophic molecules have been poorly characterized. The aim of this study was to investigate paracrine signaling effects of MSCs on skeletal myoblasts. It was found, using a biochemical and morphological approach that sphingosine 1-phosphate (S1P), a natural bioactive lipid exerting a broad range of muscle cell responses, is secreted by MSCs and represents an important factor by which these cells exert their stimulatory effects on C2C12 myoblast and satellite cell proliferation. Indeed, exposure to conditioned medium obtained from MSCs cultured in the presence of the selective sphingosine kinase inhibitor (iSK), blocked increased cell proliferation caused by the conditioned medium from untreated MSCs, and the addition of exogenous S1P in the conditioned medium from MSCs pre-treated with iSK further increased myoblast proliferation. Finally, we also demonstrated that the myoblast response to MSC-secreted vascular endothelial growth factor (VEGF) involves the release of S1P from C2C12 cells. Our data may have important implications in the optimization of cell-based strategies to promote skeletal muscle regeneration. PMID:25264785

  14. Myoblast-based cardiac repair: xenomyoblast versus allomyoblast transplantation.

    Science.gov (United States)

    Guo, Changfa; Haider, Husnain Kh; Shim, Winston S N; Tan, Ru-San; Ye, Lei; Jiang, Shujia; Law, Peter K; Wong, Philip; Sim, Eugene K W

    2007-11-01

    We sought to investigate immune cell kinetics in relation to skeletal myoblast survival and heart function improvement after nonautologous skeletal myoblast transplantation in a rat model of myocardial infarction. One week after myocardial infarction, 208 Wistar rats were grouped into group 1 (n = 24, receiving 150 muL of medium only), group 2 (n = 24, receiving 150 muL of medium and cyclosporine [INN: ciclosporin]), group 3 (n = 40, human skeletal myoblast transplantation), group 4 (n = 40, human skeletal myoblast transplantation with cyclosporine treatment), group 5 (n = 40, rat skeletal myoblast transplantation), and group 6 (n = 40, rat skeletal myoblast transplantation with cyclosporine treatment). The hearts were harvested at 10 minutes and 1, 4, 7, and 28 days after cell transplantation. Skeletal myoblast survival was confirmed by means of immunohistochemical studies and quantified by using real-time polymerase chain reaction. Host immune responses were assessed by immunostaining for macrophages and CD4+ and CD8+ lymphocytes. Heart function was evaluated by means of echocardiographic analysis. The majority of macrophages and lymphocytes infiltrated in the acute phase (from day 1 to day 7) and then subsided by day 28. The donor skeletal myoblasts survived and differentiated well in all skeletal myoblast transplantation groups. Allogeneic skeletal myoblasts showed a superior survival rate than xenogeneic skeletal myoblasts (P Cyclosporine inhibited the infiltration of the immunocytes, enhanced skeletal myoblast survival, and improved heart performance compared with that seen in the groups not receiving cyclosporine treatment (P transplantation into infarcted myocardium. After inhibition of immunocyte infiltration by means of immunosuppressive treatment, skeletal myoblast survival is enhanced, with improved heart performance. These findings suggest the feasibility of nonautologous myoblast transplantation with immunosuppressive treatment.

  15. The inhibition of cultured myoblast differentiation by the simian virus 40 large T antigen occurs after myogenin expression and Rb up-regulation and is not exerted by transformation-competent cytoplasmic mutants.

    OpenAIRE

    Tedesco, D; Caruso, M; Fischer-Fantuzzi, L; Vesco, C

    1995-01-01

    We have investigated the mechanism by which the simian virus 40 large T antigen (SVLT) interferes with the differentiation of C2 myoblasts. SVLT mutants, defective either in the Rb binding site, near the N-terminal end, in a region that affects binding to p53, or in the nuclear transport signal, were also employed to determine whether the interference was especially dependent on these functional domains. It was found that wild-type (wt) SVLT strongly inhibited the terminal differentiation of ...

  16. PLC-β1 and cell differentiation: An insight into myogenesis and osteogenesis.

    Science.gov (United States)

    Ramazzotti, Giulia; Faenza, Irene; Fiume, Roberta; Billi, Anna Maria; Manzoli, Lucia; Mongiorgi, Sara; Ratti, Stefano; McCubrey, James A; Suh, Pann-Ghill; Cocco, Lucio; Follo, Matilde Y

    2017-01-01

    Phosphoinositide-phospholipase C-β1 (PLC-β1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation and osteogenesis. During myogenic differentiation of murine C2C12 myoblasts, PLC-β1 signaling pathway involves the Inositol Polyphosphate Multikinase (IPMK) and β-catenin as downstream effectors. By means of c-jun binding to cyclin D3 promoter, the activation of PLC-β1 pathway determines cyclin D3 accumulation. However, osteogenesis requires PLC-β1 expression and up-regulation but it does not affect cyclin D3 levels, suggesting that the two processes require the activation of different mediators. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Hybrid Randomly Electrospun Poly(lactic-co-glycolic acid):Poly(ethylene oxide) (PLGA:PEO) Fibrous Scaffolds Enhancing Myoblast Differentiation and Alignment.

    Science.gov (United States)

    Evrova, Olivera; Hosseini, Vahid; Milleret, Vincent; Palazzolo, Gemma; Zenobi-Wong, Marcy; Sulser, Tullio; Buschmann, Johanna; Eberli, Daniel

    2016-11-23

    Cellular responses are regulated by their microenvironments, and engineered synthetic scaffolds can offer control over different microenvironment properties. This important relationship can be used as a tool to manipulate cell fate and cell responses for different biomedical applications. We show for the first time in this study how blending of poly(ethylene oxide) (PEO) to poly(lactic-co-glycolic acid) (PLGA) fibers to yield hybrid scaffolds changes the physical and mechanical properties of PLGA fibrous scaffolds and in turn affects cellular response. For this purpose we employed electrospinning to create fibrous scaffolds mimicking the basic structural properties of the native extracellular matrix. We introduced PEO to PLGA electrospun fibers by spinning a blend of PLGA:PEO polymer solutions in different ratios. PEO served as a sacrificial component within the fibers upon hydration, leading to pore formation in the fibers, fiber twisting, increased scaffold disintegration, and hydrophilicity, decreased Young's modulus, and significantly improved strain at break of initially electrospun scaffolds. We observed that the blended PLGA:PEO fibrous scaffolds supported myoblast adhesion and proliferation and resulted in increased myotube formation and self-alignment, when compared to PLGA-only scaffolds, even though the scaffolds were randomly oriented. The 50:50 PLGA:PEO blended scaffold showed the most promising results in terms of mechanical properties, myotube formation, and alignment, suggesting an optimal microenvironment for myoblast differentiation from the PLGA:PEO blends tested. The explored approach for tuning fiber properties can easily extend to other polymeric scaffolds and provides a valuable tool to engineer fibrillar microenvironments for several biomedical applications.

  18. Loss of MyoD Promotes Fate Transdifferentiation of Myoblasts Into Brown Adipocytes

    Directory of Open Access Journals (Sweden)

    Chao Wang

    2017-02-01

    Full Text Available Brown adipose tissue (BAT represents a promising agent to ameliorate obesity and other metabolic disorders. However, the abundance of BAT decreases with age and BAT paucity is a common feature of obese subjects. As brown adipocytes and myoblasts share a common Myf5 lineage origin, elucidating the molecular mechanisms underlying the fate choices of brown adipocytes versus myoblasts may lead to novel approaches to expand BAT mass. Here we identify MyoD as a key negative regulator of brown adipocyte development. CRISPR/CAS9-mediated deletion of MyoD in C2C12 myoblasts facilitates their adipogenic transdifferentiation. MyoD knockout downregulates miR-133 and upregulates the miR-133 target Igf1r, leading to amplification of PI3K–Akt signaling. Accordingly, inhibition of PI3K or Akt abolishes the adipogenic gene expression of MyoD null myoblasts. Strikingly, loss of MyoD converts satellite cell-derived primary myoblasts to brown adipocytes through upregulation of Prdm16, a target of miR-133 and key determinant of brown adipocyte fate. Conversely, forced expression of MyoD in brown preadipocytes blocks brown adipogenesis and upregulates the expression of myogenic genes. Importantly, miR-133a knockout significantly blunts the inhibitory effect of MyoD on brown adipogenesis. Our results establish MyoD as a negative regulator of brown adipocyte development by upregulating miR-133 to suppress Akt signaling and Prdm16.

  19. Skeletal muscle differentiation evokes endogenous XIAP to restrict the apoptotic pathway.

    Science.gov (United States)

    Smith, Michelle I; Huang, Yolanda Y; Deshmukh, Mohanish

    2009-01-01

    Myotube apoptosis occurs normally during muscle development and aging but it can lead to destruction of skeletal muscle in neuromuscular diseases. Therefore, understanding how myotube apoptosis is regulated is important for developing novel strategies for treatment of muscle loss. We investigated the regulation of apoptosis in skeletal muscle and report a striking increase in resistance to apoptosis following differentiation. We find mitotic C2C12 cells (myoblast-like cells) are sensitive to cytosolic cytochrome c microinjection. However, differentiated C2C12 cells (myotube-like cells) and primary myotubes are markedly resistant. This resistance is due to endogenous X-linked inhibitor of apoptotic protein (XIAP). Importantly, the selective difference in the ability of XIAP to block myotube but not myoblast apoptosis is not due to a change in XIAP but rather a decrease in Apaf-1 expression. This decrease in Apaf-1 links XIAP to caspase activation and death. Our findings suggest that in order for myotubes to die, they may degrade XIAP, functionally inactivate XIAP or upregulate Apaf-1. Importantly, we identify a role for endogenous Smac in overcoming XIAP to allow myotube death. However, in postmitotic cardiomyocytes, where XIAP also restricts apoptosis, endogenous Smac was not capable of overcoming XIAP to cause death. These results show that as skeletal muscle differentiate, they become resistant to apoptosis because of the ability of XIAP to regulate caspase activation. The increased restriction of apoptosis in myotubes is presumably important to ensure the long term survival of these postmitotic cells as they play a vital role in the physiology of organisms.

  20. Skeletal muscle differentiation evokes endogenous XIAP to restrict the apoptotic pathway.

    Directory of Open Access Journals (Sweden)

    Michelle I Smith

    Full Text Available Myotube apoptosis occurs normally during muscle development and aging but it can lead to destruction of skeletal muscle in neuromuscular diseases. Therefore, understanding how myotube apoptosis is regulated is important for developing novel strategies for treatment of muscle loss. We investigated the regulation of apoptosis in skeletal muscle and report a striking increase in resistance to apoptosis following differentiation. We find mitotic C2C12 cells (myoblast-like cells are sensitive to cytosolic cytochrome c microinjection. However, differentiated C2C12 cells (myotube-like cells and primary myotubes are markedly resistant. This resistance is due to endogenous X-linked inhibitor of apoptotic protein (XIAP. Importantly, the selective difference in the ability of XIAP to block myotube but not myoblast apoptosis is not due to a change in XIAP but rather a decrease in Apaf-1 expression. This decrease in Apaf-1 links XIAP to caspase activation and death. Our findings suggest that in order for myotubes to die, they may degrade XIAP, functionally inactivate XIAP or upregulate Apaf-1. Importantly, we identify a role for endogenous Smac in overcoming XIAP to allow myotube death. However, in postmitotic cardiomyocytes, where XIAP also restricts apoptosis, endogenous Smac was not capable of overcoming XIAP to cause death. These results show that as skeletal muscle differentiate, they become resistant to apoptosis because of the ability of XIAP to regulate caspase activation. The increased restriction of apoptosis in myotubes is presumably important to ensure the long term survival of these postmitotic cells as they play a vital role in the physiology of organisms.

  1. Induction of cyclins E and A in response to mitogen removal: a basic alteration associated with the arrest of differentiation of C2 myoblasts transformed by simian virus 40 large T antigen.

    OpenAIRE

    Tedesco, D; Baron, L; Fischer-Fantuzzi, L; Vesco, C

    1997-01-01

    We previously showed that C2 myoblasts transformed by simian virus 40 large T antigen (SVLT) stop the myogenic process after the induction of myogenin and of high Rb levels; the induced Rb, however, becomes notably phosphorylated. We have analyzed the protein levels and activities of cyclin-dependent kinases (cdks) in untransformed C2 cells and in transformants of either SVLT or the cytoplasmic mutant NKT1 (which permits differentiation) upon a shift from growth medium (GM) to mitogen-poor di...

  2. MiR-193b-365, a brown fat enriched microRNA cluster, is essential for brown fat differentiation

    Science.gov (United States)

    Sun, Lei; Xie, Huangming; Mori, Marcelo A; Alexander, Ryan; Yuan, Bingbing; Hattangadi, Shilpa M.; Liu, Qingqing; Kahn, C. Ronald; Lodish, Harvey F.

    2011-01-01

    Mammals have two principal types of fat. White adipose tissue (WAT) primarily serves to store extra energy as triglycerides, while brown adipose tissue (BAT) is specialized to burn lipids for heat generation and energy expenditure as a defense against cold and obesity 1, 2. Recent studies demonstrate that brown adipocytes arise in vivo from a Myf5-positive, myoblastic progenitor by the action of Prdm16 (PR domain containing 16). Here, we identified a brown fat-enriched miRNA cluster, miR-193b-365, as a key regulator of brown fat development. Blocking miR-193b and/or miR-365 in primary brown preadipocytes dramatically impaired brown adipocyte adipogenesis by enhancing Runx1t1 (runt-related transcription factor 1; translocated to, 1) expression whereas myogenic markers were significantly induced. Forced expression of miR-193b and/or miR-365 in C2C12 myoblasts blocked the entire program of myogenesis, and, in adipogenic condition, miR-193b induced myoblasts to differentiate into brown adipocytes. MiR-193b-365 was upregulated by Prdm16 partially through Pparα. Our results demonstrate that miR-193b-365 serves as an essential regulator for brown fat differentiation, in part by repressing myogenesis. PMID:21743466

  3. Static stretch promotes MEF2A nuclear translocation and expression of neonatal myosin heavy chain in C2C12 myocytes in a calcineurin- and p38-dependent manner.

    Science.gov (United States)

    Rauch, Cyril; Loughna, Paul T

    2005-03-01

    Although the effects of mechanical stimuli have been studied extensively in fully differentiated skeletal muscle and have been shown to promote changes in phenotype, including altered myosin heavy chain isoform expression, the effects of a change in mechanical environment have been poorly studied at earlier stages of skeletal muscle differentiation. In particular, the early events elicited by mechanical stimuli upon differentiating myocytes have not been investigated. In the present study, the effect of static stretch on the activation of transcriptional factors MEF2A and NFATc1, which have been shown to be involved in the differentiation and phenotype regulation of skeletal muscle, have been examined. Furthermore, putative second messenger signaling pathways that could be involved in the dephosphorylation and hence activation of these factors were also examined. We have demonstrated that static stretch application produces a robust increase in p38 phosphorylation preceding MEF2A, but not NFATc1, nuclear translocation as well as deactivation of GSK-3beta via its phosphorylation. Using SB-203580 and cyclosporine A drugs to inhibit both p38- or/and calcineurin-dependent signals, respectively, we have shown that MEF2A phosphorylation and subsequent nuclear translocation are regulated by p38 and calcineurin in a biphasic, time-dependent manner. Moreover, we also present evidence for another kinase that is involved in the stretch-related signal triggering MEF2A hyperphosphorylation, impairing its nuclear translocation, and that is related to p38. Finally, we have shown that static stretch application overnight promotes neonatal myosin heavy chain expression, which is inhibited by an inactivation of both p38 and calcineurin.

  4. Development and Growth of the Avian Pectoralis Major (Breast Muscle: Function of Syndecan-4 and Glypican-1 in Adult Myoblast Proliferation and Differentiation

    Directory of Open Access Journals (Sweden)

    Sandra G. Velleman

    2017-08-01

    Full Text Available Muscle fiber number is determined around the time hatch with continued posthatch muscle growth being mediated by the adult myoblast, satellite cell, population of cells. Satellite cells are dynamic in their expression of proteins including the cell membrane associated proteoglycans, syndecan-4 and glypican-1. These proteoglycans play roles in organizing the extracellular environment in the satellite cell niche, cytoskeletal structure, cell-to-cell adhesion, satellite cell migration, and signal transduction. This review article focuses on syndecan-4 and glypican-1 as both are capable of regulating satellite cell responsiveness to fibroblast growth factor 2. Fibroblast growth factor 2 is a potent stimulator of muscle cell proliferation and a strong inhibitor of differentiation. Proteoglycans are composed of a central core protein defined functional domains, and covalently attached glycosaminoglycans and N-glycosylation chains. The functional association of these components with satellite cell function is discussed as well as an emerging role for microRNA regulation of syndecan-4 and glypican-1.

  5. Transforming growth factor type-β inhibits Mas receptor expression in fibroblasts but not in myoblasts or differentiated myotubes; Relevance to fibrosis associated to muscular dystrophies.

    Science.gov (United States)

    Cofre, Catalina; Acuña, María José; Contreras, Osvaldo; Morales, María Gabriela; Riquelme, Cecilia; Cabello-Verrugio, Claudio; Brandan, Enrique

    2015-01-01

    Duchenne muscular dystrophy is a genetic disorder characterized by myofiber degeneration, muscle weakness, and increased fibrosis. Transforming growth factor type-β (TGF-β), a central mediator of fibrosis, is upregulated in fibrotic diseases. Angiotensin-(1-7) [Ang-(1-7)] is a peptide with actions that oppose those of angiotensin-II (Ang II). Ang-(1-7) effects are mediated by the Mas receptor. Treatment with Ang-(1-7) produce positive effects in the mdx mouse, normalizing skeletal muscle architecture, decreasing local fibrosis, and fibroblasts, and improving muscle function. Mdx mice deficient for the Mas receptor showed the opposite effects. To identify the cell type(s) responsible for Mas receptor expression, and to characterize whether profibrotic effectors had any effect on its expression, we determined the effect of profibrotic agents on Mas expression. TGF-β, but not connective tissue growth factor or Ang-II, reduced the expression of Mas receptor in fibroblasts isolated from skeletal muscle cells and fibroblasts from two established cell lines. In contrast, no effects were observed in myoblasts and differentiated myotubes. This inhibition was mediated by the Smad-dependent (canonical) and the PI3K and MEK1/2 (noncanonical) TGF-β signaling pathways. When both canonical and noncanonical inhibitors of the TGF-β-dependent pathways were added together, the inhibitory effect of TGF-β on Mas expression was lost. The decrease in Mas receptor induced by TGF-β in fibroblasts reduced the Ang-(1-7) mediated stimulation of phosphorylation of AKT pathway proteins. These results suggest that reduction of Mas receptor in fibroblasts, by TGF-β, could increase the fibrotic phenotype observed in dystrophic skeletal muscle decreasing the beneficial effect of Ang-(1-7). © 2015 International Union of Biochemistry and Molecular Biology.

  6. Effects of metal ions on proliferation of aortic smooth muscle cells and myoblastic cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Vorpahl, M.; Wiemann, M.; Bingmann, D. [Essen Univ. (Germany). Inst. fuer Physiologie; Brauer, H. [Werkstofftechnik, Univ. Essen (Germany)

    2001-12-01

    Metal ions released from implanted stents into the surrounding tissue may contribute to vascular reactions which cause restenosis in about 30%. This assumption prompted us to investigate short term effects of metal ions (Ag, Al, Cr, Fe, Mo, Ni, V, all applied as chloride salts) on proliferation of swine aortic smooth muscle cells (SMC) and a myoblastic cell line (C2C12). Cell confluence was 30 or 50% when metal ions were added and cell growth was monitored with the MTT-test after 2 days. A clear concentration dependence of acute toxicity of the different metal ions was found for both cell types. The order of toxicity indicated by IC50 values was V > Ni > Fe > Mo > Al > Cr. The nearly insoluble silverchloride exerted unclear effects. In experiments starting at high confluence, the apparent toxicity of Fe, Ni, and V was reduced. Al, which to our knowledge is not a major constituent in medical stents, was the only metal ion found here to cause a slightly increased proliferation, but this effect was restricted to the low concentration range (16-250 {mu}mol/l). In general, results for both cell types, C2C12 and SMC, were very similar. We conclude that short term effects of metal ions, which may be released in the interface of stent and vessel wall tissue, comprise a reduction rather than a stimulation of cell proliferation. However, restenosis may be initiated as a complex tissue reaction to primary toxic metal effects. (orig.)

  7. The inhibition of cultured myoblast differentiation by the simian virus 40 large T antigen occurs after myogenin expression and Rb up-regulation and is not exerted by transformation-competent cytoplasmic mutants.

    Science.gov (United States)

    Tedesco, D; Caruso, M; Fischer-Fantuzzi, L; Vesco, C

    1995-11-01

    We have investigated the mechanism by which the simian virus 40 large T antigen (SVLT) interferes with the differentiation of C2 myoblasts. SVLT mutants, defective either in the Rb binding site, near the N-terminal end, in a region that affects binding to p53, or in the nuclear transport signal, were also employed to determine whether the interference was especially dependent on these functional domains. It was found that wild-type (wt) SVLT strongly inhibited the terminal differentiation of mouse C2 myoblasts, but this arrest occurred only after the synthesis of myogenin, an initial step in biochemical differentiation. Neither the synthesis nor some basic activities of MyoD appeared to be affected by wt SVLT. In these transformants, mitogen depletion elicited an increase in the Rb level comparable to that in normal C2 cells; wt SVLT, however, promoted the phosphorylation of a large part of the induced Rb. Mutations affecting nuclear transport were far more critical for the ability to interfere with myogenic differentiation than were those affecting the transforming potential; cytoplasmic SVLT expression was fully compatible with the terminal differentiation of C2 cells, despite enabling them to grow in semisolid medium, thus showing that the myogenesis-inhibiting property can be dissociated from transforming competence. The remaining SVLT mutants presented different degrees of ability to inhibit differentiation (as shown by the expression of tissue-specific markers in transformants). The inhibiting mutants, including the Rb binding site mutant, were able to promote a higher state of Rb phosphorylation than that observed in either normal cells or cytoplasmic-SVLT transformants.

  8. Specification of skeletal muscle differentiation by repressor element-1 silencing transcription factor (REST)-regulated Kv7.4 potassium channels.

    Science.gov (United States)

    Iannotti, Fabio Arturo; Barrese, Vincenzo; Formisano, Luigi; Miceli, Francesco; Taglialatela, Maurizio

    2013-02-01

    Changes in the expression of potassium (K(+)) channels is a pivotal event during skeletal muscle differentiation. In mouse C(2)C(12) cells, similarly to human skeletal muscle cells, myotube formation increased the expression of K(v)7.1, K(v)7.3, and K(v)7.4, the last showing the highest degree of regulation. In C(2)C(12) cells, K(v)7.4 silencing by RNA interference reduced the expression levels of differentiation markers (myogenin, myosin heavy chain, troponinT-1, and Pax3) and impaired myotube formation and multinucleation. In K(v)7.4-silenced cells, the differentiation-promoting effect of the K(v)7 activator N-(2-amino-4-(4-fluorobenzylamino)-phenyl)-carbamic acid ethyl ester (retigabine) was abrogated. Expression levels for the repressor element-1 silencing transcription factor (REST) declined during myotube formation. Transcript levels for K(v)7.4, as well as for myogenin, troponinT-1, and Pax3, were reduced by REST overexpression and enhanced upon REST suppression by RNA interference. Four regions containing potential REST-binding sites in the 5' untranslated region and in the first intron of the K(v)7.4 gene were identified by bioinformatic analysis. Chromatin immunoprecipitation assays showed that REST binds to these regions, exhibiting a higher efficiency in myoblasts than in myotubes. These data suggest that K(v)7.4 plays a permissive role in skeletal muscle differentiation and highlight REST as a crucial transcriptional regulator for this K(+) channel subunit.

  9. Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Jaemin, E-mail: jmj1103@kirams.re.kr [Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720-1762 (United States); Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Conboy, Irina M., E-mail: iconboy@berkeley.edu [Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720-1762 (United States)

    2011-10-14

    Highlights: {yields} PS broadly and persistently trans-locates to the outer leaflet of plasma membrane during myoblast fusion into myotubes. {yields} Robust myotubes are formed when PS liposomes are added exogenously. {yields} PS increases the width of de novo myotubes and the numbers of myonuclei, but not the myotube length. {yields} Annexin V or PS antibody inhibits myotube formation by masking exposed PS. -- Abstract: Cell membrane consists of various lipids such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Among them, PS is a molecular marker of apoptosis, because it is located to the inner leaflet of plasma membrane generally but it is moved to the outer leaflet during programmed cell death. The process of apoptosis has been implicated in the fusion of muscle progenitor cells, myoblasts, into myotubes. However, it remained unclear whether PS regulates muscle cell differentiation directly. In this paper, localization of PS to the outer leaflet of plasma membrane in proliferating primary myoblasts and during fusion of these myoblasts into myotubes is validated using Annexin V. Moreover, we show the presence of PS clusters at the cell-cell contact points, suggesting the importance of membrane ruffling and PS exposure for the myogenic cell fusion. Confirming this conclusion, experimentally constructed PS, but not PC liposomes dramatically enhance the formation of myotubes from myoblasts, thus demonstrating a direct positive effect of PS on the muscle cell fusion. In contrast, myoblasts exposed to PC liposomes produce long myotubes with low numbers of myonuclei. Moreover, pharmacological masking of PS on the myoblast surface inhibits fusion of these cells into myotubes in a dose-dependent manner.

  10. Efficient myogenic differentiation of human adipose-derived stem cells by the transduction of engineered MyoD protein

    Energy Technology Data Exchange (ETDEWEB)

    Sung, Min Sun [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350 (Korea, Republic of); Mun, Ji-Young [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Kwon, Ohsuk [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350 (Korea, Republic of); Kwon, Ki-Sun [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Oh, Doo-Byoung, E-mail: dboh@kribb.re.kr [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350 (Korea, Republic of)

    2013-07-19

    Highlights: •MyoD was engineered to contain protein transduction domain and endosome-disruptive INF7 peptide. •The engineered MyoD-IT showed efficient nuclear targeting through an endosomal escape by INF7 peptide. •By applying MyoD-IT, human adipose-derived stem cells (hASCs) were differentiated into myogenic cells. •hASCs differentiated by applying MyoD-IT fused to myotubes through co-culturing with mouse myoblasts. •Myogenic differentiation using MyoD-IT is a safe method without the concern of altering the genome. -- Abstract: Human adipose-derived stem cells (hASCs) have great potential as cell sources for the treatment of muscle disorders. To provide a safe method for the myogenic differentiation of hASCs, we engineered the MyoD protein, a key transcription factor for myogenesis. The engineered MyoD (MyoD-IT) was designed to contain the TAT protein transduction domain for cell penetration and the membrane-disrupting INF7 peptide, which is an improved version of the HA2 peptide derived from influenza. MyoD-IT showed greatly improved nuclear targeting ability through an efficient endosomal escape induced by the pH-sensitive membrane disruption of the INF7 peptide. By applying MyoD-IT to a culture, hASCs were efficiently differentiated into long spindle-shaped myogenic cells expressing myosin heavy chains. Moreover, these cells differentiated by an application of MyoD-IT fused to myotubes with high efficiency through co-culturing with mouse C2C12 myoblasts. Because internalized proteins can be degraded in cells without altering the genome, the myogenic differentiation of hASCs using MyoD-IT would be a safe and clinically applicable method.

  11. Effect of Ultrasonic Vibration on Proliferation and Differentiation of Cells

    Directory of Open Access Journals (Sweden)

    Haruka Hino

    2016-12-01

    Full Text Available The effect of mechanical stimulation of vibration on proliferation and differentiation of cells has been studied in vitro. To apply the vibration on the cells, a piezoelectric element was attached on the outside surface of the bottom of the culture plate of six wells. The piezoelectric element was vibrated by sinusoidally alternating voltage at 1.0 MHz generated by a function generator. Five kinds of cells were used in the experiment: C2C12 (mouse myoblast cell, L929 (fibroblast connective tissue of mouse, Hepa1-6 (mouse hepatoma cell, HUVEC (human umbilical vein endothelial cell, and Neuro-2a (mouse neural crest-derived cell line. After the incubation for 24 hours, cells were exposed to the ultrasonic vibration intermittently for three days: for thirty minutes per day. At the end of the experiment, the number of cells was counted by colorimetric method with a microplate photometer. In the case of Neuro-2a, the total length of the neurite was calculated at the microscopic image. The experimental study shows following results. Cells are exfoliated by the strong vibration. Proliferation and differentiation of cells are accelerated with mild vibration. The optimum intensity of vibration depends on the kind of cells.

  12. Ubc9 negatively regulates BMP-mediated osteoblastic differentiation in cultured cells.

    Science.gov (United States)

    Yukita, Akira; Hosoya, Akihiro; Ito, Yuzuru; Katagiri, Takenobu; Asashima, Makoto; Nakamura, Hiroaki

    2012-05-01

    SUMO (small ubiquitin-related modifier) modification (SUMOylation) has been reported to regulate various biological events such as cell-cycle progression, proliferation, and survival. Bone morphogenetic proteins (BMPs) play an important role in osteoblast differentiation and maturation. Although Smad4, which acts as a transcriptional factor in the BMP signaling, is a target of SUMOylation, the involvement of SUMOylation in osteoblast differentiation remains unclear. In this report, we demonstrated spatial expression patterns of SUMO proteins and Ubc9 (ubiquitin conjugating enzyme 9), which is a unique E2-SUMOylation enzyme, in mouse tibia. Furthermore, siRNA knockdown of Ubc9 enhanced osteoblastic differentiation induced by BMP2 in C2C12 mouse myoblasts and ST2 mouse bone-marrow derived stromal cells. Ubc9 knockdown elevated the BMP signaling transduction and reduced the expression of muscle-related genes in cooperation with BMP2. Finally, a luciferase assay using an Id1 (target gene of BMP signaling) reporter revealed that Smad4 mutants prevented from SUMOylation at their Lys158 possessed more potent transcriptional activity than wild-type Smad4. Taken together, these findings suggest that Ubc9 negatively regulates osteoblastic differentiation induced by BMP via, at least in part, SUMOylation of Smad4. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Phosphorylation of Lbx1 controls lateral myoblast migration into the limb.

    Science.gov (United States)

    Masselink, Wouter; Masaki, Megumi; Sieiro, Daniel; Marcelle, Christophe; Currie, Peter D

    2017-10-15

    The migration of limb myogenic precursors from limb level somites to their ultimate site of differentiation in the limb is a paradigmatic example of a set of dynamic and orchestrated migratory cell behaviours. The homeobox containing transcription factor ladybird homeobox 1 (Lbx1) is a central regulator of limb myoblast migration, null mutations of Lbx1 result in severe disruptions to limb muscle formation, particularly in the distal region of the limb in mice (Gross et al., 2000). As such Lbx1 has been hypothesized to control lateral migration of myoblasts into the distal limb anlage. It acts as a core regulator of the limb myoblast migration machinery, controlled by Pax3. A secondary role for Lbx1 in the differentiation and commitment of limb musculature has also been proposed (Brohmann et al., 2000; Uchiyama et al., 2000). Here we show that lateral migration, but not differentiation or commitment of limb myoblasts, is controlled by the phosphorylation of three adjacent serine residues of LBX1. Electroporation of limb level somites in the chick embryo with a dephosphomimetic form of Lbx1 results in a specific defect in the lateral migration of limb myoblasts. Although the initial delamination and migration of myoblasts is unaffected, migration into the distal limb bud is severely disrupted. Interestingly, myoblasts undergo normal differentiation independent of their migratory status, suggesting that the differentiation potential of hypaxial muscle is not regulated by the phosphorylation state of LBX1. Furthermore, we show that FGF8 and ERK mediated signal transduction, both critical regulators of the developing limb bud, have the capacity to induce the phosphorylation of LBX1 at these residues. Overall, this suggests a mechanism whereby the phosphorylation of LBX1, potentially through FGF8 and ERK signalling, controls the lateral migration of myoblasts into the distal limb bud. Copyright © 2017. Published by Elsevier Inc.

  14. Induction of cyclins E and A in response to mitogen removal: a basic alteration associated with the arrest of differentiation of C2 myoblasts transformed by simian virus 40 large T antigen.

    Science.gov (United States)

    Tedesco, D; Baron, L; Fischer-Fantuzzi, L; Vesco, C

    1997-03-01

    We previously showed that C2 myoblasts transformed by simian virus 40 large T antigen (SVLT) stop the myogenic process after the induction of myogenin and of high Rb levels; the induced Rb, however, becomes notably phosphorylated. We have analyzed the protein levels and activities of cyclin-dependent kinases (cdks) in untransformed C2 cells and in transformants of either SVLT or the cytoplasmic mutant NKT1 (which permits differentiation) upon a shift from growth medium (GM) to mitogen-poor differentiation medium (DM). After the shift, cdk4 levels remained constant and cdk6 levels decreased in all cell types; cdk2 minimally increased only in SVLT cells. Cyclin D1 was downregulated in DM in all cell types, and cyclin D3 was upregulated (albeit less strongly in SVLT cells than in the others). In contrast, a dramatic difference between SVLT cells and the other cells was observed for cyclins E and A, which essentially disappeared (as protein and RNA) in normal C2 and NKT1 cells upon the shift from GM to DM, whereas they increased in SVLT cells. Concurrently, cdk2 activity ceased in C2 and NKT1 cells in DM, whereas it persisted at 20% of the GM level in SVLT cells. cdk4 activity was detectable in all cells only in GM. Cyclin E and A induction thus appeared to sustain enough Rb phosphorylation to interfere with tissue-specific expression, with cdk activity not high enough to activate cyclin self-regulation. In DM, cdk2 complexed to D3 was underphosphorylated in all cells, and SVLT allowed strong inductions of p21 and p27 without affecting their complexes with cdks.

  15. Myoblast replication is reduced in the IUGR fetus despite maintained proliferative capacity in vitro.

    Science.gov (United States)

    Soto, Susan M; Blake, Amy C; Wesolowski, Stephanie R; Rozance, Paul J; Barthel, Kristen B; Gao, Bifeng; Hetrick, Byron; McCurdy, Carrie E; Garza, Natalia G; Hay, William W; Leinwand, Leslie A; Friedman, Jacob E; Brown, Laura D

    2017-03-01

    Adults who were affected by intrauterine growth restriction (IUGR) suffer from reductions in muscle mass and insulin resistance, suggesting muscle growth may be restricted by molecular events that occur during fetal development. To explore the basis of restricted fetal muscle growth, we used a sheep model of progressive placental insufficiency-induced IUGR to assess myoblast proliferation within intact skeletal muscle in vivo and isolated myoblasts stimulated with insulin in vitro Gastrocnemius and soleus muscle weights were reduced by 25% in IUGR fetuses compared to those in controls (CON). The ratio of PAX7+ nuclei (a marker of myoblasts) to total nuclei was maintained in IUGR muscle compared to CON, but the fraction of PAX7+ myoblasts that also expressed Ki-67 (a marker of cellular proliferation) was reduced by 23%. Despite reduced proliferation in vivo, fetal myoblasts isolated from IUGR biceps femoris and cultured in enriched media in vitro responded robustly to insulin in a dose- and time-dependent manner to increase proliferation. Similarly, insulin stimulation of IUGR myoblasts upregulated key cell cycle genes and DNA replication. There were no differences in the expression of myogenic regulatory transcription factors that drive commitment to muscle differentiation between CON and IUGR groups. These results demonstrate that the molecular machinery necessary for transcriptional control of proliferation remains intact in IUGR fetal myoblasts, indicating that in vivo factors such as reduced insulin and IGF1, hypoxia and/or elevated counter-regulatory hormones may be inhibiting muscle growth in IUGR fetuses. © 2017 Society for Endocrinology.

  16. Phospholipase D1 facilitates second-phase myoblast fusion and skeletal muscle regeneration.

    Science.gov (United States)

    Teng, Shuzhi; Stegner, David; Chen, Qin; Hongu, Tsunaki; Hasegawa, Hiroshi; Chen, Li; Kanaho, Yasunori; Nieswandt, Bernhard; Frohman, Michael A; Huang, Ping

    2015-02-01

    Myoblast differentiation and fusion is a well-orchestrated multistep process that is essential for skeletal muscle development and regeneration. Phospholipase D1 (PLD1) has been implicated in the initiation of myoblast differentiation in vitro. However, whether PLD1 plays additional roles in myoblast fusion and exerts a function in myogenesis in vivo remains unknown. Here we show that PLD1 expression is up-regulated in myogenic cells during muscle regeneration after cardiotoxin injury and that genetic ablation of PLD1 results in delayed myofiber regeneration. Myoblasts derived from PLD1-null mice or treated with PLD1-specific inhibitor are unable to form mature myotubes, indicating defects in second-phase myoblast fusion. Concomitantly, the PLD1 product phosphatidic acid is transiently detected on the plasma membrane of differentiating myocytes, and its production is inhibited by PLD1 knockdown. Exogenous lysophosphatidylcholine, a key membrane lipid for fusion pore formation, partially rescues fusion defect resulting from PLD1 inhibition. Thus these studies demonstrate a role for PLD1 in myoblast fusion during myogenesis in which PLD1 facilitates the fusion of mononuclear myocytes with nascent myotubes. © 2015 Teng et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  17. Myoblasts from intrauterine growth-restricted sheep fetuses exhibit intrinsic deficiencies in proliferation that contribute to smaller semitendinosus myofibres

    Science.gov (United States)

    Yates, Dustin T; Clarke, Derek S; Macko, Antoni R; Anderson, Miranda J; Shelton, Leslie A; Nearing, Marie; Allen, Ronald E; Rhoads, Robert P; Limesand, Sean W

    2014-01-01

    Intrauterine growth restriction (IUGR) reduces skeletal muscle mass in fetuses and offspring. Our objective was to determine whether myoblast dysfunction due to intrinsic cellular deficiencies or serum factors reduces myofibre hypertrophy in IUGR fetal sheep. At 134 days, IUGR fetuses weighed 67% less (P IUGR semitendinosus muscles had similar percentages of pax7-positive nuclei and pax7 mRNA but lower (P IUGR fetuses had 34% fewer (P IUGR myoblasts also replicated less (P IUGR fetal sheep serum (FSS). Both myoblast types replicated less (P IUGR FSS-supplemented media compared to control FSS-supplemented media. In differentiation-promoting media (2% FBS), IUGR and control myoblasts had similar percentages of myogenin-positive nuclei after 5 days and formed similar-sized myotubes after 7 days. We conclude that intrinsic cellular deficiencies in IUGR myoblasts and factors in IUGR serum diminish myoblast proliferation and myofibre size in IUGR fetuses, but intrinsic myoblast deficiencies do not affect differentiation. Furthermore, the persistent reduction in IUGR myoblast replication shows adaptive deficiencies that explain poor muscle growth in IUGR newborn offspring. PMID:24860171

  18. S100B Engages RAGE or bFGF/FGFR1 in Myoblasts Depending on Its Own Concentration and Myoblast Density. Implications for Muscle Regeneration

    Science.gov (United States)

    Beccafico, Sara; Donato, Rosario

    2012-01-01

    In high-density myoblast cultures S100B enhances basic fibroblast growth factor (bFGF) receptor 1 (FGFR1) signaling via binding to bFGF and blocks its canonical receptor, receptor for advanced glycation end-products (RAGE), thereby stimulating proliferation and inhibiting differentiation. Here we show that upon skeletal muscle injury S100B is released from myofibers with maximum release at day 1 post-injury in coincidence with satellite cell activation and the beginning of the myoblast proliferation phase, and declining release thereafter in coincidence with reduced myoblast proliferation and enhanced differentiation. By contrast, levels of released bFGF are remarkably low at day 1 post-injury, peak around day 5 and decline thereafter. We also show that in low-density myoblast cultures S100B binds RAGE, but not bFGF/FGFR1 thereby simultaneously stimulating proliferation via ERK1/2 and activating the myogenic program via p38 MAPK. Clearance of S100B after a 24-h treatment of low-density myoblasts results in enhanced myotube formation compared with controls as a result of increased cell numbers and activated myogenic program, whereas chronic treatment with S100B results in stimulation of proliferation and inhibition of differentiation due to a switch of the initial low-density culture to a high-density culture. However, at relatively high doses, S100B stimulates the mitogenic bFGF/FGFR1 signaling in low-density myoblasts, provided bFGF is present. We propose that S100B is a danger signal released from injured muscles that participates in skeletal muscle regeneration by activating the promyogenic RAGE or the mitogenic bFGF/FGFR1 depending on its own concentration, the absence or presence of bFGF, and myoblast density. PMID:22276098

  19. A novel role for the RNA-binding protein FXR1P in myoblasts cell-cycle progression by modulating p21/Cdkn1a/Cip1/Waf1 mRNA stability.

    Directory of Open Access Journals (Sweden)

    Laetitia Davidovic

    2013-03-01

    Full Text Available The Fragile X-Related 1 gene (FXR1 is a paralog of the Fragile X Mental Retardation 1 gene (FMR1, whose absence causes the Fragile X syndrome, the most common form of inherited intellectual disability. FXR1P plays an important role in normal muscle development, and its absence causes muscular abnormalities in mice, frog, and zebrafish. Seven alternatively spliced FXR1 transcripts have been identified and two of them are skeletal muscle-specific. A reduction of these isoforms is found in myoblasts from Facio-Scapulo Humeral Dystrophy (FSHD patients. FXR1P is an RNA-binding protein involved in translational control; however, so far, no mRNA target of FXR1P has been linked to the drastic muscular phenotypes caused by its absence. In this study, gene expression profiling of C2C12 myoblasts reveals that transcripts involved in cell cycle and muscular development pathways are modulated by Fxr1-depletion. We observed an increase of p21--a regulator of cell-cycle progression--in Fxr1-knocked-down mouse C2C12 and FSHD human myoblasts. Rescue of this molecular phenotype is possible by re-expressing human FXR1P in Fxr1-depleted C2C12 cells. FXR1P muscle-specific isoforms bind p21 mRNA via direct interaction with a conserved G-quadruplex located in its 3' untranslated region. The FXR1P/G-quadruplex complex reduces the half-life of p21 mRNA. In the absence of FXR1P, the upregulation of p21 mRNA determines the elevated level of its protein product that affects cell-cycle progression inducing a premature cell-cycle exit and generating a pool of cells blocked at G0. Our study describes a novel role of FXR1P that has crucial implications for the understanding of its role during myogenesis and muscle development, since we show here that in its absence a reduced number of myoblasts will be available for muscle formation/regeneration, shedding new light into the pathophysiology of FSHD.

  20. The long and short of non-coding RNAs during post-natal growth and differentiation of skeletal muscles: Focus on lncRNA and miRNAs.

    Science.gov (United States)

    Butchart, Lauren C; Fox, Archa; Shavlakadze, Tea; Grounds, Miranda D

    2016-12-01

    Post-natal growth of skeletal muscle is a dynamic process involving proliferation and fusion of myoblasts with elongating myofibres (hyperplasia of myonuclei) until 3 weeks post-natally in mice, with ongoing differentiation and further increases in myofibre size mostly by hypertrophy until about 12 weeks of age. The expression of mRNAs that control these events are well described, but little is known about the in vivo roles of non-coding RNAs (ncRNAs), including both microRNAs (miRNAs) and the lesser-studied long non-coding RNAs (lncRNAs). We analysed expression patterns for a broad range of lncRNAs (including Neat1, Malat1, Sra, Meg3, LncMyoD and linc-MD1), miRNAs and mRNAs in muscles of normal male C57Bl/6J mice at 2 days and 2, 4, 6 and 12 weeks after birth. These post-natal patterns were compared with expression of these RNAs during classic C2C12 myogenesis and differentiation in tissue culture. This overview of RNAs during post-natal skeletal muscle growth provides a novel focus on ncRNAs during this often overlooked growth period, with many potential applications to normal muscle growth in humans and livestock, and to childhood muscle disorders. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  1. Effect of IR Laser on Myoblasts: Prospects of Application for Counteracting Microgravity-Induced Muscle Atrophy

    Science.gov (United States)

    Monici, Monica; Cialdai, Francesca; Romano, Giovanni; Corsetto, Paola Antonia; Rizzo, Angela Maria; Caselli, Anna; Ranaldi, Francesco

    2013-02-01

    Microgravity-induced muscle atrophy is a problem of utmost importance for the impact it may have on the health and performance of astronauts. Therefore, appropriate countermeasures are needed to prevent disuse atrophy and favour muscle recovery. Muscle atrophy is characterized by loss of muscle mass and strength, and a shift in substrate utilization from fat to glucose, that leads to a reduced metabolic efficiency and enhanced fatigability. Laser therapy is already used in physical medicine and rehabilitation to accelerate muscle recovery and in sports medicine to prevent damages produced by metabolic disturbances and inflammatory reactions after heavy exercise. The aim of the research we present was to get insights on possible benefits deriving from the application of an advanced infrared laser system to counteract deficits of muscle energy metabolism and stimulate the recovery of the hypotrophic tissue. The source used was a Multiwave Locked System (MLS) laser, which combines continuous and pulsed emissions at 808 nm and 905 nm, respectively. We studied the effect of MLS treatment on morphology and energy metabolism of C2C12 cells, a widely accepted myoblast model, previously exposed to microgravity conditions modelled by a Random Positioning Machine. The MLS laser treatment was able to restore basal levels of serine/threonine protein phosphatase activity and to counteract cytoskeletal alterations and increase in glycolytic enzymes activity that occurred following the exposure to modelled microgravity. In conclusion, the results provide interesting insights for the application of infrared laser in the treatment of muscle atrophy.

  2. Elastic hydrogel substrate supports robust expansion of murine myoblasts and enhances their engraftment

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Ke, E-mail: dk1118@yeah.net [Department of Pediatric Surgery, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu 610072 (China); Yang, Zhong [Department of Clinical Hematology, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Xu, Jian-zhong, E-mail: xjzspine@163.com [Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Liu, Wen-ying; Zeng, Qiang; Hou, Fang [Department of Pediatric Surgery, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu 610072 (China); Lin, Sen [Department of Anatomy and Histology & Embryology, Chengdu Medical College, Chengdu 610500 (China)

    2015-09-10

    The application of satellite cell-derived myoblasts in regenerative medicine has been restricted by the rapid loss of stemness during in vitro cell expansion using traditional culture systems. However, studies published in the past decade have highlighted the influence of substrate elasticity on stem cell fate and revealed that culture on a soft hydrogel substrate can promote self-renewal and prolong the regenerative potential of muscle stem cells. Whether hydrogel substrates have similar effects after long-term robust expansion remains to be determined. Herein we prepared an elastic chitosan/beta-glycerophosphate/collagen hydrogel mimicking the soft microenvironment of muscle tissues for use as the substrate for satellite cell culture and investigated its influence on long-term cell expansion. After 20 passages in culture, satellite cell-derived myoblasts cultured on our hydrogel substrate exhibited significant improvements in proliferation capability, cell viability, colony forming frequency, and potential for myogenic differentiation compared to those cultured on a routine rigid culture surface. Immunochemical staining and western blot analysis both confirmed that myoblasts cultured on the hydrogel substrate expressed higher levels of several differentiation-related markers, including Pax7, Pax3, and SSEA-1, and a lower level of MyoD compared to myoblasts cultured on rigid culture plates (all p<0.05). After transplantation into the tibialis anterior of nude mice, myoblasts that had been cultured on the hydrogel substrate demonstrated a significantly greater engraftment efficacy than those cultured on the traditional surface. Collectively, these results indicate that the elastic hydrogel substrate supported robust expansion of murine myoblasts and enhanced their engraftment in vivo. - Highlights: • An elastic hydrogel was designed to mimic the pliable muscle tissue microenvironment. • Myoblasts retained their stemness in long-term culture on the elastic

  3. Live cell imaging reveals marked variability in myoblast proliferation and fate

    Science.gov (United States)

    2013-01-01

    Background During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear. Methods We have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line. Results Analyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls. Conclusion Our results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated. PMID:23638706

  4. AP-2{alpha} suppresses skeletal myoblast proliferation and represses fibroblast growth factor receptor 1 promoter activity

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, Darrion L. [Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064 (United States); DiMario, Joseph X., E-mail: joseph.dimario@rosalindfranklin.edu [Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064 (United States)

    2010-01-15

    Skeletal muscle development is partly characterized by myoblast proliferation and subsequent differentiation into postmitotic muscle fibers. Developmental regulation of expression of the fibroblast growth factor receptor 1 (FGFR1) gene is required for normal myoblast proliferation and muscle formation. As a result, FGFR1 promoter activity is controlled by multiple transcriptional regulatory proteins during both proliferation and differentiation of myogenic cells. The transcription factor AP-2{alpha} is present in nuclei of skeletal muscle cells and suppresses myoblast proliferation in vitro. Since FGFR1 gene expression is tightly linked to myoblast proliferation versus differentiation, the FGFR1 promoter was examined for candidate AP-2{alpha} binding sites. Mutagenesis studies indicated that a candidate binding site located at - 1035 bp functioned as a repressor cis-regulatory element. Furthermore, mutation of this site alleviated AP-2{alpha}-mediated repression of FGFR1 promoter activity. Chromatin immunoprecipitation studies demonstrated that AP-2{alpha} interacted with the FGFR1 promoter in both proliferating myoblasts and differentiated myotubes. In total, these results indicate that AP-2{alpha} is a transcriptional repressor of FGFR1 gene expression during skeletal myogenesis.

  5. Characterization of human myoblast cultures for tissue engineering.

    Science.gov (United States)

    Stern-Straeter, Jens; Bran, Gregor; Riedel, Frank; Sauter, Alexander; Hörmann, Karl; Goessler, Ulrich Reinhart

    2008-01-01

    Skeletal muscle tissue engineering, a promising specialty, aims at the reconstruction of skeletal muscle loss. In vitro tissue engineering attempts to achieve this goal by creating differentiated, functional muscle tissue through a process in which stem cells are extracted from the patient, e.g. by muscle biopsies, expanded and differentiated in a controlled environment, and subsequently re-implanted. A prerequisite for this undertaking is the ability to cultivate and differentiate human skeletal muscle cell cultures. Evidently, optimal culture conditions must be investigated for later clinical utilization. We therefore analysed the proliferation of human cells in different environments and evaluated the differentiation potential of different culture media. It was shown that human myoblasts have a higher rate of proliferation in the alamarBlue assay when cultured on gelatin-coated culture flasks rather than polystyrene-coated flasks. We also demonstrated that myoblasts treated with a culture medium with a high concentration of growth factors [growth medium (GM)] showed a higher proliferation compared to cultures treated with a culture medium with lower amounts of growth factors [differentiation medium (DM)]. Differentiation of human myoblast cell cultures treated with GM and DM was analysed until day 16 and myogenesis was verified by expression of MyoD, myogenin, alpha-sarcomeric actin and myosin heavy chain by semi-quantitative RT-PCR. Immunohistochemical staining for desmin, Myf-5 and alpha-sarcomeric actin was performed to verify the myogenic phenotype of extracted satellite cells and to prove the maturation of cells. Cultures treated with DM showed positive staining for alpha-sarcomeric actin. Notably, markers of differentiation were also detected in cultures treated with GM, but there was no formation of myotubes. In the enzymatic assay of creatine phosphokinase, cultures treated with DM showed a higher activity, evidencing a higher degree of differentiation

  6. Development of a cell culture surface conversion technique using alginate thin film for evaluating effect upon cellular differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Nakashima, Y., E-mail: yuta-n@mech.kumamoto-u.ac.jp [Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 096-8555 (Japan); Graduate School of Science and Engineering, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611 (Japan); Tsusu, K.; Minami, K. [Graduate School of Science and Engineering, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611 (Japan); Nakanishi, Y. [Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 096-8555 (Japan)

    2014-06-15

    Here, we sought to develop a cell culture surface conversion technique that would not damage living cells. An alginate thin film, formed on a glass plate by spin coating of sodium alginate solution and dipping into calcium chloride solution, was used to inhibit adhesion of cells. The film could be removed by ethylenediaminetetraacetate (EDTA) at any time during cell culture, permitting observation of cellular responses to conversion of the culture surface in real time. Additionally, we demonstrated the validity of the alginate thin film coating method and the performance of the film. The thickness of the alginate thin film was controlled by varying the rotation speed during spin coating. Moreover, the alginate thin film completely inhibited the adhesion of cultured cells to the culture surface, irrespective of the thickness of the film. When the alginate thin film was removed from the culture surface by EDTA, the cultured cells adhered to the culture surface, and their morphology changed. Finally, we achieved effective differentiation of C2C12 myoblasts into myotube cells by cell culture on the convertible culture surface, demonstrating the utility of our novel technique.

  7. Coordinate regulation of RARgamma2, TBP, and TAFII135 by targeted proteolysis during retinoic acid-induced differentiation of F9 embryonal carcinoma cells

    Directory of Open Access Journals (Sweden)

    Carré Lucie

    2001-03-01

    Full Text Available Abstract Background Treatment of mouse F9 embryonal carcinoma cells with all-trans retinoic acid (T-RA induces differentiation into primitive endodermal type cells. Differentiation requires the action of the receptors for all trans, and 9cis-retinoic acid (RAR and RXR, respectively and is accompanied by growth inhibition, changes in cell morphology, increased apoptosis, proteolytic degradation of the RARγ2 receptor, and induction of target genes. Results We show that the RNA polymerase II transcription factor TFIID subunits TBP and TAFII135 are selectively depleted in extracts from differentiated F9 cells. In contrast, TBP and TAFII135 are readily detected in extracts from differentiated F9 cells treated with proteasome inhibitors showing that their disappearance is due to targeted proteolysis. This regulatory pathway is not limited to F9 cells as it is also seen when C2C12 myoblasts differentiate into myotubes. Targeting of TBP and TAFII135 for proteolysis in F9 cells takes place coordinately with that previously reported for the RARγ2 receptor and is delayed or does not take place in RAR mutant F9 cells where differentiation is known to be impaired or abolished. Moreover, ectopic expression of TAFII135 delays proteolysis of the RARγ2 receptor and impairs primitive endoderm differentiation at an early stage as evidenced by cell morphology, induction of marker genes and apoptotic response. In addition, enhanced TAFII135 expression induces a novel differentiation pathway characterised by the appearance of cells with an atypical elongated morphology which are cAMP resistant. Conclusions These observations indicate that appropriately timed proteolysis of TBP and TAFII135 is required for normal F9 cell differentiation. Hence, in addition to transactivators, targeted proteolysis of basal transcription factors also plays an important role in gene regulation in response to physiological stimuli.

  8. Gene expression profiles resulting from stable loss of p53 mirrors its role in tissue differentiation.

    Directory of Open Access Journals (Sweden)

    Oliver Couture

    Full Text Available The tumor suppressor gene p53 is involved in a variety of cellular activities such as cellular stress responses, cell cycle regulation and differentiation. In our previous studies we have shown p53's transcription activating role to be important in osteoblast differentiation. There is still a debate in the literature as to whether p53 inhibits or promotes differentiation. We have found p53 heterozygous mice to show a p53 dependency on some bone marker gene expression that is absent in knockout mice. Mice heterozygous for p53 also show a higher incidence of osteosarcomas than p53 knockout mice. This suggests that p53 is able to modify the environment within osteoblasts. In this study we compare changes in gene expression resulting after either a transient or stable reduction in p53. Accordingly we reduced p53 levels transiently and stably in C2C12 cells, which are capable of both myoblast and osteoblast differentiation, and compared the changes in gene expression of candidate genes regulated by the p53 pathway. Using a PCR array to assay for p53 target genes, we have found different expression profiles when comparing stable versus transient knockdown of p53. As expected, several genes with profound changes after transient p53 loss were related to apoptosis and cell cycle regulation. In contrast, stable p53 loss produced a greater change in MyoD and other transcription factors with tissue specific roles, suggesting that long term loss of p53 affects tissue homeostasis to a greater degree than changes resulting from acute loss of p53. These differences in gene expression were validated by measuring promoter activity of different pathway specific genes involved in differentiation. These studies suggest that an important role for p53 is context dependent, with a stable reduction in p53 expression affecting normal tissue physiology more than acute loss of p53.

  9. Injectable scaffold materials differ in their cell instructive effects on primary human myoblasts

    DEFF Research Database (Denmark)

    Hejbøl, Eva Kildall; Sellathurai, Jeeva; Nair, Prabha Damodaran

    2017-01-01

    a minimally invasive technique. In this study, we examined in vitro the cell instructive effects of three types of injectable scaffolds, fibrin, alginate, and poly(lactic-co-glycolic acid)-based microparticles on primary human myoblasts. The myoblast morphology and progression in the myogenic program differed......, depending on the type of scaffold material. In alginate gel, the cells obtained a round morphology, they ceased to proliferate, and entered quiescence. In the fibrin gels, differentiation was promoted, and myotubes were observed within a few days in culture, while poly(lactic-co-glycolic acid......)-based microparticles supported prolonged proliferation. Myoblasts released from the alginate and fibrin gels were studied, and cells released from these scaffolds had retained the ability to proliferate and differentiate. Thus, the study shows that human myogenic cells combined with injectable scaffold materials...

  10. MyD88 promotes myoblast fusion in a cell-autonomous manner.

    Science.gov (United States)

    Hindi, Sajedah M; Shin, Jonghyun; Gallot, Yann S; Straughn, Alex R; Simionescu-Bankston, Adriana; Hindi, Lubna; Xiong, Guangyan; Friedland, Robert P; Kumar, Ashok

    2017-11-20

    Myoblast fusion is an indispensable step for skeletal muscle development, postnatal growth, and regeneration. Myeloid differentiation primary response gene 88 (MyD88) is an adaptor protein that mediates Toll-like receptors and interleukin-1 receptor signaling. Here we report a cell-autonomous role of MyD88 in the regulation of myoblast fusion. MyD88 protein levels are increased during in vitro myogenesis and in conditions that promote skeletal muscle growth in vivo. Deletion of MyD88 impairs fusion of myoblasts without affecting their survival, proliferation, or differentiation. MyD88 regulates non-canonical NF-κB and canonical Wnt signaling during myogenesis and promotes skeletal muscle growth and overload-induced myofiber hypertrophy in mice. Ablation of MyD88 reduces myofiber size during muscle regeneration, whereas its overexpression promotes fusion of exogenous myoblasts to injured myofibers. Our study shows that MyD88 modulates myoblast fusion and suggests that augmenting its levels may be a therapeutic approach to improve skeletal muscle formation in degenerative muscle disorders.

  11. Thermal manipulation during embryogenesis affects myoblast proliferation and skeletal muscle growth in meat-type chickens.

    Science.gov (United States)

    Piestun, Yogev; Yahav, Shlomo; Halevy, Orna

    2015-10-01

    Thermal manipulation (TM) of 39.5°C applied during mid-embryogenesis (embryonic d 7 to 16) has been proven to promote muscle development and enhance muscle growth and meat production in meat-type chickens. This study aimed to elucidate the cellular basis for this effect. Continuous TM or intermittent TM (for 12 h/d) increased myoblast proliferation manifested by higher (25 to 48%) myoblast number in the pectoral muscles during embryonic development but also during the first week posthatch. Proliferation ability of the pectoral-muscle-derived myoblasts in vitro was significantly higher in the TM treatments until embryonic d 15 (intermittent TM) or 13 (continuous TM) compared to that of controls, suggesting increased myogenic progeny reservoir in the muscle. However, the proliferation ability of myoblasts was lower in the TM treatments vs. control during the last days of incubation. This coincided with higher levels of myogenin expression in the muscle, indicating enhanced cell differentiation in the TM muscle. A similar pattern was observed posthatch: Myoblast proliferation was significantly higher in the TM chicks relative to controls during the peak of posthatch cell proliferation until d 6, followed by lower cell number 2 wk posthatch as myoblast number sharply decreases. Higher myogenin expression was observed in the TM chicks on d 6. This resulted in increased muscle growth, manifested by significantly higher relative weight of breast muscle in the embryo and posthatch. It can be concluded that temperature elevation during mid-term embryogenesis promotes myoblast proliferation, thus increasing myogenic progeny reservoir in the muscle, resulting in enhanced muscle growth in the embryo and posthatch. © 2015 Poultry Science Association Inc.

  12. Testosterone enables growth and hypertrophy in fusion impaired myoblasts that display myotube atrophy: deciphering the role of androgen and IGF-I receptors.

    Science.gov (United States)

    Hughes, David C; Stewart, Claire E; Sculthorpe, Nicholas; Dugdale, Hannah F; Yousefian, Farzad; Lewis, Mark P; Sharples, Adam P

    2016-06-01

    We have previously highlighted the ability of testosterone (T) to improve differentiation and myotube hypertrophy in fusion impaired myoblasts that display reduced myotube hypertrophy via multiple population doublings (PD) versus their parental controls (CON); an observation which is abrogated via PI3K/Akt inhibition (Deane et al. 2013). However, whether the most predominant molecular mechanism responsible for T induced hypertrophy occurs directly via androgen receptor or indirectly via IGF-IR/PI3K/Akt pathway is currently debated. PD and CON C2C12 muscle cells were exposed to low serum conditions in the presence or absence of T (100 nM) ± inhibitors of AR (flutamide/F, 40 μm) and IGF-IR (picropodophyllin/PPP, 150 nM) for 72 h and 7 days (early/late muscle differentiation respectively). T increased AR and Akt abundance, myogenin gene expression, and myotube hypertrophy, but not ERK1/2 activity in both CON and PD cell types. Akt activity was not increased significantly in either cell type with T. Testosterone was also unable to promote early differentiation in the presence of IGF-IR inhibitor (PPP) yet still able to promote appropriate later increases in myotube hypertrophy and AR abundance despite IGF-IR inhibition. The addition of the AR inhibitor powerfully attenuated all T induced increases in differentiation and myotube hypertrophy with corresponding reductions in AR abundance, phosphorylated Akt, ERK1/2 and gene expression of IGF-IR, myoD and myogenin with increases in myostatin mRNA in both cell types. Interestingly, despite basally reduced differentiation and myotube hypertrophy, PD cells showed larger T induced increases in AR abundance vs. CON cells, a response abrogated in the presence of AR but not IGF-IR inhibitors. Furthermore, T induced increases in Akt abundance were sustained despite the presence of IGF-IR inhibition in PD cells only. Importantly, flutamide alone reduced IGF-IR mRNA in both cell types across time points, with an observed

  13. l-glutamine Improves Skeletal Muscle Cell Differentiation and Prevents Myotube Atrophy After Cytokine (TNF-α) Stress Via Reduced p38 MAPK Signal Transduction.

    Science.gov (United States)

    Girven, Matthew; Dugdale, Hannah F; Owens, Daniel J; Hughes, David C; Stewart, Claire E; Sharples, Adam P

    2016-12-01

    Tumour Necrosis Factor-Alpha (TNF-α) is chronically elevated in conditions where skeletal muscle loss occurs. As l-glutamine can dampen the effects of inflamed environments, we investigated the role of l-glutamine in both differentiating C2C12 myoblasts and existing myotubes in the absence/presence of TNF-α (20 ng · ml(-1) ) ± l-glutamine (20 mM). TNF-α reduced the proportion of cells in G1 phase, as well as biochemical (CK activity) and morphological differentiation (myotube number), with corresponding reductions in transcript expression of: Myogenin, Igf-I, and Igfbp5. Furthermore, when administered to mature myotubes, TNF-α induced myotube loss and atrophy underpinned by reductions in Myogenin, Igf-I, Igfbp2, and glutamine synthetase and parallel increases in Fox03, Cfos, p53, and Bid gene expression. Investigation of signaling activity suggested that Akt and ERK1/2 were unchanged, JNK increased (non-significantly) whereas P38 MAPK substantially and significantly increased in both myoblasts and myotubes in the presence of TNF-α. Importantly, 20 mM l-glutamine reduced p38 MAPK activity in TNF-α conditions back to control levels, with a corresponding rescue of myoblast differentiation and a reversal of atrophy in myotubes. l-glutamine resulted in upregulation of genes associated with growth and survival including; Myogenin, Igf-Ir, Myhc2 & 7, Tnfsfr1b, Adra1d, and restored atrophic gene expression of Fox03 back to baseline in TNF-α conditions. In conclusion, l-glutamine supplementation rescued suppressed muscle cell differentiation and prevented myotube atrophy in an inflamed environment via regulation of p38 MAPK. l-glutamine administration could represent an important therapeutic strategy for reducing muscle loss in catabolic diseases and inflamed ageing. J. Cell. Physiol. 9999: 231: 2720-2732, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. A composite catalyst and its utilization for aromatization of C2-C12 hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Alario, F.; Benazzi, E.; Deves, J.M.; Joly, J.F.

    1994-09-02

    A composite catalyst is proposed for aromatization reactions of hydrocarbons containing from 2 to 12 carbon atoms per molecule, and more especially from 3 to 10 C atoms per molecule; the catalyst contains: a MFI structure zeolite which skeleton is composed of silicon, aluminium and/or gallium and which crystallites external faces have been modified after synthesis; a matrix; at least one doping element chosen among gallium and zinc when the zeolite does not contain gallium in its crystal skeleton; eventually, at least one doping element chosen among gallium and zinc when the zeolite contains gallium in its skeleton; and eventually, at least one element chosen among alkaline and alkaline-earth metals. 2 tabs.

  15. A new level of plasticity: Drosophila smooth-like testes muscles compensate failure of myoblast fusion.

    Science.gov (United States)

    Kuckwa, Jessica; Fritzen, Katharina; Buttgereit, Detlev; Rothenbusch-Fender, Silke; Renkawitz-Pohl, Renate

    2016-01-15

    The testis of Drosophila resembles an individual testis tubule of mammals. Both are surrounded by a sheath of smooth muscles, which in Drosophila are multinuclear and originate from a pool of myoblasts that are set aside in the embryo and accumulate on the genital disc later in development. These muscle stem cells start to differentiate early during metamorphosis and give rise to all muscles of the inner male reproductive system. Shortly before the genital disc and the developing testes connect, multinuclear nascent myotubes appear on the anterior tips of the seminal vesicles. Here, we show that adhesion molecules are distinctly localized on the seminal vesicles; founder cell (FC)-like myoblasts express Dumbfounded (Duf) and Roughest (Rst), and fusion-competent myoblast (FCM)-like cells mainly express Sticks and stones (Sns). The smooth but multinuclear myotubes of the testes arose by myoblast fusion. RNAi-mediated attenuation of Sns or both Duf and Rst severely reduced the number of nuclei in the testes muscles. Duf and Rst probably act independently in this context. Despite reduced fusion in all of these RNAi-treated animals, myotubes migrated onto the testes, testes were shaped and coiled, muscle filaments were arranged as in the wild type and spermatogenesis proceeded normally. Hence, the testes muscles compensate for fusion defects so that the myofibres encircling the adult testes are indistinguishable from those of the wild type and male fertility is guaranteed. © 2016. Published by The Company of Biologists Ltd.

  16. Developmental Programming in Response to Intrauterine Growth Restriction Impairs Myoblast Function and Skeletal Muscle Metabolism

    Directory of Open Access Journals (Sweden)

    D. T. Yates

    2012-01-01

    Full Text Available Fetal adaptations to placental insufficiency alter postnatal metabolic homeostasis in skeletal muscle by reducing glucose oxidation rates, impairing insulin action, and lowering the proportion of oxidative fibers. In animal models of intrauterine growth restriction (IUGR, skeletal muscle fibers have less myonuclei at birth. This means that myoblasts, the sole source for myonuclei accumulation in fibers, are compromised. Fetal hypoglycemia and hypoxemia are complications that result from placental insufficiency. Hypoxemia elevates circulating catecholamines, and chronic hypercatecholaminemia has been shown to reduce fetal muscle development and growth. We have found evidence for adaptations in adrenergic receptor expression profiles in myoblasts and skeletal muscle of IUGR sheep fetuses with placental insufficiency. The relationship of β-adrenergic receptors shifts in IUGR fetuses because Adrβ2 expression levels decline and Adrβ1 expression levels are unaffected in myofibers and increased in myoblasts. This adaptive response would suppress insulin signaling, myoblast incorporation, fiber hypertrophy, and glucose oxidation. Furthermore, this β-adrenergic receptor expression profile persists for at least the first month in IUGR lambs and lowers their fatty acid mobilization. Developmental programming of skeletal muscle adrenergic receptors partially explains metabolic and endocrine differences in IUGR offspring, and the impact on metabolism may result in differential nutrient utilization.

  17. Phosphorylation of Stim1 at serine 575 via netrin-2/Cdo-activated ERK1/2 is critical for the promyogenic function of Stim1.

    Science.gov (United States)

    Lee, Hye-Jin; Bae, Gyu-Un; Leem, Young-Eun; Choi, Hyun-Kyung; Kang, Tong Mook; Cho, Hana; Kim, Seong-Tae; Kang, Jong-Sun

    2012-04-01

    The promyogenic cell surface molecule Cdo is required for activation of extracellular signal-regulated kinase (ERK) and nuclear factor of activated T cells c3 (NFATc3) induced by netrin-2 in myogenic differentiation. However, the molecular mechanism leading to NFATc3 activation is unknown. Stromal interaction molecule 1 (Stim1), an internal calcium sensor of the endoplasmic reticulum store, promotes myogenesis via activation of NFATc3. In this study we investigated the functional interaction between Cdo and Stim1 in myogenic differentiation. Overexpression and depletion of Stim1 enhanced or decreased myotube formation, respectively. Of interest, Stim1 protein levels were decreased in Cdo-deficient perinatal hindlimb muscles or primary myoblasts; this correlates with defective NFATc3 activation in Cdo(-/-) myoblasts upon differentiation. Forced activation of NFATc3 by overexpression of calcineurin restored differentiation of Cdo-depleted C2C12 myoblasts. Furthermore, Cdo and Stim1 formed a complex in 293T cells or in differentiating C2C12 myoblasts. The netrin-2-mediated NFATc3 activation was coincident with robust interactions between Cdo and Stim1 in myoblasts and the ERK-mediated Stim1 phosphorylation at serine 575. The serine 575 phosphorylation was enhanced in C2C12 cells upon differentiation, and the alanine substitution of serine 575 failed to restore differentiation of Stim1-depleted myoblasts. Taken together, the results indicate that cell adhesion signaling triggered by netrin-2/Cdo induces Stim1 phosphorylation at serine 575 by ERK, which promotes myoblast differentiation.

  18. N-methyl D-aspartate receptor synaptonuclear signaling and neuronal migration factor (Nsmf) plays a novel role in myoblast proliferation.

    Science.gov (United States)

    Moon, Hyo Youl

    2015-01-01

    Myogenesis, the formation and regeneration of muscular tissue, is a fundamental factor in embryonic development. N-methyl D-aspartate (NMDA) receptor synaptonuclear signaling and neuronal migration factor (Nsmf) mediates NMDA receptor endocytosis in GnRH neuronal cells. NMDA receptor is involved in myoblast differentiation by regulating Ca2 (+) dependent fusion of myocytes. In this study, we investigated the role of Nsmf in myoblast proliferation and differentiation. Quantitative-PCR, immunoblotting, and immunohistochemistry results showed that the Nsmf expression levels increased during both the differentiation and proliferation of myocytes. Knockdown of Nsmf in myocytes by siRNA did not affect the myocyte differentiation marker myogenin. However, flow cytometry showed that the proliferation rate of the Nsmf-knockdown cells was reduced compared to the control cells. Therefore, our results indicate that Nsmf is a novel myogenic factor that can enhance myoblast proliferation. Furthermore, Nsmf may be an important therapeutic target in diseases associated with aging, muscular dystrophy, or cachexia.

  19. Adipose Tissue-Derived Stem Cell Secreted IGF-1 Protects Myoblasts from the Negative Effect of Myostatin

    Directory of Open Access Journals (Sweden)

    Sebastian Gehmert

    2014-01-01

    Full Text Available Myostatin, a TGF-β family member, is associated with inhibition of muscle growth and differentiation and might interact with the IGF-1 signaling pathway. Since IGF-1 is secreted at a bioactive level by adipose tissue-derived mesenchymal stem cells (ASCs, these cells (ASCs provide a therapeutic option for Duchenne Muscular Dystrophy (DMD. But the protective effect of stem cell secreted IGF-1 on myoblast under high level of myostatin remains unclear. In the present study murine myoblasts were exposed to myostatin under presence of ASCs conditioned medium and investigated for proliferation and apoptosis. The protective effect of IGF-1 was further examined by using IGF-1 neutralizing and receptor antibodies as well as gene silencing RNAi technology. MyoD expression was detected to identify impact of IGF-1 on myoblasts differentiation when exposed to myostatin. IGF-1 was accountable for 43.6% of the antiapoptotic impact and 48.8% for the proliferative effect of ASCs conditioned medium. Furthermore, IGF-1 restored mRNA and protein MyoD expression of myoblasts under risk. Beside fusion and transdifferentiation the beneficial effect of ASCs is mediated by paracrine secreted cytokines, particularly IGF-1. The present study underlines the potential of ASCs as a therapeutic option for Duchenne muscular dystrophy and other dystrophic muscle diseases.

  20. Compatibility of hyaluronic acid hydrogel and skeletal muscle myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wang Wei; Zhang Li; Sun Liang; Wang Chengyue [Jinzhou Central Hospital, Jinzhou 121000 (China); Fan Ming; Liu Shuhong, E-mail: Weiwang_Ly@yahoo.com.c [Institute of Basic Medical Sciences, Academy of Military Medical Science, Beijing 100850 (China)

    2009-04-15

    Compatibility of hyaluronic acid hydrogel (HAH) and skeletal muscle myoblasts has been investigated for the first time in the present paper. Skeletal muscle myoblasts were separated from skeletons of rats and incubated with a HAH-containing culture medium. Cell morphology, hydrophilicity and cell adhesion of the HAH scaffold were investigated using optical microscopy, scanning electron microscopy, Hoechest33258 fluorescent staining, the immunocytochemistry method and water adsorption rate measurement. It was found that at a proper concentration (around 0.5%) of hyaluronic acid, the hydrogel possessed good compatibility with skeletal muscle myoblasts. The hydrogel can create a three-dimensional structure for the growth of skeletal muscle myoblasts and benefit cell attachment to provide a novel scaffold material for the tissue engineering of skeletal muscle.

  1. Reversal of Myoblast Aging by Tocotrienol Rich Fraction Posttreatment

    Directory of Open Access Journals (Sweden)

    Jing Jye Lim

    2013-01-01

    Full Text Available Skeletal muscle satellite cells are heavily involved in the regeneration of skeletal muscle in response to the aging-related deterioration of the skeletal muscle mass, strength, and regenerative capacity, termed as sarcopenia. This study focused on the effect of tocotrienol rich fraction (TRF on regenerative capacity of myoblasts in stress-induced premature senescence (SIPS. The myoblasts was grouped as young control, SIPS-induced, TRF control, TRF pretreatment, and TRF posttreatment. Optimum dose of TRF, morphological observation, activity of senescence-associated β-galactosidase (SA-β-galactosidase, and cell proliferation were determined. 50 μg/mL TRF treatment exhibited the highest cell proliferation capacity. SIPS-induced myoblasts exhibit large flattened cells and prominent intermediate filaments (senescent-like morphology. The activity of SA-β-galactosidase was significantly increased, but the proliferation capacity was significantly reduced as compared to young control. The activity of SA-β-galactosidase was significantly reduced and cell proliferation was significantly increased in the posttreatment group whereas there was no significant difference in SA-β-galactosidase activity and proliferation capacity of pretreatment group as compared to SIPS-induced myoblasts. Based on the data, we hypothesized that TRF may reverse the myoblasts aging through replenishing the regenerative capacity of the cells. However, further investigation on the mechanism of TRF in reversing the myoblast aging is needed.

  2. Impact of static magnetic fields on human myoblast cell cultures.

    Science.gov (United States)

    Stern-Straeter, Jens; Bonaterra, Gabriel Alejandro; Kassner, Stefan S; Faber, Anne; Sauter, Alexander; Schulz, Johannes D; Hörmann, Karl; Kinscherf, Ralf; Goessler, Ulrich Reinhart

    2011-12-01

    Treatment of skeletal muscle loss due to trauma or tumor ablation therapy still lacks a suitable clinical approach. Creation of functional muscle tissue in vitro using the differentiation potential of human satellite cells (myoblasts) is a promising new research field called tissue engineering. Strong differentiation stimuli, which can induce formation of myofibers after cell expansion, have to be identified and evaluated in order to create sufficient amounts of neo-tissue. The objective of this study was to determine the influence of static magnetic fields (SMF) on human satellite cell cultures as one of the preferred stem cell sources in skeletal muscle tissue engineering. Experiments were performed using human satellite cells with and without SMF stimulation after incubation with a culture medium containing low [differentiation medium (DM)] or high [growth medium (GM)] concentrations of growth factors. Proliferation analysis using the alamarBlue assay revealed no significant influence of SMF on cell division. Real-time RT-PCR of the following marker genes was investigated: myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), skeletal muscle α1 actin (ACTA1), and embryonic (MYH3), perinatal (MYH8) and adult (MYH1) skeletal muscle myosin heavy chain. We detected an influence on marker gene expression by SMF in terms of a down-regulation of the marker genes in cell cultures treated with SMF and DM, but not in cell cultures treated with SMF and GM. Immunocytochemical investigations using antibodies directed against the differentiation markers confirmed the gene expression results and showed an enhancement of maturation after stimulation with GM and SMF. Additional calculation of the fusion index also revealed an increase in myotube formation in cell cultures treated with SMF and GM. Our findings show that the effect of SMF on the process of differentiation depends on the growth factor concentration in the culture medium in human

  3. Fusion from myoblasts to myotubes is dependent on the rolling stone gene (rost) of Drosophila.

    Science.gov (United States)

    Paululat, A; Burchard, S; Renkawitz-Pohl, R

    1995-08-01

    The development and differentiation of the body wall musculature in Drosophila are accompanied by changes in gene expression and cellular architecture. We isolated a Drosophila gene, termed rolling stone (rost), which, when mutated, specifically blocks the fusion of mononucleated cells to myotubes in the body wall musculature. beta 3 tubulin, which is an early marker for the onset of mesoderm differentiation, is still expressed in these cells. Gastrulation and mesoderm formation, as well as the development of the epidermis and of the central and peripheral nervous systems, appear quite normal in homozygous rolling stone embryos. Embryonic development stops shortly before hatching in a P-element-induced mutant, as well as in 16 EMS-induced alleles. In mutant embryos, other mesodermal derivatives such as the visceral mesoderm and the dorsal vessel, develop fairly normally and defects are restricted to the body wall musculature. Myoblasts remain as single mononucleated cells, which express muscle myosin, showing that the developmental program of gene expression proceeds. These myoblasts occur at positions corresponding to the locations of dorsal, ventral and pleural muscles, showing that the gene rolling stone is involved in cell fusion, a process that is independent of cell migration in these mutants. This genetic analysis has set the stage for a molecular analysis to clarify where the rolling stone action is manifested in the fusion process and thus gives insight into the complex regulating network controlling the differentiation of the body wall musculature.

  4. Myoblast transplantation for heart failure – From bench to bedside

    Directory of Open Access Journals (Sweden)

    Eugene K.W. Sim

    2005-07-01

    Full Text Available Heart failure causes morbidity and mortality. Cell transplantation using skeletal muscle myoblast is promising for myocardial repair as it can regenerate and repair the injury. Skeletal myoblasts are unipotent progenitor cells that can be expanded and genetically modified to deliver angiogenic cytokines and growth factors to encourage angiomyogenesis. Myoblast transplantation inhibits ventricular remodelling, decreases left ventricular diastolic dimension, increases myocardial wall thickness and minimizes global ventricular dilatation in animals. Ongoing trials with skeletal myoblast transplantation show improvement in perfusion and metabolic activity. Time constraints and the problem of generating autologous skeletal myoblasts for every patient can be overcome if allogeneic skeletal myoblasts from healthy young donors can be made available. Myoblast transplantation is confronted with the problem of donor cell survival post-transplantation. Its safety and feasibility have been documented during animal and phase I studies. The only serious postoperative adverse event related to the procedure was ventricular arrhythmias. The results of phase I studies are still preliminary. Endpoint measurements highlight improvement in quality of life, reduced nitroglycerine consumption, enhanced exercise tolerance, improvement in NYHA Class and wall motion by echocardiography, and significantly reduced perfusion defects. Future directions include concerted collaborative efforts, strict inclusion and exclusion criteria, better establishment of target population. Further work needs to be done on the ideal cell type, optimal number of cells and route of administration. The most suitable time for cell transplantation after ischemic injury and optimal mode of cell delivery are evaluated. The use of cell-based techniques to assist with cardiac regeneration holds promise for the treatment of heart failure.

  5. Myostatin alters glucose transporter-4 (GLUT4) expression in bovine skeletal muscles and myoblasts isolated from double-muscled (DM) and normal-muscled (NM) Japanese shorthorn cattle.

    Science.gov (United States)

    Takahashi, H; Sato, K; Yamaguchi, T; Miyake, M; Watanabe, H; Nagasawa, Y; Kitagawa, E; Terada, S; Urakawa, M; Rose, M T; McMahon, C D; Watanabe, K; Ohwada, S; Gotoh, T; Aso, H

    2014-07-01

    The purpose of this study was to determine whether myostatin alters glucose transporter-4 (GLUT4) expression in bovine skeletal muscles and myoblasts isolated from double-muscled (DM) and normal-muscled (NM) Japanese Shorthorn cattle. Plasma concentrations of glucose were lower in DM cattle than in NM cattle (P DM cattle than in NM cattle (P DM cattle did not differ with respect to skeletal muscle expression of GLUT1 and myocyte enhancer factor-2c (MEF2c), a transcription factor of GLUT4. In differentiated myoblasts, the expression of GLUT1, GLUT4, and MEF2c mRNAs was greater in DM cattle than in NM cattle (P DM cattle relative to that of NM cattle (P DM myoblasts (P DM cattle to produce muscle relative to the NM cattle may be due to their greater sensitivity to insulin and greater use of glucose. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. CHARACTERIZATION OF P2-PURINOCEPTOR MEDIATED CYCLIC-AMP FORMATION IN MOUSE C2C12 MYOTUBES

    NARCIS (Netherlands)

    HENNING, RH; DUIN, M; DENHERTOG, A; NELEMANS, A

    1 The formation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), induced by ATP and other nucleotides was investigated in mouse C2Cl2 myotubes. 2 ATP (100 muM) and ATPgammaS (100 muM) caused a sustained increase in cyclic AMP content of the cells,

  7. IGF-1 attenuates hypoxia-induced atrophy but inhibits myoglobin expression in C2C12 skeletal muscle myotubes

    NARCIS (Netherlands)

    Peters, Eva L.; van der Linde, Sandra M.; Vogel, Ilse S.P.; Haroon, Mohammad; Offringa, Carla; de Wit, Gerard M.J.; Koolwijk, Pieter; van der Laarse, Willem J.; Jaspers, Richard T.

    2017-01-01

    Chronic hypoxia is associated with muscle wasting and decreased oxidative capacity. By contrast, training under hypoxia may enhance hypertrophy and increase oxidative capacity as well as oxygen transport to the mitochondria, by increasing myoglobin (Mb) expression. The latter may be a feasible

  8. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    Energy Technology Data Exchange (ETDEWEB)

    Zuloaga, R. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Fuentes, E.N.; Molina, A. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile); Valdés, J.A., E-mail: jvaldes@unab.cl [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile)

    2013-10-18

    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.

  9. Dynamics of the skeletal muscle secretome during myoblast differentiation

    DEFF Research Database (Denmark)

    Henningsen, Jeanette; Rigbolt, Kristoffer T G; Blagoev, Blagoy

    2010-01-01

    of protein secretion. We identified and quantitatively analyzed 635 secreted proteins, including 35 growth factors, 40 cytokines, and 36 metallopeptidases. The extensive presence of these proteins that can act as potent signaling mediators to other cells and tissues strongly highlights the important role...... of the intracellular levels of members of the semaphorin family and their corresponding secretion dynamics demonstrated that the release of secreted proteins is tightly regulated by the secretory pathway, the stability of the protein, and/or the processing of secreted proteins. Finally, we provide 299 unique...

  10. File list: InP.Myo.20.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Myo.20.AllAg.Myoblasts mm9 Input control Muscle Myoblasts SRX497480,SRX029148,S...RX497481,SRX150190,SRX497484,SRX766227,SRX497485 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Myo.20.AllAg.Myoblasts.bed ...

  11. File list: Oth.Myo.10.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.10.AllAg.Myoblasts mm9 TFs and others Muscle Myoblasts SRX030143,SRX497478,...3,SRX029144,SRX227231,SRX227233,SRX984580,SRX227229 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Myo.10.AllAg.Myoblasts.bed ...

  12. File list: Oth.Myo.20.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.20.AllAg.Myoblasts hg19 TFs and others Muscle Myoblasts SRX668234,SRX107284...,SRX1470542,SRX1470544,SRX107285,SRX341010 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Myo.20.AllAg.Myoblasts.bed ...

  13. File list: Oth.Myo.50.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.50.AllAg.Myoblasts mm9 TFs and others Muscle Myoblasts SRX030143,SRX497478,...3,SRX227233,SRX227231,SRX029143,SRX984580,SRX227229 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Myo.50.AllAg.Myoblasts.bed ...

  14. File list: Unc.Myo.20.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Myo.20.AllAg.Myoblasts mm9 Unclassified Muscle Myoblasts SRX497492,SRX497493,SR...X030145 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Myo.20.AllAg.Myoblasts.bed ...

  15. File list: ALL.Myo.10.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Myo.10.AllAg.Myoblasts hg19 All antigens Muscle Myoblasts SRX668234,SRX107284,S...RX341010,SRX1470542,SRX1470544,SRX1470543,SRX1470545,SRX107285 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Myo.10.AllAg.Myoblasts.bed ...

  16. File list: Oth.Myo.10.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.10.AllAg.Myoblasts hg19 TFs and others Muscle Myoblasts SRX668234,SRX107284...,SRX341010,SRX1470542,SRX1470544,SRX107285 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Myo.10.AllAg.Myoblasts.bed ...

  17. File list: InP.Myo.50.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Myo.50.AllAg.Myoblasts mm9 Input control Muscle Myoblasts SRX029148,SRX497481,S...RX497480,SRX766227,SRX150190,SRX497485,SRX497484 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Myo.50.AllAg.Myoblasts.bed ...

  18. File list: Unc.Myo.10.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Myo.10.AllAg.Myoblasts mm9 Unclassified Muscle Myoblasts SRX030145,SRX497492,SR...X497493 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Myo.10.AllAg.Myoblasts.bed ...

  19. File list: ALL.Myo.20.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Myo.20.AllAg.Myoblasts hg19 All antigens Muscle Myoblasts SRX668234,SRX107284,S...RX1470542,SRX1470544,SRX1470545,SRX1470543,SRX107285,SRX341010 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Myo.20.AllAg.Myoblasts.bed ...

  20. File list: Unc.Myo.50.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Myo.50.AllAg.Myoblasts mm9 Unclassified Muscle Myoblasts SRX497492,SRX497493,SR...X030145 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Myo.50.AllAg.Myoblasts.bed ...

  1. File list: InP.Myo.20.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Myo.20.AllAg.Myoblasts hg19 Input control Muscle Myoblasts SRX1470545,SRX147054...3 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/InP.Myo.20.AllAg.Myoblasts.bed ...

  2. File list: InP.Myo.05.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Myo.05.AllAg.Myoblasts mm9 Input control Muscle Myoblasts SRX029148,SRX150190,S...RX766227,SRX497480,SRX497484,SRX497485,SRX497481 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Myo.05.AllAg.Myoblasts.bed ...

  3. File list: His.Myo.20.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Myo.20.AllAg.Myoblasts mm9 Histone Muscle Myoblasts SRX766229,SRX766228,SRX7662...26,SRX766225 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Myo.20.AllAg.Myoblasts.bed ...

  4. File list: His.Myo.10.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Myo.10.AllAg.Myoblasts mm9 Histone Muscle Myoblasts SRX766228,SRX766229,SRX7662...25,SRX766226 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Myo.10.AllAg.Myoblasts.bed ...

  5. File list: InP.Myo.05.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Myo.05.AllAg.Myoblasts hg19 Input control Muscle Myoblasts SRX1470545,SRX147054...3 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/InP.Myo.05.AllAg.Myoblasts.bed ...

  6. File list: InP.Myo.50.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Myo.50.AllAg.Myoblasts hg19 Input control Muscle Myoblasts SRX1470545,SRX147054...3 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/InP.Myo.50.AllAg.Myoblasts.bed ...

  7. File list: ALL.Myo.05.AllAg.Myoblasts [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Myo.05.AllAg.Myoblasts hg19 All antigens Muscle Myoblasts SRX668234,SRX107284,S...RX341010,SRX1470542,SRX1470544,SRX1470545,SRX1470543,SRX107285 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Myo.05.AllAg.Myoblasts.bed ...

  8. Myoblast transplantation for cardiac repair: from automyoblast to allomyoblast transplantation.

    Science.gov (United States)

    Guo, Changfa; Haider, Husnain Kh; Wang, Chunsheng; Tan, Ru-San; Shim, Winston S N; Wong, Philip; Sim, Eugene K W

    2008-12-01

    We sought to compare host immune cell kinetics, survival profile of donor skeletal myoblasts, and skeletal myoblast graft efficacy after autologous and allogeneic skeletal myoblast transplantation into a rat model of myocardial infarction. One week after myocardial infarction, 128 animals were divided into four groups: group 1 (n = 24, receiving medium only), group 2 (n = 24, receiving medium and cyclosporine), group 3 (n = 40, autologous skeletal myoblast transplantation), and group 4 (n = 40, allogeneic skeletal myoblast transplantation with cyclosporine treatment). Rats were euthanized 10 minutes, 1 day, and 4, 7, and 28 days later. Host immune cell kinetics were assessed by immunohistochemical studies for macrophages, and CD4+ and CD8+ lymphocytes. Donor skeletal myoblast survival was confirmed by tracking prelabeled signals, and quantified by beta-gal assay. Heart function was evaluated by echocardiography. A transient immune cell infiltration was demonstrated in group 3, with macrophage infiltration on day 1 and day 4, CD8+ cell infiltration on day 4 and day 7, and CD4+ cell infiltration on day 4. In group 4, immunocyte infiltration was slightly more severe than that in group 3. Automyoblasts and allomyoblasts showed no significant difference of survival from day 1 to day 7 (p > 0.10); however, on day 28, automyoblasts showed better survival than allomyoblasts (p Transplantation of allomyoblasts increased systolic heart function and limited heart dilation after myocardial injury to a similar degree as automyoblasts (p > 0.10). The use of allomyoblasts is feasible and effective for cardiac repair with immunosuppressive treatment as compared with automyoblasts.

  9. Grooved PLGA films incorporated with RGD/YIGSR peptides for potential application on skeletal muscle tissue engineering.

    Science.gov (United States)

    Wang, Peng-Yuan; Wu, Tsung-Han; Tsai, Wei-Bor; Kuo, Wei-Hsuan; Wang, Meng-Jiy

    2013-10-01

    Alignment of myocytes or myotubes is critical for skeletal muscle tissue engineering. In this study, grooved PLGA films (800nm in width of ridge/groove and 600nm in depth) incorporated with RGD or YIGSR peptides were fabricated to evaluate its efficacy for skeletal muscle tissue engineering. The growth and differentiation of C2C12 myoblasts were enhanced by the presentation of RGD or YIGSR compared with the untreated PLGA control. On the other hand, cell morphology was guided by the grooved structure, i.e. alignment of myoblasts and myotubes with the direction of grooves. This study elucidates the effects of both surface biochemical and topographic cues on the proliferation and differentiation of C2C12 myoblasts on biodegradable polymer films. Combination of surface topography and peptide presentation has a great potential in designing scaffolds for skeletal muscle tissue engineering. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Modelling human myoblasts survival upon xenotransplantation into immunodeficient mouse muscle.

    Science.gov (United States)

    Praud, Christophe; Vauchez, Karine; Zongo, Pascal; Vilquin, Jean-Thomas

    2018-03-15

    Cell transplantation has been challenged in several clinical indications of genetic or acquired muscular diseases, but therapeutic success were mitigated. To understand and improve the yields of tissue regeneration, we aimed at modelling the fate of CD56-positive human myoblasts after transplantation. Using immunodeficient severe combined immunodeficiency (SCID) mice as recipients, we assessed the survival, integration and satellite cell niche occupancy of human myoblasts by a triple immunohistochemical labelling of laminin, dystrophin and human lamin A/C. The counts were integrated into a classical mathematical decline equation. After injection, human cells were essentially located in the endomysium, then they disappeared progressively from D0 to D28. The final number of integrated human nuclei was grossly determined at D2 after injection, suggesting that no more efficient fusion between donor myoblasts and host fibers occurs after the resolution of the local damages created by needle insertion. Almost 1% of implanted human cells occupied a satellite-like cell niche. Our mathematical model validated by histological counting provided a reliable quantitative estimate of human myoblast survival and/or incorporation into SCID muscle fibers. Informations brought by histological labelling and this mathematical model are complementary. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Effect of atrophy and contractions on myogenin mRNA concentration in chick and rat myoblast omega muscle cells

    Science.gov (United States)

    Krebs, J. M.; Denney, R. M.

    1997-01-01

    The skeletal rat myoblast omega (RMo) cell line forms myotubes that exhibit spontaneous contractions under appropriate conditions in culture. We examined if the RMo cells would provide a model for studying atrophy and muscle contraction. To better understand how to obtain contractile cultures, we examined levels of contraction under different growing conditions. The proliferation medium and density of plating affected the subsequent proportion of spontaneously contracting myotubes. Using a ribonuclease protection assay, we found that exponentially growing RMo myoblasts contained no detectable myogenin or herculin mRNA, while differentiating myoblasts contained high levels of myogenin mRNA but no herculin mRNA. There was no increase in myogenin mRNA concentration in either primary chick or RMo myotubes whose contractions were inhibited by depolarizing concentrations of potassium (K+). Thus, altered myogenin mRNA concentrations are not involved in atrophy of chick myotubes. Depolarizing concentrations of potassium inhibited spontaneous contractions in both RMo cultures and primary chick myotube cultures. However, we found that the myosin concentration of 6-d-old contracting RMo cells fed medium plus AraC was 11 +/- 3 micrograms myosin/microgram DNA, not significantly different from 12 +/- 4 micrograms myosin/microgram DNA (n = 3), the myosin concentration of noncontracting RMo cells (treated with 12 mM K+ for 6 d). Resolving how RMo cells maintained their myosin content when contraction is inhibited may be important for understanding atrophy.

  12. Marked heterogeneity in growth characteristics of myoblast clonal cultures and myoblast mixed cultures obtained from the same individual

    NARCIS (Netherlands)

    Maier, Andrea B.; Cohen, Ron; Blom, Joke; Van Heemst, Diana; Westendorp, Rudi G.J.

    Background: Sarcopenia is defined as an age-related decrease in skeletal muscle mass and function while adjacent satellite cells are unable to compensate for this loss. However, myoblast cultures can be established even in the presence of sarcopenia. Objective: It is yet unknown whether satellite

  13. Myogenesis in the sea urchin embryo: the molecular fingerprint of the myoblast precursors

    Science.gov (United States)

    2013-01-01

    Background In sea urchin larvae the circumesophageal fibers form a prominent muscle system of mesodermal origin. Although the morphology and later development of this muscle system has been well-described, little is known about the molecular signature of these cells or their precise origin in the early embryo. As an invertebrate deuterostome that is more closely related to the vertebrates than other commonly used model systems in myogenesis, the sea urchin fills an important phylogenetic gap and provides a unique perspective on the evolution of muscle cell development. Results Here, we present a comprehensive description of the development of the sea urchin larval circumesophageal muscle lineage beginning with its mesodermal origin using high-resolution localization of the expression of several myogenic transcriptional regulators and differentiation genes. A few myoblasts are bilaterally distributed at the oral vegetal side of the tip of the archenteron and first appear at the late gastrula stage. The expression of the differentiation genes Myosin Heavy Chain, Tropomyosin I and II, as well as the regulatory genes MyoD2, FoxF, FoxC, FoxL1, Myocardin, Twist, and Tbx6 uniquely identify these cells. Interestingly, evolutionarily conserved myogenic factors such as Mef2, MyoR and Six1/2 are not expressed in sea urchin myoblasts but are found in other mesodermal domains of the tip of the archenteron. The regulatory states of these domains were characterized in detail. Moreover, using a combinatorial analysis of gene expression we followed the development of the FoxF/FoxC positive cells from the onset of expression to the end of gastrulation. Our data allowed us to build a complete map of the Non-Skeletogenic Mesoderm at the very early gastrula stage, in which specific molecular signatures identify the precursors of different cell types. Among them, a small group of cells within the FoxY domain, which also express FoxC and SoxE, have been identified as plausible myoblast

  14. Myogenesis in the sea urchin embryo: the molecular fingerprint of the myoblast precursors.

    Science.gov (United States)

    Andrikou, Carmen; Iovene, Edmondo; Rizzo, Francesca; Oliveri, Paola; Arnone, Maria Ina

    2013-12-02

    In sea urchin larvae the circumesophageal fibers form a prominent muscle system of mesodermal origin. Although the morphology and later development of this muscle system has been well-described, little is known about the molecular signature of these cells or their precise origin in the early embryo. As an invertebrate deuterostome that is more closely related to the vertebrates than other commonly used model systems in myogenesis, the sea urchin fills an important phylogenetic gap and provides a unique perspective on the evolution of muscle cell development. Here, we present a comprehensive description of the development of the sea urchin larval circumesophageal muscle lineage beginning with its mesodermal origin using high-resolution localization of the expression of several myogenic transcriptional regulators and differentiation genes. A few myoblasts are bilaterally distributed at the oral vegetal side of the tip of the archenteron and first appear at the late gastrula stage. The expression of the differentiation genes Myosin Heavy Chain, Tropomyosin I and II, as well as the regulatory genes MyoD2, FoxF, FoxC, FoxL1, Myocardin, Twist, and Tbx6 uniquely identify these cells. Interestingly, evolutionarily conserved myogenic factors such as Mef2, MyoR and Six1/2 are not expressed in sea urchin myoblasts but are found in other mesodermal domains of the tip of the archenteron. The regulatory states of these domains were characterized in detail. Moreover, using a combinatorial analysis of gene expression we followed the development of the FoxF/FoxC positive cells from the onset of expression to the end of gastrulation. Our data allowed us to build a complete map of the Non-Skeletogenic Mesoderm at the very early gastrula stage, in which specific molecular signatures identify the precursors of different cell types. Among them, a small group of cells within the FoxY domain, which also express FoxC and SoxE, have been identified as plausible myoblast precursors. Together

  15. BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation.

    Directory of Open Access Journals (Sweden)

    Takashi Matsumoto

    Full Text Available Bone morphogenetic proteins (BMPs regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3 which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophilaaristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp, Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.

  16. Human myotubes from myoblast cultures undergoing senescence exhibit defects in glucose and lipid metabolism

    DEFF Research Database (Denmark)

    Nehlin, Jan O; Just, Marlene; Rustan, Arild C

    2011-01-01

    Adult stem cells are known to have a finite replication potential. Muscle biopsy-derived human satellite cells (SCs) were grown at different passages and differentiated to human myotubes in culture to analyze the functional state of various carbohydrate and lipid metabolic pathways...... number and could be explained by reduced incorporation into diacyl- and triacylglycerols. The levels of long-chain acyl-CoA esters decreased with increased passage number. Late-passage, non-proliferating, myoblast cultures showed strong senescence-associated β-galactosidase activity indicating...... that the observed metabolic defects accompany the induction of a senescent state. The main function of SCs is regeneration and skeletal muscle-build up. Thus, the metabolic defects observed during aging of SC-derived myotubes could have a role in sarcopenia, the gradual age-related loss of muscle mass and strength....

  17. Transplanted myoblasts can migrate several millimeters to fuse with damaged myofibers in nonhuman primate skeletal muscle.

    Science.gov (United States)

    Skuk, Daniel; Goulet, Marlyne; Tremblay, Jacques P

    2011-09-01

    A major restriction of the intramuscular transplantation of myoblasts is that the grafted cells fuse mostly with myofibers along the injection trajectories. This has been attributed to a "lack of migration ability" of the grafted myoblasts. It has been assumed that grafted myoblasts remain motionless in the sites of delivery and fuse only with myofibers with which they come into contact. In the present study, we analyzed this phenomenon in 17 cynomolgus monkeys. We found that intramuscularly injected myoblasts within 1 hour after their injection are mainly located in the perimysium and not distributed along the injection trajectories. This suggested that the grafted myoblasts later migrate from the perimysium to fuse with myofibers that are damaged by the injections. Therefore, we analyzed whether β-galactosidase-labeled myoblasts injected subcutaneously over skeletal muscles migrate in needle-damaged and nondamaged muscle regions. We observed that grafted myoblasts migrated up to 1cm in depth from the muscle surface into the muscles, although they seemingly fused mainly with damaged myofibers. Our findings suggest that myoblast transplantation is not necessarily restricted bya "lack of migration ability" of the grafted cells but by the fact that myoblasts fuse with regenerating myofibers and not with undamaged myofibers.

  18. Regulation of mitochondrial biogenesis during myogenesis

    OpenAIRE

    Remels, A.H.V.; Langen, R.C.J.; Schrauwen, P.; Schaart, G.; Schols, A.M.W.J.; Gosker, H. R.

    2009-01-01

    Abstract Pathways involved in mitochondrial biogenesis associated with myogenic differentiation are poorly defined. Therefore, C2C12 myoblasts were differentiated into multi-nucleated myotubes and parameters/regulators of mitochondrial biogenesis were investigated. Mitochondrial respiration, citrate synthase- and ?-hydroxyacyl-CoA dehydrogenase activity as well as protein content of complexes I, II, III and V of the mitochondrial respiratory chain increased 4-8 fold during differen...

  19. Scaffold protein enigma homolog 1 overcomes the repression of myogenesis activation by inhibitor of DNA binding 2

    Energy Technology Data Exchange (ETDEWEB)

    Nakatani, Miyuki [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan); Ito, Jumpei [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan); Japan Society for the Promotion of Science, Tokyo, 102-0083 (Japan); Koyama, Riko [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan); Iijima, Masumi; Yoshimoto, Nobuo [The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047 (Japan); Niimi, Tomoaki [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan); Kuroda, Shun' ichi [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan); The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047 (Japan); Maturana, Andrés D., E-mail: maturana@agr.nagoya-u.ac.jp [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan)

    2016-05-27

    Enigma Homolog 1 (ENH1) is a scaffold protein for signaling proteins and transcription factors. Previously, we reported that ENH1 overexpression promotes the differentiation of C2C12 myoblasts. However, the molecular mechanism underlying the role of ENH1 in the C2C12 cells differentiation remains elusive. ENH1 was shown to inhibit the proliferation of neuroblastoma cells by sequestering Inhibitor of DNA binding protein 2 (Id2) in the cytosol. Id2 is a repressor of basic Helix-Loop-Helix transcription factors activity and prevents myogenesis. Here, we found that ENH1 overcome the Id2 repression of C2C12 cells myogenic differentiation and that ENH1 overexpression promotes mice satellite cells activation, the first step toward myogenic differentiation. In addition, we show that ENH1 interacted with Id2 in C2C12 cells and mice satellite cells. Collectively, our results suggest that ENH1 plays an important role in the activation of myogenesis through the repression of Id2 activity. -- Highlights: •Enigma Homolog 1 (ENH1) is a scaffold protein. •ENH1 binds to inhibitor of DNA binding 2 (Id2) in myoblasts. •ENH1 overexpression overcomes the Id2's repression of myogenesis. •The Id2-ENH1 complex play an important role in the activation of myogenesis.

  20. Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System

    NARCIS (Netherlands)

    Chaturvedi, V.; Dye, D.E.; Kinnear, B.F.; Kuppevelt, T.H. van; Grounds, M.D.; Coombe, D.R.

    2015-01-01

    Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast

  1. A Pitx2-MicroRNA Pathway Modulates Cell Proliferation in Myoblasts and Skeletal-Muscle Satellite Cells and Promotes Their Commitment to a Myogenic Cell Fate

    Science.gov (United States)

    Lozano-Velasco, Estefanía; Vallejo, Daniel; Esteban, Francisco J.; Doherty, Chris; Hernández-Torres, Francisco; Franco, Diego

    2015-01-01

    The acquisition of a proliferating-cell status from a quiescent state as well as the shift between proliferation and differentiation are key developmental steps in skeletal-muscle stem cells (satellite cells) to provide proper muscle regeneration. However, how satellite cell proliferation is regulated is not fully understood. Here, we report that the c-isoform of the transcription factor Pitx2 increases cell proliferation in myoblasts by downregulating microRNA 15b (miR-15b), miR-23b, miR-106b, and miR-503. This Pitx2c-microRNA (miRNA) pathway also regulates cell proliferation in early-activated satellite cells, enhancing Myf5+ satellite cells and thereby promoting their commitment to a myogenic cell fate. This study reveals unknown functions of several miRNAs in myoblast and satellite cell behavior and thus may have future applications in regenerative medicine. PMID:26055324

  2. Evaluation of a thermoresponsive polycaprolactone scaffold for in vitro three-dimensional stem cell differentiation.

    Science.gov (United States)

    Hruschka, Veronika; Saeed, Aram; Slezak, Paul; Cheikh Al Ghanami, Racha; Feichtinger, Georg Alexander; Alexander, Cameron; Redl, Heinz; Shakesheff, Kevin; Wolbank, Susanne

    2015-01-01

    Tissue engineering (TE) strategies aim at imitating the natural process of regeneration by using bioresorbable scaffolds that support cellular attachment, migration, proliferation, and differentiation. Based on the idea of combining a fully degradable polymer [poly(ɛ-caprolactone)] with a thermoresponsive polymer (polyethylene glycol methacrylate), a scaffold was developed, which liquefies below 20°C and solidifies at 37°C. In this study, this scaffold was evaluated for its ability to support C2C12 cells and human adipose-derived stem cells (ASCs) to generate an expandable three-dimensional (3D) construct for soft or bone TE. As a first step, biomaterial seeding was optimized and cellular attachment, survival, distribution, and persistence within the 3D material were characterized. C2C12 cells were differentiated toward the osteogenic as well as myogenic lineage, while ASCs were cultured in control, adipogenic, or osteogenic differentiation media. Differentiation was examined using quantitative real-time PCR for the expression of osteogenic, myogenic, and adipogenic markers and by enzyme activity and immunoassays. Both cell types attached and were found evenly distributed within the material. C2C12 cells and ASCs demonstrated the potential to differentiate in all tested lineages under 2D conditions. Under 3D osteogenic conditions for C2C12 cells, only osteocalcin expression (fold induction: 16.3±0.2) and alkaline phosphatase (ALP) activity (pdifferentiation of ASC was limited and donor dependent. Only one donor showed an increase in the osteogenic markers osteocalcin (p=0.027) and osteopontin (p=0.038). In contrast, differentiation toward the myogenic or adipogenic lineage showed expression of specific markers in 3D, at least at the level of the 2D culture. In 3D culture, strong induction of myogenin (pdifferentiation of one donor showed greater expression of peroxisome proliferative-activated receptor gamma (PPARγ) (p=0.004), fatty acid binding protein 4

  3. Effects of myasthenia gravis patient sera on human myoblast cultures.

    Science.gov (United States)

    Luckman, S P; Skeie, G O; Helgeland, G; Gilhus, N E

    2006-01-01

    To investigate the role of acetylcholine receptor (AChR) and other anti-muscle autoantibodies in myasthenia gravis (MG). Since many of these autoantibodies target proteins with structural or signalling functions, we examined the effect of MG sera on muscle cell morphology. Primary human myoblast cultures were exposed to MG sera and morphological changes observed by light and fluorescence microscopy. MG patient sera caused changes in cell shape (cell retraction) and led to the formation of inclusion bodies and intracellular vesicles. A disordered arrangement of actin microfilaments was also observed. The effects were not complement-mediated, were both dose- and time-dependent, and appeared to correlate with disease severity of the MG donor. The factors responsible for these effects in vitro may also play a role in the pathogenesis of MG in vivo. Further study of these factors may improve our understanding of MG pathogenesis.

  4. An HMGA2-IGF2BP2 axis regulates myoblast proliferation and myogenesis.

    Science.gov (United States)

    Li, Zhizhong; Gilbert, Jason A; Zhang, Yunyu; Zhang, Minsi; Qiu, Qiong; Ramanujan, Krishnan; Shavlakadze, Tea; Eash, John K; Scaramozza, Annarita; Goddeeris, Matthew M; Kirsch, David G; Campbell, Kevin P; Brack, Andrew S; Glass, David J

    2012-12-11

    A group of genes that are highly and specifically expressed in proliferating skeletal myoblasts during myogenesis was identified. Expression of one of these genes, Hmga2, increases coincident with satellite cell activation, and later its expression significantly declines correlating with fusion of myoblasts into myotubes. Hmga2 knockout mice exhibit impaired muscle development and reduced myoblast proliferation, while overexpression of HMGA2 promotes myoblast growth. This perturbation in proliferation can be explained by the finding that HMGA2 directly regulates the RNA-binding protein IGF2BP2. Add-back of IGF2BP2 rescues the phenotype. IGF2BP2 in turn binds to and controls the translation of a set of mRNAs, including c-myc, Sp1, and Igf1r. These data demonstrate that the HMGA2-IGF2BP2 axis functions as a key regulator of satellite cell activation and therefore skeletal muscle development. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. TNF-α inhibits SATB2 expression and osteoblast differentiation through NF-κB and MAPK pathways.

    Science.gov (United States)

    Zuo, Chijian; Zhao, Xiaoying; Shi, Yu; Wu, Wen; Zhang, Ning; Xu, Jiake; Wang, Chuandong; Hu, Guoli; Zhang, Xiaoling

    2018-01-12

    Although the mechanisms of Tumor necrosis factor alpha (TNF-α) on facilitating osteoclast differentiation and bone resorption is well known, the mechanisms behind the suppression of the osteoblast differentiation from mesenchymal stem cells (MSCs) are still poorly understood. In this study, we observed a negative correlation between TNF-α levels and the expression of special AT-rich sequence-binding protein 2 (SATB2), a critical osteoblastogenesis transcription factor, in ovariectomy (OVX)-induced bone loss and IL-1-induced arthritis animal model. We found that TNF-α treatment inhibited mesenchymal cell line C2C12 osteoblast differentiation and sharply decreased BMP2-induced SATB2 expression. Upon TNF-α treatment, the activity of smad1/5/8 was inhibited, by contrast, extracellular signal-regulated kinase-1/2 (ERK1/2) and P38 was increased in C2C12 cells, the inhibitor of ERK1/2 (U0126) was found to abrogate the TNF-α inhibition of SATB2 expression. Furthermore, the NF-κB signaling pathway in C2C12 cells was significantly activated by the treatment of TNF-α, and TNF-α induced NF-κB directly binds to SATB2 promoter to suppress its expression. These results suggest that TNF-α suppresses SATB2 expression through activating NF-κB and MAPK signaling and depressing smad1/5/8 signaling, which contributes to the inhibition of osteoblast differentiation and might be potential therapeutic targets for inflammation-induced bone loss.

  6. Myoblast Fusion in Fly and Vertebrates: New Genes, New Processes and New Perspectives

    OpenAIRE

    Richardson, Brian E.; Nowak, Scott J.; Baylies, Mary K.

    2008-01-01

    Muscle formation and repair depends critically on the fusion of myoblasts. Despite the importance of this process, little is known about the cellular and molecular mechanisms regulating fusion. Forward genetic screens in Drosophila melanogaster have uncovered genes that, when mutated, prevent myoblast fusion. Analyses of these gene products have indicated that the actin cytoskeleton and its regulation play a central role in the fusion process. In this review, we discuss recent advances in the...

  7. Balance between S-nitrosylation and denitrosylation modulates myoblast proliferation independently of soluble guanylyl cyclase activation.

    Science.gov (United States)

    Yamashita, Aline M S; Ancillotti, Maryana T C; Rangel, Luciana P; Fontenele, Marcio; Figueiredo-Freitas, Cicero; Possidonio, Ana C; Soares, Carolina P; Sorenson, Martha M; Mermelstein, Claudia; Nogueira, Leonardo

    2017-07-01

    Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form S-nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of S-nitrosoglutathione reductase (GSNOR) during myogenesis is understood. Here, we used primary cultures of chick embryonic skeletal muscle cells to investigate whether changes in intracellular RSNO alter proliferation and fusion of myoblasts in the presence and absence of cGMP. Cultures were grown to fuse most of the myoblasts into myotubes, with and without S-nitrosocysteine (CysNO), 8-Br-cGMP, DETA-NO, or inhibitors for NO synthase (NOS), GSNOR, soluble guanylyl cyclase (sGC), or a combination of these, followed by analysis of GSNOR activity, protein expression, RSNO, cGMP, and cell morphology. Although the activity of GSNOR increased progressively over 72 h, inhibiting GSNOR (by GSNOR inhibitor - GSNORi - or by knocking down GSNOR with siRNA) produced an increase in RSNO and in the number of myoblasts and fibroblasts, accompanied by a decrease in myoblast fusion index. This was also detected with CysNO supplementation. Enhanced myoblast number was proportional to GSNOR inhibition. Effects of the GSNORi and GSNOR knockdown were blunted by NOS inhibition, suggesting their dependence on NO synthesis. Interestingly, GSNORi and GSNOR knockdown reversed the attenuated proliferation obtained with sGC inhibition in myoblasts, but not in fibroblasts. Hence myoblast proliferation is enhanced by increasing RSNO, and regulated by GSNOR activity, independently of cGMP production and signaling. Copyright © 2017 the American Physiological Society.

  8. Serum from human burn victims impairs myogenesis and protein synthesis in primary myoblasts

    Science.gov (United States)

    Corrick, Katie L.; Stec, Michael J.; Merritt, Edward K.; Windham, Samuel T.; Thomas, Steven J.; Cross, James M.; Bamman, Marcas M.

    2015-01-01

    The pathophysiological response to a severe burn injury involves a robust increase in circulating inflammatory/endocrine factors and a hypermetabolic state, both of which contribute to prolonged skeletal muscle atrophy. In order to characterize the role of circulating factors in muscle atrophy following a burn injury, human skeletal muscle satellite cells were grown in culture and differentiated to myoblasts/myotubes in media containing serum from burn patients or healthy, age, and sex-matched controls. While incubation in burn serum did not affect NFκB signaling, cells incubated in burn serum displayed a transient increase in STAT3 phosphorlyation (Tyr705) after 48 h of treatment with burn serum (≈ + 70%; P < 0.01), with these levels returning to normal by 96 h. Muscle cells differentiated in burn serum displayed reduced myogenic fusion signaling (phospho-STAT6 (Tyr641), ≈−75%; ADAM12, ≈-20%; both P < 0.01), and reduced levels of myogenin (≈−75%; P < 0.05). Concomitantly, myotubes differentiated in burn serum demonstrated impaired myogenesis (assessed by number of nuclei/myotube). Incubation in burn serum for 96 h did not increase proteolytic signaling (assessed via caspase-3 and ubiquitin levels), but reduced anabolic signaling [p-p70S6k (Ser421/Thr424), −30%; p-rpS6 (Ser240/244), ≈-50%] and impaired protein synthesis (−24%) (P < 0.05). This resulted in a loss of total protein content (−18%) and reduced cell size (−33%) (P < 0.05). Overall, incubation of human muscle cells in serum from burn patients results in impaired myogenesis and reduced myotube size, indicating that circulating factors may play a significant role in muscle loss and impaired muscle recovery following burn injury. PMID:26136691

  9. The small G-proteins Rac1 and Cdc42 are essential for myoblast fusion in the mouse

    DEFF Research Database (Denmark)

    Vasyutina, Elena; Martarelli, Benedetta; Brakebusch, Cord

    2009-01-01

    Rac1 and Cdc42 are small G-proteins that regulate actin dynamics and affect plasma membrane protrusion and vesicle traffic. We used conditional mutagenesis in mice to demonstrate that Rac1 and Cdc42 are essential for myoblast fusion in vivo and in vitro. The deficit in fusion of Rac1 or Cdc42...... mutant myoblasts correlates with a deficit in the recruitment of actin fibers and vinculin to myoblast contact sites. Comparison of the changes observed in mutant myogenic cells indicates that Rac1 and Cdc42 function in a nonredundant and not completely overlapping manner during the fusion process. Our...... genetic analysis demonstrates thus that the function of Rac in myoblast fusion is evolutionarily conserved from insects to mammals and that Cdc42, a molecule hitherto not implicated in myoblast fusion, is essential for the fusion of murine myoblasts....

  10. Insulin-like growth factor-1 suppresses the Myostatin signaling pathway during myogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Retamales, A.; Zuloaga, R.; Valenzuela, C.A. [Laboratorio de Biotecnología Molecular, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago (Chile); Gallardo-Escarate, C. [Laboratory of Biotechnology and Aquatic Genomics, Universidad de Concepción, Concepción (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), P.O. Box 160-C, Concepción (Chile); Molina, A. [Laboratorio de Biotecnología Molecular, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), P.O. Box 160-C, Concepción (Chile); Valdés, J.A., E-mail: jvaldes@unab.cl [Laboratorio de Biotecnología Molecular, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), P.O. Box 160-C, Concepción (Chile)

    2015-08-21

    Myogenic differentiation is a complex and well-coordinated process for generating mature skeletal muscle fibers. This event is autocrine/paracrine regulated by growth factors, principally Myostatin (MSTN) and Insulin-like Growth Factor-1 (IGF-1). Myostatin, a member of the transforming growth factor-β superfamily, is a negative regulator of skeletal muscle growth in vertebrates that exerts its inhibitory function by activating Smad transcription factors. In contrast, IGF-1 promotes the differentiation of skeletal myoblasts by activating the PI3K/Akt signaling pathway. This study reports on a novel functional crosstalk between the IGF-1 and MSTN signaling pathways, as mediated through interaction between PI3K/Akt and Smad3. Stimulation of skeletal myoblasts with MSTN resulted in a transient increase in the pSmad3:Smad3 ratio and Smad-dependent transcription. Moreover, MSTN inhibited myod gene expression and myoblast fusion in an Activin receptor-like kinase/Smad3-dependent manner. Preincubation of skeletal myoblasts with IGF-1 blocked MSTN-induced Smad3 activation, promoting myod expression and myoblast differentiation. This inhibitory effect of IGF-1 on the MSTN signaling pathway was dependent on IGF-1 receptor, PI3K, and Akt activities. Finally, immunoprecipitation assay analysis determined that IGF-1 pretreatment increased Akt and Smad3 interaction. These results demonstrate that the IGF-1/PI3K/Akt pathway may inhibit MSTN signaling during myoblast differentiation, providing new insight to existing knowledge on the complex crosstalk between both growth factors. - Highlights: • IGF-1 inhibits Myostatin canonical signaling pathway through IGF-1R/PI3K/Akt pathway. • IGF-1 promotes myoblast differentiation through a direct blocking of Myostatin signaling pathway. • IGF-1 induces the interaction of Akt with Smad3 in skeletal myoblast.

  11. Polyurethane scaffolds seeded with genetically engineered skeletal myoblasts: a promising tool to regenerate myocardial function.

    Science.gov (United States)

    Blumenthal, Britta; Golsong, Peter; Poppe, Annika; Heilmann, Claudia; Schlensak, Christian; Beyersdorf, Friedhelm; Siepe, Matthias

    2010-02-01

    In animal models, intramyocardial injection of primary skeletal myoblasts is supposed to promote tissue regeneration and to improve cardiac function after myocardial infarction. The usage of genetically engineered myoblasts overexpressing the paracrine factors involved in tissue repair is believed to enhance these effects. However, cell therapy via injection is always accompanied by a high death rate of the injected cells. Here, we describe the construction of a growth factor-producing myoblast-seeded scaffold to overcome this limitation. Skeletal myoblasts were isolated and expanded from newborn Lewis rats. Cells were seeded on polyurethane (PU) scaffolds (Artelon) and transfected with DNA of VEGF-A, HGF, SDF-1, or Akt1 using the lipid-based Metafectene Pro method. Overexpression was verified by ELISA, RT-PCR (VEGF-A, HGF, and SDF-1) and Western blot analysis (Akt1). The seeded scaffolds were transplanted onto damaged myocardium of Lewis rats 2 weeks after myocardial infarction. Six weeks later, their therapeutic potential in vivo was analyzed by measurement of infarction size and capillary density. Primary rat skeletal myoblasts seeded on PU scaffolds were efficiently transfected, achieving transfection rates of 20%. In vitro, we noted a significant increase in expression of VEGF-A, HGF, SDF-1, and Akt1 after transfection. In vivo, transplantation of growth factor-producing myoblast-seeded scaffolds resulted in enhanced angiogenesis (VEGF-A, HGF, and Akt1) or a reduced infarction zone (SDF-1 and Akt1) in the ischemically damaged myocardium. In summary, we constructed a growth factor-producing myoblast-seeded scaffold which combines the beneficial potential of stem cell transplantation with the promising effects of gene-therapeutic approaches. Because this matrix also allows us to circumvent previous cell application drawbacks, it may represent a promising tool for tissue regeneration and the re-establishment of cardiac function after myocardial infarction.

  12. hHGF overexpression in myoblast sheets enhances their angiogenic potential in rat chronic heart failure.

    Directory of Open Access Journals (Sweden)

    Antti Siltanen

    Full Text Available After severe myocardial infarction (MI, heart failure results from ischemia, fibrosis, and remodeling. A promising therapy to enhance cardiac function and induce therapeutic angiogenesis via a paracrine mechanism in MI is myoblast sheet transplantation. We hypothesized that in a rat model of MI-induced chronic heart failure, this therapy could be further improved by overexpression of the antiapoptotic, antifibrotic, and proangiogenic hepatocyte growth factor (HGF in the myoblast sheets. We studied the ability of wild type (L6-WT and human HGF-expressing (L6-HGF L6 myoblast sheet-derived paracrine factors to stimulate cardiomyocyte, endothelial cell, or smooth muscle cell migration in culture. Further, we studied the autocrine effect of hHGF-expression on myoblast gene expression profiles by use of microarray analysis. We induced MI in Wistar rats by left anterior descending coronary artery (LAD ligation and allowed heart failure to develop for 4 weeks. Thereafter, we administered L6-WT (n = 15 or L6-HGF (n = 16 myoblast sheet therapy. Control rats (n = 13 underwent LAD ligation and rethoracotomy without therapy, and five rats underwent a sham operation in both surgeries. We evaluated cardiac function with echocardiography at 2 and 4 weeks after therapy, and analyzed cardiac angiogenesis and left ventricular architecture from histological sections at 4 weeks. Paracrine mediators from L6-HGF myoblast sheets effectively induced migration of cardiac endothelial and smooth muscle cells but not cardiomyocytes. Microarray data revealed that hHGF-expression modulated myoblast gene expression. In vivo, L6-HGF sheet therapy effectively stimulated angiogenesis in the infarcted and non-infarcted areas. Both L6-WT and L6-HGF therapies enhanced cardiac function and inhibited remodeling in a similar fashion. In conclusion, L6-HGF therapy effectively induced angiogenesis in the chronically failing heart. Cardiac function, however, was not further

  13. Culture Conditions Affect Expression of DUX4 in FSHD Myoblasts

    Directory of Open Access Journals (Sweden)

    Sachchida Nand Pandey

    2015-05-01

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is believed to be caused by aberrant expression of double homeobox 4 (DUX4 due to epigenetic changes of the D4Z4 region at chromosome 4q35. Detecting DUX4 is challenging due to its stochastic expression pattern and low transcription level. In this study, we examined different cDNA synthesis strategies and the sensitivity for DUX4 detection. In addition, we investigated the effects of dexamethasone and knockout serum replacement (KOSR on DUX4 expression in culture. Our data showed that DUX4 was consistently detected in cDNA samples synthesized using Superscript III. The sensitivity of DUX4 detection was higher in the samples synthesized using oligo(dT primers compared to random hexamers. Adding dexamethasone to the culture media significantly suppressed DUX4 expression in immortalized (1.3 fold, p < 0.01 and primary (4.7 fold, p < 0.01 FSHD myoblasts, respectively. Culture medium with KOSR increased DUX4 expression and the response is concentration dependent. The findings suggest that detection strategies and culture conditions should be carefully considered when studying DUX4 in cultured cells.

  14. A composite catalyst containing an halogen, a noble metal and at least an additional metal, and its utilization for aromatization of C2-C12 hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Alario, F.; Benazzi, E.; Deves, J.M.

    1994-09-02

    A composite catalyst is proposed for aromatization reactions of hydrocarbons containing from 2 to 12 carbon atoms per molecule, and more especially from 3 to 10 C atoms per molecule; the catalyst contains: a MFI structure zeolite which skeleton is composed of silicon, aluminium and/or gallium and which crystallites external faces have been modified after synthesis; a matrix; at least a noble metal from the platinum family and at least an additional metal chosen among tin, germanium, indium, copper, iron, molybdenum, gallium, thallium, gold, silver, ruthenium, chromium, tungsten and lead; at least an halogen (chlorine); eventually gallium and/or zinc; and eventually, at least one element chosen among alkaline and alkaline-earth metals. 2 tabs.

  15. Long-Term Endurance Exercise in Humans Stimulates Cell Fusion of Myoblasts along with Fusogenic Endogenous Retroviral Genes In Vivo.

    Science.gov (United States)

    Frese, Sebastian; Ruebner, Matthias; Suhr, Frank; Konou, Thierry M; Tappe, Kim A; Toigo, Marco; Jung, Hans H; Henke, Christine; Steigleder, Ruth; Strissel, Pamela L; Huebner, Hanna; Beckmann, Matthias W; van der Keylen, Piet; Schoser, Benedikt; Schiffer, Thorsten; Frese, Laura; Bloch, Wilhelm; Strick, Reiner

    2015-01-01

    Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV) envelope genes due to their involvement in cell fusion of osteoclasts and syncytiotrophoblasts. Comparing muscle biopsies from post- with the pre-competitive seasons a significant 2.25-fold increase of myonuclei/mm fiber, a 2.38-fold decrease of fiber area/nucleus and a 3.1-fold decrease of satellite cells (SCs) occurred. We propose that during the pre-competitive season SC proliferation occurred following with increased cell fusion during the competitive season. Expression of twenty-two envelope genes of muscle biopsies demonstrated a significant increase of putative muscle-cell fusogenic genes Syncytin-1 and Syncytin-3, but also for the non-fusogenic erv3. Immunohistochemistry analyses showed that Syncytin-1 mainly localized to the sarcolemma of myofibers positive for myosin heavy-chain isotypes. Cellular receptors SLC1A4 and SLC1A5 of Syncytin-1 showed significant decrease of expression in post-competitive muscles compared with the pre-competitive season, but only SLC1A4 protein expression localized throughout the myofiber. Erv3 protein was strongly expressed throughout the myofiber, whereas envK1-7 localized to SC nuclei and myonuclei. Syncytin-1 transcription factors, PPARγ and RXRα, showed no protein expression in the myofiber, whereas the pCREB-Ser133 activator of Syncytin-1 was enriched to SC nuclei and myonuclei. Syncytin-1, Syncytin-3, SLC1A4 and PAX7 gene regulations along with MyoD1 and myogenin were verified during proliferating or actively-fusing human primary myoblast cell

  16. Long-Term Endurance Exercise in Humans Stimulates Cell Fusion of Myoblasts along with Fusogenic Endogenous Retroviral Genes In Vivo.

    Directory of Open Access Journals (Sweden)

    Sebastian Frese

    Full Text Available Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV envelope genes due to their involvement in cell fusion of osteoclasts and syncytiotrophoblasts. Comparing muscle biopsies from post- with the pre-competitive seasons a significant 2.25-fold increase of myonuclei/mm fiber, a 2.38-fold decrease of fiber area/nucleus and a 3.1-fold decrease of satellite cells (SCs occurred. We propose that during the pre-competitive season SC proliferation occurred following with increased cell fusion during the competitive season. Expression of twenty-two envelope genes of muscle biopsies demonstrated a significant increase of putative muscle-cell fusogenic genes Syncytin-1 and Syncytin-3, but also for the non-fusogenic erv3. Immunohistochemistry analyses showed that Syncytin-1 mainly localized to the sarcolemma of myofibers positive for myosin heavy-chain isotypes. Cellular receptors SLC1A4 and SLC1A5 of Syncytin-1 showed significant decrease of expression in post-competitive muscles compared with the pre-competitive season, but only SLC1A4 protein expression localized throughout the myofiber. Erv3 protein was strongly expressed throughout the myofiber, whereas envK1-7 localized to SC nuclei and myonuclei. Syncytin-1 transcription factors, PPARγ and RXRα, showed no protein expression in the myofiber, whereas the pCREB-Ser133 activator of Syncytin-1 was enriched to SC nuclei and myonuclei. Syncytin-1, Syncytin-3, SLC1A4 and PAX7 gene regulations along with MyoD1 and myogenin were verified during proliferating or actively-fusing human

  17. Scaffold protein enigma homolog 1 overcomes the repression of myogenesis activation by inhibitor of DNA binding 2.

    Science.gov (United States)

    Nakatani, Miyuki; Ito, Jumpei; Koyama, Riko; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2016-05-27

    Enigma Homolog 1 (ENH1) is a scaffold protein for signaling proteins and transcription factors. Previously, we reported that ENH1 overexpression promotes the differentiation of C2C12 myoblasts. However, the molecular mechanism underlying the role of ENH1 in the C2C12 cells differentiation remains elusive. ENH1 was shown to inhibit the proliferation of neuroblastoma cells by sequestering Inhibitor of DNA binding protein 2 (Id2) in the cytosol. Id2 is a repressor of basic Helix-Loop-Helix transcription factors activity and prevents myogenesis. Here, we found that ENH1 overcome the Id2 repression of C2C12 cells myogenic differentiation and that ENH1 overexpression promotes mice satellite cells activation, the first step toward myogenic differentiation. In addition, we show that ENH1 interacted with Id2 in C2C12 cells and mice satellite cells. Collectively, our results suggest that ENH1 plays an important role in the activation of myogenesis through the repression of Id2 activity. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. The MARVEL domain protein, Singles Bar, is required for progression past the pre-fusion complex stage of myoblast fusion.

    Science.gov (United States)

    Estrada, Beatriz; Maeland, Anne D; Gisselbrecht, Stephen S; Bloor, James W; Brown, Nicholas H; Michelson, Alan M

    2007-07-15

    Multinucleated myotubes develop by the sequential fusion of individual myoblasts. Using a convergence of genomic and classical genetic approaches, we have discovered a novel gene, singles bar (sing), that is essential for myoblast fusion. sing encodes a small multipass transmembrane protein containing a MARVEL domain, which is found in vertebrate proteins involved in processes such as tight junction formation and vesicle trafficking where--as in myoblast fusion--membrane apposition occurs. sing is expressed in both founder cells and fusion competent myoblasts preceding and during myoblast fusion. Examination of embryos injected with double-stranded sing RNA or embryos homozygous for ethane methyl sulfonate-induced sing alleles revealed an identical phenotype: replacement of multinucleated myofibers by groups of single, myosin-expressing myoblasts at a stage when formation of the mature muscle pattern is complete in wild-type embryos. Unfused sing mutant myoblasts form clusters, suggesting that early recognition and adhesion of these cells are unimpaired. To further investigate this phenotype, we undertook electron microscopic ultrastructural studies of fusing myoblasts in both sing and wild-type embryos. These experiments revealed that more sing mutant myoblasts than wild-type contain pre-fusion complexes, which are characterized by electron-dense vesicles paired on either side of the fusing plasma membranes. In contrast, embryos mutant for another muscle fusion gene, blown fuse (blow), have a normal number of such complexes. Together, these results lead to the hypothesis that sing acts at a step distinct from that of blow, and that sing is required on both founder cell and fusion-competent myoblast membranes to allow progression past the pre-fusion complex stage of myoblast fusion, possibly by mediating fusion of the electron-dense vesicles to the plasma membrane.

  19. The real estate of myoblast cardiac transplantation: negative remodeling is associated with location.

    Science.gov (United States)

    McCue, Jonathan D; Swingen, Cory; Feldberg, Tanya; Caron, Gabe; Kolb, Adam; Denucci, Christopher; Prabhu, Somnath; Motilall, Randy; Breviu, Brian; Taylor, Doris A

    2008-01-01

    Skeletal myoblast transplantation has been proposed as a therapy for ischemic cardiomyopathy owing to its possible role in myogenesis. The relative safety and efficacy based on location within scar is not known. We hypothesized that skeletal myoblasts transplanted into peripheral scar (compared with central scar) would more effectively attenuate negative left ventricular (LV) remodeling but at the risk of arrhythmia. New Zealand White rabbits (n = 34) underwent mid-left anterior descending artery (LAD) ligation to produce a transmural LV infarction. One month after LAD ligation, skeletal myoblasts were injected either in the scar center (n = 13) or scar periphery (n = 10) and compared with saline injection (n = 11). Holter monitoring and magnetic resonance imaging (MRI) was performed pre-injection; Holter monitoring was continued until 2 weeks after injection, with follow-up MRI at 1 month. The centrally treated animals demonstrated increased LV end-systolic volume, end-diastolic volume, and mass that correlated with the number of injected cells. There was a trend toward attenuation of negative LV remodeling in peripherally treated animals compared with vehicle. Significant late ectopy was seen in several centrally injected animals, with no late ectopy seen in peripherally injected animals. We noted untoward effects with respect to negative LV remodeling after central injection, suggesting that transplanted cell location with respect to scar may be a key factor in the safety and efficacy of skeletal myoblast cardiac transplantation. Administration of skeletal myoblasts into peripheral scar appears safe, with a trend toward improved function in comparison with sham injection.

  20. Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders

    Directory of Open Access Journals (Sweden)

    Mamchaoui Kamel

    2011-11-01

    Full Text Available Abstract Background Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies. Methods Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders. Results The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both in vitro and in vivo after transplantation into regenerating muscle of immunodeficient mice. Conclusions Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess in vivo the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.

  1. The physical interaction of myoblasts with the microenvironment during remodeling of the cytoarchitecture.

    Directory of Open Access Journals (Sweden)

    Daniel J Modulevsky

    Full Text Available Integrins, focal adhesions, the cytoskeleton and the extracellular matrix, form a structural continuum between the external and internal environment of the cell and mediate the pathways associated with cellular mechanosensitivity and mechanotransduction. This continuum is important for the onset of muscle tissue generation, as muscle precursor cells (myoblasts require a mechanical stimulus to initiate myogenesis. The ability to sense a mechanical cue requires an intact cytoskeleton and strong physical contact and adhesion to the microenvironment. Importantly, myoblasts also undergo reorientation, alignment and large scale remodeling of the cytoskeleton when they experience mechanical stretch and compression in muscle tissue. It remains unclear if such dramatic changes in cell architecture also inhibit physical contact and adhesion with the tissue microenvironment that are clearly important to myoblast physiology. In this study, we employed interference reflection microscopy to examine changes in the close physical contact of myoblasts with a substrate during induced remodeling of the cytoarchitecture (de-stabilization of the actin and microtubule cytoskeleton and inhibition of acto-myosin contractility. Our results demonstrate that while each remodeling pathway caused distinct effects on myoblast morphology and sub-cellular structure, we only observed a ~13% decrease in close physical contact with the substrate, regardless of the pathway inhibited. However, this decrease did not correlate well with changes in cell adhesion strength. On the other hand, there was a close correlation between cell adhesion and β1-integrin expression and the presence of cell-secreted fibronectin, but not with the presence of intact focal adhesions. In this study, we have shown that myoblasts are able to maintain a large degree of physical contact and adhesion to the microenvironment, even during shot periods (<60 min of large scale remodeling and physiological

  2. HGF potentiates extracellular matrix-driven migration of human myoblasts: involvement of matrix metalloproteinases and MAPK/ERK pathway.

    Science.gov (United States)

    González, Mariela Natacha; de Mello, Wallace; Butler-Browne, Gillian S; Silva-Barbosa, Suse Dayse; Mouly, Vincent; Savino, Wilson; Riederer, Ingo

    2017-10-10

    The hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration. We evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting. We demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin. We demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving

  3. The Dlx5-FGF10 signaling cascade controls cranial neural crest and myoblast interaction during oropharyngeal patterning and development.

    Science.gov (United States)

    Sugii, Hideki; Grimaldi, Alexandre; Li, Jingyuan; Parada, Carolina; Vu-Ho, Thach; Feng, Jifan; Jing, Junjun; Yuan, Yuan; Guo, Yuxing; Maeda, Hidefumi; Chai, Yang

    2017-11-01

    Craniofacial development depends on cell-cell interactions, coordinated cellular movement and differentiation under the control of regulatory gene networks, which include the distal-less (Dlx) gene family. However, the functional significance of Dlx5 in patterning the oropharyngeal region has remained unknown. Here, we show that loss of Dlx5 leads to a shortened soft palate and an absence of the levator veli palatini, palatopharyngeus and palatoglossus muscles that are derived from the 4th pharyngeal arch (PA); however, the tensor veli palatini, derived from the 1st PA, is unaffected. Dlx5-positive cranial neural crest (CNC) cells are in direct contact with myoblasts derived from the pharyngeal mesoderm, and Dlx5 disruption leads to altered proliferation and apoptosis of CNC and muscle progenitor cells. Moreover, the FGF10 pathway is downregulated in Dlx5-/- mice, and activation of FGF10 signaling rescues CNC cell proliferation and myogenic differentiation in these mutant mice. Collectively, our results indicate that Dlx5 plays crucial roles in the patterning of the oropharyngeal region and development of muscles derived from the 4th PA mesoderm in the soft palate, likely via interactions between CNC-derived and myogenic progenitor cells. © 2017. Published by The Company of Biologists Ltd.

  4. c-MET regulates myoblast motility and myocyte fusion during adult skeletal muscle regeneration.

    Science.gov (United States)

    Webster, Micah T; Fan, Chen-Ming

    2013-01-01

    Adult muscle stem cells, satellite cells (SCs), endow skeletal muscle with tremendous regenerative capacity. Upon injury, SCs activate, proliferate, and migrate as myoblasts to the injury site where they become myocytes that fuse to form new muscle. How migration is regulated, though, remains largely unknown. Additionally, how migration and fusion, which both require dynamic rearrangement of the cytoskeleton, might be related is not well understood. c-MET, a receptor tyrosine kinase, is required for myogenic precursor cell migration into the limb for muscle development during embryogenesis. Using a genetic system to eliminate c-MET function specifically in adult mouse SCs, we found that c-MET was required for muscle regeneration in response to acute muscle injury. c-MET mutant myoblasts were defective in lamellipodia formation, had shorter ranges of migration, and migrated slower compared to control myoblasts. Surprisingly, c-MET was also required for efficient myocyte fusion, implicating c-MET in dual functions of regulating myoblast migration and myocyte fusion.

  5. Electrotransfection and lipofection show comparable efficiency for in vitro gene delivery of primary human myoblasts.

    Science.gov (United States)

    Mars, Tomaz; Strazisar, Marusa; Mis, Katarina; Kotnik, Nejc; Pegan, Katarina; Lojk, Jasna; Grubic, Zoran; Pavlin, Mojca

    2015-04-01

    Transfection of primary human myoblasts offers the possibility to study mechanisms that are important for muscle regeneration and gene therapy of muscle disease. Cultured human myoblasts were selected here because muscle cells still proliferate at this developmental stage, which might have several advantages in gene therapy. Gene therapy is one of the most sought-after tools in modern medicine. Its progress is, however, limited due to the lack of suitable gene transfer techniques. To obtain better insight into the transfection potential of the presently used techniques, two non-viral transfection methods--lipofection and electroporation--were compared. The parameters that can influence transfection efficiency and cell viability were systematically approached and compared. Cultured myoblasts were transfected with the pEGFP-N1 plasmid either using Lipofectamine 2000 or with electroporation. Various combinations for the preparation of the lipoplexes and the electroporation media, and for the pulsing protocols, were tested and compared. Transfection efficiency and cell viability were inversely proportional for both approaches. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for lipofection, while for electroporation, RPMI medium and a pulsing protocol using eight pulses of 2 ms at E = 0.8 kV/cm proved to be the optimal combination. The transfection efficiencies for the optimal lipofection and optimal electrotransfection protocols were similar (32 vs. 32.5%, respectively). Both of these methods are effective for transfection of primary human myoblasts; however, electroporation might be advantageous for in vivo application to skeletal muscle.

  6. Analysis of Mitochondrial Network Morphology in Cultured Myoblasts from Patients with Mitochondrial Disorders

    Czech Academy of Sciences Publication Activity Database

    Sládková, J.; Spáčilová, J.; Čapek, Martin; Tesařová, M.; Hansíková, H.; Honzík, T.; Martínek, J.; Zámečník, J.; Kostková, O.; Zeman, J.

    2015-01-01

    Roč. 39, č. 5 (2015), s. 340-350 ISSN 0191-3123 R&D Projects: GA ČR(CZ) GB14-36804G; GA MŠk(CZ) LH13028 Institutional support: RVO:67985823 Keywords : cristae * Fiji * image analysis * mitochondrial disorders * myoblasts * ultrastructure Subject RIV: EA - Cell Biology Impact factor: 0.828, year: 2015

  7. Laminin-Coated Poly(Methyl Methacrylate) (PMMA) Nanofiber Scaffold Facilitates the Enrichment of Skeletal Muscle Myoblast Population

    National Research Council Canada - National Science Library

    Nor Kamalia Zahari; Ruszymah Binti Haji Idrus; Shiplu Roy Chowdhury

    2017-01-01

    .... To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate) (PMMA) nanofiber (PM...

  8. Basal Lamina Mimetic Nanofibrous Peptide Networks for Skeletal Myogenesis

    Science.gov (United States)

    Yasa, I. Ceren; Gunduz, Nuray; Kilinc, Murat; Guler, Mustafa O.; Tekinay, Ayse B.

    2015-11-01

    Extracellular matrix (ECM) is crucial for the coordination and regulation of cell adhesion, recruitment, differentiation and death. Therefore, equilibrium between cell-cell and cell-matrix interactions and matrix-associated signals are important for the normal functioning of cells, as well as for regeneration. In this work, we describe importance of adhesive signals for myoblast cells’ growth and differentiation by generating a novel ECM mimetic peptide nanofiber scaffold system. We show that not only structure but also composition of bioactive signals are important for cell adhesion, growth and differentiation by mimicking the compositional and structural properties of native skeletal muscle basal lamina. We conjugated laminin-derived integrin binding peptide sequence, “IKVAV”, and fibronectin-derived well known adhesive sequence, “RGD”, into peptide nanostructures to provide adhesive and myogenic cues on a nanofibrous morphology. The myogenic and adhesive signals exhibited a synergistic effect on model myoblasts, C2C12 cells. Our results showed that self-assembled peptide nanofibers presenting laminin derived epitopes support adhesion, growth and proliferation of the cells and significantly promote the expression of skeletal muscle-specific marker genes. The functional peptide nanofibers used in this study present a biocompatible and biodegradable microenvironment, which is capable of supporting the growth and differentiation of C2C12 myoblasts into myotubes.

  9. Necdin enhances myoblasts survival by facilitating the degradation of the mediator of apoptosis CCAR1/CARP1.

    Directory of Open Access Journals (Sweden)

    Stephanie François

    Full Text Available Regeneration of muscle fibers, lost during pathological muscle degeneration or after injuries, is sustained by the production of new myofibers by means of the satellite cells. Survival of the satellite cells is a critical requirement for efficient muscle reconstitution. Necdin, a member of the MAGE proteins family, is expressed in satellite cell-derived myogenic precursors during perinatal growth and in the adult upon activation during muscle regeneration, where it plays an important role both in myoblast differentiation and survival. We show here that necdin exerts its pro-survival activity by counteracting the action of the pro-apoptotic protein Cell Cycle Apoptosis Regulatory Protein (CCAR1/CARP1 that we have identified as a new molecular interactor of necdin by two-hybrid screening. Necdin is responsible for the maintenance of CCAR1 protein levels, by implementing its ubiquitination and degradation through the proteasome. Taken together, these data shed new light on the molecular mechanism of necdin anti-apoptotic activity in myogenesis.

  10. Tocotrienol-Rich Fraction Ameliorates Antioxidant Defense Mechanisms and Improves Replicative Senescence-Associated Oxidative Stress in Human Myoblasts

    Directory of Open Access Journals (Sweden)

    Shy Cian Khor

    2017-01-01

    Full Text Available During aging, oxidative stress affects the normal function of satellite cells, with consequent regeneration defects that lead to sarcopenia. This study aimed to evaluate tocotrienol-rich fraction (TRF modulation in reestablishing the oxidative status of myoblasts during replicative senescence and to compare the effects of TRF with other antioxidants (α-tocopherol (ATF and N-acetyl-cysteine (NAC. Primary human myoblasts were cultured to young, presenescent, and senescent phases. The cells were treated with antioxidants for 24 h, followed by the assessment of free radical generation, lipid peroxidation, antioxidant enzyme mRNA expression and activities, and the ratio of reduced to oxidized glutathione. Our data showed that replicative senescence increased reactive oxygen species (ROS generation and lipid peroxidation in myoblasts. Treatment with TRF significantly diminished ROS production and decreased lipid peroxidation in senescent myoblasts. Moreover, the gene expression of superoxide dismutase (SOD2, catalase (CAT, and glutathione peroxidase (GPX1 was modulated by TRF treatment, with increased activity of superoxide dismutase and catalase and reduced glutathione peroxidase in senescent myoblasts. In comparison to ATF and NAC, TRF was more efficient in heightening the antioxidant capacity and reducing free radical insults. These results suggested that TRF is able to ameliorate antioxidant defense mechanisms and improves replicative senescence-associated oxidative stress in myoblasts.

  11. FHL1 reduces dystrophy in transgenic mice overexpressing FSHD muscular dystrophy region gene 1 (FRG1.

    Directory of Open Access Journals (Sweden)

    Sandra J Feeney

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.

  12. Milk-derived ribonuclease 5 preparations induce myogenic differentiation in vitro and muscle growth in vivo.

    Science.gov (United States)

    Knight, Matthew I; Tester, Angus M; McDonagh, Matthew B; Brown, Andrew; Cottrell, Jeremy; Wang, Jianghui; Hobman, Peter; Cocks, Benjamin G

    2014-12-01

    Ribonuclease 5, also known as angiogenin, is a stable and abundant ribonuclease in milk whey protein, which is able to regulate several cellular functions, including capillary formation, neuron survival, and epithelial cell growth. Ribonuclease 5 is important for protein synthesis directly stimulating rRNA synthesis in the nucleolus. Here, we show that biologically active RNase5 can be purified from bovine milk. Furthermore, we show that milk-derived RNase5 directly stimulates muscle cell differentiation in vitro, inducing C2C12 cell differentiation and myogenesis. When supplemented into the diet of healthy adult mice, milk-derived RNase5 preparations promoted muscle weight gain and grip strength. Collectively, these data indicate that milk-derived RNase5 preparations exhibit a novel role in skeletal muscle cell function. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  13. Cultured senescent myoblasts derived from human vastus lateralis exhibit normal mitochondrial ATP synthesis capacities with correlating concomitant ROS production while whole cell ATP production is decreased

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2012-01-01

    satellite cells at early and late passage numbers. We show that cultured muscle satellite cells undergoing senescence express a reduced mitochondrial mass, decreased whole cell ATP level, normal to increased mitochondrial ATP production under ATP utilization, increased mitochondrial membrane potential......The free radical theory of aging says that increased oxidative stress and mitochondrial dysfunction are associated with old age. In the present study we have investigated the effects of cellular senescence on muscle energetic by comparing mitochondrial content and function in cultured muscle...... and increased superoxide/mitochondrial mass and hydrogen peroxide/mitochondrial mass ratios. Moreover, the increased ROS production correlates with the corresponding mitochondrial ATP production. Thus, myotubes differentiated from human myoblasts undergoing senescence have a reduced mitochondrial content...

  14. Cargo delivery to adhering myoblast cells from liposome-containing poly(dopamine) composite coatings

    DEFF Research Database (Denmark)

    Madsen, Martin Elias Lynge; Mian Teo, Boon; Laursen, Marie Bækgaard

    2013-01-01

    fluorescence over time reveals a decrease in both parameters over 48 h. Our results demonstrate the ability to affect the cell response depending on the properties of the films, opening up a variety of opportunities for biomedical applications in substrate-mediated drug delivery....... a quartz crystal microbalance with dissipation. We assess the response of adhering myoblast cells to these films containing fluorescent lipids in terms of uptake efficiency and cell mean fluorescence using flow cytometry. The viability of adhering myoblast cells, when the hydrophobic cytotoxic compound...... thiocoraline is entrapped in the lipid membrane, is assessed for different films. Coatings with one or two liposome deposition steps are considered. Further, the effect of the polymer separation layers between the liposome layers is determined. We found that it is possible to use the different nano...

  15. In vitro culture of primary human myoblasts by using the dextran microcarriers Cytodex3®.

    Science.gov (United States)

    Rozwadowska, Natalia; Malcher, Agnieszka; Baumann, Ewa; Kolanowski, Tomasz Jan; Rucinski, Marek; Mietkiewski, Tomasz; Fiedorowicz, Katarzyna; Kurpisz, Maciej

    2016-01-01

    Primary cells in vitro culture scale-up is a crucial issue in cell-based tissue and organ regeneration therapy. Reducing costs and space occupied by the cells cultured in vitro has been an important target. Cells cultured in vitro with the use of bioreactor with dextran microcarriers (Cytodex®) have potentially a chance to meet many of the cell therapy requirements. We used collagen-coated carriers (Cytodex3®) and a spinner flask bioreactor to develop environment suitable for human myoblast proliferation. In parallel, standard adherent in vitro culture conditions for myoblasts propagation (T-flask) were conducted. Cell cycle characterization, senescence, myogenic gene ex-pression and cell apoptosis were evaluated in order to find differences between two culture systems under study. The number of cells obtained in bioreactor per 106 of starting cells population was approximately ten times lower in comparison with T-flask culture system. The microcarriers cultured adult myoblasts in compari-son with the regular T-flask culture showed faster and more advanced replicative aging and lower proliferative potential. Moreover, the percentage of the cells that entailed an irreversible cell arrest (G0 phase) was also significantly (p < 0.0001) increased. Our results suggest that population of primary human myoblasts obtained from adult individuals and propagated on dextran microcarriers did not meet the requirements of the regenerative medicine regarding quantity and quality of the cells obtained. Nonetheless, further optimization of the cell scaling up process including both microcarriers and/or bioreactor program is still an important option.

  16. PKC-Mediated ZYG1 Phosphorylation Induces Fusion of Myoblasts as well as of Dictyostelium Cells

    Science.gov (United States)

    Amagai, Aiko; MacWilliams, Harry; Isono, Takahiro; Omatsu-Kanbe, Mariko; Urano, Shinya; Yamamoto, Kazuo; Maeda, Yasuo

    2012-01-01

    We have previously demonstrated that a novel protein ZYG1 induces sexual cell fusion (zygote formation) of Dictyostelium cells. In the process of cell fusion, involvements of signal transduction pathways via Ca2+ and PKC (protein kinase C) have been suggested because zygote formation is greatly enhanced by PKC activators. In fact, there are several deduced sites phosphorylated by PKC in ZYG1 protein. Thereupon, we designed the present work to examine whether or not ZYG1 is actually phosphorylated by PKC and localized at the regions of cell-cell contacts where cell fusion occurs. These were ascertained, suggesting that ZYG1 might be the target protein for PKC. A humanized version of zyg1 cDNA (mzyg1) was introduced into myoblasts to know if ZYG1 is also effective in cell fusion of myoblasts. Quite interestingly, enforced expression of ZYG1 in myoblasts was found to induce markedly their cell fusion, thus strongly suggesting the existence of a common signaling pathway for cell fusion beyond the difference of species. PMID:22505931

  17. PKC-Mediated ZYG1 Phosphorylation Induces Fusion of Myoblasts as well as of Dictyostelium Cells

    Directory of Open Access Journals (Sweden)

    Aiko Amagai

    2012-01-01

    Full Text Available We have previously demonstrated that a novel protein ZYG1 induces sexual cell fusion (zygote formation of Dictyostelium cells. In the process of cell fusion, involvements of signal transduction pathways via Ca2+ and PKC (protein kinase C have been suggested because zygote formation is greatly enhanced by PKC activators. In fact, there are several deduced sites phosphorylated by PKC in ZYG1 protein. Thereupon, we designed the present work to examine whether or not ZYG1 is actually phosphorylated by PKC and localized at the regions of cell-cell contacts where cell fusion occurs. These were ascertained, suggesting that ZYG1 might be the target protein for PKC. A humanized version of zyg1 cDNA (mzyg1 was introduced into myoblasts to know if ZYG1 is also effective in cell fusion of myoblasts. Quite interestingly, enforced expression of ZYG1 in myoblasts was found to induce markedly their cell fusion, thus strongly suggesting the existence of a common signaling pathway for cell fusion beyond the difference of species.

  18. Targeted Gene Addition of Microdystrophin in Mice Skeletal Muscle via Human Myoblast Transplantation

    Directory of Open Access Journals (Sweden)

    Basma F Benabdallah

    2013-01-01

    Full Text Available Zinc finger nucleases (ZFN can facilitate targeted gene addition to the genome while minimizing the risks of insertional mutagenesis. Here, we used a previously characterized ZFN pair targeting the chemokine (C-C motif receptor 5 (CCR5 locus to introduce, as a proof of concept, the enhanced green fluorescent protein (eGFP or the microdystrophin genes into human myoblasts. Using integrase-defective lentiviral vectors (IDLVs and chimeric adenoviral vectors to transiently deliver template DNA and ZFN respectively, we achieved up to 40% targeted gene addition in human myoblasts. When the O6-methylguanine-DNA methyltransferaseP140K gene was co-introduced with eGFP, the frequency of cells with targeted integration could be increased to over 90% after drug selection. Importantly, gene-targeted myoblasts retained their mitogenic activity and potential to form myotubes both in vitro and in vivo when injected into the tibialis anterior of immune-deficient mice. Altogether, our results could lead to the development of improved cell therapy transplantation protocols for muscular diseases.

  19. Myokines (muscle-derived cytokines and chemokines) including ciliary neurotrophic factor (CNTF) inhibit osteoblast differentiation.

    Science.gov (United States)

    Johnson, Rachelle W; White, Jason D; Walker, Emma C; Martin, T John; Sims, Natalie A

    2014-07-01

    Muscle and bone are intimately linked by bi-directional signals regulating both muscle and bone cell gene expression and proliferation. It is generally accepted that muscle cells secrete factors (myokines) that influence adjacent bone cells, but these myokines are yet to be identified. We have previously shown that osteocyte-specific deletion of the co-receptor subunit utilized by IL-6 family cytokines, glycoprotein 130 (gp130), resulted in impaired bone formation in the trabecular bone, but enhanced periosteal expansion, suggesting a gp130-dependent periosteum-specific inhibition of osteoblast function, potentially induced by the local muscle fibres. We report here that differentiated primary calvarial osteoblasts cultured in myotube-conditioned media (CM) from myogenic C2C12 cells show reduced mRNA levels of genes associated with osteoblast differentiation. Alkaline phosphatase protein activity and all mRNA markers of osteoblast differentiation in the tested panel (runx2, osterix, alkaline phosphatase, parathyroid hormone (PTH) receptor, osteoprotegerin, osteocalcin, sclerostin) were reduced following culture with myotube CM. The exception was RANKL, which was significantly elevated in differentiated primary osteoblast cultures expressing osteocytic genes. A cytokine array of the C2C12 myotube-conditioned media identified TIMP-1 and MCP-1 as the most abundant myokines, but treatment with recombinant TIMP-1 or MCP-1 did not inhibit osteoblast gene expression. Rather, the IL-6 family cytokine ciliary neurotrophic factor (CNTF), which we found abundantly expressed by mouse muscle at the transcript and protein level, reduced osteoblast gene expression, although not to the same extent as the myotube-conditioned media. These data indicate that muscle cells secrete abundant TIMP-1, MCP-1, and CNTF, and that of these, only CNTF has the ability to suppress osteoblast function and gene expression in a similar manner to myotube-conditioned medium. This suggests that CNTF is

  20. G Protein-Coupled Estrogen Receptor 1 (GPER1)/GPR30 Localizes in the Plasma Membrane and Trafficks Intracellularly on Cytokeratin Intermediate Filaments

    National Research Council Canada - National Science Library

    Sandén, Caroline; Broselid, Stefan; Cornmark, Louise; Andersson, Krister; Daszkiewicz-Nilsson, Joanna; Mårtensson, Ulrika; Olde, Björn; Leeb-Lundberg, Fredrik

    2011-01-01

    .... In native cells, including mouse myoblast C(2)C(12) cells, Madine-Darby canine kidney (MDCK) epithelial cells, and human ductal breast epithelial tumor T47-D cells, G-1, a GPER1 agonist, and 17...

  1. Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Erica M. [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Niu, MengMeng; Bergholz, Johann [Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China); Jim Xiao, Zhi-Xiong, E-mail: jxiao@bu.edu [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China)

    2015-05-29

    The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation. - Highlights: • Nutlin-3 inhibits myoblast proliferation and prevents differentiation into myotubes. • Nutlin-3 increases MDM2 expression and down-regulates Rb protein levels. • This study has implication in nutlin-3 treatment of rhabdomyosarcomas.

  2. G-protein coupled receptor 56 promotes myoblast fusion through serum response factor- and nuclear factor of activated T-cell-mediated signalling but is not essential for muscle development in vivo.

    Science.gov (United States)

    Wu, Melissa P; Doyle, Jamie R; Barry, Brenda; Beauvais, Ariane; Rozkalne, Anete; Piao, Xianhua; Lawlor, Michael W; Kopin, Alan S; Walsh, Christopher A; Gussoni, Emanuela

    2013-12-01

    Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the gene for G-protein coupled receptor 56 (GPR56) was transiently up-regulated during the early fusion of human myoblasts. Human mutations in the gene for GPR56 cause the disease bilateral frontoparietal polymicrogyria; however, the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR56 in skeletal muscle. GPR56(-/-) myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, a loss of GPR56 expression in muscle cells results in decreases or delays in the expression of myogenic differentiation 1, myogenin and nuclear factor of activated T-cell (NFAT)c2. Our data suggest that these abnormalities result from decreased GPR56-mediated serum response element and NFAT signalling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase compared to wild-type. In agreement with these findings, clinical data from 13 bilateral frontoparietal polymicrogyria patients revealed mild serum creatine kinase increase in only two patients. In summary, targeted disruption of GPR56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR56 knockout mice and human patients suggests that other factors may compensate for the lack of this G-protein coupled receptor during muscle development and that the motor delay observed in these patients is likely not a result of primary muscle abnormalities. © 2013 FEBS.

  3. Inducing myoblast re-entry into the cell cycle: a potential mechanism for laser-enhanced skeletal muscle regeneration

    Science.gov (United States)

    Liu, T.; Fang, Y.; Zhang, C. P.; Chen, P.; Wang, C. Z.; Kang, H. X.; Shen, B. J.; Liang, J.; Fu, X. B.

    2014-09-01

    This study investigated the effect of low-level laser irradiation (LLLI) on the cell cycle and proliferative activity of cultured myoblasts, and sought to elucidate the possible cellular mechanism by which LLLI promotes the regeneration of skeletal muscle in vivo. Primary myoblasts isolated from rat hindlegs were irradiated with helium-neon laser light at different energy densities. Distributions of cell-cycle subpopulations and the expression of cell-cycle regulatory proteins in myoblasts were assessed using flow cytometric analysis and western blot assay. It was found that laser irradiation stimulated cell-cycle entry; induced the expression of cyclin A and cyclin D; and increased cell proliferation index and bromodeoxyuridine incorporation as compared to the unirradiated control cells, indicating LLLI augmented the number of proliferative myoblasts in the S phase and G2/M phase of the cell cycle. These results suggest that LLLI at certain fluxes and wavelengths could activate quiescent myoblasts, leading to cell division and facilitating new myofiber formation. This could contribute to the improvement of skeletal muscle regeneration following trauma and myopathic diseases.

  4. Designing of a Si-MEMS device with an integrated skeletal muscle cell-based bio-actuator.

    Science.gov (United States)

    Fujita, Hideaki; Van Dau, Thanh; Shimizu, Kazunori; Hatsuda, Ranko; Sugiyama, Susumu; Nagamori, Eiji

    2011-02-01

    With the aim of designing a mechanical drug delivery system involving a bio-actuator, we fabricated a Micro Electro Mechanical Systems (MEMS) device that can be driven through contraction of skeletal muscle cells. The device is composed of a Si-MEMS with springs and ratchets, UV-crosslinked collagen film for cell attachment, and C2C12 muscle cells. The Si-MEMS device is 600 μm x 1000 μm in size and the width of the collagen film is 250 ~ 350 μm, which may allow the device to go through small blood vessels. To position the collagen film on the MEMS device, a thermo-sensitive polymer was used as the sacrifice-layer which was selectively removed with O₂ plasma at the positions where the collagen film was glued. The C2C12 myoblasts were seeded on the collagen film, where they proliferated and formed myotubes after induction of differentiation. When C2C12 myotubes were stimulated with electric pulses, contraction of the collagen film-C2C12 myotube complex was observed. When the edge of the Si-MEMS device was observed, displacement of ~8 μm was observed, demonstrating the possibility of locomotive movement when the device is placed on a track of adequate width. Here, we propose that the C2C12-collagen film complex is a new generation actuator for MEMS devices that utilize glucose as fuel, which will be useful in environments in which glucose is abundant such as inside a blood vessel.

  5. The omega-3 fatty acid, eicosapentaenoic acid (EPA, prevents the damaging effects of tumour necrosis factor (TNF-alpha during murine skeletal muscle cell differentiation

    Directory of Open Access Journals (Sweden)

    Pearson Stephen

    2008-07-01

    Full Text Available Abstract Background Eicosapentaenoic acid (EPA is a ώ-3 polyunsaturated fatty acid with anti-inflammatory and anti-cachetic properties that may have potential benefits with regards to skeletal muscle atrophy conditions where inflammation is present. It is also reported that pathologic levels of the pro-inflammatory cytokine tumour necrosis factor (TNF-α are associated with muscle wasting, exerted through inhibition of myogenic differentiation and enhanced apoptosis. These findings led us to hypothesize that EPA may have a protective effect against skeletal muscle damage induced by the actions of TNF-α. Results The deleterious effects of TNF-α on C2C12 myogenesis were completely inhibited by co-treatment with EPA. Thus, EPA prevented the TNF-mediated loss of MyHC expression and significantly increased myogenic fusion (p p p p p p Conclusion In conclusion, EPA has a protective action against the damaging effects of TNF-α on C2C12 myogenesis. These findings support further investigations of EPA as a potential therapeutic agent during skeletal muscle regeneration following injury.

  6. Choice of cell-delivery route for skeletal myoblast transplantation for treating post-infarction chronic heart failure in rat.

    Directory of Open Access Journals (Sweden)

    Satsuki Fukushima

    2008-08-01

    Full Text Available Intramyocardial injection of skeletal myoblasts (SMB has been shown to be a promising strategy for treating post-infarction chronic heart failure. However, insufficient therapeutic benefit and occurrence of ventricular arrhythmias are concerns. We hypothesised that the use of a retrograde intracoronary route for SMB-delivery might favourably alter the behaviour of the grafted SMB, consequently modulating the therapeutic effects and arrhythmogenicity.Three weeks after coronary artery ligation in female wild-type rats, 5x10(6 GFP-expressing SMB or PBS only (control were injected via either the intramyocardial or retrograde intracoronary routes. Injection of SMB via either route similarly improved cardiac performance and physical activity, associated with reduced cardiomyocyte-hypertrophy and fibrosis. Grafted SMB via either route were only present in low numbers in the myocardium, analysed by real-time PCR for the Y-chromosome specific gene, Sry. Cardiomyogenic differentiation of grafted SMB was extremely rare. Continuous ECG monitoring by telemetry revealed that only intramyocardial injection of SMB produced spontaneous ventricular tachycardia up to 14 days, associated with local myocardial heterogeneity generated by clusters of injected SMB and accumulated inflammatory cells. A small number of ventricular premature contractions with latent ventricular tachycardia were detected in the late-phase of SMB injection regardless of the injection-route.Retrograde intracoronary injection of SMB provided significant therapeutic benefits with attenuated early-phase arrhythmogenicity in treating ischaemic cardiomyopathy, indicating the promising utility of this route for SMB-delivery. Late-phase arrhythmogenicity remains a concern, regardless of the delivery route.

  7. A novel in vitro model for studying quiescence and activation of primary isolated human myoblasts

    DEFF Research Database (Denmark)

    Sellathurai, Jeeva; Cheedipudi, Sirisha; Dhawan, Jyotsna

    2013-01-01

    term exponentially proliferating cultures normally used for in vitro studies. Human myoblasts cultured through many passages inevitably consist of a mixture of proliferating and non-proliferating cells, while cells activated from G0 are in a synchronously proliferating phase, and therefore may...... be a better model for in vivo proliferating satellite cells. Furthermore, the temporal propagation of proliferation in these synchronized cultures resembles the pattern seen in vivo during regeneration. We therefore present this culture model as a useful and novel condition for molecular analysis...

  8. Laminin-Coated Poly(Methyl Methacrylate (PMMA Nanofiber Scaffold Facilitates the Enrichment of Skeletal Muscle Myoblast Population

    Directory of Open Access Journals (Sweden)

    Nor Kamalia Zahari

    2017-10-01

    Full Text Available Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate (PMMA nanofiber (PM scaffolds were fabricated using electrospinning techniques and coated with extracellular matrix (ECM proteins, such as collagen or laminin, in the presence or absence of genipin. A mixed population of myoblasts and fibroblasts was isolated from human skeletal muscle tissues and cultured on plain surfaces, as well as coated and non-coated PM scaffolds. PMMA can produce smooth fibers with an average diameter of 360 ± 50 nm. Adsorption of collagen and laminin on PM scaffolds is significantly enhanced in the presence of genipin, which introduces roughness to the nanofiber surface without affecting fiber diameter and mechanical properties. It was also demonstrated that laminin-coated PM scaffolds significantly enhance myoblast proliferation (0.0081 ± 0.0007 h−1 and migration (0.26 ± 0.04 μm/min, while collagen-coated PM scaffolds favors fibroblasts proliferation (0.0097 ± 0.0009 h−1 and migration (0.23 ± 0.03 μm/min. Consequently, the myoblast population was enriched on laminin-coated PM scaffolds throughout the culture process. Therefore, laminin coating of nanofiber scaffolds could be a potential scaffold for the development of a tissue-engineered muscle substitute.

  9. Laminin-Coated Poly(Methyl Methacrylate) (PMMA) Nanofiber Scaffold Facilitates the Enrichment of Skeletal Muscle Myoblast Population.

    Science.gov (United States)

    Zahari, Nor Kamalia; Idrus, Ruszymah Binti Haji; Chowdhury, Shiplu Roy

    2017-10-30

    Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate) (PMMA) nanofiber (PM) scaffolds were fabricated using electrospinning techniques and coated with extracellular matrix (ECM) proteins, such as collagen or laminin, in the presence or absence of genipin. A mixed population of myoblasts and fibroblasts was isolated from human skeletal muscle tissues and cultured on plain surfaces, as well as coated and non-coated PM scaffolds. PMMA can produce smooth fibers with an average diameter of 360 ± 50 nm. Adsorption of collagen and laminin on PM scaffolds is significantly enhanced in the presence of genipin, which introduces roughness to the nanofiber surface without affecting fiber diameter and mechanical properties. It was also demonstrated that laminin-coated PM scaffolds significantly enhance myoblast proliferation (0.0081 ± 0.0007 h-1) and migration (0.26 ± 0.04 μm/min), while collagen-coated PM scaffolds favors fibroblasts proliferation (0.0097 ± 0.0009 h-1) and migration (0.23 ± 0.03 μm/min). Consequently, the myoblast population was enriched on laminin-coated PM scaffolds throughout the culture process. Therefore, laminin coating of nanofiber scaffolds could be a potential scaffold for the development of a tissue-engineered muscle substitute.

  10. CLP-1 associates with MyoD and HDAC to restore skeletal muscle cell regeneration.

    Science.gov (United States)

    Galatioto, Josephine; Mascareno, Eduardo; Siddiqui, M A Q

    2010-11-01

    Emerging evidence suggests that eukaryotic gene transcription is regulated primarily at the elongation stage by association and dissociation of the inhibitory protein cardiac lineage protein 1 (CLP-1/HEXIM1) from the positive transcription elongation factor b (P-TEFb) complex. It was reported recently that P-TEFb interacts with skeletal muscle-specific regulatory factor, MyoD, suggesting a linkage between CLP-1-mediated control of transcription and skeletal myogenesis. To examine this, we produced CLP-1 knockdown skeletal muscle C2C12 cells by homologous recombination, and demonstrated that the C2C12 CLP-1 +/- cells failed to differentiate when challenged by low serum in the medium. We also showed that CLP-1 interacts with both MyoD and histone deacetylases (HDACs) maximally at the early stage of differentiation of C2C12 cells. This led us to hypothesize that the association might be crucial to inhibition of MyoD-target proliferative genes. Chromatin immunoprecipitation analysis revealed that the CLP-1/MyoD/HDAC complex binds to the promoter of the cyclin D1 gene, which is downregulated in differentiated muscle cells. These findings suggest a novel transcriptional paradigm whereby CLP-1, in conjunction with MyoD and HDAC, acts to inhibit growth-related gene expression, a requirement for myoblasts to exit the cell cycle and transit to myotubes.

  11. Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest

    DEFF Research Database (Denmark)

    Sellathurai, Jeeva; Nielsen, Joachim; Hejbøl, Eva Kildall

    2016-01-01

    and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β...

  12. Action of lovastatin, simvastatin, and pravastatin on sterol synthesis and their antiproliferative effect in cultured myoblasts from human striated muscle

    NARCIS (Netherlands)

    Vliet, A.K. van; Nègre-Arrariou, P.; Thiel, G.C.F. van; Bolhuis, P.A.; Cohen, L.H.

    1996-01-01

    Lovastatin, simvastatin, and pravastatin are fairly strong inhibitors of sterol synthesis in human myoblasts in culture. Lovastatin and simvastatin have IC50 values of 19 ± 6 nM and 4.0 ± 2.3 nM, respectively. Pravastatin is a weaker inhibitor of sterol synthesis (IC50 value of 110 ± 38 nM). Through

  13. Biodistribution studies of {sup 99m}Tc-labeled myoblasts in a murine model of muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Colombo, F.R. E-mail: colombof@policlinico.mi.it; Torrente, Y.; Casati, R.; Benti, R.; Corti, S.; Salani, S.; D' Angelo, M.G.; DeLiso, A.; Scarlato, G.; Bresolin, N.; Gerundini, P

    2001-11-01

    The purpose of this study was twofold: first, to evaluate the myoblast labeling of various {sup 99m}Tc complexes and to select the complex that best accomplishes this labeling, and second to evaluate the biodistribution of myoblasts labeled with this complex using mice with MDX muscular dystrophy (the murine homologue of Duchenne's muscular dystrophy). The following ligands were used to prepare the corresponding {sup 99m}Tc complexes: hexakis-methoxy-isobutyl-isonitrile (MIBI), bis(2-ethoxyethyl)diphosphinoethane (Tf), (RR,SS)-4,8-diaza-3,6,6,9-tetramethyl-undecane-2,10-dione-bisoxime (HM-PAO), bis(N-ethyl)dithiocarbamate (NEt), and bis(N-ethoxy, N-ethyl)dithiocarbamate (NOEt). One million murine myoblasts were incubated for 30-60 minutes with 5 mCi of each of the 99mTc complexes prepared from the above ligands. Viability was assessed by microscopic counting after trypan blue staining, and the radioactivity absorbed in the cells was measured after centrifugation. The compound with the highest uptake in cellular pellets was [{sup 99m}Tc]N-NOEt. The biodistribution of myoblasts labeled with this complex was evaluated after intraaortic injection in dystrophic mice. Such an approach has the potential of effecting widespread gene transfer through the bloodstream to muscles lacking dystrophin.

  14. The muscle integrin binding protein (MIBP) interacts with alpha7beta1 integrin and regulates cell adhesion and laminin matrix deposition.

    Science.gov (United States)

    Li, Ji; Rao, Hongwei; Burkin, Dean; Kaufman, Stephen J; Wu, Chuanyue

    2003-09-01

    Integrins are alphabeta transmembrane receptors that function in key cellular processes, including cell adhesion, differentiation, and extracellular matrix deposition through interactions with extracellular, membrane, and cytoplasmic proteins. We previously identified and cloned a muscle beta1 integrin cytoplasmic binding protein termed MIBP and found that the expression level of MIBP is critical in the decision-making process of terminal myogenic differentiation. We report here that MIBP interacts with the alpha7beta1 integrin but not the alpha5beta1 integrin in C2C12 myoblasts, suggesting an important role of integrin alpha chains in the regulation of the beta1-MIBP interaction. Furthermore, consistent with its selective binding activity toward the alpha7beta1 laminin receptor, we have found that overexpression of MIBP in C2C12 myoblasts resulted in a significant reduction of cell adhesion to laminin and inhibition of laminin matrix deposition. By contrast, neither cell adhesion to fibronectin nor fibronectin matrix deposition was significantly altered in cells overexpressing MIBP. Finally, we show that both the protein level and tyrosine phosphorylation of paxillin, a key signaling molecule involved in the cellular control of myogenic differentiation, are increased by MIBP. These results suggest that MIBP functions in the control of myogenic differentiation by regulating alpha7beta1 integrin-mediated cell interactions with laminin matrix and intracellular signaling through paxillin.

  15. Osterix acetylation at K307 and K312 enhances its transcriptional activity and is required for osteoblast differentiation

    DEFF Research Database (Denmark)

    Lu, Jianlei; Qu, Shuang; Yao, Bing

    2016-01-01

    increased after treatment with histone deacetylase inhibitors Trichostatin A and hydroxamic acid. Meanwhile, the results of immunoprecipitation indicated that Osx was an acetylated protein and that the CREB binding protein (CBP), and less efficiently p300, acetylated Osx. The interaction and colocalization...... of CBP and Osx were demonstrated by Co-immunoprecipitation and immunofluorescence, respectively. In addition, K307 and K312 were identified as the acetylated sites of Osx. By contrast, HDAC4, a histone deacetylase (HDAC), was observed to interact and co-localize with Osx. HDAC4 was demonstrated...... to mediate the deacetylation of Osx. Moreover, we found that acetylation of Osx enhanced its stability, DNA binding ability and transcriptional activity. Finally, we demonstrated that acetylation of Osx was required for the osteogenic differentiation of C2C12 cells. Taken together, our results provide...

  16. Mast cell tryptase stimulates myoblast proliferation; a mechanism relying on protease-activated receptor-2 and cyclooxygenase-2

    Directory of Open Access Journals (Sweden)

    Côté Claude H

    2011-10-01

    Full Text Available Abstract Background Mast cells contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase which activates protease-activated receptor-2 (PAR-2. The possibility that a tryptase/PAR-2 signaling pathway exists in skeletal muscle cell has never been investigated. The aim of this study was to evaluate whether tryptase can stimulate myoblast proliferation and determine the downstream cascade. Methods Proliferation of L6 rat skeletal myoblasts stimulated with PAR-2 agonists (tryptase, trypsin and SLIGKV was assessed. The specificity of the tryptase effect was evaluated with a specific inhibitor, APC-366. Western blot analyses were used to evaluate the expression and functionality of PAR-2 receptor and to assess the expression of COX-2. COX-2 activity was evaluated with a commercial activity assay kit and by measurement of PGF2α production. Proliferation assays were also performed in presence of different prostaglandins (PGs. Results Tryptase increased L6 myoblast proliferation by 35% above control group and this effect was completely inhibited by APC-366. We confirmed the expression of PAR-2 receptor in vivo in skeletal muscle cells and in satellite cells and in vitro in L6 cells, where PAR-2 was found to be functional. Trypsin and SLIGKV increased L6 cells proliferation by 76% and 26% above control, respectively. COX-2 activity was increased following stimulation with PAR-2 agonist but its expression remained unchanged. Inhibition of COX-2 activity by NS-398 abolished the stimulation of cell proliferation induced by tryptase and trypsin. Finally, 15-deoxy-Δ-12,14-prostaglandin J2 (15Δ-PGJ2, a product of COX-2-derived prostaglandin D2, stimulated myoblast proliferation, but not PGE2 and PGF2α. Conclusions Taken together, our data show that tryptase can stimulate myoblast proliferation and this effect is part of a signaling cascade dependent on PAR-2 activation and on the downstream

  17. Hepatocyte growth factor-transfected skeletal myoblasts to limit the development of postinfarction heart failure.

    Science.gov (United States)

    Poppe, Annika; Golsong, Peter; Blumenthal, Britta; von Wattenwyl, Robert; Blanke, Philipp; Beyersdorf, Friedhelm; Schlensak, Christian; Siepe, Matthias

    2012-03-01

    Stem cells transplanted to an injured heart affect the host myocardium indirectly. The cytokine hepatocyte growth factor (HGF) may play a key role in this paracrine activity. We hypothesized that HGF-overexpressing stem cells would restore cardiac function after myocardial infarction (MI). Because there is a high rate of cell death when injecting the cells intramyocardially, we used scaffold-based cell transfer. Skeletal myoblasts (SkMs) were isolated and expanded from newborn Lewis rats. Cells were transfected with pcDNA3-huHGF and seeded on polyurethane (PU) scaffolds or diluted in medium for cell injection. The seeded scaffolds were transplanted in rats two weeks after MI (group: PU-HGF-SkM) or the infection solution was intramyocardially injected (group: Inj-HGF-SkM). Two groups (Inj-SkM and PU-SkM) have been prepared with untransfected cells and sham group without any cell therapy served as control (n = 10 each group). At the beginning of treatment (baseline) and six weeks later, hemodynamic parameters were assessed. At the end of the study, histological analysis was employed. In sham animals we detected a decrease in systolic and diastolic function during the observation time. Treatment with untransfected myoblasts did not lead to any significant changes in hemodynamic parameters between the intervention and six weeks later. In group PU-HGF-SkM, systolic parameters like dP/dt(max), dP/dt(min) and isovolumic contraction improved significantly from baseline to study end. Some diastolic parameters were inferior as compared to baseline (SB-Ked, pressure half time [PHT], Tau). In group Inj-HGF-SkM, only PHT was impaired as compared to preinterventional values. Histological analysis showed significantly more capillaries in the infarction border zone in groups PU-HGF-SkM than in sham and Inj-SkM group. The infarction size was not affected by the therapy. Transplanting HGF-transfected myoblasts after MI can limit the development of ventricular dysfunction

  18. YB-1 gene expression is kept constant during myocyte differentiation through replacement of different transcription factors and then falls gradually under the control of neural activity.

    Science.gov (United States)

    Kobayashi, Shunsuke; Tanaka, Toru; Moue, Masamitsu; Ohashi, Sachiyo; Nishikawa, Taishi

    2015-11-01

    We have previously reported that translation of acetylcholine receptor α-subunit (AChR α) mRNA in skeletal muscle cells is regulated by Y-box binding protein 1 (YB-1) in response to neural activity, and that in the postnatal mouse developmental changes in the amount of YB-1 mRNA are similar to those of AChR α mRNA, which is known to be regulated by myogenic transcription factors. Here, we examined transcriptional regulation of the YB-1 gene in mouse skeletal muscle and differentiating C2C12 myocytes. Although neither YB-1 nor AChR α was detected at either the mRNA or protein level in adult hind limb muscle, YB-1 expression was transiently activated in response to denervation of the sciatic nerve and completely paralleled that of AChR α, suggesting that these genes are regulated by the same transcription factors. However, during differentiation of C2C12 cells to myotubes, the level of YB-1 remained constant even though the level of AChR α increased markedly. Reporter gene, gel mobility shift and ChIP assays revealed that in the initial stage of myocyte differentiation, transcription of the YB-1 gene was regulated by E2F1 and Sp1, and was then gradually replaced under the control of both MyoD and myogenin through an E-box sequence in the proximal region of the YB-1 gene promoter. These results suggest that transcription factors for the YB-1 gene are exchanged during skeletal muscle cell differentiation, perhaps playing a role in translational control of mRNAs by YB-1 in both myotube formation and the response of skeletal muscle tissues to neural stimulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Biocompatible, Biodegradable, and Electroactive Polyurethane-Urea Elastomers with Tunable Hydrophilicity for Skeletal Muscle Tissue Engineering.

    Science.gov (United States)

    Chen, Jing; Dong, Ruonan; Ge, Juan; Guo, Baolin; Ma, Peter X

    2015-12-30

    It remains a challenge to develop electroactive and elastic biomaterials to mimic the elasticity of soft tissue and to regulate the cell behavior during tissue regeneration. We designed and synthesized a series of novel electroactive and biodegradable polyurethane-urea (PUU) copolymers with elastomeric property by combining the properties of polyurethanes and conducting polymers. The electroactive PUU copolymers were synthesized from amine capped aniline trimer (ACAT), dimethylol propionic acid (DMPA), polylactide, and hexamethylene diisocyanate. The electroactivity of the PUU copolymers were studied by UV-vis spectroscopy and cyclic voltammetry. Elasticity and Young's modulus were tailored by the polylactide segment length and ACAT content. Hydrophilicity of the copolymer films was tuned by changing DMPA content and doping of the copolymer. Cytotoxicity of the PUU copolymers was evaluated by mouse C2C12 myoblast cells. The myogenic differentiation of C2C12 myoblasts on copolymer films was also studied by analyzing the morphology of myotubes and relative gene expression during myogenic differentiation. The chemical structure, thermal properties, surface morphology, and processability of the PUU copolymers were characterized by NMR, FT-IR, gel permeation chromatography (GPC), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), scanning electron microscopy (SEM), atomic force microscopy (AFM), and solubility testing, respectively. Those biodegradable electroactive elastic PUU copolymers are promising materials for repair of soft tissues such as skeletal muscle, cardiac muscle, and nerve.

  20. The Effect of Differentiation Induction on FAK and Src Activity in Live HMSCs Visualized by FRET

    Science.gov (United States)

    Wu, Yiqian; Xu, Wenfeng; Zhuo, Yue; Peng, Qin; Li, Bo; Zhang, Ling; Wang, Yingxiao

    2013-01-01

    FAK and Src signaling play important roles in cell differentiation, survival and migration. However, it remains unclear how FAK and Src activities are regulated at the initial stage of stem cell differentiation. We utilized fluorescence resonance energy transfer (FRET)-based FAK and Src biosensors to visualize these kinase activities at the plasma membrane of human mesenchymal stem cells (HMSCs) under the stimulation of osteogenic, myoblastic, or neural induction reagents. Our results indicate that the membrane FAK and Src activities are distinctively regulated by these differentiation induction reagents. FAK and Src activities were both up-regulated with positive feedback upon osteogenic induction, while myoblastic induction only activated Src, but not FAK. Neural induction, however, transiently activated FAK and subsequently Src, which triggered a negative feedback to partially inhibit FAK activity. These results unravel distinct regulation mechanisms of FAK and Src activities during HMSC fate decision, which should advance our understanding of stem cell differentiation in tissue engineering. PMID:24015220

  1. Effect of oxygen tension on bioenergetics and proteostasis in young and old myoblast precursor cells

    Directory of Open Access Journals (Sweden)

    M. Konigsberg

    2013-01-01

    Full Text Available In the majority of studies using primary cultures of myoblasts, the cells are maintained at ambient oxygen tension (21% O2, despite the fact that physiological O2 at the tissue level in vivo is much lower (~1–5% O2. We hypothesized that the cellular response in presence of high oxygen concentration might be particularly important in studies comparing energetic function or oxidative stress in cells isolated from young versus old animals. To test this, we asked whether oxygen tension plays a role in mitochondrial bioenergetics (oxygen consumption, glycolysis and fatty acid oxidation or oxidative damage to proteins (protein disulfides, carbonyls and aggregates in myoblast precursor cells (MPCs isolated from young (3–4 m and old (29–30 m C57BL/6 mice. MPCs were grown under physiological (3% or ambient (21% O2 for two weeks prior to exposure to an acute oxidative insult (H2O2. Our results show significantly higher basal mitochondrial respiration in young versus old MPCs, an increase in basal respiration in young MPCs maintained at 3% O2 compared to cells maintained at 21% O2, and a shift toward glycolytic metabolism in old MPCs grown at 21% O2. H2O2 treatment significantly reduced respiration in old MPCs grown at 3% O2 but did not further repress respiration at 21% O2 in old MPCs. Oxidative damage to protein was higher in cells maintained at 21% O2 and increased in response to H2O2 in old MPCs. These data underscore the importance of understanding the effect of ambient oxygen tension in cell culture studies, in particular studies measuring oxidative damage and mitochondrial function.

  2. Irisin Enhances Osteoblast Differentiation In Vitro

    Directory of Open Access Journals (Sweden)

    Graziana Colaianni

    2014-01-01

    Full Text Available It has been recently demonstrated that exercise activity increases the expression of the myokine Irisin in skeletal muscle, which is able to drive the transition of white to brown adipocytes, likely following a phenomenon of transdifferentiation. This new evidence supports the idea that muscle can be considered an endocrine organ, given its ability to target adipose tissue by promoting energy expenditure. In accordance with these new findings, we hypothesized that Irisin is directly involved in bone metabolism, demonstrating its ability to increase the differentiation of bone marrow stromal cells into mature osteoblasts. Firstly, we confirmed that myoblasts from mice subjected to 3 weeks of free wheel running increased Irisin expression compared to nonexercised state. The conditioned media (CM collected from myoblasts of exercised mice induced osteoblast differentiation in vitro to a greater extent than those of mice housed in resting conditions. Furthermore, the differentiated osteoblasts increased alkaline phosphatase and collagen I expression by an Irisin-dependent mechanism. Our results show, for the first time, that Irisin directly targets osteoblasts, enhancing their differentiation. This finding advances notable perspectives in future studies which could satisfy the ongoing research of exercise-mimetic therapies with anabolic action on the skeleton.

  3. Binding of the chemokine CXCL12α to its natural extracellular matrix ligand heparan sulfate enables myoblast adhesion and facilitates cell motility.

    Science.gov (United States)

    Thakar, Dhruv; Dalonneau, Fabien; Migliorini, Elisa; Lortat-Jacob, Hugues; Boturyn, Didier; Albiges-Rizo, Corinne; Coche-Guerente, Liliane; Picart, Catherine; Richter, Ralf P

    2017-04-01

    The chemokine CXCL12α is a potent chemoattractant that guides the migration of muscle precursor cells (myoblasts) during myogenesis and muscle regeneration. To study how the molecular presentation of chemokines influences myoblast adhesion and motility, we designed multifunctional biomimetic surfaces as a tuneable signalling platform that enabled the response of myoblasts to selected extracellular cues to be studied in a well-defined environment. Using this platform, we demonstrate that CXCL12α, when presented by its natural extracellular matrix ligand heparan sulfate (HS), enables the adhesion and spreading of myoblasts and facilitates their active migration. In contrast, myoblasts also adhered and spread on CXCL12α that was quasi-irreversibly surface-bound in the absence of HS, but were essentially immotile. Moreover, co-presentation of the cyclic RGD peptide as integrin ligand along with HS-bound CXCL12α led to enhanced spreading and motility, in a way that indicates cooperation between CXCR4 (the CXCL12α receptor) and integrins (the RGD receptors). Our findings reveal the critical role of HS in CXCL12α induced myoblast adhesion and migration. The biomimetic surfaces developed here hold promise for mechanistic studies of cellular responses to different presentations of biomolecules. They may be broadly applicable for dissecting the signalling pathways underlying receptor cross-talks, and thus may guide the development of novel biomaterials that promote highly specific cellular responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Experiment list: SRX344977 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available gged ING1 || cell type=Growing C1C12 myoblasts || chromatin preparation method=So...ource_name=Input_ING1_flag_myoblasts || cell line=C2C12 || genotype/variation=ectopically expressing Flag-ta

  5. Experiment list: SRX451714 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ue Diagnosis=NOS 81447171,94.3,36.2,943 GSM1316961: MLL3 MB; Mus musculus; ChIP-Seq source_name=Growing C1C1...2 myoblasts || cell line=C2C12 || cell type=growing myoblasts (MB) || chromatin preparation method=Sonicatio

  6. Experiment list: SRX344978 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ged ING1 || cell type=Growing C1C12 myoblasts || chromatin preparation method=Son...urce_name=Input_ING1_flag_myoblasts || cell line=C2C12 || genotype/variation=ectopically expressing Flag-tag

  7. Experiment list: SRX451712 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ue Diagnosis=NOS 2016249,43.5,11.1,312 GSM1316959: LSD1 MB; Mus musculus; ChIP-Seq source_name=Growing C1C12... myoblasts || cell line=C2C12 || cell type=growing myoblasts (MB) || chromatin preparation method=Sonication

  8. Age-associated repression of type 1 inositol 1, 4, 5-triphosphate receptor impairs muscle regeneration

    Science.gov (United States)

    Lee, Bora; Lee, Seung-Min; Bahn, Young Jae; Lee, Kwang-Pyo; Kang, Moonkyung; Kim, Yeon-Soo; Woo, Sun-Hee; Lim, Jae-Young; Kim, Eunhee; Kwon, Ki-Sun

    2016-01-01

    Skeletal muscle mass and power decrease with age, leading to impairment of mobility and metabolism in the elderly. Ca2+ signaling is crucial for myoblast differentiation as well as muscle contraction through activation of transcription factors and Ca2+-dependent kinases and phosphatases. Ca2+ channels, such as dihydropyridine receptor (DHPR), two-pore channel (TPC) and inositol 1,4,5-triphosphate receptor (ITPR), function to maintain Ca2+ homeostasis in myoblasts. Here, we observed a significant decrease in expression of type 1 IP3 receptor (ITPR1), but not types 2 and 3, in aged mice skeletal muscle and isolated myoblasts, compared with those of young mice. ITPR1 knockdown using shRNA-expressing viruses in C2C12 myoblasts and tibialis anterior muscle of mice inhibited myotube formation and muscle regeneration after injury, respectively, a typical phenotype of aged muscle. This aging phenotype was associated with repression of muscle-specific genes and activation of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. ERK inhibition by U0126 not only induced recovery of myotube formation in old myoblasts but also facilitated muscle regeneration after injury in aged muscle. The conserved decline in ITPR1 expression in aged human skeletal muscle suggests utility as a potential therapeutic target for sarcopenia, which can be treated using ERK inhibition strategies. PMID:27658230

  9. Age-associated repression of type 1 inositol 1, 4, 5-triphosphate receptor impairs muscle regeneration.

    Science.gov (United States)

    Choi, Jeong Yi; Hwang, Chae Young; Lee, Bora; Lee, Seung-Min; Bahn, Young Jae; Lee, Kwang-Pyo; Kang, Moonkyung; Kim, Yeon-Soo; Woo, Sun-Hee; Lim, Jae-Young; Kim, Eunhee; Kwon, Ki-Sun

    2016-09-21

    Skeletal muscle mass and power decrease with age, leading to impairment of mobility and metabolism in the elderly. Ca(2+) signaling is crucial for myoblast differentiation as well as muscle contraction through activation of transcription factors and Ca(2+)-dependent kinases and phosphatases. Ca(2+) channels, such as dihydropyridine receptor (DHPR), two-pore channel (TPC) and inositol 1,4,5-triphosphate receptor (ITPR), function to maintain Ca(2+) homeostasis in myoblasts. Here, we observed a significant decrease in expression of type 1 IP3 receptor (ITPR1), but not types 2 and 3, in aged mice skeletal muscle and isolated myoblasts, compared with those of young mice. ITPR1 knockdown using shRNA-expressing viruses in C2C12 myoblasts and tibialis anterior muscle of mice inhibited myotube formation and muscle regeneration after injury, respectively, a typical phenotype of aged muscle. This aging phenotype was associated with repression of muscle-specific genes and activation of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. ERK inhibition by U0126 not only induced recovery of myotube formation in old myoblasts but also facilitated muscle regeneration after injury in aged muscle. The conserved decline in ITPR1 expression in aged human skeletal muscle suggests utility as a potential therapeutic target for sarcopenia, which can be treated using ERK inhibition strategies.

  10. Options for tracking GFP-Labeled transplanted myoblasts using in vivo fluorescence imaging: implications for tracking stem cell fate.

    Science.gov (United States)

    Yang, Zhong; Wang, Yaming; Li, Yanan; Liu, Qiang; Zeng, Qing; Xu, Xiaoyin

    2014-06-12

    Green fluorescent protein (GFP) is a useful biomarker, widely used in biomedical research to track stem cells after transplantation and/or to assess therapeutic transgene expression. However, both GFP and therapeutic gene products themselves may be immunogenic to the recipient. The main aim of this study was to use animal models to evaluate potential impact of GFP on the cell engraftment and to optimize tracking strategies prior to transplantation. By using a fluorescent imaging (FLI) system, we investigated the dynamic cell behavior of GFP-transduced myoblasts in tibialis anterior (TA) muscles of immunocompetent mdx mice and immuno-compromised nude mice over a period of three months. The results suggested an apparent underlying host immunorejection in the mdx mice. Dystrophin immunostaining showed that the engraftment of wild type myoblasts was much more effective than that of the GFP-labeled counterparts in the mdx mice, further confirming an antigen role of GFP in this process. We tracked the GFP-transduced myoblasts in C57BL/6 mice and found GFP to be minimally immunogenic in these animals, as indicated by the GFP signal maintaining a much stronger level than that found in mdx and BALB/c mice at parallel time points. We also compared the in vivo cell behavior differences between myoblasts from virally GFP-transduced and GFP transgenic mice. The latter displayed much better engraftment, as determined both biomaging and histological observations. Our results not only demonstrated the immunogenicity of GFP in immunocompetent mice, but determined the optimized conditions for GFP-based in vivo stem cells tracking, that can potentially be extrapolated to human biomedical research.

  11. The Mesodermal Expression of rolling stone (rost) Is Essential for Myoblast Fusion in Drosophila and Encodes a Potential Transmembrane Protein

    OpenAIRE

    Paululat, Achim; Goubeaud, Anette; Damm, Christine; Knirr, Stefan; Burchard, Susanne; Renkawitz-Pohl, Renate

    1997-01-01

    In homozygous rolling stone embryos, the fusion of myoblasts to syncytial myotubes is diminished. Nevertheless, the visceral mesoderm, the heart mesoderm, and few somatic muscles are properly formed. Thus, we postulate a central role of rolling stone for the fusion process within the somatic mesoderm. We have cloned the rolling stone gene, and the deduced protein sequence is in accordance with a transmembrane protein, which agrees with the enrichment of Rost in the membrane fraction of Drosop...

  12. Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR

    Directory of Open Access Journals (Sweden)

    Haohan Wang

    2016-03-01

    Full Text Available Abstract As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway.

  13. The mesodermal expression of rolling stone (rost) is essential for myoblast fusion in Drosophila and encodes a potential transmembrane protein.

    Science.gov (United States)

    Paululat, A; Goubeaud, A; Damm, C; Knirr, S; Burchard, S; Renkawitz-Pohl, R

    1997-07-28

    In homozygous rolling stone embryos, the fusion of myoblasts to syncytial myotubes is diminished. Nevertheless, the visceral mesoderm, the heart mesoderm, and few somatic muscles are properly formed. Thus, we postulate a central role of rolling stone for the fusion process within the somatic mesoderm. We have cloned the rolling stone gene, and the deduced protein sequence is in accordance with a transmembrane protein, which agrees with the enrichment of Rost in the membrane fraction of Drosophila embryos. No homologous genes have been described so far. rolling stone is expressed in the embryonic nervous system and cells of the somatic mesoderm, most notable in muscle founder cells. To elucidate the function of rolling stone for myoblast fusion, we applied a knock-out strategy. The expression of an antisense rolling stone transcript specifically within the mesoderm of wild-type embryos results in fusion defects of myoblasts, proving that the rolling stone expression in the mesoderm is responsible for the rolling stone phenotype. We suggest that rolling stone is a member of a group of genes that are necessary for the fusion process during myogenesis.

  14. siRNA delivery into cultured primary human myoblasts--optimization of electroporation parameters and theoretical analysis.

    Science.gov (United States)

    Lojk, Jasna; Mis, Katarina; Pirkmajer, Sergej; Pavlin, Mojca

    2015-12-01

    Introduction of genetic material into muscle tissue has been extensively researched, including isolation and in vitro expansion of primary myoblasts as a potential source of cells for skeletal and heart muscle tissue engineering applications. In this study, we optimized the electroporation protocol for introduction of short interfering ribonucleic acid (siRNA) against messenger RNA for Hypoxia Inducible Factor 1α (HIF-1α) into cultured primary human myoblasts. We established optimal pulsing protocol for siRNA electro transfection, and theoretically analyzed the effect of electric field and pulse duration on silencing efficiency and electrophoretic displacement of siRNA. Silencing of HIF-1α was determined with quantitative polymerase chain reaction and Western Blot. The most efficient silencing (71% knockdown) was achieved with 8 × 2 ms pulses, E = 0.6 kV/cm. Viability was determined immediately, 1 h and 48 h after electroporation. In general, there was a trade-off between efficient silencing and preserved viability. Electric field and pulse duration are crucial parameters for silencing, since both increase membrane permeabilization and electrophoretic transfer of siRNA. Short-term viability showed immediate toxicity of pulses due to membrane damage, while indirect effects on cell proliferation were observed after 48 h. Presented results are important for faster optimization of electroporation parameters for ex vivo electrotransfer of short RNA molecules into primary human myoblasts. © 2015 Wiley Periodicals, Inc.

  15. Methionine Regulates mTORC1 via the T1R1/T1R3-PLC?-Ca2+-ERK1/2 Signal Transduction Process in C2C12 Cells

    OpenAIRE

    Yuanfei Zhou; Jiao Ren; Tongxing Song; Jian Peng; Hongkui Wei

    2016-01-01

    The mammalian target of rapamycin complex 1 (mTORC1) integrates amino acid (AA) availability to support protein synthesis and cell growth. Taste receptor type 1 member (T1R) is a G protein-coupled receptor that functions as a direct sensor of extracellular AA availability to regulate mTORC1 through Ca2+ stimulation and extracellular signal–regulated kinases 1 and 2 (ERK1/2) activation. However, the roles of specific AAs in T1R1/T1R3-regulated mTORC1 are poorly defined. In this study, T1R1 and...

  16. Dual roles of palladin protein in in vitro myogenesis: inhibition of early induction but promotion of myotube maturation.

    Directory of Open Access Journals (Sweden)

    Ngoc-Uyen-Nhi Nguyen

    Full Text Available Palladin is a microfilament-associated phosphoprotein whose function in skeletal muscle has rarely been studied. Therefore, we investigate whether myogenesis is influenced by the depletion of palladin expression known to interfere with the actin cytoskeleton dynamic required for skeletal muscle differentiation. The inhibition of palladin in C2C12 myoblasts leads to precocious myogenic differentiation with a concomitant reduction in cell apoptosis. This premature myogenesis is caused, in part, by an accelerated induction of p21, myogenin, and myosin heavy chain, suggesting that palladin acts as a negative regulator in early differentiation phases. Paradoxically, palladin-knockdown myoblasts are unable to differentiate terminally, despite their ability to perform some initial steps of differentiation. Cells with attenuated palladin expression form thinner myotubes with fewer myonuclei compared to those of the control. It is noteworthy that a negative regulator of myogenesis, myostatin, is activated in palladin-deficient myotubes, suggesting the palladin-mediated impairment of late-stage myogenesis. Additionally, overexpression of 140-kDa palladin inhibits myoblast differentiation while 200-kDa and 90-kDa palladin-overexpressed cells display an enhanced differentiation rate. Together, our data suggest that palladin might have both positive and negative roles in maintaining the proper skeletal myogenic differentiation in vitro.

  17. Disruption of myoblast alignment by highly motile rhabdomyosarcoma cell in tissue structure.

    Science.gov (United States)

    Li, Menglu; Nagamori, Eiji; Kino-Oka, Masahiro

    2017-02-01

    Rhabdomyosarcoma (RMS) is a highly malignant tumor type of skeletal muscle origin, hallmarked by local invasion. Interaction between invasive tumor cells and normal cells plays a major role in tumor invasion and metastasis. Culturing tumor cells in a three-dimensional (3D) model can translate tumor malignancy relevant cell-cell interaction. To mimic tumor heterogeneity in vitro, a co-culture system consisting of a malignant embryonal rhabdomyosarcoma (ERMS) cell line RD and a normal human skeletal muscle myoblast (HSMM) cell line was established by cell sheet technology. Various ratios of RDs to HSMMs were employed to understand the quantitative effect on intercellular interactions. Disruption of sheet structure was observed in heterogeneous cell sheets having a low ratio of RDs to HSMMs, whereas homogeneous HSMM or RD sheets maintained intact structure. Deeper exploration of dynamic tumor cell behavior inside HSMM sheets revealed that HSMM cell alignment was disrupted by highly motile RDs. This study demonstrated that RMS cells are capable of compromising their surrounding environment through induced decay of HSMMs alignment in a cell-based 3D system. This suggests that muscle disruption might be a major consequence of RMS cell invasion into muscles, which could be a promising target to preventing tumor invasion. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  18. Edible Scaffolds Based on Non-Mammalian Biopolymers for Myoblast Growth

    Directory of Open Access Journals (Sweden)

    Javier Enrione

    2017-12-01

    Full Text Available In vitro meat has recently emerged as a new concept in food biotechnology. Methods to produce in vitro meat generally involve the growth of muscle cells that are cultured on scaffolds using bioreactors. Suitable scaffold design and manufacture are critical to downstream culture and meat production. Most current scaffolds are based on mammalian-derived biomaterials, the use of which is counter to the desire to obviate mammal slaughter in artificial meat production. Consequently, most of the knowledge is related to the design and control of scaffold properties based on these mammalian-sourced materials. To address this, four different scaffold materials were formulated using non-mammalian sources, namely, salmon gelatin, alginate, and additives including gelling agents and plasticizers. The scaffolds were produced using a freeze-drying process, and the physical, mechanical, and biological properties of the scaffolds were evaluated. The most promising scaffolds were produced from salmon gelatin, alginate, agarose, and glycerol, which exhibited relatively large pore sizes (~200 μm diameter and biocompatibility, permitting myoblast cell adhesion (~40% and growth (~24 h duplication time. The biodegradation profiles of the scaffolds were followed, and were observed to be less than 25% after 4 weeks. The scaffolds enabled suitable myogenic response, with high cell proliferation, viability, and adequate cell distribution throughout. This system composed of non-mammalian edible scaffold material and muscle-cells is promising for the production of in vitro meat.

  19. Edible Scaffolds Based on Non-Mammalian Biopolymers for Myoblast Growth.

    Science.gov (United States)

    Enrione, Javier; Blaker, Jonny J; Brown, Donald I; Weinstein-Oppenheimer, Caroline R; Pepczynska, Marzena; Olguín, Yusser; Sánchez, Elizabeth; Acevedo, Cristian A

    2017-12-08

    In vitro meat has recently emerged as a new concept in food biotechnology. Methods to produce in vitro meat generally involve the growth of muscle cells that are cultured on scaffolds using bioreactors. Suitable scaffold design and manufacture are critical to downstream culture and meat production. Most current scaffolds are based on mammalian-derived biomaterials, the use of which is counter to the desire to obviate mammal slaughter in artificial meat production. Consequently, most of the knowledge is related to the design and control of scaffold properties based on these mammalian-sourced materials. To address this, four different scaffold materials were formulated using non-mammalian sources, namely, salmon gelatin, alginate, and additives including gelling agents and plasticizers. The scaffolds were produced using a freeze-drying process, and the physical, mechanical, and biological properties of the scaffolds were evaluated. The most promising scaffolds were produced from salmon gelatin, alginate, agarose, and glycerol, which exhibited relatively large pore sizes (~200 μm diameter) and biocompatibility, permitting myoblast cell adhesion (~40%) and growth (~24 h duplication time). The biodegradation profiles of the scaffolds were followed, and were observed to be less than 25% after 4 weeks. The scaffolds enabled suitable myogenic response, with high cell proliferation, viability, and adequate cell distribution throughout. This system composed of non-mammalian edible scaffold material and muscle-cells is promising for the production of in vitro meat.

  20. Increasing alpha 7 beta 1-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis without changing gene expression.

    Science.gov (United States)

    Liu, Jianming; Burkin, Dean J; Kaufman, Stephen J

    2008-02-01

    The dystrophin-glycoprotein complex maintains the integrity of skeletal muscle by associating laminin in the extracellular matrix with the actin cytoskeleton. Several human muscular dystrophies arise from defects in the components of this complex. The alpha(7)beta(1)-integrin also binds laminin and links the extracellular matrix with the cytoskeleton. Enhancement of alpha(7)-integrin levels alleviates pathology in mdx/utrn(-/-) mice, a model of Duchenne muscular dystrophy, and thus the integrin may functionally compensate for the absence of dystrophin. To test whether increasing alpha(7)-integrin levels affects transcription and cellular functions, we generated alpha(7)-integrin-inducible C2C12 cells and transgenic mice that overexpress the integrin in skeletal muscle. C2C12 myoblasts with elevated levels of integrin exhibited increased adhesion to laminin, faster proliferation when serum was limited, resistance to staurosporine-induced apoptosis, and normal differentiation. Transgenic expression of eightfold more integrin in skeletal muscle did not result in notable toxic effects in vivo. Moreover, high levels of alpha(7)-integrin in both myoblasts and in skeletal muscle did not disrupt global gene expression profiles. Thus increasing integrin levels can compensate for defects in the extracellular matrix and cytoskeleton linkage caused by compromises in the dystrophin-glycoprotein complex without triggering apparent overt negative side effects. These results support the use of integrin enhancement as a therapy for muscular dystrophy.

  1. Diabetes-Related Ankyrin Repeat Protein (DARP/Ankrd23 Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle.

    Directory of Open Access Journals (Sweden)

    Yoshiaki Shimoda

    Full Text Available Skeletal muscle is the major site for glucose disposal, the impairment of which closely associates with the glucose intolerance in diabetic patients. Diabetes-related ankyrin repeat protein (DARP/Ankrd23 is a member of muscle ankyrin repeat proteins, whose expression is enhanced in the skeletal muscle under diabetic conditions; however, its role in energy metabolism remains poorly understood. Here we report a novel role of DARP in the regulation of glucose homeostasis through modulating AMP-activated protein kinase (AMPK activity. DARP is highly preferentially expressed in skeletal muscle, and its expression was substantially upregulated during myotube differentiation of C2C12 myoblasts. Interestingly, DARP-/- mice demonstrated better glucose tolerance despite similar body weight, while their insulin sensitivity did not differ from that in wildtype mice. We found that phosphorylation of AMPK, which mediates insulin-independent glucose uptake, in skeletal muscle was significantly enhanced in DARP-/- mice compared to that in wildtype mice. Gene silencing of DARP in C2C12 myotubes enhanced AMPK phosphorylation, whereas overexpression of DARP in C2C12 myoblasts reduced it. Moreover, DARP-silencing increased glucose uptake and oxidation in myotubes, which was abrogated by the treatment with AICAR, an AMPK activator. Of note, improved glucose tolerance in DARP-/- mice was abolished when mice were treated with AICAR. Mechanistically, gene silencing of DARP enhanced protein expression of LKB1 that is a major upstream kinase for AMPK in myotubes in vitro and the skeletal muscle in vivo. Together with the altered expression under diabetic conditions, our data strongly suggest that DARP plays an important role in the regulation of glucose homeostasis under physiological and pathological conditions, and thus DARP is a new therapeutic target for the treatment of diabetes mellitus.

  2. Apparent elastic modulus and hysteresis of skeletal muscle cells throughout differentiation

    Science.gov (United States)

    Collinsworth, Amy M.; Zhang, Sarah; Kraus, William E.; Truskey, George A.

    2002-01-01

    The effect of differentiation on the transverse mechanical properties of mammalian myocytes was determined by using atomic force microscopy. The apparent elastic modulus increased from 11.5 +/- 1.3 kPa for undifferentiated myoblasts to 45.3 +/- 4.0 kPa after 8 days of differentiation (P muscle cells exhibited viscoelastic behavior that changed during differentiation, yielding an increase in the transverse elastic modulus. Major contributors to changes in the transverse elastic modulus during differentiation were actin and myosin.

  3. Stem Cell Differentiation Toward the Myogenic Lineage for Muscle Tissue Regeneration: A Focus on Muscular Dystrophy.

    Science.gov (United States)

    Ostrovidov, Serge; Shi, Xuetao; Sadeghian, Ramin Banan; Salehi, Sahar; Fujie, Toshinori; Bae, Hojae; Ramalingam, Murugan; Khademhosseini, Ali

    2015-12-01

    Skeletal muscle tissue engineering is one of the important ways for regenerating functionally defective muscles. Among the myopathies, the Duchenne muscular dystrophy (DMD) is a progressive disease due to mutations of the dystrophin gene leading to progressive myofiber degeneration with severe symptoms. Although current therapies in muscular dystrophy are still very challenging, important progress has been made in materials science and in cellular technologies with the use of stem cells. It is therefore useful to review these advances and the results obtained in a clinical point of view. This article focuses on the differentiation of stem cells into myoblasts, and their application in muscular dystrophy. After an overview of the different stem cells that can be induced to differentiate into the myogenic lineage, we introduce scaffolding materials used for muscular tissue engineering. We then described some widely used methods to differentiate different types of stem cell into myoblasts. We highlight recent insights obtained in therapies for muscular dystrophy. Finally, we conclude with a discussion on stem cell technology. We discussed in parallel the benefits brought by the evolution of the materials and by the expansion of cell sources which can differentiate into myoblasts. We also discussed on future challenges for clinical applications and how to accelerate the translation from the research to the clinic in the frame of DMD.

  4. Stimulative Effects of Low Intensity He-Ne Laser Irradiation on the Proliferative Potential and Cell-Cycle Progression of Myoblasts in Culture

    Directory of Open Access Journals (Sweden)

    Cui-Ping Zhang

    2014-01-01

    Full Text Available Low intensity laser irradiation (LILI was found to promote the regeneration of skeletal muscle in vivo but the cellular mechanisms are not fully understood. Myoblasts, normally quiescent and inactivated in adult skeletal muscle, are a type of myogenic progenitor cells and considered as the major candidates responsible for muscle regeneration. The aim of the present study was to study the effect of LILI on the growth potential and cell-cycle progression of the cultured myoblasts. Primary myoblasts isolated from rat hind legs were cultured in nutrient-deficient medium for 36 hours and then irradiated by helium-neon laser at a certain energy density. Immunohistochemical and flow cytometric analysis revealed that laser irradiation could increase the expression of cellular proliferation marker and the amount of cell subpopulations in the proliferative phase as compared with the nonirradiated control group. Meanwhile, the expressions of cell-cycle regulatory proteins in the laser-treated myoblasts were markedly upregulated as compared to the unirradiated cells, indicating that LILI could promote the reentry of quiescent myoblasts into the cell division cycle. These results suggest that LILI at certain fluences could promote their proliferation, thus contributing to the skeletal muscle regeneration following trauma and myopathic diseases.

  5. In-depth analysis of the secretome identifies three major independent secretory pathways in differentiating human myoblasts

    DEFF Research Database (Denmark)

    Le Bihan, Marie-Catherine; Bigot, Anne; Jensen, Søren Skov

    2012-01-01

    Efficient muscle regeneration requires cross talk between multiple cell types via secreted signaling molecules. However, as yet there has been no comprehensive analysis of this secreted signaling network in order to understand how it regulates myogenesis in humans. Using integrated proteomic...... and genomic strategies, we show that human muscle cells release not only soluble secreted proteins through conventional secretory mechanisms but also complex protein and nucleic acid cargos via membrane microvesicle shedding. The soluble secretome of muscle cells contains 253 conventionally secreted signaling...

  6. Quantitative profiling of the rat heart myoblast secretome reveals differential responses to hypoxia and re-oxygenation stress.

    Science.gov (United States)

    Li, Xin; Ren, Yan; Sorokin, Vitaly; Poh, Kian Keong; Ho, Hee Hwa; Lee, Chuen Neng; de Kleijn, Dominique; Lim, Sai Kiang; Tam, James P; Sze, Siu Kwan

    2014-02-26

    Secretion of bioactive mediators regulates cell interactions with the microenvironment in tissue homeostasis and wound healing processes. We assessed the cardiomyocyte secretory response to hypoxia with the aim of identifying key mediators of tissue pathology and repair after ischemic heart attack. We profiled the secretome of rat H9C2 cardiomyoblast cells subjected to 16h hypoxia followed by 24h re-oxygenation using iTRAQ and label-free quantitative proteomics. A total of 860 and 2007 proteins were identified in the iTRAQ and label-free experiments respectively. Among these proteins, 1363 were identified as being secreted proteins, including mediators of critical cellular functions that were modulated by hypoxia/re-oxygenation stress (SerpinH1, Ppia, Attractin, EMC1, Postn, Thbs1, Timp1, Stip1, Robo2, Fat1). Further analysis indicated that hypoxia is associated with angiogenesis, inflammation, and remodeling of the extracellular matrix (ECM), whereas subsequent re-oxygenation was instead associated with modified secretion of proteins involved in suppression of inflammation, ECM modification, and decreased output of anti-apoptosis proteins. These data indicate that hypoxia and subsequent re-oxygenation modify the cardiomyocyte secretome in order to mitigate cellular injury and promote healing. The identified changes in cardiomyocyte secretome advance our current understanding of cardiac biology in ischemia/reperfusion injury and may lead to the identification of novel prognostic biomarker. Cardiovascular diseases (CVDs) are the leading cause of death globally. Myocardial infarction (MI) resulting from ischemic heart disease represents a substantial component of CVD-associated mortality, and is associated with obstruction of blood flow to the myocardium. Restoration of blood flow through the occluded coronary artery is the current most effective therapy to limit infarct size and preserve cardiac function after acute myocardial infarction. However, this treatment does not prevent subsequent development of heart failure in some patients. Reperfusion following ischemia causes additional cell death and increase in infarct size, a phenomenon called myocardial ischemia/reperfusion (I/R) injury. In order to advance our current understanding of cardiac biology in ischemia/reperfusion injury, we assessed the cardiomyocyte secretory response to hypoxia with the aim of identifying key mediators of tissue pathology and repair after ischemic heart attack. We profiled the secretome of rat H9C2 cardiomyoblast cells subjected to 16h hypoxia followed by 24h re-oxygenation using LC-MS/MS-based iTRAQ and label-free quantitative proteomics approaches. We identified many secreted proteins as mediators of critical cellular functions that were modulated by hypoxia and re-oxygenation stress. Further analysis of these modulated secretory proteins indicated that hypoxia is associated with angiogenesis, inflammation, and remodeling of the extracellular matrix (ECM), whereas subsequent re-oxygenation/reperfusion was instead associated with modified secretion of proteins involved in suppression of inflammation, ECM modification, and decreased output of anti-apoptosis proteins. These data indicate that hypoxia and subsequent re-oxygenation modify the cardiomyocyte secretome in order to mitigate cellular injury and promote healing. The identified changes in cardiomyocyte secretome may lead to the identification of novel prognostic biomarkers secreted from injured heart tissues into the circulation of patients with cardiovascular disease. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Insulin-like growth factor-1 (IGF-1) promotes myoblast proliferation and skeletal muscle growth of embryonic chickens via the PI3K/Akt signalling pathway.

    Science.gov (United States)

    Yu, Minli; Wang, Huan; Xu, Yali; Yu, Debing; Li, Dongfeng; Liu, Xiuhong; Du, Wenxing

    2015-08-01

    During embryonic development, IGF-1 fulfils crucial roles in skeletal myogenesis. However, the involvement of IGF-1-induced myoblast proliferation in muscle growth is still unclear. In the present study, we have characterised the role of IGF-1 in myoblast proliferation both in vitro and in vivo and have revealed novel details of how exogenous IGF-1 influences myogenic genes in chicken embryos. The results show that IGF-1 significantly induces the proliferation of cultured myoblasts in a dose-dependent manner. Additionally, the IGF-1 treatment significantly promoted myoblasts entering a new cell cycle and increasing the mRNA expression levels of cell cycle-dependent genes. However, these effects were inhibited by the PI3K inhibitor LY294002 and the Akt inhibitor KP372-1. These data indicated that the pro-proliferative effect of IGF-1 was mediated in response to the PI3K/Akt signalling pathway. Moreover, we also showed that exogenous IGF-1 stimulated myoblast proliferation in vivo. IGF-1 administration obviously promoted the incorporation of BrdU and remarkably increased the number of PAX7-positive cells in the skeletal muscle of chicken embryos. Administration of IGF-1 also significantly induced the upregulation of myogenic factors gene, the enhancement of c-Myc and the inhibition of myostatin (Mstn) expression. These findings demonstrate that IGF-1 has strong activity as a promoter of myoblast expansion and muscle fiber formation during early myogenesis. Therefore, this study offers insight into the mechanisms responsible for IGF-1-mediated stimulation of embryonic skeletal muscle development, which could have important implications for the improvement of chicken meat production. © 2015 International Federation for Cell Biology.

  8. BMP antagonists enhance myogenic differentiation and ameliorate the dystrophic phenotype in a DMD mouse model.

    Science.gov (United States)

    Shi, SongTing; Hoogaars, Willem M H; de Gorter, David J J; van Heiningen, Sandra H; Lin, Herbert Y; Hong, Charles C; Kemaladewi, Dwi U; Aartsma-Rus, Annemieke; ten Dijke, Peter; 't Hoen, Peter A C

    2011-02-01

    Duchenne Muscular Dystrophy (DMD) is an X-linked lethal muscle wasting disease characterized by muscle fiber degeneration and necrosis. The progressive pathology of DMD can be explained by an insufficient regenerative response resulting in fibrosis and adipose tissue formation. BMPs are known to inhibit myogenic differentiation and in a previous study we found an increased expression of a BMP family member BMP4 in DMD myoblasts. The aim of the current study was therefore to investigate whether inhibition of BMP signaling could be beneficial for myoblast differentiation and muscle regeneration processes in a DMD context. All tested BMP inhibitors, Noggin, dorsomorphin and LDN-193189, were able to accelerate and enhance myogenic differentiation. However, dorsomorphin repressed both BMP and TGFβ signaling and was found to be toxic to primary myoblast cell cultures. In contrast, Noggin was found to be a potent and selective BMP inhibitor and was therefore tested in vivo in a DMD mouse model. Local adenoviral-mediated overexpression of Noggin in muscle resulted in an increased expression of the myogenic regulatory genes Myog and Myod1 and improved muscle histology. In conclusion, our results suggest that repression of BMP signaling may constitute an attractive adjunctive therapy for DMD patients. Copyright © 2010 Elsevier Inc. All rights reserved.

  9. High-content phenotypic screening and triaging strategy to identify small molecules driving oligodendrocyte progenitor cell differentiation.

    Science.gov (United States)

    Peppard, Jane V; Rugg, Catherine A; Smicker, Matthew A; Powers, Elaine; Harnish, Erica; Prisco, Joy; Cirovic, Dragan; Wright, Paul S; August, Paul R; Chandross, Karen J

    2015-03-01

    Multiple Sclerosis is a demyelinating disease of the CNS and the primary cause of neurological disability in young adults. Loss of myelinating oligodendrocytes leads to neuronal dysfunction and death and is an important contributing factor to this disease. Endogenous oligodendrocyte precursor cells (OPCs), which on differentiation are responsible for replacing myelin, are present in the adult CNS. As such, therapeutic agents that can stimulate OPCs to differentiate and remyelinate demyelinated axons under pathologic conditions may improve neuronal function and clinical outcome. We describe the details of an automated, cell-based, morphometric-based, high-content screen that is used to identify small molecules eliciting the differentiation of OPCs after 3 days. Primary screening was performed using rat CG-4 cells maintained in culture conditions that normally support a progenitor cell-like state. From a library of 73,000 diverse small molecules within the Sanofi collection, 342 compounds were identified that increased OPC morphological complexity as an indicator of oligodendrocyte maturation. Subsequent to the primary high-content screen, a suite of cellular assays was established that identified 22 nontoxic compounds that selectively stimulated primary rat OPCs but not C2C12 muscle cell differentiation. This rigorous triaging yielded several chemical series for further expansion and bio- or cheminformatics studies, and their compelling biological activity merits further investigation. © 2014 Society for Laboratory Automation and Screening.

  10. Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis.

    Science.gov (United States)

    Goh, Qingnian; Dearth, Christopher L; Corbett, Jacob T; Pierre, Philippe; Chadee, Deborah N; Pizza, Francis X

    2015-02-15

    We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast-myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube-myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube-myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method

    Directory of Open Access Journals (Sweden)

    Jean-Paul Iyombe-Engembe

    2016-01-01

    Full Text Available The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD patients, expression of dystrophin (DYS protein is disrupted because exon deletions result in a frame shift. We present here the CRISPR-induced deletion (CinDel, a new promising genome-editing technology to correct the DMD gene. This strategy is based on the use of two gRNAs targeting specifically exons that precede and follow the patient deletion in the DMD gene. This pair of gRNAs induced a precise large additional deletion leading to fusion of the targeted exons. Using an adequate pair of gRNAs, the deletion of parts of these exons and the intron separating them restored the DMD reading frame in 62% of the hybrid exons in vitro in DMD myoblasts and in vivo in electroporated hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted DYS protein was detected following the formation of myotubes by the unselected, treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene, this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides.

  12. Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method.

    Science.gov (United States)

    Iyombe-Engembe, Jean-Paul; Ouellet, Dominique L; Barbeau, Xavier; Rousseau, Joël; Chapdelaine, Pierre; Lagüe, Patrick; Tremblay, Jacques P

    2016-01-26

    The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. We present here the CRISPR-induced deletion (CinDel), a new promising genome-editing technology to correct the DMD gene. This strategy is based on the use of two gRNAs targeting specifically exons that precede and follow the patient deletion in the DMD gene. This pair of gRNAs induced a precise large additional deletion leading to fusion of the targeted exons. Using an adequate pair of gRNAs, the deletion of parts of these exons and the intron separating them restored the DMD reading frame in 62% of the hybrid exons in vitro in DMD myoblasts and in vivo in electroporated hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted DYS protein was detected following the formation of myotubes by the unselected, treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene, this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides.

  13. In mouse myoblasts nuclear prosomes are associated with the nuclear matrix and accumulate preferentially in the perinucleolar areas.

    Science.gov (United States)

    De Conto, F; Pilotti, E; Razin, S V; Ferraglia, F; Géraud, G; Arcangeletti, C; Scherrer, K

    2000-07-01

    Prosomes are the core of 26S proteasomes, although they were originally observed as 20S particles associated with cytoplasmic mRNPs. Here we show for the first time that prosomes are also genuine constituents of the nuclear matrix, chromatin and the nuclear RNP networks. Using mouse myoblasts we tested three monoclonal antibodies recognising the prosomal subunits p23K, p27K and p30K, and found that the corresponding prosome subclasses are characterised by a variable distribution pattern within the nuclei. Their presence on the nuclear matrix, and most abundantly in the perinucleolar area, is of particular importance. When myoblasts fuse into myotubes, the distribution pattern of certain types of prosomes on the nuclear matrix changes drastically. Surprisingly, DNA strongly interferes with the detection of prosomal antigens by immunofluorescence methods, whereas RNA, histones and other proteins soluble in 2 M NaCl have no such effect. This 'masking' of prosomes can be completely overcome by extensive or even mild digestion with DNase I or restriction enzymes. Many nuclear prosomes can be solubilized by combined treatment with 0.5% Triton X-100 and 2 M NaCl, and others can be released by digestion of DNA and/or RNA, and about 10-20% of nuclear prosomes remain tightly bound to the protein-based nuclear matrix.

  14. Rbfox2-coordinated alternative splicing of Mef2d and Rock2 controls myoblast fusion during myogenesis

    Science.gov (United States)

    Bland, Christopher S.; Kalsotra, Auinash; Scavuzzo, Marissa A.; Curk, Tomaz; Ule, Jernej; Li, Wei; Cooper, Thomas A.

    2014-01-01

    Summary Alternative splicing plays important regulatory roles during periods of physiological change. During development a large number of genes coordinately express protein isoform transitions regulated by alternative splicing, however, the mechanisms that coordinate splicing and the functional integration of the resultant tissue-specific protein isoforms are typically unknown. Here we show that the conserved Rbfox2 RNA binding protein regulates 30% of the splicing transitions observed during myogenesis and is required for the specific step of myoblast fusion. Integration of Rbfox2-dependent splicing outcomes from RNA-seq with Rbfox2 iCLIP data identified Mef2d and Rock2 as Rbfox2 splicing targets. Restored activities of Mef2d and Rock2 rescued myoblast fusion in Rbfox2 depleted cultures demonstrating functional cooperation of protein isoforms generated by coordinated alterative splicing. The results demonstrate that coordinated alternative splicing by a single RNA binding protein modulates transcription (Mef2d) and cell signaling (Rock2) programs to drive tissue-specific functions (cell fusion) to promote a developmental transition. PMID:25087874

  15. Keap1 redox-dependent regulation of doxorubicin-induced oxidative stress response in cardiac myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Nordgren, Kendra K.S., E-mail: knordgre@d.umn.edu; Wallace, Kendall B., E-mail: kwallace@d.umn.edu

    2014-01-01

    Doxorubicin (DOX) is a widely prescribed treatment for a broad scope of cancers, but clinical utility is limited by the cumulative, dose-dependent cardiomyopathy that occurs with repeated administration. DOX-induced cardiotoxicity is associated with the production of reactive oxygen species (ROS) and oxidation of lipids, DNA and proteins. A major cellular defense mechanism against such oxidative stress is activation of the Keap1/Nrf2-antioxidant response element (ARE) signaling pathway, which transcriptionally regulates expression of antioxidant genes such as Nqo1 and Gstp1. In the present study, we address the hypothesis that an initial event associated with DOX-induced oxidative stress is activation of the Keap1/Nrf2-dependent expression of antioxidant genes and that this is regulated through drug-induced changes in redox status of the Keap1 protein. Incubation of H9c2 rat cardiac myoblasts with DOX resulted in a time- and dose-dependent decrease in non-protein sulfhydryl groups. Associated with this was a near 2-fold increase in Nrf2 protein content and enhanced transcription of several of the Nrf2-regulated down-stream genes, including Gstp1, Ugt1a1, and Nqo1; the expression of Nfe2l2 (Nrf2) itself was unaltered. Furthermore, both the redox status and the total amount of Keap1 protein were significantly decreased by DOX, with the loss of Keap1 being due to both inhibited gene expression and increased autophagic, but not proteasomal, degradation. These findings identify the Keap1/Nrf2 pathway as a potentially important initial response to acute DOX-induced oxidative injury, with the primary regulatory events being the oxidation and autophagic degradation of the redox sensor Keap1 protein. - Highlights: • DOX caused a ∼2-fold increase in Nrf2 protein content. • DOX enhanced transcription of several Nrf2-regulated down-stream genes. • Redox status and total amount of Keap1 protein were significantly decreased by DOX. • Loss of Keap1 protein was due to

  16. Zinc promotes proliferation and activation of myogenic cells via the PI3K/Akt and ERK signaling cascade

    Energy Technology Data Exchange (ETDEWEB)

    Ohashi, Kazuya, E-mail: asuno10k@yahoo.co.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Wada, Eiji, E-mail: gacchu1@yahoo.co.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Zammit, Peter S., E-mail: peter.zammit@kcl.ac.uk [Randall Division of Cell and Molecular Biophysics, King' s College London, London SE1 1UL (United Kingdom); Shiozuka, Masataka, E-mail: cmuscle@mail.ecc.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Matsuda, Ryoichi, E-mail: cmatsuda@mail.ecc.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan)

    2015-05-01

    Skeletal muscle stem cells named muscle satellite cells are normally quiescent but are activated in response to various stimuli, such as injury and overload. Activated satellite cells enter the cell cycle and proliferate to produce a large number of myogenic progenitor cells, and these cells then differentiate and fuse to form myofibers. Zinc is one of the essential elements in the human body, and has multiple roles, including cell growth and DNA synthesis. However, the role of zinc in myogenic cells is not well understood, and is the focus of this study. We first examined the effects of zinc on differentiation of murine C2C12 myoblasts and found that zinc promoted proliferation, with an increased number of cells incorporating EdU, but inhibited differentiation with reduced myogenin expression and myotube formation. Furthermore, we used the C2C12 reserve cell model of myogenic quiescence to investigate the role of zinc on activation of myogenic cells. The number of reserve cells incorporating BrdU was increased by zinc in a dose dependent manner, with the number dramatically further increased using a combination of zinc and insulin. Akt and extracellular signal-regulated kinase (ERK) are downstream of insulin signaling, and both were phosphorylated after zinc treatment. The zinc/insulin combination-induced activation involved the phosphoinositide 3-kinase (PI3K)/Akt and ERK cascade. We conclude that zinc promotes activation and proliferation of myogenic cells, and this activation requires phosphorylation of PI3K/Akt and ERK as part of the signaling cascade. - Highlights: • Zinc has roles for promoting proliferation and inhibition differentiation of C2C12. • Zinc promotes activation of reserve cells. • Insulin and zinc synergize activation of reserve cells. • PI3K/Akt and ERK cascade affect zinc/insulin-mediated activation of reserve cells.

  17. Concordant but Varied Phenotypes among Duchenne Muscular Dystrophy Patient-Specific Myoblasts Derived using a Human iPSC-Based Model

    Directory of Open Access Journals (Sweden)

    In Young Choi

    2016-06-01

    Full Text Available Duchenne muscular dystrophy (DMD remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs. Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our “chemical-compound-based” strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFβ signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological “dual-SMAD” inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form “rescued” multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human “DMD-in-a-dish” model using hiPSC-based disease modeling.

  18. Concordant but Varied Phenotypes among Duchenne Muscular Dystrophy Patient-Specific Myoblasts Derived using a Human iPSC-Based Model.

    Science.gov (United States)

    Choi, In Young; Lim, HoTae; Estrellas, Kenneth; Mula, Jyothi; Cohen, Tatiana V; Zhang, Yuanfan; Donnelly, Christopher J; Richard, Jean-Philippe; Kim, Yong Jun; Kim, Hyesoo; Kazuki, Yasuhiro; Oshimura, Mitsuo; Li, Hongmei Lisa; Hotta, Akitsu; Rothstein, Jeffrey; Maragakis, Nicholas; Wagner, Kathryn R; Lee, Gabsang

    2016-06-07

    Duchenne muscular dystrophy (DMD) remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs). Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our "chemical-compound-based" strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFβ signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological "dual-SMAD" inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form "rescued" multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human "DMD-in-a-dish" model using hiPSC-based disease modeling. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. A double-blind, randomized, controlled, multicenter study to assess the safety and cardiovascular effects of skeletal myoblast implantation by catheter delivery in patients with chronic heart failure after myocardial infarction.

    Science.gov (United States)

    Povsic, Thomas J; O'Connor, Christopher M; Henry, Timothy; Taussig, Andrew; Kereiakes, Dean J; Fortuin, F David; Niederman, Alan; Schatz, Richard; Spencer, Richard; Owens, Douglas; Banks, Missy; Joseph, Diane; Roberts, Rhonda; Alexander, John H; Sherman, Warren

    2011-10-01

    We sought to determine the safety and preliminary efficacy of transcatheter intramyocardial administration of myoblasts in patients with heart failure (HF). MARVEL is a randomized placebo-controlled trial of image-guided, catheter-based intramyocardial injection of placebo or myoblasts (400 or 800 million) in patients with class II to IV HF and ejection fraction stopping the study for financial reasons. At 6 months, similar numbers of events occurred in each group: 8 (placebo), 7 (low dose), and 8 (high dose), without deaths. Ventricular tachycardia responsive to amiodarone was more frequent in myoblast-treated patients: 1 (placebo), 3 (low dose), and 4 (high dose). A trend toward improvement in functional capacity was noted in myoblast-treated groups (Δ6-minute walk test of -3.6 vs +95.6 vs +85.5 m [placebo vs low dose vs high dose; P = .50]) without significant changes in Minnesota Living With HF scores. In HF patients with chronic postinfarction cardiomyopathy, transcatheter administration of myoblasts in doses of 400 to 800 million cells is feasible and may lead to important clinical benefits. Ventricular tachycardia may be provoked by myoblast injection but appears to be a transient and treatable problem. A large-scale outcome trial of myoblast administration in HF patients with postinfarction cardiomyopathy is feasible and warranted. Copyright © 2011 Mosby, Inc. All rights reserved.

  20. Notch pathway repression by vestigial is required to promote indirect flight muscle differentiation in Drosophila melanogaster.

    Science.gov (United States)

    Bernard, F; Dutriaux, A; Silber, J; Lalouette, A

    2006-07-01

    Drosophila dorsal longitudinal muscles develop during metamorphosis by fusion of myoblasts with larval templates. It has been shown that both vestigial and Notch are crucial for correct formation of these muscles. We investigated the relationship between vestigial and the Notch pathway during this process. Using Enhancer of Split Region Transcript m6 gene expression as a reporter of Notch pathway activity, we were able to demonstrate that this pathway is only active in myoblasts. Moreover, close examination of the cellular location of several of the main actors of the N pathway (Notch, Delta, neuralized, Serrate, Mind bomb1 and fringe) during dorsal longitudinal muscle development enabled us to find that Notch receptor can play multiple roles in adult myogenesis. We report that the locations of the two Notch ligands (Delta and Serrate) are different. Interestingly, we found that fringe, which encodes a glycosyltransferase that modifies the affinity of the Notch receptor for its ligands, is expressed in muscle fibers and in a subset of myoblasts. In addition, we demonstrate that fringe expression is essential for Notch pathway inhibition and muscle differentiation. Lastly, we report that, in vestigial mutants, fringe expression is lost, and when fringe is overexpressed, a significant rescue of indirect flight muscle degeneration is obtained. Altogether, our data show that a vestigial-differentiating function is achieved through the inhibition of the Notch pathway.

  1. Dock mediates Scar- and WASp-dependent actin polymerization through interaction with cell adhesion molecules in founder cells and fusion-competent myoblasts.

    Science.gov (United States)

    Kaipa, Balasankara Reddy; Shao, Huanjie; Schäfer, Gritt; Trinkewitz, Tatjana; Groth, Verena; Liu, Jianqi; Beck, Lothar; Bogdan, Sven; Abmayr, Susan M; Önel, Susanne-Filiz

    2013-01-01

    The formation of the larval body wall musculature of Drosophila depends on the asymmetric fusion of two myoblast types, founder cells (FCs) and fusion-competent myoblasts (FCMs). Recent studies have established an essential function of Arp2/3-based actin polymerization during myoblast fusion, formation of a dense actin focus at the site of fusion in FCMs, and a thin sheath of actin in FCs and/or growing muscles. The formation of these actin structures depends on recognition and adhesion of myoblasts that is mediated by cell surface receptors of the immunoglobulin superfamily. However, the connection of the cell surface receptors with Arp2/3-based actin polymerization is poorly understood. To date only the SH2-SH3 adaptor protein Crk has been suggested to link cell adhesion with Arp2/3-based actin polymerization in FCMs. Here, we propose that the SH2-SH3 adaptor protein Dock, like Crk, links cell adhesion with actin polymerization. We show that Dock is expressed in FCs and FCMs and colocalizes with the cell adhesion proteins Sns and Duf at cell-cell contact points. Biochemical data in this study indicate that different domains of Dock are involved in binding the cell adhesion molecules Duf, Rst, Sns and Hbs. We emphasize the importance of these interactions by quantifying the enhanced myoblast fusion defects in duf dock, sns dock and hbs dock double mutants. Additionally, we show that Dock interacts biochemically and genetically with Drosophila Scar, Vrp1 and WASp. Based on these data, we propose that Dock links cell adhesion in FCs and FCMs with either Scar- or Vrp1-WASp-dependent Arp2/3 activation.

  2. Splicing transitions of the anchoring protein ENH during striated muscle development.

    Science.gov (United States)

    Ito, Jumpei; Hashimoto, Taiki; Nakamura, Sho; Aita, Yusuke; Yamazaki, Tomoko; Schlegel, Werner; Takimoto, Koichi; Maturana, Andrés D

    2012-05-04

    The ENH (PDLIM5) protein acts as a scaffold to tether various functional proteins at subcellular sites via PDZ and three LIM domains. Splicing of the ENH primary transcript generates various products with different repertories of protein interaction modules. Three LIM-containing ENH predominates in neonatal cardiac tissue, whereas LIM-less ENHs are abundant in adult hearts, as well as skeletal muscles. Here we examine the timing of splicing transitions of ENH gene products during postnatal heart development and C2C12 myoblast differentiation. Real-time PCR analysis shows that LIM-containing ENH1 mRNA is gradually decreased during postnatal heart development, whereas transcripts with the short exon 5 appear in the late postnatal period and continues to increase until at least one month after birth. The splicing transition from LIM-containing ENH1 to LIM-less ENHs is also observed during the early period of C2C12 differentiation. This transition correlates with the emergence of ENH transcripts with the short exon 5, as well as the expression of myogenin mRNA. In contrast, the shift from the short exon 5 to the exon 7 occurs in the late differentiation period. The timing of this late event corresponds to the appearance of mRNA for the skeletal myosin heavy chain MYH4. Thus, coordinated and stepwise splicing transitions result in the production of specific ENH transcripts in mature striated muscles. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) as a novel regulator of myogenic cell differentiation.

    Science.gov (United States)

    Attia, Mohamed; Mohamed, Attia; Huet, Eric; Eric, Huet; Delbé, Jean; Jean, Delbé; Ledoux, Dominique; Dominique, Ledoux; Menashi, Suzanne; Suzanne, Menashi; Martelly, Isabelle; Isabelle, Martelly

    2011-01-01

    Matrix metalloproteinases (MMPs) are thought to play an important role in skeletal muscle cell growth and differentiation. In view of the MMP inducing function of EMMPRIN/CD147, its role in myogenic cell differentiation was investigated. EMMPRIN level increased during differentiation of both rat primary myoblasts derived from satellite cells and mouse C2.7 myogenic cells and was associated with an alteration in its molecular forms. In parallel, expression of pro-MMP-9 gradually decreased and that of pro-MMP-2 and active MMP-2 increased. While small interfering RNA (siRNA) inhibition of EMMPRIN expression accelerated cell differentiation, exogenously added recombinant EMMPRIN inhibited differentiation by an MMP-mediated mechanism, as the MMP inhibitor marimastat abrogated EMMPRIN's effect. Our results further suggest that EMMPRIN regulates differentiation through an MMP activation of transforming growth factor beta (TGFβ), a known inhibitor of myoblast's differentiation, as the increased activation and signaling of TGFβ by EMMPRIN was attenuated in the presence of marimastat. EMMPRIN inhibition may thus represent a novel strategy in the treatment of muscular degenerative disorders.

  4. Catechins activate muscle stem cells by Myf5 induction and stimulate muscle regeneration.

    Science.gov (United States)

    Kim, A Rum; Kim, Kyung Min; Byun, Mi Ran; Hwang, Jun-Ha; Park, Jung Il; Oh, Ho Taek; Kim, Hyo Kyeong; Jeong, Mi Gyeong; Hwang, Eun Sook; Hong, Jeong-Ho

    2017-07-22

    Muscle weakness is one of the most common symptoms in aged individuals and increases risk of mortality. Thus, maintenance of muscle mass is important for inhibiting aging. In this study, we investigated the effect of catechins, polyphenol compounds in green tea, on muscle regeneration. We found that (-)-epicatechin gallate (ECG) and (-)-epigallocatechin-3-gallate (EGCG) activate satellite cells by induction of Myf5 transcription factors. For satellite cell activation, Akt kinase was significantly induced after ECG treatment and ECG-induced satellite cell activation was blocked in the presence of Akt inhibitor. ECG also promotes myogenic differentiation through the induction of myogenic markers, including Myogenin and Muscle creatine kinase (MCK), in satellite and C2C12 myoblast cells. Finally, EGCG administration to mice significantly increased muscle fiber size for regeneration. Taken together, the results suggest that catechins stimulate muscle stem cell activation and differentiation for muscle regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Detection of Pancreatic Cancer-Induced Cachexia Using a Fluorescent Myoblast Reporter System and Analysis of Metabolite Abundance.

    Science.gov (United States)

    Winnard, Paul T; Bharti, Santosh K; Penet, Marie-France; Marik, Radharani; Mironchik, Yelena; Wildes, Flonne; Maitra, Anirban; Bhujwalla, Zaver M

    2016-03-15

    The dire effects of cancer-induced cachexia undermine treatment and contribute to decreased survival rates. Therapeutic options for this syndrome are limited, and therefore efforts to identify signs of precachexia in cancer patients are necessary for early intervention. The applications of molecular and functional imaging that would enable a whole-body "holistic" approach to this problem may lead to new insights and advances for diagnosis and treatment of this syndrome. Here we have developed a myoblast optical reporter system with the purpose of identifying early cachectic events. We generated a myoblast cell line expressing a dual tdTomato:GFP construct that was grafted onto the muscle of mice-bearing human pancreatic cancer xenografts to provide noninvasive live imaging of events associated with cancer-induced cachexia (i.e., weight loss). Real-time optical imaging detected a strong tdTomato fluorescent signal from skeletal muscle grafts in mice with weight losses of only 1.2% to 2.7% and tumor burdens of only approximately 79 to 170 mm(3). Weight loss in cachectic animals was also associated with a depletion of lipid, cholesterol, valine, and alanine levels, which may provide informative biomarkers of cachexia. Taken together, our findings demonstrate the utility of a reporter system that is capable of tracking tumor-induced weight loss, an early marker of cachexia. Future studies incorporating resected tissue from human pancreatic ductal adenocarcinoma into a reporter-carrying mouse may be able to provide a risk assessment of cachexia, with possible implications for therapeutic development. ©2015 American Association for Cancer Research.

  6. Automated high-content assay for compounds selectively toxic to Trypanosoma cruzi in a myoblastic cell line.

    Directory of Open Access Journals (Sweden)

    Julio Alonso-Padilla

    2015-01-01

    Full Text Available Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease.Genetically engineered parasitic strains are used for high throughput screening (HTS of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6 and was validated against a series of known anti-trypanosomatid drugs.We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite.

  7. Morinda citrifolia Leaf Extract Enhances Osteogenic Differentiation Through Activation of Wnt/β-Catenin Signaling.

    Science.gov (United States)

    Gu, Hanna; Boonanantanasarn, Kanitsak; Kang, Moonkyu; Kim, Ikhwi; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa

    2017-10-05

    Morinda citrifolia (Noni) leaf is an herbal medicine with application in the domestic treatment of a broad range of conditions, including bone fracture and luxation. However, the basic mechanism underlying the stimulation of osteogenic differentiation by Noni leaf extract remains poorly understood. This study aimed to examine the effect of this extract on osteogenic differentiation and the mechanism by which Noni leaf extract enhances osteogenic differentiation. Aqueous extract of Noni leaves was prepared, and rutin and kaempferol-3-O-rutinoside were identified to be two of its major components. C2C12 and human periodontal ligament (hPDL) cells were used to study the effect of Noni. Noni did not show cytotoxicity at a concentration range of 0.015%-1.0% (w/v%) and significantly enhanced the activity of alkaline phosphatase (ALP) and expression levels of osteoblast differentiation markers, including Runx2, ALP, osterix, and osteocalcin, bone morphogenetic protein 2, Wnt3a, and β-catenin. In addition, Noni enhanced the matrix mineralization of hPDL cells. In the signaling pathways, Noni increased the phosphorylation levels of Akt and GSK3β and nuclear translocation and transcriptional activity of β-catenin, which were attenuated by the addition of Dkk-1, a Wnt inhibitor, or LY294002, a PI3K inhibitor. These results suggest that Noni leaf extract enhances osteogenic differentiation through the PI3K/Akt-dependent activation of Wnt/β-catenin signaling. Noni leaf extract might be a novel alternative medicine for bone and periodontal regeneration in patients with periodontal diseases.

  8. Omega-3 fatty acids differentially modulate enzymatic anti-oxidant systems in skeletal muscle cells.

    Science.gov (United States)

    da Silva, E P; Nachbar, R T; Levada-Pires, A C; Hirabara, S M; Lambertucci, R H

    2016-01-01

    During physical activity, increased reactive oxygen species production occurs, which can lead to cell damage and in a decline of individual's performance and health. The use of omega-3 polyunsaturated fatty acids as a supplement to protect the immune system has been increasing; however, their possible benefit to the anti-oxidant system is not well described. Thus, the aim of this study was to evaluate whether the omega-3 fatty acids (docosahexaenoic acid and eicosapentaenoic acid) can be beneficial to the anti-oxidant system in cultured skeletal muscle cells. C2C12 myocytes were differentiated and treated with either eicosapentaenoic acid or docosahexaenoic acid for 24 h. Superoxide content was quantified using the dihydroethidine oxidation method and superoxide dismutase, catalase, and glutathione peroxidase activity, and expression was quantified. We observed that the docosahexaenoic fatty acids caused an increase in superoxide production. Eicosapentaenoic acid induced catalase activity, while docosahexaenoic acid suppressed superoxide dismutase activity. In addition, we found an increased protein expression of the total manganese superoxide dismutase and catalase enzymes when cells were treated with eicosapentaenoic acid. Taken together, these data indicate that the use of eicosapentaenoic acid may present both acute and chronic benefits; however, the treatment with DHA may not be beneficial to muscle cells.

  9. Estrogen/ERR-α signaling axis is associated with fiber-type conversion of upper airway muscles in patients with obstructive sleep apnea hypopnea syndrome

    Science.gov (United States)

    Chen, H. H.; Lu, J.; Guan, Y. F.; Li, S. J.; Hu, T. T.; Xie, Z. S.; Wang, F.; Peng, X. H.; Liu, X.; Xu, X.; Zhao, F. P.; Yu, B. L.; Li, X. P.

    2016-01-01

    Estrogen is related with the low morbidity associated with obstructive sleep apnea hypopnea syndrome (OSAS) in women, but the underlying mechanisms remain largely unknown. In this study, we examined the relationship between OSAS and estrogen related receptor-α (ERR-α). We found that the expression levels of ERR-α and Myh7 were both downregulated in palatopharyngeal tissues from OSAS patients. In addition, we report that ERR-α is dynamically expressed during differentiation of C2C12 myoblasts. Knockdown of ERR-α via instant siRNA resulted in reduced expression of Myh7, but not Myh4. Furthermore, differentiation of C2C12 cells under 3% chronic intermittent hypoxia, a model resembling human OSAS, was impaired and accompanied by a obvious reduction in Myh7 expression levels. Moreover, activation of ERR-α with 17β-estradiol (E2) increased the expression of Myh7, whereas pretreatment with the ERR-α antagonist XCT790 reversed the E2-induced slow fiber-type switch. A rat ovariectomy model also demonstrated the switch to fast fiber type. Collectively, our findings suggest that ERR-α is involved in estrogen-mediated OSAS by regulating Myhc-slow expression. The present study illustrates an important role of the estrogen/ERR-α axis in the pathogenesis of OSAS, and may represent an attractive therapeutic target, especially in postmenopausal women. PMID:27250523

  10. Potential Therapeutic Role of L-Carnitine in Skeletal Muscle Oxidative Stress and Atrophy Conditions

    Directory of Open Access Journals (Sweden)

    Anna Montesano

    2015-01-01

    Full Text Available The targeting of nutraceutical treatment to skeletal muscle damage is an emerging area of research, driven by the need for new therapies for a range of muscle-associated diseases. L-Carnitine (CARN is an essential nutrient and plays a key role in mitochondrial β-oxidation and in the ubiquitin-proteasome system regulation. As a dietary supplement to improve athletic performance, CARN has been studied for its potential to enhance β-oxidation. However, CARN effects on myogenesis, mitochondrial activity, and hypertrophy process are not completely elucidated. This in vitro study aims to investigate CARN role on skeletal muscle remodeling, differentiation process, and myotubes formation. We analyzed muscle differentiation and morphological features in C2C12 myoblasts exposed to 5 mM CARN. Our results showed that CARN was able to accelerate C2C12 myotubes formation and induce morphological changes, characterizing the start of hypertrophy process. In addition, CARN improved AKT activation and downstream cellular signaling pathways involved in skeletal muscle atrophy process prevention. Also, CARN positively regulated the pathways involved in oxidative stress defense. In this work, we provide an interesting novel mechanism of the potential therapeutic use of CARN to treat pathological conditions characterized by skeletal muscle morphological and functional impairment, oxidative stress production, and atrophy process in aging.

  11. Metformin Treatment Prevents Sedentariness Related Damages in Mice

    Directory of Open Access Journals (Sweden)

    Pamela Senesi

    2016-01-01

    Full Text Available Metformin (METF, historical antihyperglycemic drug, is a likely candidate for lifespan extension, treatment and prevention of sedentariness damages, insulin resistance, and obesity. Skeletal muscle is a highly adaptable tissue, capable of hypertrophy response to resistance training and of regeneration after damage. Aims of this work were to investigate METF ability to prevent sedentariness damage and to enhance skeletal muscle function. Sedentary 12-week-old C57BL/6 mice were treated with METF (250 mg/kg per day, in drinking water for 60 days. METF role on skeletal muscle differentiation was studied in vitro using murine C2C12 myoblasts. Muscular performance evaluation revealed that METF enhanced mice physical performance (Estimated VO2max. Biochemical analyses of hepatic and muscular tissues indicated that in liver METF increased AMPK and CAMKII signaling. In contrast, METF inactivated ERKs, the principal kinases involved in hepatic stress. In skeletal muscle, METF activated AKT, key kinase in skeletal muscle mass maintenance. In in vitro studies, METF did not modify the C2C12 proliferation capacity, while it positively influenced the differentiation process and myotube maturation. In conclusion, our novel results suggest that METF has a positive action not only on the promotion of healthy aging but also on the prevention of sedentariness damages.

  12. Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T. [Department of Kinesiology, The University of Toledo, Toledo, OH (United States); Pierre, Philippe [Centre d’Immunologie de Marseille-Luminy U2M, Aix-Marseille Université, Marseille (France); INSERM U631, Institut National de la Santé et Recherche Médicale, Marseille (France); CNRS UMR6102, Centre National de la Recherche Scientifique, Marseille (France); Chadee, Deborah N. [Department of Biological Sciences, The University of Toledo, Toledo, OH (United States); Pizza, Francis X., E-mail: Francis.Pizza@utoledo.edu [Department of Kinesiology, The University of Toledo, Toledo, OH (United States)

    2015-02-15

    We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast–myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube–myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube–myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. - Highlights: • We examined mechanisms through which skeletal muscle cell expression of ICAM-1 facilitates events of in vitro myogenesis. • Expression of ICAM-1 by cultured myoblasts did not influence their ability to proliferate or differentiate. • Skeletal muscle cell expression of ICAM-1 augmented myoblast fusion, myotube alignment, myotube–myotube fusion, and myotube size. • ICAM-1 augmented myogenic processes through

  13. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

    Energy Technology Data Exchange (ETDEWEB)

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany); Renkawitz-Pohl, Renate, E-mail: renkawit@biologie.uni-marburg.de [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany)

    2013-02-15

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity.

  14. DNA replication timing is maintained genome-wide in primary human myoblasts independent of D4Z4 contraction in FSH muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Benjamin D Pope

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is linked to contraction of an array of tandem 3.3-kb repeats (D4Z4 at 4q35.2 from 11-100 copies to 1-10 copies. The extent to which D4Z4 contraction at 4q35.2 affects overall 4q35.2 chromatin organization remains unclear. Because DNA replication timing is highly predictive of long-range chromatin interactions, we generated genome-wide replication-timing profiles for FSHD and control myogenic precursor cells. We compared non-immortalized myoblasts from four FSHD patients and three control individuals to each other and to a variety of other human cell types. This study also represents the first genome-wide comparison of replication timing profiles in non-immortalized human cell cultures. Myoblasts from both control and FSHD individuals all shared a myoblast-specific replication profile. In contrast, male and female individuals were readily distinguished by monoallelic differences in replication timing at DXZ4 and other regions across the X chromosome affected by X inactivation. We conclude that replication timing is a robust cell-type specific feature that is unaffected by FSHD-related D4Z4 contraction.

  15. Retention of the Structure and Function of Heparan Sulfate Biomaterials After Gamma Irradiation.

    Science.gov (United States)

    Smith, Raymond A A; Chua, R J E; Carnachan, Susan M; Tan, Clarissa L L; Sims, Ian M; Hinkley, Simon F R; Nurcombe, Victor; Cool, Simon M

    2017-11-30

    Heparan sulfate (HS) is a highly heterogeneous polysaccharide implicated in many important biological processes. Our previous work has demonstrated that a particular affinity-selected HS (referred to henceforth as "HS3") is capable of enhancing the osteogenic effects of bone morphogenetic protein 2 (BMP2). Here, we gamma-irradiated HS with 26 kGy of ionizing radiation to determine how this affected the structure, composition, and function. Initial structural studies were performed on a commercial preparation of HS as a proof-of-concept. Gamma irradiation of this HS preparation did not significantly alter its structure or composition compared to nonirradiated material, as demonstrated by proton nuclear magnetic resonance spectroscopy, molecular weight analysis using size exclusion chromatography, and disaccharide compositional analysis. When HS3 was gamma irradiated, no significant effect on binding affinity toward BMP2 was observed, based on competitive surface plasmon resonance and differential scanning fluorimetry assays. Furthermore, irradiation did not significantly affect HS3's ability to synergistically enhance the osteogenic effects of BMP2 in vitro; as measured by the relative abundance of osteogenic transcripts in transdifferentiating C2C12 murine myoblasts. Additionally, no significant differences were observed in the levels of alkaline phosphatase (ALP) or calcium deposition in C2C12s treated with BMP2, together with the irradiated, or nonirradiated HS3. Irradiation of HS3 incorporated into collagen type I sponges did not affect its ability to enhance BMP2-mediated ALP expression in C2C12 cells. Our data confirm that gamma irradiation is a cost-effective and viable solution for the sterilization of HS species that allows the retention of its structure and biological function. The work suggests an effective way to incorporate clinically compatible HS species into orthotic implants, scaffolds, and other medical devices for use in the treatment of a range

  16. Discoidin domain receptor 1 is activated independently of beta(1) integrin

    DEFF Research Database (Denmark)

    Vogel, W; Brakebusch, C; Fässler, R

    2000-01-01

    with kinase activity or membrane-anchored proteins serving as coreceptors. In particular, the role of the collagen-binding integrins alpha(1)beta(1) or alpha(2)beta(1) in the DDR activation process is undefined. Here, we provide three lines of evidence suggesting that DDR1 signaling is distinct from integrin...... for alpha(2)beta(1) integrin or in cells with a targeted deletion of the beta(1) integrin gene. Finally, we show that overexpression of dominant negative DDR1 in the myoblast cell line C2C12 blocks cellular differentiation and the formation of myofibers....... activation. First we demonstrate that the enzymatic activity of DDR1 is essential for receptor tyrosine phosphorylation. Collagen-induced DDR receptor autophosphorylation can be blocked either by a dominant negative mutant or by a preparation of recombinant extracellular domain. Second, we show DDR1 signals...

  17. Caffeine induces hyperacetylation of histones at the MEF2 site on the Glut4 promoter and increases MEF2A binding to the site via a CaMK-dependent mechanism.

    Science.gov (United States)

    Mukwevho, Emmanuel; Kohn, Tertius A; Lang, Dirk; Nyatia, Edward; Smith, James; Ojuka, Edward O

    2008-03-01

    This study was conducted to explore the mechanism by which caffeine increases GLUT4 expression in C(2)C(12) myotubes. Myoblasts were differentiated in DMEM containing 2% horse serum for 13 days and the resultant myotubes exposed to 10 mM caffeine in the presence or absence of 25 microM KN93 or 10 mM dantrolene for 2 h. After the treatment, cells were kept in serum-free medium and harvested between 0 and 6 h later, depending on the assay. Chromatin immunoprecipitation (ChIP) assays revealed that caffeine treatment caused hyperacetylation of histone H3 at the myocyte enhancer factor 2 (MEF2) site on the Glut4 promoter (P CaMK II), attenuated all the above caffeine-induced changes. These data indicate that caffeine increases GLUT4 expression by acetylating the MEF2 site to increase MEF2A binding via a mechanism that involves CaMK II.

  18. TRAF6 promotes myogenic differentiation via the TAK1/p38 mitogen-activated protein kinase and Akt pathways.

    Directory of Open Access Journals (Sweden)

    Fang Xiao

    Full Text Available p38 mitogen-activated protein kinase (MAPK is an essential kinase involved in myogenic differentiation. Although many substrates of p38 MAPK have been identified, little is known about its upstream activators during myogenic differentiation. TRAF6 is known to function in cytokine signaling during inflammatory responses. However, not much is known about its role in myogenic differentiation and muscle regeneration. We showed here that TRAF6 and its intrinsic ubiquitin E3 ligase activity are required for myogenic differentiation. In mouse myoblasts, knockdown of TRAF6 compromised the p38 MAPK and Akt pathways, while deliberate activation of either pathway rescued the differentiation defect caused by TRAF6 knockdown. TAK1 acted as a key signal transducer downstream of TRAF6 in myogenic differentiation. In vivo, knockdown of TRAF6 in mouse muscles compromised the injury-induced muscle regeneration without impairing macrophage infiltration and myoblast proliferation. Collectively, we demonstrated that TRAF6 promotes myogenic differentiation and muscle regeneration via the TAK1/p38 MAPK and Akt pathways.

  19. The parafibromin tumor suppressor protein interacts with actin-binding proteins actinin-2 and actinin-3

    Directory of Open Access Journals (Sweden)

    Marx Stephen J

    2008-08-01

    Full Text Available Abstract Background Germline and somatic inactivating mutations in the HRPT2 gene occur in the inherited hyperparathyroidism-jaw tumor syndrome, in some cases of parathyroid cancer and in some cases of familial hyperparathyroidism. HRPT2 encodes parafibromin. To identify parafibromin interacting proteins we used the yeast two-hybrid system for screening a heart cDNA library with parafibromin as the bait. Results Fourteen parafibromin interaction positive preys representing 10 independent clones encoding actinin-2 were isolated. Parafibromin interacted with muscle alpha-actinins (actinin-2 and actinin-3, but not with non-muscle alpha-actinins (actinin-1 and actinin-4. The parafibromin-actinin interaction was verified by yeast two-hybrid, GST pull-down, and co-immunoprecipitation. Yeast two-hybrid analysis revealed that the N-terminal region of parafibromin interacted with actinins. In actin sedimentation assays parafibromin did not dissociate skeletal muscle actinins from actin filaments, but interestingly, parafibromin could also bundle/cross-link actin filaments. Parafibromin was predominantly nuclear in undifferentiated proliferating myoblasts (C2C12 cells, but in differentiated C2C12 myotubes parafibromin co-localized with actinins in the cytoplasmic compartment. Conclusion These data support a possible contribution of parafibromin outside the nucleus through its interaction with actinins and actin bundling/cross-linking. These data also suggest that actinins (and actin participate in sequestering parafibromin in the cytoplasmic compartment.

  20. A Chemically Polymerized Electrically Conducting Composite of Polypyrrole Nanoparticles and Polyurethane for Tissue Engineering

    Science.gov (United States)

    Broda, Christopher R.; Lee, Jae Y.; Sirivisoot, Sirinrath; Schmidt, Christine E.; Harrison, Benjamin S.

    2011-01-01

    A variety of cell types respond to electrical stimuli, accordingly many conducting polymers (CPs) have been used as tissue engineering (TE) scaffolds, one such CP is polypyrrole (PPy). PPy is a well studied biomaterial with potential TE applications due to its electrical conductivity and many other beneficial properties. Combining its characteristics with an elastomeric material, such as polyurethane (PU), may yield a hybrid scaffold with electrical activity and significant mechanical resilience. Pyrrole was in situ polymerized within a PU emulsion mixture in weight ratios of 1:100, 1:20, 1:10 and 1:5, respectively. Morphology, electrical conductivity, mechanical properties and cytocompatibility with C2C12 myoblast cells were characterized. The polymerization resulted in a composite with a principle base of PU interspersed with an electrically percolating network of PPy nanoparticles. As the mass ratio of PPy to PU increased so did electrical conductivity of the composites. In addition, as the mass ratio of PPy to PU increased, stiffness of the composite increased while maximum elongation length decreased. Ultimate tensile strength was reduced by approximately 47% across all samples with the addition of PPy to the PU base. Cytocompatibility assay data indicated no significant cytotoxic effect from the composites. Static cellular seeding of C2C12 cells and subsequent differentiation showed myotube formation on the composite materials. PMID:21681943

  1. Control of Cell Alignment and Morphology by Redesigning ECM-Mimetic Nanotopography on Multilayer Membranes.

    Science.gov (United States)

    Sousa, Maria P; Caridade, Sofia G; Mano, João F

    2017-08-01

    Inspired by native extracellular matrix (ECM) together with the multilevel architecture observed in nature, a material which topography recapitulates topographic features of the ECM and the internal architecture mimics the biological materials organization is engineered. The nanopatterned design along the XY plane is combined with a nanostructured organization along the Z axis on freestanding membranes prepared by layer-by-layer deposition of chitosan and chondroitin sulfate. Cellular behavior is monitored using two different mammalian cell lines, fibroblasts (L929) and myoblasts (C2C12), in order to perceive the response to topography. Viability, proliferation, and morphology of L929 are sensitively controlled by topography; also differentiation of C2C12 into myotubes is influenced by the presence of nanogrooves. This kind of nanopatterned structure has also been associated with strong cellular alignment. To the best of the knowledge, it is the first time that such a straightforward and inexpensive strategy is proposed to produce nanopatterned freestanding multilayer membranes. Controlling cellular alignment plays a critical role in many human tissues, such as muscles, nerves, or blood vessels, so these membranes can be potentially useful in specific tissue regeneration strategies. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Cell type-specific and common characteristics of exosomes derived from mouse cell lines: Yield, physicochemical properties, and pharmacokinetics.

    Science.gov (United States)

    Charoenviriyakul, Chonlada; Takahashi, Yuki; Morishita, Masaki; Matsumoto, Akihiro; Nishikawa, Makiya; Takakura, Yoshinobu

    2017-01-01

    Exosomes are small membrane vesicles secreted from cells and are expected to be used as drug delivery systems. Important characteristics of exosomes, such as yield, physicochemical properties, and pharmacokinetics, may be different among different cell types. However, there is limited information about the effect of cell type on these characteristics. In the present study, we evaluated these characteristics of exosomes derived from five different types of mouse cell lines: B16BL6 murine melanoma cells, C2C12 murine myoblast cells, NIH3T3 murine fibroblasts cells, MAEC murine aortic endothelial cells, and RAW264.7 murine macrophage-like cells. Exosomes were collected using a differential ultracentrifugation method. The exosomes collected from all the cell types were negatively charged globular vesicles with a diameter of approximately 100nm. C2C12 and RAW264.7 cells produced more exosomes than the other types of cells. The exosomes were labeled with a fusion protein of Gaussia luciferase and lactadherin to evaluate their pharmacokinetics. After intravenous injection into mice, all the exosomes rapidly disappeared from the systemic circulation and mainly distributed to the liver. In conclusion, the exosome yield was significantly different among the cell types, and all the exosomes evaluated in this study showed comparable physicochemical and pharmacokinetic properties. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Simplified microenvironments and reduced cell culture size influence the cell differentiation outcome in cellular microarrays.

    Science.gov (United States)

    Rodríguez-Seguí, Santiago A; Ortuño, María José; Ventura, Francesc; Martínez, Elena; Samitier, Josep

    2013-01-01

    Cellular microarrays present a promising tool for multiplex evaluation of the signalling effect of substrate-immobilized factors on cellular differentiation. In this paper, we compare the early myoblast-to-osteoblast cell commitment steps in response to a growth factor stimulus using standard well plate differentiation assays or cellular microarrays. Our results show that restraints on the cell culture size, inherent to cellular microarrays, impair the differentiation outcome. Also, while cells growing on spots with immobilised BMP-2 are early biased towards the osteoblast fate, longer periods of cell culturing in the microarrays result in cell proliferation and blockage of osteoblast differentiation. The results presented here raise concerns about the efficiency of cell differentiation when the cell culture dimensions are reduced to a simplified microspot environment. Also, these results suggest that further efforts should be devoted to increasing the complexity of the microspots composition, aiming to replace signalling cues missing in this system.

  4. PI(4,5)P2 regulates myob