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Sample records for c2c12 myoblasts expressing

  1. Mechanical stretch regulates microRNA expression profile via NF-κB activation in C2C12 myoblasts

    PubMed Central

    Hua, Wenxi; Zhang, Mahui; Wang, Yongkui; Yu, Lei; Zhao, Tingting; Qiu, Xiaozhong; Wang, Leyu

    2016-01-01

    MicroRNAs (miRNAs/miRs) and nuclear factor (NF)-κB activation are involved in mechanical stretch-induced skeletal muscle regeneration. However, there are a small number of miRNAs that have been reported to be associated with NF-κB activation during mechanical stretch-induced myogenesis. In the present study, C2C12 myoblasts underwent cyclic mechanical stretch in vitro, to explore the relationship between miRNA expression and NF-κB activation during stretch-mediated myoblast proliferation. The results revealed that 10% deformation, 0.125 Hz cyclic mechanical stretch could promote myoblast proliferation. The miRNA expression profile was subsequently altered; miR-500, −1934, −31, −378, −331 and −5097 were downregulated, whereas miR-1941 was upregulated. These miRNAs were all involved in stretch-mediated myoblast proliferation. Notably, the expression of these miRNAs was reversed following treatment of 0.125 Hz mechanically stretched C2C12 cells with NF-κB inhibitors, which was accompanied by C2C12 cell growth suppression. Therefore, the present study is the first, to the best of our knowledge, to demonstrate that the NF-κB-dependent miRNA profile is associated with mechanical stretch-induced myoblast proliferation. PMID:27840929

  2. Wnt/{beta}-catenin signaling changes C2C12 myoblast proliferation and differentiation by inducing Id3 expression

    SciTech Connect

    Zhang, Long; Shi, Songting; Zhang, Juan; Zhou, Fangfang; Dijke, Peter ten

    2012-03-02

    Highlights: Black-Right-Pointing-Pointer Expression of Id3 but not Id1 is induced by Wnt3a stimulation in C2C12 cells. Black-Right-Pointing-Pointer Wnt3a induces Id3 expression via canonical Wnt/{beta}-catenin pathway. Black-Right-Pointing-Pointer Wnt3a-induced Id3 expression does not depend on BMP signaling activation. Black-Right-Pointing-Pointer Induction of Id3 expression is critical determinant in Wnt3a-induced cell proliferation and differentiation. -- Abstract: Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a {beta}-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However, Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/{beta}-catenin induced gene in myoblast cell fate determination.

  3. Selective androgen receptor modulator, YK11, regulates myogenic differentiation of C2C12 myoblasts by follistatin expression.

    PubMed

    Kanno, Yuichiro; Ota, Rumi; Someya, Kousuke; Kusakabe, Taichi; Kato, Keisuke; Inouye, Yoshio

    2013-01-01

    The myogenic differentiation of C2C12 myoblast cells is induced by the novel androgen receptor (AR) partial agonist, (17α,20E)-17,20-[(1-methoxyethylidene)bis-(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11), as well as by dihydrotestosterone (DHT). YK11 is a selective androgen receptor modulator (SARM), which activates AR without the N/C interaction. In this study, we further investigated the mechanism by which YK11 induces myogenic differentiation of C2C12 cells. The induction of key myogenic regulatory factors (MRFs), such as myogenic differentiation factor (MyoD), myogenic factor 5 (Myf5) and myogenin, was more significant in the presence of YK11 than in the presence of DHT. YK11 treatment of C2C12 cells, but not DHT, induced the expression of follistatin (Fst), and the YK11-mediated myogenic differentiation was reversed by anti-Fst antibody. These results suggest that the induction of Fst is important for the anabolic effect of YK11.

  4. Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts.

    PubMed

    Ferri, Paola; Barbieri, Elena; Burattini, Sabrina; Guescini, Michele; D'Emilio, Alessandra; Biagiotti, Laura; Del Grande, Paolo; De Luca, Antonio; Stocchi, Vilberto; Falcieri, Elisabetta

    2009-12-15

    It is known that the MyoD family members (MyoD, Myf5, myogenin, and MRF4) play a pivotal role in the complex mechanism of skeletal muscle cell differentiation. However, fragmentary information on transcription factor-specific regulation is available and data on their post-transcriptional and post-translational behavior are still missing. In this work, we combined mRNA and protein expression analysis with their subcellular localization. Each myogenic regulator factor (MRF) revealed a specific mRNA trend and a protein quantitative analysis not overlapping, suggesting the presence of post-transcriptional mechanisms. In addition, each MRF showed a specific behavior in situ, characterized by a differentiation stage-dependent localization suggestive of a post-translational regulation also. Consistently with their transcriptional activity, immunogold electron microscopy data revealed MRFs distribution in interchromatin domains. Our results showed a MyoD and Myf5 contrasting expression profile in proliferating myoblasts, as well as myogenin and MRF4 opposite distribution in the terminally differentiated myotubes. Interestingly, MRFs expression and subcellular localization analysis during C2C12 cell differentiation stages showed two main MRFs regulation mechanisms: (i) the protein half-life regulation to modulate the differentiation stage-dependent transcriptional activity and (ii) the cytoplasmic retention, as a translocation process, to inhibit the transcriptional activity. Therefore, our results exhibit that MRFs nucleo-cytoplasmic trafficking is involved in muscle differentiation and suggest that, besides the MRFs expression level, also MRFs subcellular localization, related to their functional activity, plays a key role as a regulatory step in transcriptional control mechanisms.

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

    SciTech Connect

    Eom, Young Woo; Lee, Jong Eun; Yang, Mal Sook; Jang, In Keun; Kim, Hyo Eun; Lee, Doo Hoon; Kim, Young Jin; Park, Won Jin; Kong, Jee Hyun; Shim, Kwang Yong; Lee, Jong In; Kim, Hyun Soo

    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.

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

    SciTech Connect

    Hong, Gia-Ming

    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

  7. α-Linolenic Acid Reduces TNF-Induced Apoptosis in C2C12 Myoblasts by Regulating Expression of Apoptotic Proteins

    PubMed Central

    Carotenuto, Felicia; Coletti, Dario; Di Nardo, Paolo; Teodori, Laura

    2016-01-01

    Impaired regeneration and consequent muscle wasting is a major feature of muscle degenerative diseases. Nutritional interventions such as adjuvant strategy for preventing these conditions are recently gaining increasing attention. Ingestion of n3-polyunsaturated fatty acids has been suggested as having a positive impact on muscle diseases. We recently demonstrated that a diet enriched with plant derived n3-fatty acid, α-linolenic acid (ALA), exerts potent beneficial effects in preserving skeletal muscle regeneration in models of muscle dystrophy. To better elucidate the underlying mechanism we here investigate on the expression level of the anti- and pro-apoptotic proteins, as well as caspase-3 activity, in C2C12 myoblasts challenged with pathological levels of tumor necrosis factor-α (TNF). The results demonstrated that ALA protective effect on C2C12 myoblasts was associated with a decrease in caspase-3 activity and an increase of the Bcl-2/Bax ratio. Indeed, the effect of ALA was directed to rescuing Bcl-2 expression and to revert Bax translocation to mitochondria both affected in an opposite way by TNF, a major pro-inflammatory cytokine expressed in damaged skeletal muscle. Therefore, ALA counteracts inflammatory signals in the muscle microenvironment and may represent a valuable strategy for ameliorating skeletal muscle pathologies. PMID:28078067

  8. Berberine Hydrochloride Protects C2C12 Myoblast Cells Against Oxidative Stress-Induced Damage via Induction of Nrf-2-Mediated HO-1 Expression.

    PubMed

    Choi, Yung Hyun

    2016-09-01

    Preclinical Research The aim of the present study was to evaluate the effects of berberine hydrochloride (BBH), an isoquinoline alkaloid that can be isolated from a variety of herbs, on hydrogen peroxide (H2 O2 )-induced oxidative stress in C2C12 myoblasts and to investigate the molecular mechanisms involved in this process, especially the expression of the Nrf2/HO-1 pathway. BBH preconditioning attenuated H2 O2 -induced growth inhibition and DNA damage as well as apoptosis in C2C12 cells via suppression of the accumulation of intracellular reactive oxygen species (ROS). Treatment with BBHride alone effectively upregulated the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) and elevated HO-1 activity. However, the protective effects of BBH against H2 O2 -induced ROS generation and cell growth reduction were abolished by an HO-1 inhibitor. Moreover, BBH-mediated induction and activation of HO-1 were reduced by genetic silencing of Nrf2 using small interfering RNA (siRNA). In addition, the effects of BBH against H2 O2 -induced ROS accumulation and growth inhibition were abrogated in C2C12 cells transfected with Nrf2 siRNA. Therefore, the present study demonstrated that BBH could protect C2C12 cells against oxidative stress-induced injury and this effect involved activation of the Nrf2/HO-1 pathway. Drug Dev Res, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

    PubMed

    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.

  10. Bioenergetic Profile Experiment using C2C12 Myoblast Cells

    PubMed Central

    Nicholls, David G.; Darley-Usmar, Victor M.; Wu, Min; Jensen, Per Bo; Rogers, George W.; Ferrick, David A.

    2010-01-01

    The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity. Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment. Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR. In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell. This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and

  11. Bioenergetic profile experiment using C2C12 myoblast cells.

    PubMed

    Nicholls, David G; Darley-Usmar, Victor M; Wu, Min; Jensen, Per Bo; Rogers, George W; Ferrick, David A

    2010-12-06

    The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity. Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO(2) into the extracellular environment. Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration--the Oxygen Consumption Rate--or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate--or ECAR. In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell. This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and

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

    PubMed

    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.

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

    SciTech Connect

    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven

    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.

  14. Electrical Impedance Monitoring of C2C12 Myoblast Differentiation on an Indium Tin Oxide Electrode

    PubMed Central

    Park, Ilhwan; Hong, Yeonhee; Jun, Young-Hoo; Lee, Ga-Yeon; Jun, Hee-Sook; Pyun, Jae-Chul; Choi, Jeong-Woo; Cho, Sungbo

    2016-01-01

    Electrical cell-substrate impedance sensing is increasingly being used for label-free and real-time monitoring of changes in cell morphology and number during cell growth, drug screening, and differentiation. In this study, we evaluated the feasibility of using ECIS to monitor C2C12 myoblast differentiation using a fabricated indium tin oxide (ITO) electrode-based chip. C2C12 myoblast differentiation on the ITO electrode was validated based on decreases in the mRNA level of MyoD and increases in the mRNA levels of myogenin and myosin heavy chain (MHC). Additionally, MHC expression and morphological changes in myoblasts differentiated on the ITO electrode were comparable to those in cells in the control culture dish. From the monitoring the integration of the resistance change at 21.5 kHz, the cell differentiation was label-free and real-time detectable in 30 h of differentiation (p < 0.05). PMID:27929401

  15. Morin exerts cytoprotective effects against oxidative stress in C2C12 myoblasts via the upregulation of Nrf2-dependent HO-1 expression and the activation of the ERK pathway.

    PubMed

    Lee, Moon Hee; Han, Min Ho; Lee, Dae-Sung; Park, Cheol; Hong, Su-Hyun; Kim, Gi-Young; Hong, Sang Hoon; Song, Kyoung Seob; Choi, Il-Whan; Cha, Hee-Jae; Choi, Yung Hyun

    2017-02-01

    In the present study, we investigated the cytoprotective efficacy of morin, a natural flavonoid, against oxidative stress and elucidated the underlying mechanisms in C2C12 myoblasts. Our results indicated that morin treatment prior to hydrogen peroxide (H2O2) exposure significantly increased cell viability and prevented the generation of reactive oxygen species. H2O2-induced comet-like DNA formation and γH2AX phosphorylation were also markedly suppressed by morin with a parallel inhibition of apoptosis in C2C12 myoblasts, suggesting that morin prevented H2O2-induced cellular DNA damage. Furthermore, morin markedly enhanced the expression of heme oxygenase-1 (HO-1) associated with the induction and phosphorylation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and the inhibition of Kelch-like ECH-associated protein 1 (Keap1) expression. Notably, these events were eliminated by transient transfection with Nrf2‑specific small interfering RNA. Additional experiments demonstrated that the activation of the Nrf2/HO-1 pathway by morin was mediated by the extracellular signal‑regulated kinase (ERK) signaling cascade. This phenomenon was confirmed with suppressed Nrf2 phosphorylation and consequently diminished HO-1 expression in cells treated with a pharmacological inhibitor of ERK. Collectively, these results demonstrated that morin augments the cellular antioxidant defense capacity through the activation of Nrf2/HO‑1 signaling, which involves the activation of the ERK pathway, thereby protecting C2C12 myoblasts from H2O2-induced oxidative cytotoxicity.

  16. Antioxidative effects of diallyl trisulfide on hydrogen peroxide-induced cytotoxicity through regulation of nuclear factor-E2-related factor-mediated thioredoxin reductase 1 expression in C2C12 skeletal muscle myoblast cells.

    PubMed

    Kang, Ji Sook; Kim, Gi-Yiung; Kim, Byung Woo; Choi, Yung Hyun

    2017-04-01

    Diallyl trisulfide (DATS) is one of the major sulfur-containing compounds in garlic oil. In this study, we analyzed the effects of DATS against hydrogen peroxide (H2O2)-induced oxidative stress in C2C12 myoblasts. DATS preconditioning significantly attenuated H2O2-induced growth inhibition and DNA damage, as well as apoptosis by decreasing the generation of ROS. Treatment with DATS alone effectively upregulated the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) and thioredoxin reductase 1 (TrxR1), which was associated with the increased phosphorylation of Nrf2. However, the protective effects of DATS against H2O2-induced growth reduction and ROS accumulation were significantly abolished by auranofin, an inhibitor of TrxR activity. Moreover, DATS-mediated phosphorylation of Nrf2 and induction of TrxR1 were markedly reduced by genetic silencing of Nrf2. DATS treatment also induced the phosphorylation extracellular signal-regulating kinase (ERK), and analysis using specific inhibitors of cellular signaling pathways demonstrated that only ERK activation was involved in Nrf2 phosphorylation and TrxR1 induction. In addition, the cytoprotective potentials were abrogated in C2C12 cells pretreated with an ERK specific inhibitor. The results demonstrate that DATS protects against oxidative stress-induced DNA damage and apoptosis in C2C12 cells in part through the activation of Nrf2-mediated TrxR1 induction via the ERK signaling pathway.

  17. Fad24, a Positive Regulator of Adipogenesis, Is Required for S Phase Re-entry of C2C12 Myoblasts Arrested in G0 Phase and Involved in p27(Kip1) Expression at the Protein Level.

    PubMed

    Ochiai, Natsuki; Nishizuka, Makoto; Osada, Shigehiro; Imagawa, Masayoshi

    2016-05-01

    Factor for adipocyte differentiation 24 (fad24) is a positive regulator of adipogenesis. We previously found that human fad24 is abundantly expressed in skeletal muscle. However, the function of fad24 in skeletal muscle remains largely unknown. Because skeletal muscle is a highly regenerative tissue, we focused on the function of fad24 in skeletal muscle regeneration. In this paper, we investigated the role of fad24 in the cell cycle re-entry of quiescent C2C12 myoblasts-mimicked satellite cells. The expression levels of fad24 and histone acetyltransferase binding to ORC1 (hbo1), a FAD24-interacting factor, were elevated at the early phase of the regeneration process in response to cardiotoxin-induced muscle injury. The knockdown of fad24 inhibited the proliferation of quiescent myoblasts, whereas fad24 knockdown did not affect differentiation. S phase entry following serum activation is abrogated by fad24 knockdown in quiescent cells. Furthermore, fad24 knockdown cells show a marked accumulation of p27(Kip1) protein. These results suggest that fad24 may have an important role in the S phase re-entry of quiescent C2C12 cells through the regulation of p27(Kip1) at the protein level.

  18. Chromatin plasticity as a differentiation index during muscle differentiation of C2C12 myoblasts

    SciTech Connect

    Watanabe, Tomonobu M.; Higuchi, Sayaka; Kawauchi, Keiko; Tsukasaki, Yoshikazu; Ichimura, Taro; Fujita, Hideaki

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer Change in the epigenetic landscape during myogenesis was optically investigated. Black-Right-Pointing-Pointer Mobility of nuclear proteins was used to state the epigenetic status of the cell. Black-Right-Pointing-Pointer Mobility of nuclear proteins decreased as myogenesis progressed in C2C12. Black-Right-Pointing-Pointer Differentiation state diagram was developed using parameters obtained. -- Abstract: Skeletal muscle undergoes complicated differentiation steps that include cell-cycle arrest, cell fusion, and maturation, which are controlled through sequential expression of transcription factors. During muscle differentiation, remodeling of the epigenetic landscape is also known to take place on a large scale, determining cell fate. In an attempt to determine the extent of epigenetic remodeling during muscle differentiation, we characterized the plasticity of the chromatin structure using C2C12 myoblasts. Differentiation of C2C12 cells was induced by lowering the serum concentration after they had reached full confluence, resulting in the formation of multi-nucleated myotubes. Upon induction of differentiation, the nucleus size decreased whereas the aspect ratio increased, indicating the presence of force on the nucleus during differentiation. Movement of the nucleus was also suppressed when differentiation was induced, indicating that the plasticity of chromatin changed upon differentiation. To evaluate the histone dynamics during differentiation, FRAP experiment was performed, which showed an increase in the immobile fraction of histone proteins when differentiation was induced. To further evaluate the change in the histone dynamics during differentiation, FCS was performed, which showed a decrease in histone mobility on differentiation. We here show that the plasticity of chromatin decreases upon differentiation, which takes place in a stepwise manner, and that it can be used as an index for the differentiation stage

  19. Chromatin plasticity as a differentiation index during muscle differentiation of C2C12 myoblasts.

    PubMed

    Watanabe, Tomonobu M; Higuchi, Sayaka; Kawauchi, Keiko; Tsukasaki, Yoshikazu; Ichimura, Taro; Fujita, Hideaki

    2012-02-24

    Skeletal muscle undergoes complicated differentiation steps that include cell-cycle arrest, cell fusion, and maturation, which are controlled through sequential expression of transcription factors. During muscle differentiation, remodeling of the epigenetic landscape is also known to take place on a large scale, determining cell fate. In an attempt to determine the extent of epigenetic remodeling during muscle differentiation, we characterized the plasticity of the chromatin structure using C2C12 myoblasts. Differentiation of C2C12 cells was induced by lowering the serum concentration after they had reached full confluence, resulting in the formation of multi-nucleated myotubes. Upon induction of differentiation, the nucleus size decreased whereas the aspect ratio increased, indicating the presence of force on the nucleus during differentiation. Movement of the nucleus was also suppressed when differentiation was induced, indicating that the plasticity of chromatin changed upon differentiation. To evaluate the histone dynamics during differentiation, FRAP experiment was performed, which showed an increase in the immobile fraction of histone proteins when differentiation was induced. To further evaluate the change in the histone dynamics during differentiation, FCS was performed, which showed a decrease in histone mobility on differentiation. We here show that the plasticity of chromatin decreases upon differentiation, which takes place in a stepwise manner, and that it can be used as an index for the differentiation stage during myogenesis using the state diagram developed with the parameters obtained in this study.

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

    PubMed Central

    da Silva, Aline; Vieira, Rodolfo Paula; Mesquita-Ferrari, Raquel Agnelli; Cogo, José Carlos; Zamuner, Stella Regina

    2016-01-01

    Background Snakebites is a neglected disease and in Brazil is considered a serious health problem, with the majority of the snakebites caused by the genus Bothrops. Antivenom therapy and other first-aid treatments do not reverse local myonecrose which is the main sequel caused by the envenomation. 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. In this in vitro study, we aimed to analyze the effect of LLL irradiation against cytotoxicity induced by Bothrops jararacussu venom on myoblast C2C12 cells. Methodology C2C12 were utilized as a model target and were incubated with B. jararacussu venom (12.5 μg/mL) and immediately irradiated with LLL at wavelength of red 685 nm or infrared 830 nm with energy density of 2.0, 4.6 and 7.0 J/cm2. Effects of LLL on cellular responses of venom-induced cytotoxicity were examined, including cell viability, measurement of cell damage and intra and extracellular ATP levels, expression of myogenic regulatory factors, as well as cellular differentiation. Results In non-irradiated cells, the venom caused a decrease in cell viability and a massive release of LDH and CK levels indicating myonecrosis. Infrared and red laser at all energy densities were able to considerably decrease venom-induced cytotoxicity. Laser irradiation induced myoblasts to differentiate into myotubes and this effect was accompanied by up regulation of MyoD and specially myogenin. Moreover, LLL was able to reduce the extracellular while increased the intracellular ATP content after venom exposure. In addition, no difference in the intensity of cytotoxicity was shown by non-irradiated and irradiated venom. Conclusion LLL irradiation caused a protective effect on C2C12 cells against the cytotoxicity caused by B. jararacussu venom and promotes differentiation of these cells by up regulation of myogenic factors. A modulatory

  1. Astragalus Polysaccharide Inhibits Autophagy and Apoptosis from Peroxide-Induced Injury in C2C12 Myoblasts.

    PubMed

    Yin, Yi; Lu, Lu; Wang, Dongtao; Shi, Ying; Wang, Ming; Huang, Yanfeng; Chen, Dexiu; Deng, Cong; Chen, Jiebin; Lv, Peijia; Wang, Yanjing; Li, Chengjie; Wei, Lian-Bo

    2015-11-01

    The aim is to study the effects and underlying mechanisms of astragalus polysaccharide (APS) on the peroxide-induced injury in C2C12 myoblasts in vitro. Cell viability in the presence or absence of APS was detected by the methyl thiazolyl tetrazolium colorimetric assay. The autophagosomes were observed by electron microscopy to examine the influence of APS on autophagy caused by H2O2 in C2C12 cells, and the percentage of apoptosis cells was measured by flow cytometry. To further confirm the effect of H2O2 on C2C12 cells, the protein expression of LC3 and RARP, which are the markers of autophagy and apoptosis, respectively, was analyzed by Western blot, as well as the expression levels of p-p70S6K, p70S6K, Bcl-2, Bax, cyto-C, and Caspase-3, to reveal the underlying mechanisms. We observed multiple effects of APS on C2C12 functionality. APS treatment of C2C12 cells at 1 mg/mL reduced cell viability to less than 70 %, and analysis by electron microscopy revealed that APS also reduced the number of H2O2-induced autophagosome formation. Similarly, APS abated the H2O2-mediated increase in cell apoptosis, which was accompanied by the inhibition of LC3 II and RARP that are normally upregulated by H2O2. The expression of p-p70S6K and p70S6K, however, remained unchanged in C2C12 cells in the Control, H2O2 and H2O2 + APS groups. In addition, APS promoted the expression of protein Bcl-2 in H2O2-treated C2C12 cells, but did not change Bax, thus reducing the Bax/Bcl-2 ratio that in turn prevented the release of cytochrome c and the activation of caspase-3. APS inhibits the autophagy and apoptosis induced by peroxide injury in C2C12 myoblasts through two independent signaling pathways: the mTOR-independent pathway for the inhibition of autophagy, and the caspase-3-dependent pathway for the suppression of apoptosis.

  2. Rab8A regulates insulin-stimulated GLUT4 translocation in C2C12 myoblasts.

    PubMed

    Li, Hanbing; Ou, Liting; Fan, Jiannan; Xiao, Mei; Kuang, Cuifang; Liu, Xu; Sun, Yonghong; Xu, Yingke

    2017-02-01

    Rab proteins are important regulators of GLUT4 trafficking in muscle and adipose cells. It is still unclear which Rabs are involved in insulin-stimulated GLUT4 translocation in C2C12 myoblasts. In this study, we detect the colocalization of Rab8A with GLUT4 and the presence of Rab8A at vesicle exocytic sites by TIRFM imaging. Overexpression of dominant-negative Rab8A (T22N) diminishes insulin-stimulated GLUT4 translocation, while constitutively active Rab8A (Q67L) augments it. In addition, knockdown of Rab8A inhibits insulin-stimulated GLUT4 translocation, which is rescued by replenishment of RNAi-resistant Rab8A. Together, these results indicate an indispensable role for Rab8A in insulin-regulated GLUT4 trafficking in C2C12 cells.

  3. Arecoline inhibits myogenic differentiation of C2C12 myoblasts by reducing STAT3 phosphorylation.

    PubMed

    Chang, Yung-Fu; Liu, Ting-Yuan; Liu, Shao-Tung; Tseng, Chao-Neng

    2012-10-01

    Areca nut (Areca catechu) is chewed regularly as a medical and psychoactive food by about 10% of the world population, in countries including India, Taiwan and parts of Southern Asia. Areca nut chewing during pregnancy has been associated with both lower birth weight and premature birth. Animals of low birth weights showed retardation of muscle development. Our previous study showed that arecoline, the major areca alkaloid, decreased the number of implanted embryos. Here we sought to determine the effects of arecoline in myogenic differentiation by in vitro assays using C2C12 myoblast cells. The results showed that arecoline higher than 0.4mM significantly increased apoptosis and decreased viability of C2C12 cells. Morphometric measurements of myotube formation and analyses of myogenic markers, myosin heavy chain and myogenin, revealed that myogenic differentiation was inhibited by 0.04-0.08 mM arecoline. Moreover, phosphorylated but not total STAT3 was significantly inhibited by arecoline during myotube formation. These results indicate that arecoline inhibits the myogenic differentiation of C2C12 cells by reducing the activation of STAT3, an upstream regulator of myogenesis. Improved understanding of the effects of arecoline during myogenic differentiation may help to establish public health policies and to develop potential treatments for such patients. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Nandrolone, an anabolic steroid, stabilizes Numb protein through inhibition of mdm2 in C2C12 myoblasts.

    PubMed

    Liu, Xin-Hua; Yao, Shen; Levine, Alice C; Kirschenbaum, Alexander; Pan, Jiangping; Wu, Yong; Qin, Weiping; Collier, Lauren; Bauman, William A; Cardozo, Christopher P

    2012-01-01

    Nandrolone, an anabolic steroid, slows denervation atrophy of rat muscle, prevents denervation-induced nuclear accumulation of intracellular domain of the Notch receptor, and elevates expression of Numb. Numb acts as an inhibitor of Notch signaling and promotes myogenic differentiation of satellite cells. Turnover of Numb is regulated by mdm2, an E3 ubiquitin ligase. With these considerations in mind, we investigated the effects of nandrolone on the expression of Numb and mdm2 proteins and determined the effect of mdm2 on nandrolone-induced alterations in Numb protein in C2C12 myoblasts. When C2C12 cells were cultured in a medium favoring differentiation (Dulbecco modified Eagle medium containing 2% horse serum), nandrolone up-regulated Numb protein levels in a time-dependent manner and prolonged Numb protein half-life from 10 to 18 hours. In contrast, nandrolone reduced the expression of mdm2 protein. To determine whether the decreased mdm2 expression induced by nandrolone was responsible for the increased levels and prolonged half-life of Numb protein in this cell line, mdm2-small interfering RNA (siRNA) was employed to inhibit mdm2 expression. Compared to cells transfected with scrambled siRNA (negative control), transfection with mdm2-siRNA increased basal Numb protein expression but abolished the further increase in Numb protein levels by nandrolone. In addition, transfection of mdm2-siRNA mimicked the effect of nandrolone to prolong the half-life of Numb protein. Moreover, when C2C12 cells were forced to overexpress mdm2, there was a significant decline in the expression of both basal and inducible Numb protein. Our data suggest that nandrolone, by a novel mechanism for this agent in a muscle cell type, increases Numb protein levels in C2C12 myoblasts by stabilizing Numb protein against degradation, at least in part, via suppression of mdm2 expression.

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

    PubMed Central

    Washio, Kohei; Kobayashi, Mao; Saito, Natsuko; Amagasa, Misato; Kitamura, Hiroshi

    2015-01-01

    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. PMID:26604971

  6. Shengmai-san enhances antioxidant potential in C2C12 myoblasts through the induction of intracellular glutathione peroxidase.

    PubMed

    Nishida, Hiroshi; Ichikawa, Haruyo; Konishi, Tetsuya

    2007-12-01

    Cellular and tissue injury associated with reactive oxygen species (ROS) has been reported in many kinds of disorders. While the antioxidant enzymes play critical roles in inhibiting the ROS-mediated injury, glutathione peroxidase (GPx) is scavenging hydroperoxides including H(2)O(2). We previously reported that Shengmai-san (SMS), a traditional Chinese medicine, prevented ischemia/reperfusion injury of the brain and other organs in rats. To clarify the effect of SMS on intracellular responses of muscle cells against oxidative stress, C2C12 myoblasts were subjected to H(2)O(2) abuse. SMS pre-incubation prevented the decreasing cell viability after H(2)O(2) treatment. The accumulations of cellular protein carbonyl associated with apoptotic cell death were also inhibited by the SMS pre-incubation prior to oxidative damage induction. At the same time, enhanced activity, protein, and mRNA expression levels of GPx were observed in cells pre-incubated with SMS prior to H(2)O(2) abuse. Moreover, intracellular GSH was subsequently decreased after H(2)O(2) treatment. These findings suggest that SMS improved the antioxidant capacity against acute oxidative stress through the constitutive enhancement of GPx expression in C2C12 myoblasts. Because of its antioxidative property, SMS might be useful not only for the oxidative damage associated diseases but also for the transplantation of myoblasts into muscular dystrophy patients.

  7. E-cadherin cytoplasmic domain inhibits cell surface localization of endogenous cadherins and fusion of C2C12 myoblasts.

    PubMed

    Ozawa, Masayuki

    2015-10-09

    Myoblast fusion is a highly regulated process that is essential for skeletal muscle formation during muscle development and regeneration in mammals. Much remains to be elucidated about the molecular mechanism of myoblast fusion although cadherins, which are Ca(2+)-dependent cell-cell adhesion molecules, are thought to play a critical role in this process. Mouse myoblasts lacking either N-cadherin or M-cadherin can still fuse to form myotubes, indicating that they have no specific function in this process and may be functionally replaced by either M-cadherin or N-cadherin, respectively. In this study, we show that expressing the E-cadherin cytoplasmic domain ectopically in C2C12 myoblasts inhibits cell surface localization of endogenous M-cadherin and N-cadherin, as well as cell-cell fusion. This domain, however, does not inhibit myoblast differentiation according to microarray-based gene expression analysis. In contrast, expressing a dominant-negative β-catenin mutant ectopically, which suppresses Wnt/β-catenin signaling, did not inhibit cell-cell fusion. Therefore, the E-cadherin cytoplasmic domain inhibits cell-cell fusion by inhibiting cell surface localization of endogenous cadherins and not by inhibiting Wnt/β-catenin signaling.

  8. The cytoprotective effect of isorhamnetin against oxidative stress is mediated by the upregulation of the Nrf2-dependent HO-1 expression in C2C12 myoblasts through scavenging reactive oxygen species and ERK inactivation.

    PubMed

    Choi, Yung Hyun

    2016-04-01

    This study was designed to confirm the protective effects of isorhamnetin against oxidative stress-induced cellular damage. Our results indicated that isorhamnetin inhibited the hydrogen peroxide (H2O2)-induced growth inhibition and exhibited scavenging activity against the intracellular reactive oxygen species (ROS) in mouse-derived C2C12 myoblasts. Isorhamnetin also significantly attenuated H2O2-induced DNA damage and apoptosis, and increased the levels of the nuclear factor erythroid 2-related factor 2 (Nrf2) and its phosphorylation associated with the induction of heme oxygenase-1 (HO-1). However, the protective effects of isorhamnetin on H2O2-induced ROS and growth inhibition were significantly abolished by an HO-1 competitive inhibitor. Moreover, the potential of isorhamnetin to mediate HO-1 induction and protect against H2O2-mediated growth inhibition was abrogated by transient transfection with Nrf2-specific small interfering RNA. Additionally, isorhamnetin induced the activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. However, the specific inhibitor of ERK, but not JNK and p38 MAPK, was able to abolish the HO-1 upregulation and the Nrf2 phosphorylation. Collectively, these results demonstrate that isorhamnetin augments the cellular antioxidant defense capacity by activating the Nrf2/HO-1 pathway involving the activation of the ERK pathway, thus protecting the C2C12 cells from H2O2-induced cytotoxicity.

  9. MicroRNAs Regulate Cellular ATP Levels by Targeting Mitochondrial Energy Metabolism Genes during C2C12 Myoblast Differentiation.

    PubMed

    Siengdee, Puntita; Trakooljul, Nares; Murani, Eduard; Schwerin, Manfred; Wimmers, Klaus; Ponsuksili, Siriluck

    2015-01-01

    In our previous study, we identified an miRNA regulatory network involved in energy metabolism in porcine muscle. To better understand the involvement of miRNAs in cellular ATP production and energy metabolism, here we used C2C12 myoblasts, in which ATP levels increase during differentiation, to identify miRNAs modulating these processes. ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation into myotubes were assessed. The results suggest 14 miRNAs (miR-423-3p, miR-17, miR-130b, miR-301a/b, miR-345, miR-15a, miR-16a, miR-128, miR-615, miR-1968, miR-1a/b, and miR-194) as cellular ATP regulators targeting genes involved in mitochondrial energy metabolism (Cox4i2, Cox6a2, Ndufb7, Ndufs4, Ndufs5, and Ndufv1) during C2C12 differentiation. Among these, miR-423-3p showed a high inverse correlation with increasing ATP levels. Besides having implications in promoting cell growth and cell cycle progression, its function in cellular ATP regulation is yet unknown. Therefore, miR-423-3p was selected and validated for the function together with its potential target, Cox6a2. Overexpression of miR-423-3p in C2C12 myogenic differentiation lead to decreased cellular ATP level and decreased expression of Cox6a2 compared to the negative control. These results suggest miR-423-3p as a novel regulator of ATP/energy metabolism by targeting Cox6a2.

  10. MicroRNAs Regulate Cellular ATP Levels by Targeting Mitochondrial Energy Metabolism Genes during C2C12 Myoblast Differentiation

    PubMed Central

    Siengdee, Puntita; Trakooljul, Nares; Murani, Eduard; Schwerin, Manfred; Wimmers, Klaus; Ponsuksili, Siriluck

    2015-01-01

    In our previous study, we identified an miRNA regulatory network involved in energy metabolism in porcine muscle. To better understand the involvement of miRNAs in cellular ATP production and energy metabolism, here we used C2C12 myoblasts, in which ATP levels increase during differentiation, to identify miRNAs modulating these processes. ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation into myotubes were assessed. The results suggest 14 miRNAs (miR-423-3p, miR-17, miR-130b, miR-301a/b, miR-345, miR-15a, miR-16a, miR-128, miR-615, miR-1968, miR-1a/b, and miR-194) as cellular ATP regulators targeting genes involved in mitochondrial energy metabolism (Cox4i2, Cox6a2, Ndufb7, Ndufs4, Ndufs5, and Ndufv1) during C2C12 differentiation. Among these, miR-423-3p showed a high inverse correlation with increasing ATP levels. Besides having implications in promoting cell growth and cell cycle progression, its function in cellular ATP regulation is yet unknown. Therefore, miR-423-3p was selected and validated for the function together with its potential target, Cox6a2. Overexpression of miR-423-3p in C2C12 myogenic differentiation lead to decreased cellular ATP level and decreased expression of Cox6a2 compared to the negative control. These results suggest miR-423-3p as a novel regulator of ATP/energy metabolism by targeting Cox6a2. PMID:26010876

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

    NASA Technical Reports Server (NTRS)

    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.

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

    NASA Technical Reports Server (NTRS)

    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.

  13. Cyclic Stretch Facilitates Myogenesis in C2C12 Myoblasts and Rescues Thiazolidinedione-Inhibited Myotube Formation

    PubMed Central

    Chang, Ya-Ju; Chen, Yun-Ju; Huang, Chia-Wei; Fan, Shih-Chen; Huang, Bu-Miin; Chang, Wen-Tsan; Tsai, Yau-Sheng; Su, Fong-Chin; Wu, Chia-Ching

    2016-01-01

    Thiazolidinedione (TZD), a specific peroxisome proliferator-activated receptor γ (PPARγ) 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 α-smooth muscle actin (α-SMA) protein expression. The PPARγ protein and PPAR response element (PPRE) promoter activity decreased during MM induction. Cyclic stretch further facilitated the myogenesis in MM with increased α-SMA and decreased PPARγ protein expression and inhibited PPRE promoter activity. Adding a PPARγ agonist (TZD) to the MM stopped the myogenesis and restored the PPRE promoter activity, whereas a PPARγ 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 PPARγ and its target drugs. PMID:27047938

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

    PubMed

    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.

  15. An adaptable stage perfusion incubator for the controlled cultivation of C2C12 myoblasts.

    PubMed

    Kurth, Felix; Franco-Obregón, Alfredo; Bärtschi, Christoph A; Dittrich, Petra S

    2015-01-07

    Here we present a stage perfusion incubation system that allows for the cultivation of mammalian cells within PDMS microfluidic devices for long-term microscopic examination and analysis. The custom-built stage perfusion incubator is adaptable to any x-y microscope stage and is enabled for temperature, gas and humidity control as well as equipped with chip and tubing holder. The applied double-layered microfluidic chip allows the predetermined positioning and concentration of cells while the gas permeable PDMS material facilitates pH control via CO2 levels throughout the chip. We demonstrate the functionality of this system by culturing C2C12 murine myoblasts in buffer free medium within its confines for up to 26 hours. We moreover demonstrated the system's compatibility with various chip configurations, other cells lines (HEK-293 cells) and for longer-term culturing. The cost-efficient system are applicable for any type of PDMS-based cell culture system. Detailed technical drawings and specification to reproduce this perfusion incubation system is provided in the ESI.

  16. The sphingosine kinase activator K6PC-5 stimulates C2C12 myoblast differentiation.

    PubMed

    Bernacchioni, C; Cencetti, F; Kwon, M J; Gwak, H S; Jeong, S K; Bruni, P; Donati, C

    2011-01-01

    Previously, K6PC-5, a synthetic derivative of ceramide, was demonstrated to activate sphingosine kinase (SK)-1 in keratinocytes. In this study its potential biological effect in mouse myoblasts was examined. The obtained results show that K6PC-5 promotes myogenic differentiation by enhancing myogenic marker expression, differentiation index and fusion index. Interestingly, its biological action was prevented by pharmacological inhibition of SK or S1P2 receptor, in full agreement with their recognized role in myoblast differentiation. This is the first evidence that pharmacological activation of SK accelerates myogenesis and suggests that this new therapeutic strategy could be possibly employed in skeletal muscle disorders where muscle regeneration is deficient.

  17. Low Glucose but Not Galactose Enhances Oxidative Mitochondrial Metabolism in C2C12 Myoblasts and Myotubes

    PubMed Central

    Elkalaf, Moustafa; Anděl, Michal; Trnka, Jan

    2013-01-01

    Background Substituting galactose for glucose in cell culture media has been suggested to enhance mitochondrial metabolism in a variety of cell lines. We studied the effects of carbohydrate availability on growth, differentiation and metabolism of C2C12 myoblasts and myotubes. Methodology/Principal Findings We measured growth rates, ability to differentiate, citrate synthase and respiratory chain activities and several parameters of mitochondrial respiration in C2C12 cells grown in media with varying carbohydrate availability (5 g/l glucose, 1 g/l glucose, 1 g/l galactose, and no added carbohydrates). C2C12 myoblasts grow more slowly without glucose irrespective of the presence of galactose, which is not consumed by the cells, and they fail to differentiate without glucose in the medium. Cells grown in a no-glucose medium (with or without galactose) have lower maximal respiration and spare respiratory capacity than cells grown in the presence of glucose. However, increasing glucose concentration above physiological levels decreases the achievable maximal respiration. C2C12 myotubes differentiated at a high glucose concentration showed higher dependency on oxidative respiration under basal conditions but had lower maximal and spare respiratory capacity when compared to cells differentiated under low glucose condition. Citrate synthase activity or mitochondrial yield were not significantly affected by changes in the available substrate concentration but a trend towards a higher respiratory chain activity was observed at reduced glucose levels. Conclusions/Significance Our results show that using galactose to increase oxidative metabolism may not be applicable to every cell line, and the changes in mitochondrial respiratory parameters associated with treating cells with galactose are mainly due to glucose deprivation. Moderate concentrations of glucose (1 g/l) in a growth medium are optimal for mitochondrial respiration in C2C12 cell line while supraphysiological

  18. Polyphenols prevent clinorotation-induced expression of atrogenes in mouse C2C12 skeletal myotubes.

    PubMed

    Hemdan, Dalia Ismaeil Ibrahim; Hirasaka, Katsuya; Nakao, Reiko; Kohno, Shohei; Kagawa, Sachiko; Abe, Tomoki; Harada-Sukeno, Akiko; Okumura, Yuushi; Nakaya, Yutaka; Terao, Junji; Nikawa, Takeshi

    2009-02-01

    Oxidative stress is a key factor in stimulating the expression of atrogenes, which are muscle atrophy-related ubiquitin ligases, in skeletal muscle, and it induces muscle atrophy during unloading. However, the effects of antioxidative nutrients on atrogene expression have not been demonstrated. We report on the inhibitory effects of polyphenols, such as epicatechin (EC), epicatechin gallate (ECg) and epigallocatechin gallate (EGCg) and quercetin, on atrogene expression up-regulated by three dimensional (3D)-clinorotation or glucocorticoid. These treatments markedly elevated the expression of atrogenes, including atrogin-1 and MuRF-1, in mouse C2C12 myoblasts and myotubes. Interestingly, EC, ECg, EGCg and quercetin significantly decreased the expression of atrogin-1 and MuRF-1 up-regulated by 3D-clinorotation, whereas they hardly affected atrogene expression induced by dexamethasone. ERK signaling is a well known MAPK pathway to mediate oxidative stress. Therefore, we also investigated the effect of these polyphenols on phosphorylation of ERK in C2C12 myotubes. As expected, EC, ECg, EGCg, and quercetin significantly suppressed phosphorylation of ERK, corresponding to the up-regulation of atrogenes induced by 3D-clinorotation. These results suggest that antioxidative nutrients, such as catechins and quercetin, suppress atrogene expression in skeletal muscle cells, possibly through the inhibition of ERK signaling. Thus, catechins and quercetin may prevent unloading-mediated muscle atrophy.

  19. Hypomorphic Smn knockdown C2C12 myoblasts reveal intrinsic defects in myoblast fusion and myotube morphology

    SciTech Connect

    Shafey, Dina; Cote, Patrice D.; Kothary, Rashmi . E-mail: rkothary@ohri.ca

    2005-11-15

    Dosage of the survival motor neuron (SMN) protein has been directly correlated with the severity of disease in patients diagnosed with spinal muscular atrophy (SMA). It is also clear that SMA is a neurodegenerative disorder characterized by the degeneration of the {alpha}-motor neurons in the anterior horn of the spinal cord and atrophy of the associated skeletal muscle. What is more controversial is whether it is neuronal and/or muscle-cell-autonomous defects that are responsible for the disease per se. Although motor neuron degeneration is generally accepted as the primary event in SMA, intrinsic muscle defects in this disease have not been ruled out. To gain a better understanding of the influence of SMN protein dosage in muscle, we have generated a hypomorphic series of myoblast (C2C12) stable cell lines with variable Smn knockdown. We show that depletion of Smn in these cells resulted in a decrease in the number of nuclear 'gems' (gemini of coiled bodies), reduced proliferation with no increase in cell death, defects in myoblast fusion, and malformed myotubes. Importantly, the severity of these abnormalities is directly correlated with the decrease in Smn dosage. Taken together, our work supports the view that there is an intrinsic defect in skeletal muscle cells of SMA patients and that this defect contributes to the overall pathogenesis in this devastating disease.

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

    SciTech Connect

    Ma, Zhiyuan; Sun, Xiaorui; Xu, Dequan; Xiong, Yuanzhu; Zuo, Bo

    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.

  1. Transformation by myc prevents fusion but not biochemical differentiation of C2C12 myoblasts: mechanisms of phenotypic correction in mixed culture with normal cells

    PubMed Central

    1994-01-01

    To study the effects of myc oncogene on muscle differentiation, we infected the murine skeletal muscle cell line C2C12 with retroviral vectors encoding various forms of avian c- or v-myc oncogene. myc expression induced cell transformation but, unlike many other oncogenes, prevented neither biochemical differentiation, nor commitment (irreversible withdrawal from the cell cycle). Yet, myotube formation by fusion of differentiated cells was strongly inhibited. Comparison of uninfected C2C12 myotubes with differentiated myc- expressing C2C12 did not reveal consistent differences in the expression of several muscle regulatory or structural genes. The present results lead us to conclude that transformation by myc is compatible with differentiation in C2C12 cells. myc expression induced cell death under growth restricting conditions. Differentiated cells escaped cell death despite continuing expression of myc, suggesting that the muscle differentiation programme interferes with the mechanism of myc-induced cell death. Cocultivation of v-myc-transformed C2C12 cells with normal fibroblasts or myoblasts restored fusion competence and revealed two distinguishable mechanisms that lead to correction of the fusion defect. PMID:8195295

  2. Thimerosal-Induced Apoptosis in Mouse C2C12 Myoblast Cells Occurs through Suppression of the PI3K/Akt/Survivin Pathway

    PubMed Central

    Li, Wen-Xue; Chen, Si-Fan; Chen, Li-Ping; Yang, Guang-Yu; Li, Jun-Tao; Liu, Hua-Zhang; Zhu, Wei

    2012-01-01

    Background Thimerosal, a mercury-containing preservative, is one of the most widely used preservatives and found in a variety of biological products. Concerns over its possible toxicity have reemerged recently due to its use in vaccines. Thimerosal has also been reported to be markedly cytotoxic to neural tissue. However, little is known regarding thimerosal-induced toxicity in muscle tissue. Therefore, we investigated the cytotoxic effect of thimerosal and its possible mechanisms on mouse C2C12 myoblast cells. Methodology/Principal Findings The study showed that C2C12 myoblast cells underwent inhibition of proliferation and apoptosis after exposure to thimerosal (125–500 nM) for 24, 48 and 72 h. Thimerosal caused S phase arrest and induced apoptosis as assessed by flow cytometric analysis, Hoechst staining and immunoblotting. The data revealed that thimerosal could trigger the leakage of cytochrome c from mitochondria, followed by cleavage of caspase-9 and caspase-3, and that an inhibitor of caspase could suppress thimerosal-induced apoptosis. Thimerosal inhibited the phosphorylation of Aktser473 and survivin expression. Wortmannin, a PI3K inhibitor, inhibited Akt activity and decreased survivin expression, resulting in increased thimerosal-induced apoptosis in C2C12 cells, while the activation of PI3K/Akt pathway by mIGF-I (50 ng/ml) increased the expression of survivin and attenuated apoptosis. Furthermore, the inhibition of survivin expression by siRNA enhanced thimerosal-induced cell apoptosis, while overexpression of survivin prevented thimerosal-induced apoptosis. Taken together, the data show that the PI3K/Akt/survivin pathway plays an important role in the thimerosal-induced apoptosis in C2C12 cells. Conclusions/Significance Our results suggest that in C2C12 myoblast cells, thimerosal induces S phase arrest and finally causes apoptosis via inhibition of PI3K/Akt/survivin signaling followed by activation of the mitochondrial apoptotic pathway. PMID

  3. Nrf2-Mediated HO-1 Induction Contributes to Antioxidant Capacity of a Schisandrae Fructus Ethanol Extract in C2C12 Myoblasts

    PubMed Central

    Kang, Ji Sook; Han, Min Ho; Kim, Gi-Young; Kim, Cheol Min; Kim, Byung Woo; Hwang, Hye Jin; Choi, Yung Hyun

    2014-01-01

    This study was designed to confirm the protective effect of Schisandrae Fructus, which are the dried fruits of Schisandra chinensis (Turcz.) Baill, against oxidative stress-induced cellular damage and to elucidate the underlying mechanisms in C2C12 myoblasts. Preincubating C2C12 cells with a Schisandrae Fructus ethanol extract (SFEE) significantly attenuated hydrogen peroxide (H2O2)-induced inhibition of growth and induced scavenging activity against intracellular reactive oxygen species (ROS) induced by H2O2. SFEE also inhibited comet tail formation and phospho-histone γH2A.X expression, suggesting that it prevents H2O2-induced cellular DNA damage. Furthermore, treating C2C12 cells with SFEE significantly induced heme oxygenase-1 (HO-1) and phosphorylation of nuclear factor-erythroid 2 related factor 2 (Nrf2). However, zinc protoporphyrin IX, a potent inhibitor of HO-1 activity, significantly reversed the protective effects of SFEE against H2O2-induced growth inhibition and ROS generation in C2C12 cells. Additional experiments revealed that the potential of the SFEE to induce HO-1 expression and protect against H2O2-mediated cellular damage was abrogated by transient transfection with Nrf2-specific small interfering RNA, suggesting that the SFEE protected C2C12 cells against oxidative stress-induced injury through the Nrf2/HO-1 pathway. PMID:25493944

  4. The histone demethylase KDM4B interacts with MyoD to regulate myogenic differentiation in C2C12 myoblast cells.

    PubMed

    Choi, Jang Hyun; Song, Young Joon; Lee, Hansol

    2015-01-24

    Enzymes that mediate posttranslational modifications of histone and nonhistone proteins have been implicated in regulation of skeletal muscle differentiation. However, functions of histone demethylases that could counter the actions of H3-K9 specific histone methyltransferases remain still obscure. Here we present evidences that KDM4B histone demethylase regulates expression of myogenic regulators such as MyoD and thereby controls myogenic differentiation of C2C12 myoblast cells. We demonstrate that expression of KDM4B gradually increases during myogenic differentiation and depletion of KDM4B using shRNA results in inhibition of differentiation in C2C12 myoblast cells, which is correlated with decreased expression of MyoD and myogenin. In addition, we find that KDM4B shRNA represses expression of MyoD promoter-driven luciferase reporter and exogenous expression of MyoD rescues myogenic potential in KDM4B-depleted myoblast cells. We further show that KDM4B interacts with MyoD, binds to MyoD and myogenin promoters in vivo, and finally, is involved in demethylation of tri-methylated H3-K9 on promoters of MyoD and myogenin. Taken together, our data suggest that KDM4B plays key roles in myogenic differentiation of C2C12 cells, presumably by its function as a H3-K9 specific histone demethylase.

  5. Creatine supplementation prevents the inhibition of myogenic differentiation in oxidatively injured C2C12 murine myoblasts.

    PubMed

    Sestili, Piero; Barbieri, Elena; Martinelli, Chiara; Battistelli, Michela; Guescini, Michele; Vallorani, Luciana; Casadei, Lucia; D'Emilio, Alessandra; Falcieri, Elisabetta; Piccoli, Giovanni; Agostini, Deborah; Annibalini, Giosuè; Paolillo, Marco; Gioacchini, Anna Maria; Stocchi, Vilberto

    2009-09-01

    Creatine (Cr), one of the most popular nutritional supplements among athletes, has been recently shown to prevent the cytotoxicity caused by different oxidative stressors in various mammalian cell lines, including C2C12 myoblasts, via a direct antioxidant activity. Here, the effect of Cr on the differentiating capacity of C2C12 cells exposed to H(2)O(2) has been investigated. Differentiation into myotubes was monitored using morphological, ultrastructural, and molecular techniques. Treatment with H(2)O(2) (1 h) not only caused a significant (30%) loss of cell viability, but also abrogated the myogenic ability of surviving C2C12. Cr-supplementation (24 h prior to H(2)O(2) treatment) was found to prevent these effects. Interestingly, H(2)O(2)-challenged cells preconditioned with the established antioxidants trolox or N-acetyl-cysteine, although cytoprotected, did not display the same differentiating ability characterizing oxidatively-injured, Cr-supplemented cells. Besides acting as an antioxidant, Cr increased the level of muscle regulatory factors and IGF1 (an effect partly refractory to oxidative stress), the cellular availability of phosphocreatine and seemed to exert some mitochondrially-targeted protective activity. It is concluded that Cr preserves the myogenic ability of oxidatively injured C2C12 via a pleiotropic mechanism involving not only its antioxidant capacity, but also the contribution to cell energy charge and effects at the transcriptional level which common bona fide antioxidants lack.

  6. Differentiation of C2C12 myoblasts expressing lamin A mutated at a site responsible for Emery-Dreifuss muscular dystrophy is improved by inhibition of the MEK-ERK pathway and stimulation of the PI3-kinase pathway

    SciTech Connect

    Favreau, Catherine; Delbarre, Erwan; Courvalin, Jean-Claude; Buendia, Brigitte

    2008-04-01

    Mutation R453W in A-type lamins, that are major nuclear envelope proteins, generates Emery-Dreifuss muscular dystrophy. We previously showed that mouse myoblasts expressing R453W-lamin A incompletely exit the cell cycle and differentiate into myocytes with a low level of multinucleation. Here we attempted to improve differentiation by treating these cells with a mixture of PD98059, an extracellular-regulated kinase (ERK) kinase (also known as mitogen-activated kinase, MEK) inhibitor, and insulin-like growth factor-II, an activator of phosphoinositide 3-kinase. We show that mouse myoblasts expressing R453W-lamin A were sensitive to the drug treatment as shown by (i) an increase in multinucleation, (ii) downregulation of proliferation markers (cyclin D1, hyperphosphorylated Rb), (iii) upregulation of myogenin, and (iv) sustained activation of p21 and cyclin D3. However, nuclear matrix anchorage of p21 and cyclin D3 in a complex with hypophosphorylated Rb that is critical to trigger cell cycle arrest and myogenin induction was deficient and incompletely restored by drug treatment. As the turn-over of R453W-lamin A at the nuclear envelope was greatly enhanced, we propose that R453W-lamin A impairs the capacity of the nuclear lamina to serve as scaffold for substrates of the MEK-ERK pathway and for MyoD-induced proteins that play a role in the differentiation process.

  7. Proteomic analysis of C2C12 myoblast and myotube exosome-like vesicles: a new paradigm for myoblast-myotube cross talk?

    PubMed

    Forterre, Alexis; Jalabert, Audrey; Berger, Emmanuelle; Baudet, Mathieu; Chikh, Karim; Errazuriz, Elisabeth; De Larichaudy, Joffrey; Chanon, Stéphanie; Weiss-Gayet, Michèle; Hesse, Anne-Marie; Record, Michel; Geloen, Alain; Lefai, Etienne; Vidal, Hubert; Couté, Yohann; Rome, Sophie

    2014-01-01

    Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. We found that these vesicles displayed the classical properties of exosomes isolated from other cell types containing components of the ESCRT machinery of the MVBs, as well as numerous tetraspanins. Specific muscle proteins were also identified confirming that ELV composition also reflects their muscle origin. Furthermore quantitative analysis revealed stage-preferred expression of 31 and 78 proteins in ELV-MB and ELV-MT respectively. We found that myotube-secreted ELVs, but not ELV-MB, reduced myoblast proliferation and induced differentiation, through, respectively, the down-regulation of Cyclin D1 and the up-regulation of myogenin. We also present evidence that proteins from ELV-MT can be incorporated into myoblasts by using the GFP protein as cargo within ELV-MT. Taken together, our data provide a useful database of proteins from C2C12-released ELVs throughout myogenesis and reveals the importance of exosome-like vesicles in skeletal muscle biology.

  8. An siRNA-based screen in C2C12 myoblasts identifies novel genes involved in myogenic differentiation.

    PubMed

    Alwan, Rayan; Bruel, Ange-Line; Da Silva, Anne; Blanquet, Véronique; Bouhouche, Khaled

    2017-10-01

    Myogenesis is a highly regulated multi-step process involving myoblast proliferation and differentiation. Although studies over the last decades have identified several factors governing these distinct major phases, many of them are not yet known. In order to identify novel genes, we took advantage of the C2C12 myoblastic line to establish a functional siRNA screen combined with quantitative-imaging analysis of a large amount of differentiated myoblasts. We knocked down 100 preselected mouse genes without a previously characterized role in muscle. Using image analysis, we tracked gene-silencing phenotypes by quantitative assessment of cellular density, myotube quantity, myotube morphology and fusion index. Our results have revealed six genes involved in both stages of C2C12 myogenesis and 13 genes specific to the differentiation stage. These findings prove that our RNAi-based screen could be an efficient tool to detect clear and subtle phenotypes allowing the identification of new myogenic regulators in mammals. Copyright © 2017. Published by Elsevier Inc.

  9. An exploration of the antioxidant effects of garlic saponins in mouse-derived C2C12 myoblasts.

    PubMed

    Kang, Ji Sook; Kim, Sung Ok; Kim, Gi-Young; Hwang, Hye Jin; Kim, Byung Woo; Chang, Young-Chae; Kim, Wun-Jae; Kim, Cheol Min; Yoo, Young Hyun; Choi, Yung Hyun

    2016-01-01

    In this study, we aimed to confirm the protective effects of garlic saponins against oxidative stress-induced cellular damage and to further elucidate the underlying mechanisms in mouse-derived C2C12 myoblasts. Relative cell viability was determined by 3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyltetrazolium bromide assay. Comet assay was used to measure DNA damage and oxidative stress was determined using 2',7'-dichlorofluorescein diacetate to measure intracellular reactive oxygen species (ROS) generation. Western blot analysis and small interfering RNA (siRNA)-based knockdown were used in order to investigate the possible molecular mechanisms. Our results revealed that garlic saponins prevented hydrogen peroxide (H2O2)-induced growth inhibition and exhibited scavenging activity against intracellular ROS. We also observed that garlic saponins prevented H2O2-induced comet tail formation and decreased the phosphorylation levels of γH2AX expression, suggesting that they can prevent H2O2-induced DNA damage. In addition, garlic saponins increased the levels of heme oxygenase-1 (HO-1), a potent antioxidant enzyme associated with the induction and phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the translocation of Nrf2 from the cytosol into the nucleus. However, the protective effects of garlic saponins on H2O2-induced ROS generation and growth inhibition were significantly reduced by zinc protoporphyrin Ⅸ, an HO-1 competitive inhibitor. In addition, the potential of garlic saponins to mediate HO-1 induction and protect against H2O2‑mediated growth inhibition was adversely affected by transient transfection with Nrf2-specific siRNA. Garlic saponins activated extracellular signal‑regulated kinase (ERK) signaling, whereas a specific ERK inhibitor was able to inhibit HO-1 upregulation, as well as Nrf2 induction and phosphorylation. Taken together, the findings of our study suggest that garlic saponins activate the Nrf2/HO-1 pathway by enabling

  10. Flavones Inhibit LPS-Induced Atrogin-1/MAFbx Expression in Mouse C2C12 Skeletal Myotubes.

    PubMed

    Shiota, Chieko; Abe, Tomoki; Kawai, Nobuhiko; Ohno, Ayako; Teshima-Kondo, Shigetada; Mori, Hiroyo; Terao, Junji; Tanaka, Eiji; Nikawa, Takeshi

    2015-01-01

    Muscle atrophy is a complex process that occurs as a consequence of various stress events. Muscle atrophy-associated genes (atrogenes) such as atrogin-1/MAFbx and MuRF-1 are induced early in the atrophy process, and the increase in their expression precedes the loss of muscle weight. Although antioxidative nutrients suppress atrogene expression in skeletal muscle cells, the inhibitory effects of flavonoids on inflammation-induced atrogin-1/MAFbx expression have not been clarified. Here, we investigated the inhibitory effects of flavonoids on lipopolysaccharide (LPS)-induced atrogin-1/MAFbx expression. We examined whether nine flavonoids belonging to six flavonoid categories inhibited atrogin-1/MAFbx expression in mouse C2C12 myotubes. Two major flavones, apigenin and luteolin, displayed potent inhibitory effects on atrogin-1/MAFbx expression. The pretreatment with apigenin and luteolin significantly prevented the decrease in C2C12 myotube diameter caused by LPS stimulation. Importantly, the pretreatment of LPS-stimulated myoblasts with these flavones significantly inhibited LPS-induced JNK phosphorylation in C2C12 myotubes, resulting in the significant suppression of atrogin-1/MAFbx promoter activity. These results suggest that apigenin and luteolin, prevent LPS-mediated atrogin-1/MAFbx expression through the inhibition of the JNK signaling pathway in C2C12 myotubes. Thus, these flavones, apigenin and luteolin, may be promising agents to prevent LPS-induced muscle atrophy.

  11. Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts.

    PubMed

    Sin, Jon; Andres, Allen M; Taylor, David J R; Weston, Thomas; Hiraumi, Yoshimi; Stotland, Aleksandr; Kim, Brandon J; Huang, Chengqun; Doran, Kelly S; Gottlieb, Roberta A

    2016-01-01

    Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNM1L/DRP1 (dynamin 1-like)-mediated fragmentation and subsequent removal of mitochondria via SQSTM1 (sequestosome 1)-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha)-mediated biogenesis. Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.

  12. Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts

    PubMed Central

    Sin, Jon; Andres, Allen M.; Taylor, David J. R.; Weston, Thomas; Hiraumi, Yoshimi; Stotland, Aleksandr; Kim, Brandon J.; Huang, Chengqun; Doran, Kelly S.; Gottlieb, Roberta A.

    2016-01-01

    ABSTRACT Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNM1L/DRP1 (dynamin 1-like)-mediated fragmentation and subsequent removal of mitochondria via SQSTM1 (sequestosome 1)-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha)-mediated biogenesis. Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation. PMID:26566717

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

    PubMed

    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 < P < 0.025, t-test) and direct cell counting (P < 0.0005, t-test) showed that static 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.

  14. G-CSF does not influence C2C12 myogenesis despite receptor expression in healthy and dystrophic skeletal muscle

    PubMed Central

    Wright, Craig R.; Brown, Erin L.; Della-Gatta, Paul A.; Ward, Alister C.; Lynch, Gordon S.; Russell, Aaron P.

    2014-01-01

    Granulocyte-colony stimulating factor (G-CSF) increases recovery of rodent skeletal muscles after injury, and increases muscle function in rodent models of neuromuscular disease. However, the mechanisms by which G-CSF mediates these effects are poorly understood. G-CSF acts by binding to the membrane spanning G-CSFR and activating multiple intracellular signaling pathways. Expression of the G-CSFR within the haematopoietic system is well known, but more recently it has been demonstrated to be expressed in other tissues. However, comprehensive characterization of G-CSFR expression in healthy and diseased skeletal muscle, imperative before implementing G-CSF as a therapeutic agent for skeletal muscle conditions, has been lacking. Here we show that the G-CSFR is expressed in proliferating C2C12 myoblasts, differentiated C2C12 myotubes, human primary skeletal muscle cell cultures and in mouse and human skeletal muscle. In mdx mice, a model of human Duchenne muscular dystrophy (DMD), G-CSF mRNA and protein was down-regulated in limb and diaphragm muscle, but circulating G-CSF ligand levels were elevated. G-CSFR mRNA in the muscles of mdx mice was up-regulated however steady-state levels of the protein were down-regulated. We show that G-CSF does not influence C2C12 myoblast proliferation, differentiation or phosphorylation of Akt, STAT3, and Erk1/2. Media change alone was sufficient to elicit increases in Akt, STAT3, and Erk1/2 phosphorylation in C2C12 muscle cells and suggest previous observations showing a G-CSF increase in phosphoprotein signaling be viewed with caution. These results suggest that the actions of G-CSF may require the interaction with other cytokines and growth factors in vivo, however these data provides preliminary evidence supporting the investigation of G-CSF for the management of muscular dystrophy. PMID:24822049

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

    PubMed Central

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

    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. PMID:27257813

  16. Expression of Glucose Transporter 4 (GLUT4) is Increased by Cinnamaldehyde in C2C12 Mouse Muscle Cells

    PubMed Central

    Nikzamir, Abdolrahim; Palangi, Alireza; Kheirollaha, Alireza; Tabar, Hashemi; Malakaskar, Alimohamad; Shahbazian, Hajieh; Fathi, Mohammad

    2014-01-01

    Background: In diabetes mellitus because of the absence or insufficient sensitivity to insulin, glucose transporter protein in cell membrane, glucose transporter 4, is decreased. GLUT4 is the major glucose transporter in skeletal muscle and adipose tissue, which is under control of insulin. It remains, however, unclear whether cinnamaldehyde plays a regulatory role(s) or not. Objectives: The objective of this study was to investigate the effects of cinnamaldehyde on GLUT4 gene expression. Materials and Methods: This study was an experimental trial. Tests were performed in triplicates. This study examined effects of cinnamaldehyde on Glut4 gene expression in C2C12 skeletal muscle cells by using Real Time PCR. C2C12 myoblasts were cultured in DMEM + 10 % FBS. After differentiation of myoblasts to myotubes, the cells were serum deprived for 5 hours and then treated with 10, 20, or 50 µM of cinnamaldehyde for 1 hour. Results: Our data revealed a significant increase in the expression of Glut4 in cinnamaldehyde treated cells. In addition, GLUT4 mRNA level was increased in a dose dependent manner. Analyses were performed using the SPSS 16 for Windows software. Differences between the groups were determined by one-way ANOVA. Conclusions: These results demonstrate that cinnamaldehyde up regulates the expression of mouse skeletal muscle GLUT4 gene expression. PMID:24719730

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

    SciTech Connect

    Kodaka, Manami; Yang, Zeyu; Nakagawa, Kentaro; Maruyama, Junichi; Xu, Xiaoyin; Sarkar, Aradhan; Ichimura, Ayana; Nasu, Yusuke; Ozawa, Takeaki; Iwasa, Hiroaki; Ishigami-Yuasa, Mari; Ito, Shigeru; Kagechika, Hiroyuki; 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.

  18. Sodium arsenite delays the differentiation of C2C12 mouse myoblast cells and alters methylation patterns on the transcription factor myogenin

    SciTech Connect

    Steffens, Amanda A.; Hong Giaming; Bain, Lisa J.

    2011-01-15

    Epidemiological studies have correlated arsenic exposure with cancer, skin diseases, and adverse developmental outcomes such as spontaneous abortions, neonatal mortality, low birth weight, and delays in the use of musculature. The current study used C2C12 mouse myoblast cells to examine whether low concentrations of arsenic could alter their differentiation into myotubes, indicating that arsenic can act as a developmental toxicant. Myoblast cells were exposed to 20 nM sodium arsenite, allowed to differentiate into myotubes, and expression of the muscle-specific transcription factor myogenin, along with the expression of tropomyosin, suppressor of cytokine signaling 3 (Socs3), prostaglandin I2 synthesis (Ptgis), and myocyte enhancer 2 (Mef2), was investigated using QPCR and immunofluorescence. Exposing C2C12 cells to 20 nM sodium arsenite delayed the differentiation process, as evidenced by a significant reduction in the number of multinucleated myotubes, a decrease in myogenin mRNA expression, and a decrease in the total number of nuclei expressing myogenin protein. The expression of mRNA involved in myotube formation, such as Ptgis and Mef2 mRNA, was also significantly reduced by 1.6-fold and 4-fold during differentiation. This was confirmed by immunofluorescence for Mef2, which showed a 2.6-fold reduction in nuclear translocation. Changes in methylation patterns in the promoter region of myogenin (-473 to + 90) were examined by methylation-specific PCR and bisulfite genomic sequencing. Hypermethylated CpGs were found at -236 and -126 bp, whereas hypomethylated CpGs were found at -207 bp in arsenic-exposed cells. This study indicates that 20 nM sodium arsenite can alter myoblast differentiation by reducing the expression of the transcription factors myogenin and Mef2c, which is likely due to changes in promoter methylation patterns. The delay in muscle differentiation may lead to developmental abnormalities.

  19. Degree of Suppression of Mouse Myoblast Cell Line C2C12 Differentiation Varies According to Chondroitin Sulfate Subtype

    PubMed Central

    Warita, Katsuhiko; Oshima, Nana; Takeda-Okuda, Naoko; Tamura, Jun-ichi; Hosaka, Yoshinao Z.

    2016-01-01

    Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a factor involved in the suppression of myogenic differentiation. CS comprises two repeating sugars and has different subtypes depending on the position and number of bonded sulfate groups. However, the effect of each subtype on myogenic differentiation remains unclear. In this study, we spiked cultures of C2C12 myoblasts, cells which are capable of undergoing skeletal muscle differentiation, with one of five types of CS (CS-A, -B, -C, -D, or -E) and induced differentiation over a fixed time. After immunostaining of the formed myotubes with an anti-MHC antibody, we counted the number of nuclei in the myotubes and then calculated the fusion index (FI) as a measure of myotube differentiation. The FI values of all the CS-treated groups were lower than the FI value of the control group, especially the group treated with CS-E, which displayed notable suppression of myotube formation. To confirm that the sugar chain in CS-E is important in the suppression of differentiation, chondroitinase ABC (ChABC), which catabolizes CS, was added to the media. The addition of ChABC led to the degradation of CS-E, and neutralized the suppression of myotube formation by CS-E. Collectively, it can be concluded that the degree of suppression of differentiation depends on the subtype of CS and that CS-E strongly suppresses myogenic differentiation. We conclude that the CS sugar chain has inhibitory action against myoblast cell fusion. PMID:27775651

  20. Effects of myogenin on muscle fiber types and key metabolic enzymes in gene transfer mice and C2C12 myoblasts.

    PubMed

    Zhu, Lin-Na; Ren, Yang; Chen, Jing-Qing; Wang, Yi-Zhen

    2013-12-15

    Skeletal muscle fiber type composition is one of the important factors influencing muscle growth and meat quality. As a member of the myogenic transcription factors, myogenin (MyoG) is required for embryonic myoblast differentiation, but the expression of MyoG continues in mature muscle tissue of adult animals, especially in oxidative metabolic muscle, which suggests that MyoG may play a more extended role. Therefore, using MyoG gene transfer mice and C2C12 myoblasts as in vivo and in vitro models, respectively, we elected to study the role of MyoG in muscle fiber types and oxidative metabolism by using overexpression and siRNA suppression strategies. The overexpression of MyoG by DNA electroporation in mouse gastrocnemius muscle had no significant effect on fiber type composition but upregulated the mRNA expression (P<0.01) and enzyme activity (P<0.05) of oxidative succinic dehydrogenase (SDH). In addition, downregulation of the activity of the glycolytic enzymes lactate dehydrogenase (LDH, P<0.05) and pyruvate kinase (PK, P<0.05) was observed in MyoG gene transfer mice. In vitro experiments verified the results obtained in mice. Stable MyoG-transfected differentiating C2C12 cells showed higher mRNA expression levels of myosin heavy chain (MyHC) isoform IIX (P<0.01) and SDH (P<0.05), while the LDH mRNA was attenuated. The enzyme activities of SDH (P<0.01) and LDH (P<0.05) were similarly altered at the mRNA level. When MyoG was knocked down in C2C12 cells, MyHC IIX expression (P<0.05) was decreased, but the mRNA level (P<0.05) and the enzyme activity (P<0.05) of SDH were increased. Downregulating MyoG also increased the activity of the glycolytic enzymes PK (P<0.05) and hexokinase (HK, P<0.05). Based on those results, we concluded that MyoG barely changes the MyHC isoforms, except MyHC IIX, in differentiating myoblasts but probably influences the shift from glycolytic metabolism towards oxidative metabolism both in vivo and in vitro. These results contribute to

  1. Protein O-fucosyltransferase 1 expression impacts myogenic C2C12 cell commitment via the Notch signaling pathway.

    PubMed

    Der Vartanian, Audrey; Audfray, Aymeric; Al Jaam, Bilal; Janot, Mathilde; Legardinier, Sébastien; Maftah, Abderrahman; Germot, Agnès

    2015-01-01

    The Notch signaling pathway plays a crucial role in skeletal muscle regeneration in mammals by controlling the transition of satellite cells from quiescence to an activated state, their proliferation, and their commitment toward myotubes or self-renewal. O-fucosylation on Notch receptor epidermal growth factor (EGF)-like repeats is catalyzed by the protein O-fucosyltransferase 1 (Pofut1) and primarily controls Notch interaction with its ligands. To approach the role of O-fucosylation in myogenesis, we analyzed a murine myoblastic C2C12 cell line downregulated for Pofut1 expression by short hairpin RNA (shRNA) inhibition during the time course of differentiation. Knockdown of Pofut1 affected the signaling pathway activation by a reduction of the amount of cleaved Notch intracellular domain and a decrease in downstream Notch target gene expression. Depletion in Pax7(+)/MyoD(-) cells and earlier myogenic program entrance were observed, leading to an increase in myotube quantity with a small number of nuclei, reflecting fusion defects. The rescue of Pofut1 expression in knockdown cells restored Notch signaling activation and a normal course in C2C12 differentiation. Our results establish the critical role of Pofut1 on Notch pathway activation during myogenic differentiation.

  2. Effects of 1,25(OH)2D3 and 25(OH)D3 on C2C12 Myoblast Proliferation, Differentiation, and Myotube Hypertrophy

    PubMed Central

    van der Meijden, K.; Bravenboer, N.; Dirks, N.F.; Heijboer, A.C.; den Heijer, M.; de Wit, G.M.J.; Offringa, C.; Lips, P.

    2016-01-01

    An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)2D by 1α‐hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)2D3 and 25(OH)D3 affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D3 and 1,25(OH)2D3. We show that myoblasts not only responded to 1,25(OH)2D3, but also to the precursor 25(OH)D3 by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)2D3 as well as 25(OH)D3 stimulated VDR mRNA expression and in myotubes 1,25(OH)2D3 also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D3 metabolism. Although 1α‐hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)2D3 or 25(OH)D3 myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D3 to 24R,25(OH)2D3 which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D3 metabolites, but is also able to metabolize 25(OH)D3 and 1,25(OH)2D3. J. Cell. Physiol. 231: 2517–2528, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:27018098

  3. Effects of 1,25(OH)2 D3 and 25(OH)D3 on C2C12 Myoblast Proliferation, Differentiation, and Myotube Hypertrophy.

    PubMed

    van der Meijden, K; Bravenboer, N; Dirks, N F; Heijboer, A C; den Heijer, M; de Wit, G M J; Offringa, C; Lips, P; Jaspers, R T

    2016-11-01

    An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)2 D by 1α-hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)2 D3 and 25(OH)D3 affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D3 and 1,25(OH)2 D3 . We show that myoblasts not only responded to 1,25(OH)2 D3 , but also to the precursor 25(OH)D3 by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)2 D3 as well as 25(OH)D3 stimulated VDR mRNA expression and in myotubes 1,25(OH)2 D3 also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D3 metabolism. Although 1α-hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)2 D3 or 25(OH)D3 myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D3 to 24R,25(OH)2 D3 which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D3 metabolites, but is also able to metabolize 25(OH)D3 and 1,25(OH)2 D3 . J. Cell. Physiol. 231: 2517-2528, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

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

    PubMed

    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.

  5. Response of C2C12 myoblasts to hypoxia: the relative roles of glucose and oxygen in adaptive cellular metabolism.

    PubMed

    Li, Wei; Hu, Zhen-Fu; Chen, Bin; Ni, Guo-Xin

    2013-01-01

    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. 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. 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. 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.

  6. FoxO1 regulates muscle fiber-type specification and inhibits calcineurin signaling during C2C12 myoblast differentiation.

    PubMed

    Yuan, Yuan; Shi, Xin-e; Liu, Yue-guang; Yang, Gong-she

    2011-02-01

    Adult skeletal muscle fibers can be categorized into slow-oxidative and fast-glycolytic subtypes based on specialized metabolic and contractile properties. The Forkhead box O1 (FoxO1) transcription factor governs muscle growth, metabolism, and cell differentiation, and has been shown to be involved in regulating muscle fiber type specification. However, to date, the mechanism behind FoxO1-mediated fiber type diversity is still unclear. In this article, FoxO1 being expressed preferentially in fast twitch fiber enriched muscles is reported. Moreover, the autors also detected that FoxO1 expression decreased in both fast and slow muscles from mice undergoing endurance exercise which induced a fast-to-slow fiber type transition. Using C2C12 myoblast, constitutively active FoxO1 mutant altered the proportion of muscle fiber type composition toward a fast-glycolytic phenotype and attenuated calcineurin phosphatase activity. In addition, a transcriptionally inactive FoxO1 by resveratrol triggered the expression of genes related to slow-oxidative muscle but not sufficient to induce a complete slow fiber transformation. Taken together, these results suggest that FoxO1 up-regulates fast fiber-type formation and down-regulates muscle oxidative capacity at least in part through inhibition of the calcineurin pathway.

  7. Role of androgen receptor on cyclic mechanical stretch-regulated proliferation of C2C12 myoblasts and its upstream signals: IGF-1-mediated PI3K/Akt and MAPKs pathways.

    PubMed

    Ma, Yiming; Fu, Shaoting; Lu, Lin; Wang, Xiaohui

    2017-07-15

    To detect the effects of androgen receptor (AR) on cyclic mechanical stretch-modulated proliferation of C2C12 myoblasts and its pathways: roles of IGF-1, PI3K and MAPK. C2C12 were randomly divided into five groups: un-stretched control, six or 8 h of fifteen percent stretch, and six or 8 h of twenty percent stretch. Cyclic mechanical stretch of C2C12 were completed using a computer-controlled FlexCell Strain Unit. Cell proliferation and IGF-1 concentration in medium were detected by CCK8 and ELISA, respectively. Expressions of AR and IGF-1R, and expressions and activities of PI3K, p38 and ERK1/2 in stretched C2C12 cells were determined by Western blot. ①The proliferation of C2C12 cells, IGF-1 concentration in medium, expressions of AR and IGF-1R, and activities of PI3K, p38 and ERK1/2 were increased by 6 h of fifteen percent stretch, while decreased by twenty percent stretch for six or 8 h ②The fifteen percent stretch-increased proliferation of C2C12 cells was reversed by AR inhibitor, Flutamide. ③The increases of AR expression, activities of PI3K, p38 and ERK1/2 resulted from fifteen percent stretch were attenuated by IGF-1 neutralizing antibody, while twenty percent stretch-induced decreases of the above indicators were enhanced by recombinant IGF-1. ④Specific inhibitors of p38, ERK1/2 and PI3K all decreased the expression of AR in fifteen percent and twenty percent of stretched C2C12 cells. Cyclic mechanical stretch modulated the proliferation of C2C12 cells, which may be attributed to the alterations of AR via IGF-1-PI3K/Akt and IGF-1-MAPK (p38, ERK1/2) pathways in C2C12 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Influence of the extracellular matrix and integrins on volume-sensitive osmolyte anion channels in C2C12 myoblasts

    PubMed Central

    Neveux, Iva; Doe, Jinger; Leblanc, Normand

    2010-01-01

    The purpose of this study was to determine whether extracellular matrix (ECM) composition through integrin receptors modulated the volume-sensitive osmolyte anion channels (VSOACs) in skeletal muscle-derived C2C12 cells. Cl− currents were recorded in whole cell voltage-clamped cells grown on laminin (LM), fibronectin (FN), or in the absence of a defined ECM (NM). Basal membrane currents recorded in isotonic media (300 mosmol/kg) were larger in cells grown on FN (3.8-fold at +100 mV) or LM (8.8-fold at +100 mV) when compared with NM. VSOAC currents activated by cell exposure to hypotonic solution were larger in cells grown on LM (1.72-fold at +100 mV) or FN (1.75-fold at +100 mV) compared with NM. Additionally, the kinetics of VSOAC activation was ≈27% quicker on FN and LM. These currents were tamoxifen sensitive, displayed outward rectification, reversed at the equilibrium potential of Cl− and inactivated at potentials >+60 mV. Specific knockdown of β1-integrin by short hairpin RNA interference strongly inhibited the VSOAC Cl− currents in cells plated on FN. In conclusion, ECM composition and integrins profoundly influence the biophysical properties and mechanisms of onset of VSOACs. PMID:20164377

  9. Cartap-induced cytotoxicity in mouse C2C12 myoblast cell line and the roles of calcium ion and oxidative stress on the toxic effects.

    PubMed

    Liao, Jiunn-Wang; Kang, Jaw-Jou; Jeng, Chian-Ren; Chang, Shao-Kuang; Kuo, Ming-Jang; Wang, Shun-Cheng; Liu, Michael R S; Pang, Victor Fei

    2006-02-15

    Our previous study has demonstrated that instead of neuromuscular blockage cartap, an organonitrogen insecticide, could cause a marked irreversible Ca2+-dependent contracture in both isolated mouse and rabbit phrenic nerve-diaphragms. We further examined the potential of direct myocytotoxicity of cartap and the possible roles of calcium ion and oxidative stress on cartap-induced muscle cell injury using the mouse myoblast cell line, C2C12. Cartap exerted a dose- and time-dependent cytotoxic effect in C2C12 cells measured by MTT colorimetric assay and trypan blue dye exclusion. The extracellular activities of both creatine kinase (CK) and lactate dehydrogenase (LDH) were elevated in the cartap-treated groups at or greater than 100 microM. The isoenzymatic profiles showed that the elevations were mainly due to CK-3, LDH-3, and LDH-4. Following the addition of 0.5-2.5mM EGTA, a Ca2+ chelator, or 30-100 microM verapamil, an L-type Ca2+ channel blocker, the cartap-induced reduction in MTT metabolic rate of C2C12 cells was significantly restored in a dose-dependent manner in both EGTA and verapamil-treated cells. Furthermore, EGTA could significantly reduce the cartap-induced elevation in the levels of total extracellular CK and LDH activities. Additionally, cartap significantly increased the level of endogenous reactive oxygen species (ROS) in C2C12 cells in a dose- and time-dependent manner. The cartap-induced ROS generation could be significantly inhibited by antioxidants, including Vitamins C and E, catalase, and superoxide dismutase, with catalase the most effective. EGTA could significantly inhibit cartap-induced ROS generation in a dose-dependent manner. The results suggested that cartap could induce ROS generation in C2C12 cells via a Ca2+-dependent mechanism resulting in subsequent cytotoxicity, at least partially, to C2C12 cells. It is speculated that both Ca2+ and Ca2+-induced ROS may also play the central role on the myogenic contracture and myofiber injury

  10. The combination of ursolic acid and leucine potentiates the differentiation of C2C12 murine myoblasts through the mTOR signaling pathway.

    PubMed

    Kim, Minjung; Sung, Bokyung; Kang, Yong Jung; Kim, Dong Hwan; Lee, Yujin; Hwang, Seong Yeon; Yoon, Jeong-Hyun; Yoo, Mi-Ae; Kim, Cheol Min; Chung, Hae Young; Kim, Nam Deuk

    2015-03-01

    Aging causes phenotypic changes in skeletal muscle progenitor cells that lead to the progressive loss of myogenic differentiation and thus a decrease in muscle mass. The naturally occurring triterpene, ursolic acid, has been reported to be an effective agent for the prevention of muscle loss by suppressing degenerative muscular dystrophy. Leucine, a branched-chain amino acid, and its metabolite, β-hydroxy-β-methylbutyric acid, have been reported to enhance protein synthesis in skeletal muscle. Therefore, the aim of the present study was to investigate whether the combination of ursolic acid and leucine promotes greater myogenic differentiation compared to either agent alone in C2C12 murine myoblasts. Morphological changes were observed and creatine kinase (CK) activity analysis was performed to determine the conditions through which the combination of ursolic acid and leucine would exert the most prominent effects on muscle cell differentiation. The effect of the combination of ursolic acid and leucine on the expression of myogenic differentiation marker genes was examined by RT-PCR and western blot analysis. The combination of ursolic acid (0.5 µM) and leucine (10 µM) proved to be the most effective in promoting myogenic differentiation. The combination of ursolic acid and leucine significantly increased CK activity than treatment with either agent alone. The level of myosin heavy chain, a myogenic differentiation marker protein, was also enhanced by the combination of ursolic acid and leucine. The combination of ursolic acid and leucine significantly induced the expression of myogenic differentiation marker genes, such as myogenic differentiation 1 (MyoD) and myogenin, at both the mRNA and protein level. In addition, the number of myotubes and the fusion index were increased. These findings indicate that the combination of ursolic acid and leucine promotes muscle cell differentiation, thus suggesting that this combination of agents may prove to be beneficial

  11. Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage [published erratum appears in J Cell Biol 1995 Feb;128(4):following 713

    PubMed Central

    1994-01-01

    The implantation of bone morphogenetic protein (BMP) into muscular tissues induces ectopic bone formation at the site of implantation. To investigate the mechanism underlying this process, we examined whether recombinant bone morphogenetic protein-2 (BMP-2) converts the differentiation pathway of the clonal myoblastic cell line, C2C12, into that of osteoblast lineage. Incubating the cells with 300 ng/ml of BMP- 2 for 6 d almost completely inhibited the formation of the multinucleated myotubes expressing troponin T and myosin heavy chain, and induced the appearance of numerous alkaline phosphatase (ALP)- positive cells. BMP-2 dose dependently induced ALP activity, parathyroid hormone (PTH)-dependent 3',5'-cAMP production, and osteocalcin production at concentrations above 100 ng/ml. The concentration of BMP-2 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubating primary muscle cells with 300 ng/ml of BMP-2 for 6 d also inhibited myotube formation, whereas induced ALP activity and osteocalcin production. Incubation with 300 ng/ml of BMP-2 suppressed the expression of mRNA for muscle creatine kinase within 6 h, whereas it induced mRNA expression for ALP, PTH/PTH-related protein (PTHrP) receptors, and osteocalcin within 24-48 h. BMP-2 completely inhibited the expression of myogenin mRNA by day 3. By day 3, BMP-2 also inhibited the expression of MyoD mRNA, but it was transiently stimulated 12 h after exposure to BMP-2. Expression of Id-1 mRNA was greatly stimulated by BMP-2. When C2C12 cells pretreated with BMP-2 for 6 d were transferred to a colony assay system in the absence of BMP-2, more than 84% of the colonies generated became troponin T-positive and ALP activity disappeared. TGF-beta 1 also inhibited myotube formation in C2C12 cells, and suppressed the expression of myogenin and MyoD mRNAs without inducing that of Id-1 mRNA. However, no osteoblastic phenotype was induced by TGF

  12. Palmitate increases musclin gene expression through activation of PERK signaling pathway in C2C12 myotubes.

    PubMed

    Gu, Ning; Guo, Qian; Mao, Ke; Hu, Hailong; Jin, Sanli; Zhou, Ying; He, Hongjuan; Oh, Yuri; Liu, Chuanpeng; Wu, Qiong

    2015-11-20

    Musclin is a type of muscle-secreted cytokine and its increased gene expression induces insulin resistance in type 2 diabetes. However, the mechanism underlying increased musclin gene expression is currently unclear. Excessive saturated fatty acids (SFA) can activate the secretion of several muscle-secreted cytokines as well as endoplasmic reticulum (ER) stress pathway, thereby contributing to the development of type 2 diabetes. The purpose of this study was to investigate the mechanisms responsible for the effect of palmitate, the most abundant SFA in the plasma, on the gene expression of musclin in C2C12 myotubes. Treatment of C2C12 myotubes with palmitate or tunicamycin significantly increased the expression of musclin as well as ER stress-related genes, but treatment with oleate did not. Pre-treatment of C2C12 myotubes with 4-phenyl butyrate suppressed the expression of ER stress-related genes, simultaneously, resulting in decreased expression of the musclin gene induced by palmitate or tunicamycin. These results indicate that ER stress is related to palmitate-induced musclin gene expression. Moreover, palmitate-induced musclin gene expression was significantly inhibited in C2C12 myotubes when PERK pathway signaling was suppressed by knockdown of the PERK gene or treatment with GSK2656157, a PERK autophosphorylation inhibitor. However, there was no difference in the palmitate-induced musclin gene expression when IRE1 and ATF6 signaling pathways were suppressed by knockdown of the IRE1 and ATF6 genes. These findings suggest that palmitate increases musclin gene expression via the activation of the PERK signaling pathway in C2C12 myotubes.

  13. Transcription factor TEAD4 regulates expression of myogenin and the unfolded protein response genes during C2C12 cell differentiation.

    PubMed

    Benhaddou, A; Keime, C; Ye, T; Morlon, A; Michel, I; Jost, B; Mengus, G; Davidson, I

    2012-02-01

    The TEAD (1-4) transcription factors comprise the conserved TEA/ATTS DNA-binding domain recognising the MCAT element in the promoters of muscle-specific genes. Despite extensive genetic analysis, the function of TEAD factors in muscle differentiation has proved elusive due to redundancy among the family members. Expression of the TEA/ATTS DNA-binding domain that acts as a dominant negative repressor of TEAD factors in C2C12 myoblasts inhibits their differentiation, whereas selective shRNA knockdown of TEAD4 results in abnormal differentiation characterised by the formation of shortened myotubes. Chromatin immunoprecipitation coupled to array hybridisation shows that TEAD4 occupies 867 promoters including those of myogenic miRNAs. We show that TEAD factors directly induce Myogenin, CDKN1A and Caveolin 3 expression to promote myoblast differentiation. RNA-seq identifies a set of genes whose expression is strongly reduced upon TEAD4 knockdown among which are structural and regulatory proteins and those required for the unfolded protein response. In contrast, TEAD4 represses expression of the growth factor CTGF (connective tissue growth factor) to promote differentiation. Together these results show that TEAD factor activity is essential for normal C2C12 cell differentiation and suggest a role for TEAD4 in regulating expression of the unfolded protein response genes.

  14. Transcription factor TEAD4 regulates expression of Myogenin and the unfolded protein response genes during C2C12 cell differentiation

    PubMed Central

    Benhaddou, A; Keime, C; Ye, T; Morlon, A; Michel, I; Jost, B; Mengus, G; Davidson, I

    2012-01-01

    The TEAD (1–4) transcription factors comprise the conserved TEA/ATTS DNA-binding domain recognising the MCAT element in the promoters of muscle-specific genes. Despite extensive genetic analysis, the function of TEAD factors in muscle differentiation has proved elusive due to redundancy among the family members. Expression of the TEA/ATTS DNA-binding domain that acts as a dominant negative repressor of TEAD factors in C2C12 myoblasts inhibits their differentiation, whereas selective shRNA knockdown of TEAD4 results in abnormal differentiation characterised by the formation of shortened myotubes. Chromatin immunoprecipitation coupled to array hybridisation shows that TEAD4 occupies 867 promoters including those of myogenic miRNAs. We show that TEAD factors directly induce Myogenin, CDKN1A and Caveolin 3 expression to promote myoblast differentiation. RNA-seq identifies a set of genes whose expression is strongly reduced upon TEAD4 knockdown among which are structural and regulatory proteins and those required for the unfolded protein response. In contrast, TEAD4 represses expression of the growth factor CTGF (connective tissue growth factor) to promote differentiation. Together these results show that TEAD factor activity is essential for normal C2C12 cell differentiation and suggest a role for TEAD4 in regulating expression of the unfolded protein response genes. PMID:21701496

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

    SciTech Connect

    Liu, Xin-Hua; Bauman, William A.; Cardozo, Christopher

    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

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

    PubMed

    Liu, Xin-Hua; Bauman, William A; Cardozo, Christopher

    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.

  17. Role of dynamin-related protein 1-mediated mitochondrial fission in resistance of mouse C2C12 myoblasts to heat injury.

    PubMed

    Yu, Tianzheng; Deuster, Patricia; Chen, Yifan

    2016-12-15

    Understanding how skeletal muscles respond to high temperatures may help develop strategies for improving exercise tolerance and preventing heat injury. Mitochondria regulate cell survival by constantly changing their morphology through fusion and fission in response to environmental stimuli. Little is known about the involvement of mitochondrial dynamics in tolerance of skeletal muscle against heat stress. Mild heat acclimation and moderate heat shock appear to have different effects on the mitochondrial morphology and fission protein Drp1 in skeletal muscle cells. Mitochondrial integrity plays a key role in cell survival under heat stress. The regulation of mitochondrial morphology is closely coupled to cell survival during stress. We examined changes in the mitochondrial morphology of mouse C2C12 skeletal muscle cells in response to heat acclimation and heat shock exposure. Acclimated cells showed a greater survival rate during heat shock exposure than non-acclimated cells, and were characterized by long interconnected mitochondria and reduced expression of dynamin-related protein 1 (Drp1) for their mitochondrial fractions. Exposure of C2C12 muscle cells to heat shock led to apoptotic death featuring activation of caspase 3/7, release of cytochrome c and loss of cell membrane integrity. Heat shock also caused excessive mitochondrial fragmentation, loss of mitochondrial membrane potential and production of reactive oxygen species in C2C12 cells. Western blot and immunofluorescence image analysis revealed translocation of Drp1 to mitochondria from the cytosol in C2C12 cells exposed to heat shock. Mitochondrial division inhibitor 1 or Drp1 gene silencer reduced mitochondrial fragmentation and increased cell viability during exposure to heat shock. These results suggest that Drp1-dependent mitochondrial fission may regulate susceptibility to heat-induced apoptosis in muscle cells and that Drp1 may serve as a target for the prevention of heat-related injury

  18. Cytotoxic action in myoblasts and myotubes (C2C12) and enzymatic characterization of a new phospholipase A2 isoform (Bj-V) from Bothrops jararacussu venom.

    PubMed

    Bonfim, Vera Luis; Ponce-Soto, Luis Alberto; Novello, Jose Camilo; Marangoni, Sergio

    2006-01-01

    A new PLA2 Bj-V from Bothrops jararacussu (14039.49 Da determined by MALDI-TOF mass spectrometry) was isolated in only one chromatographic step by HPLC ion-exchange and its purity was confirmed by reverse phase. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWQFGQMIL KETGKIPFPY YGAYGCYCGW GGRGGKPKDG TDRCCYVHD...) showed a high degree of homology with basic D49 PLA2 myotoxins from other Bothrops venoms. Bj V showed discrete sigmoidal enzymatic behavior, with maximal activity at pH 8.4 and 35-40 degrees C. Full PLA2 activity required Ca2+ (10 mM) and there was little catalytic activity in the presence of 1 mM Ca2+. The addition of Mn2+ or Mg2+ (10 mM) in the presence of low (1 mM) Ca2+ slightly increased the enzyme activity, whereas Zn2+ and Cu2+ (10 mM) diminished the activity. The substitution of Ca2+ for Mg2+ or Cu2+ also reduced the enzymatic activity. Bj V had PLA2 activity and produced cytotoxicity in murine C2C12 skeletal muscle myoblasts and myotubes. The isolation of these isoforms Bj-IV [1] and Bj-V (described herein) found in a fraction previously described as homogeneous shows us the importance of optimization in purification techniques in order to better understand their biological behavior.

  19. O-GlcNAc protein modification in C2C12 myoblasts exposed to oxidative stress indicates parallels with endogenous antioxidant defense.

    PubMed

    Peternelj, Tina Tinkara; Marsh, Susan A; Morais, Christudas; Small, David M; Dalbo, Vincent J; Tucker, Patrick S; Coombes, Jeff S

    2015-02-01

    A growing body of evidence demonstrates the involvement of protein modification with O-linked β-N-acetylglucosamine (O-GlcNAc) in the stress response and its beneficial effects on cell survival. Here we investigated protein O-GlcNAcylation in skeletal muscle cells exposed to oxidative stress and the crosstalk with endogenous antioxidant system. The study focused on antioxidant enzymes superoxide dismutase 2 (SOD2), catalase (CAT), and glutathione peroxidase 1 (GPX1), and transcriptional regulators proliferator-activated receptor gamma coactivator 1-α (PGC-1α) and forkhead box protein O1 (FOXO1), which play important roles in oxidative stress response and are known to be O-GlcNAc-modified. C2C12 myoblasts were subjected to 24 h incubation with different reagents, including hydrogen peroxide, diethyl maleate, high glucose, and glucosamine, and the inhibitors of O-GlcNAc cycling enzymes. Surprisingly, O-GlcNAc levels were significantly increased only with glucosamine, whilst other treatments showed no effect. Significant changes at the mRNA level were observed with concomitant upregulation of the genes for O-GlcNAc enzymes and stress-related proteins with oxidizing agents and downregulation of these genes with agents promoting O-GlcNAcylation. Our findings suggest a role of O-GlcNAc in the stress response and indicate an inhibitory mechanism controlling O-GlcNAc levels in the muscle cells. This could represent an important homeostatic regulation of the cellular defense system.

  20. IGF-1 Attenuates Hypoxia-Induced Atrophy but Inhibits Myoglobin Expression in C2C12 Skeletal Muscle Myotubes.

    PubMed

    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

    2017-09-01

    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 strategy to prevent atrophy under hypoxia and enhance an eventual hypertrophic response to anabolic stimulation. Mb expression may be further enhanced by lipid supplementation. We investigated individual and combined effects of hypoxia, insulin-like growth factor (IGF)-1 and lipids, in mouse skeletal muscle C2C12 myotubes. Differentiated C2C12 myotubes were cultured for 24 h under 20%, 5% and 2% oxygen with or without IGF-1 and/or lipid treatment. In culture under 20% oxygen, IGF-1 induced 51% hypertrophy. Hypertrophy was only 32% under 5% and abrogated under 2% oxygen. This was not explained by changes in expression of genes involved in contractile protein synthesis or degradation, suggesting a reduced rate of translation rather than of transcription. Myoglobin mRNA expression increased by 75% under 5% O₂ but decreased by 50% upon IGF-1 treatment under 20% O₂, compared to control. Inhibition of mammalian target of rapamycin (mTOR) activation using rapamycin restored Mb mRNA expression to control levels. Lipid supplementation had no effect on Mb gene expression. Thus, IGF-1-induced anabolic signaling can be a strategy to improve muscle size under mild hypoxia, but lowers Mb gene expression.

  1. IGF-1 Attenuates Hypoxia-Induced Atrophy but Inhibits Myoglobin Expression in C2C12 Skeletal Muscle Myotubes

    PubMed Central

    Vogel, Ilse S. P.; 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 strategy to prevent atrophy under hypoxia and enhance an eventual hypertrophic response to anabolic stimulation. Mb expression may be further enhanced by lipid supplementation. We investigated individual and combined effects of hypoxia, insulin-like growth factor (IGF)-1 and lipids, in mouse skeletal muscle C2C12 myotubes. Differentiated C2C12 myotubes were cultured for 24 h under 20%, 5% and 2% oxygen with or without IGF-1 and/or lipid treatment. In culture under 20% oxygen, IGF-1 induced 51% hypertrophy. Hypertrophy was only 32% under 5% and abrogated under 2% oxygen. This was not explained by changes in expression of genes involved in contractile protein synthesis or degradation, suggesting a reduced rate of translation rather than of transcription. Myoglobin mRNA expression increased by 75% under 5% O2 but decreased by 50% upon IGF-1 treatment under 20% O2, compared to control. Inhibition of mammalian target of rapamycin (mTOR) activation using rapamycin restored Mb mRNA expression to control levels. Lipid supplementation had no effect on Mb gene expression. Thus, IGF-1-induced anabolic signaling can be a strategy to improve muscle size under mild hypoxia, but lowers Mb gene expression. PMID:28862673

  2. Dietary isoflavone daidzein promotes Tfam expression that increases mitochondrial biogenesis in C2C12 muscle cells.

    PubMed

    Yoshino, Makiko; Naka, Ayano; Sakamoto, Yuri; Shibasaki, Ayako; Toh, Mariko; Tsukamoto, Sakuka; Kondo, Kazuo; Iida, Kaoruko

    2015-11-01

    Mitochondrial dysfunction in muscles leads to a wide range of metabolic and age-related disorders. Recently, it has been reported that a natural polyphenol, resveratrol, affects mitochondrial biogenesis. This study aimed to identify other natural polyphenolic compounds that regulate mitochondrial biogenesis in muscles. For this purpose, we used the C2C12 murine muscle cell line. Screening involved a reporter assay based on the promoter of mitochondrial transcription factor A (Tfam). We found that several polyphenols exhibited the ability to increase Tfam promoter activity and that the soy isoflavone daidzein was a most potent candidate that regulated mitochondrial biogenesis. When C2C12 myotubes were treated with 25-50 μM daidzein for 24h, there were significant increases in the expression of Tfam and mitochondrial genes such as COX1 and Cytb as well as the mitochondrial content. Using several mutant Tfam promoter fragments, we found that the transcription factor, nuclear respiratory factor (NRF) and its coactivator, PGC1α, were necessary for the effect of daidzein on Tfam expression. Finally, silencing of sirtuin-1 (SIRT1) by shRNA resulted in inhibition of the daidzein effects on mitochondrial gene expression. In conclusion, daidzein regulates mitochondrial biogenesis in muscle cells by regulating transcriptional networks through a SIRT1-associated pathway. These results suggest that daidzein would be beneficial to protect against a wide range of diseases caused by muscle mitochondrial dysfunction. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Sphingosine-1-phosphate pretreatment amends hypoxia-induced metabolic dysfunction and impairment of myogenic potential in differentiating C2C12 myoblasts by stimulating viability, calcium homeostasis and energy generation.

    PubMed

    Rahar, Babita; Chawla, Sonam; Pandey, Sanjay; Bhatt, Anant Narayan; Saxena, Shweta

    2017-01-09

    Sphingosine-1-phosphate (S1P) has a role in transpiration in patho-physiological signaling in skeletal muscles. The present study evaluated the pre-conditioning efficacy of S1P in facilitating differentiation of C2C12 myoblasts under a normoxic/hypoxic cell culture environment. Under normoxia, exogenous S1P significantly promoted C2C12 differentiation as evident from morphometric descriptors and differentiation markers of the mature myotubes, but it could facilitate only partial recovery from hypoxia-induced compromised differentiation. Pretreatment of S1P optimized the myokine secretion, intracellular calcium release and energy generation by boosting the aerobic/anaerobic metabolism and mitochondrial mass. In the hypoxia-exposed cells, there was derangement of the S1PR1-3 expression patterns, while the same could be largely restored with S1P pretreatment. This is being proposed as a plausible underlying mechanism for the observed pro-myogenic efficacy of exogenous S1P preconditioning. The present findings are an invaluable addition to the existing knowledge on the pro-myogenic potential of S1P and may prove beneficial in the field of hypoxia-related myo-pathologies.

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

    SciTech Connect

    Yokokawa, Takumi; Sato, Koji; Iwanaka, Nobumasa; Honda, Hiroki; Higashida, Kazuhiko; Iemitsu, Motoyuki; Hayashi, Tatsuya; Hashimoto, Takeshi

    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.

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

    PubMed

    Bai, X; Wang, X L; Tang, B; Shi, H N; Boireau, P; Rosenthal, B; Wu, X P; Liu, M Y; Liu, X L

    2016-11-15

    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 deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for skeletal muscle repair, notably, via their actions on myogenic precursor cells. However, these interactions during T. spiralis infection have not been characterized. In the present study, the ability of conditioned medium (CM) from J774A.1 macrophages treated with ML-ESPs to influence the differentiation of murine myoblasts, and the mechanisms of this influence, were investigated in vitro. The results showed that the expression of Myogenic Regulatory Factors (MRFs) MyoD and myogenin, myosin heavy chain (MyHC), and the p21 cyclin-dependent kinase inhibitor were reduced in CM treated cells compared to their expression in the control group. These findings indicated that CM inhibited myoblast differentiation. Conversely, CM promoted myoblast proliferation and increased cyclin D1 levels. Taken together, results of our study suggested that CM can indirectly influence myoblast differentiation and proliferation, which provides a new method for the elucidation of the complex mechanisms involved in cell-parasite and cell-cell interactions during T. spiralis infection.

  6. Ankyrin repeat and suppressor of cytokine signaling (SOCS) box-containing protein (ASB) 15 alters differentiation of mouse C2C12 myoblasts and phosphorylation of mitogen-activated protein kinase and Akt.

    PubMed

    McDaneld, T G; Spurlock, D M

    2008-11-01

    Ankyrin repeat and suppressor of cytokine signaling box-containing protein (ASB) 15 is a novel ASB gene family member predominantly expressed in skeletal muscle. We have previously reported that overexpression of ASB15 delays differentiation and alters protein turnover in mouse C(2)C(12) myoblasts. However, the extent of ASB15 regulation of differentiation and molecular pathways underlying this activity are unknown. The extracellular signal-regulated kinase (Erk) 1/2 and phosphatidylinositol-3 kinase-Akt (PI3K/Akt; Akt is also known as protein kinase B) signaling pathways have a role in skeletal muscle growth. Activation (phosphorylation) of the Erk1/2 signaling pathway promotes proliferation, whereas activation of the PI3K/Akt signaling pathway promotes myoblast differentiation. Accordingly, we tested the hypothesis that ASB15 controls myoblast differentiation through its regulation of these kinases. Stably transfected myoblasts overexpressing ASB15 (ASB15+) demonstrated decreased differentiation, whereas attenuation of ASB15 expression (ASB15-) increased differentiation. However, ASB15+ cells had less abundance of the phosphorylated mitogen-activated protein kinase (active) form, despite decreased differentiation relative to control myoblasts (ASB15Con). The mitogen-activated protein kinase kinase inhibitor, U0126, effectively decreased mitogen-activated protein kinase phosphorylation and stimulated differentiation in ASB15- and ASB15Con cells. However, inhibition of the Erk1/2 pathway was unable to overcome the inhibitory effect of overexpressing ASB15 on differentiation (ASB15+), suggesting that the Erk1/2 pathway is likely not the predominant mediator of ASB15 activity on differentiation. Expression of ASB15 also altered phosphorylation of the PI3K/Akt pathway, as ASB15+ and ASB15- cells had decreased and increased Akt phosphorylation, respectively. These data were consistent with observed differences in differentiation. Administration of IGF-I, a PI3K

  7. Co-culture of C2C12 and 3T3-L1 preadipocyte cells alters the gene expression of calpains, caspases and heat shock proteins.

    PubMed

    Pandurangan, Muthuraman; Jeong, Dawoon; Amna, Touseef; Van Ba, Hoa; Hwang, Inho

    2012-10-01

    The present study was carried out to understand the co-culture effect of C2C12 and 3T3-L1 preadipocyte cells on calpain, caspase, and heat shock protein (Hsp) systems. Calpains, caspases, and heat shock proteins play critical roles in the growth and development of mammalian cells. Cells were co-cultured using transwell inserts with a 0.4-μm porous membrane to separate C2C12 and 3T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates and inserts containing C2C12 transferred to 3T3-L1 plates. Following co-culture for 24 and 48 h, the cells in the lower well were harvested for analysis. Calpains include μ-calpain, m-calpain, and their specific inhibitor calpastatin. The expression pattern of μ-calpain did not change in the co-cultured C2C12 and 3T3-L1 cells, whereas m-capain mRNA expression significantly reduced in the 48-h co-cultured 3T3-L1 cells. Calpastatin mRNA expression significantly increased in the 48-h co-cultured C2C12 cells. Caspase-7 mRNA expression did not change in the 24- and 48-h co-cultured C2C12 and 3T3-L1 cells. Caspase-3 mRNA expression significantly reduced in the 24- and 48-h co-cultured 3T3-L1 cells; caspase-9 mRNA had a significant reduction only at 48 h, whereas caspase-9 mRNA expression significantly increased in the 48-h co-cultured C2C12 cells. Hsp27 and Hsp90 mRNA expressions are significantly reduced in the 24- and 48-h co-cultured C2C12 and 3T3-L1 cells, whereas Hsp70 mRNA expression significantly increased in the 48-h co-cultured 3T3-L1 cells. The co-culture reflects three-dimensional views of C2C12 and 3T3-L1 cell types as in vivo, which is quite distinct from the one-dimensional monocultured C2C12 and 3T3-L1 cells.

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

    PubMed

    Yokokawa, Takumi; Sato, Koji; Iwanaka, Nobumasa; Honda, Hiroki; Higashida, Kazuhiko; Iemitsu, Motoyuki; Hayashi, Tatsuya; Hashimoto, Takeshi

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    SciTech Connect

    Chu, Weiwei; Wei, Wei; Yu, Shigang; Han, Haiyin; Shi, Xiaoli; Sun, Wenxing; Gao, Ying; Zhang, Lifan; Chen, Jie

    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.

  10. Cytotoxicity of zinc oxide nanoparticles on antioxidant enzyme activities and mRNA expression in the cocultured C2C12 and 3T3-L1 cells.

    PubMed

    Pandurangan, Muthuraman; Veerappan, Muthuviveganandavel; Kim, Doo Hwan

    2015-02-01

    The present study was aimed to investigate the dose-dependent effect of zinc oxide (ZnO) nanoparticles on antioxidant enzyme activities and messenger RNA (mRNA) expression in the cocultured C2C12 and 3T3-L1 cells. Coculturing experiments are 3D and more reliable compared to mono-culture (2D) experiment. Even though, there are several studies on ZnO nanoparticle-mediated cytotoxicity, but there are no studies on the effect of ZnO nanoparticle on antioxidant enzyme activities and mRNA expression in the cocultured C2C12 and 3T3-L1 cells. A cytotoxicity assay was carried out to determine the effect of ZnO nanoparticles on the C2C12 and 3T3-L1 cell viability. At higher concentration of ZnO nanoparticles, C2C12 and 3T3-L1 cells almost die. ZnO nanoparticles increased reactive oxygen species (ROS) and lipid peroxidation and reduced glutathione (GSH) levels in a dose-dependent manner in the C2C12 and 3T3-L1 cells. In addition, ZnO nanoparticles increased antioxidant enzyme activities and their mRNA expression in the C2C12 and 3T3-L1 cells. In conclusion, the present study showed that ZnO nanoparticles increased oxidative stress, antioxidant enzyme activities, and their mRNA expression in the cocultured C2C12 and 3T3-L1 cells.

  11. Androgens Up-regulate Transcription of the Notch Inhibitor Numb in C2C12 Myoblasts via Wnt/β-Catenin Signaling to T Cell Factor Elements in the Numb Promoter*

    PubMed Central

    Liu, Xin-Hua; Wu, Yong; Yao, Shen; Levine, Alice C.; Kirschenbaum, Alexander; Collier, Lauren; Bauman, William A.; Cardozo, Christopher P.

    2013-01-01

    Androgen signaling via the androgen receptor is a key pathway that contributes to development, cell fate decisions, and differentiation, including that of myogenic progenitors. Androgens and synthetic steroids have well established anabolic actions on skeletal muscle. Wnt and Notch signaling pathways are also essential to myogenic cell fate decisions during development and tissue repair. However, the interactions among these pathways are largely unknown. Androgenic regulation of Wnt signaling has been reported. Nandrolone, an anabolic steroid, has been shown to inhibit Notch signaling and up-regulate Numb, a Notch inhibitor. To elucidate the mechanisms of interaction between nandrolone and Wnt/Notch signaling, we investigated the effects of nandrolone on Numb expression and Wnt signaling and determined the roles of Wnt signaling in nandrolone-induced Numb expression in C2C12 myoblasts. Nandrolone increased Numb mRNA and protein levels and T cell factor (Tcf) transcriptional activity via inhibition of glycogen synthase kinase 3β. Up-regulation of Numb expression by nandrolone was blocked by the Wnt inhibitors, sFRP1 and DKK1, whereas Wnt3a increased Numb mRNA and protein expression. In addition, we observed that the proximal promoter of the Numb gene had functional Tcf binding elements to which β-catenin was recruited in a manner enhanced by both nandrolone and Wnt3a. Moreover, site-directed mutagenesis indicated that the Tcf binding sites in the Numb promoter are required for the nandrolone-induced Numb transcriptional activation in this cell line. These results reveal a novel molecular mechanism underlying up-regulation of Numb transcription with a critical role for increased canonical Wnt signaling. In addition, the data identify Numb as a novel target gene of the Wnt signaling pathway by which Wnts would be able to inhibit Notch signaling. PMID:23649620

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

    PubMed

    Chu, Weiwei; Wei, Wei; Yu, Shigang; Han, Haiyin; Shi, Xiaoli; Sun, Wenxing; Gao, Ying; Zhang, Lifan; Chen, Jie

    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.

  13. Sustained and therapeutic delivery of factor IX in nude haemophilia B mice by encapsulated C2C12 myoblasts: concurrent tumourigenesis.

    PubMed

    Hortelano, G; Wang, L; Xu, N; Ofosu, F A

    2001-03-01

    This study reports the generation of an immunodeficient murine model for haemophilia B, obtained by breeding factor IX-deficient mice with an immunodeficient mouse strain, and use of this mouse model to evaluate the long-term efficacy and safety of a gene therapy strategy for treating haemophilia B. Nude haemophilic mice were implanted with biocompatible microcapsules enclosing recombinant myoblasts secreting human factor IX. The activated partial thromboplastin time (APTT) of plasma of mice thus treated was invariably shortened 3 weeks after microcapsule implantation, and remained shortened for at least 77 days. Shortening of the APTT of the haemophilia mice coincided with the appearance of human factor IX in mice plasmas (up to 600 ng mL(-1) on day 77), and normalization of the tail-bleeding time. Thus, the microencapsulated myoblasts reversed the clinical phenotype of haemophilia B. In contrast, plasmas of immunocompetent haemophilic mice similarly implanted with microcapsules only showed a transient shortening of APTT, and coincident transient delivery of human factor IX antigen. Rapid disappearance of human factor IX from plasmas of immunocompetent mice also coincided with production of antibodies to the human transgene. Significantly, 86% of the nude haemophilia mice developed tumours of myoblast origin. Thus, while this study revealed the feasibility of this gene therapy approach to treat severe haemophilia B, it also highlights the importance of using safer cell lines to prevent tumour development.

  14. Expression and purification of TAT-fused carbonic anhydrase III and its effect on C2C12 cell apoptosis induced by hypoxia/reoxygenation

    PubMed Central

    Shang, Xiliang; Bao, Yuanyuan; Ren, Huimin; Huang, He; Li, Yunxia

    2012-01-01

    Introduction Carbonic anhydrase III (CAIII) is remarkably abundant in slow skeletal muscles. It has multiple biological activities which could dissipate or resist some fatigue-related substances. In this study, we purified trans-activating transcriptional activator (TAT) fused CAIII protein and investigated its effect on C2C12 cell apoptosis induced by hypoxia/reoxygenation. Material and methods The CAIII and TAT-CAIII genes were constructed, cloned into plasmid pET28a and expressed in Escherichia coli BL21 (DE3). The fusion proteins were purified with a nickel-nitrilotriacetic acid affinity chromatography column and then verified by Western blot and phosphatase activity staining subsequently. The C2C12 cells were treated respectively with serum-free medium containing 1 μM TAT-CAIII or 1 μM CAIII for 1 h and the intracellular distributions of fusion proteins were observed by indirect immunofluorescence. The effect of TAT-CAIII on C2C12 cell apoptosis induced by hypoxia/reoxygenation was detected by flow cytometry. Results The CAIII and TAT-CAIII fusion proteins were expressed and purified successfully. After being cultured for 1 h, green fluorescence was visible in TAT-CAIII group cells under the fluorescence microscope, while no fluorescence was found in the CAIII group. Compared with the oxygen-glucose deprivation group, the apoptosis rate of C2C12 cells induced by hypoxia/reoxygenation in the TAT-CAIII group decreased significantly (p < 0.001). Conclusions The purified TAT-CAIII could be transferred into cells efficiently and clearly decreased the apoptosis rate of C2C12 cells induced by hypoxia/reoxygenation, which indicated that it had antioxidative activity. This study lays an experimental basis for future research on the relationship between CAIII and muscle fatigue. PMID:23056085

  15. Combined Effects of Androgen and Growth Hormone on Osteoblast Marker Expression in Mouse C2C12 and MC3T3-E1 Cells Induced by Bone Morphogenetic Protein

    PubMed Central

    Kimura, Kosuke; Terasaka, Tomohiro; Iwata, Nahoko; Katsuyama, Takayuki; Komatsubara, Motoshi; Nagao, Ryota; Inagaki, Kenichi; Otsuka, Fumio

    2017-01-01

    Osteoblasts undergo differentiation in response to various factors, including growth factors and steroids. Bone mass is diminished in androgen- and/or growth hormone (GH)-deficient patients. However the functional relationship between androgen and GH, and their combined effects on bone metabolism, remains unclear. Here we investigated the mutual effects of androgen and GH on osteoblastic marker expression using mouse myoblastic C2C12 and osteoblast-like MC3T3-E1 cells. Combined treatment with dihydrotestosterone (DHT) and GH enhanced BMP-2-induced expression of Runx2, ALP, and osteocalcin mRNA, compared with the individual treatments in C2C12 cells. Co-treatment with DHT and GH activated Smad1/5/8 phosphorylation, Id-1 transcription, and ALP activity induced by BMP-2 in C2C12 cells but not in MC3T3-E1 cells. The insulin-like growth factor (IGF-I) mRNA level was amplified by GH and BMP-2 treatment and was restored by co-treatment with DHT in C2C12 cells. The mRNA level of the IGF-I receptor was not significantly altered by GH or DHT, while it was increased by IGF-I. In addition, IGF-I treatment increased collagen-1 mRNA expression, whereas blockage of endogenous IGF-I activity using an anti-IGF-I antibody failed to suppress the effect of GH and DHT on BMP-2-induced Runx2 expression in C2C12 cells, suggesting that endogenous IGF-I was not substantially involved in the underlying GH actions. On the other hand, androgen receptor and GH receptor mRNA expression was suppressed by BMP-2 in both cell lines, implying the existence of a feedback action. Collectively the results showed that the combined effects of androgen and GH facilitated BMP-2-induced osteoblast differentiation at an early stage by upregulating BMP receptor signaling. PMID:28067796

  16. Ndrg2 is a PGC-1α/ERRα target gene that controls protein synthesis and expression of contractile-type genes in C2C12 myotubes.

    PubMed

    Foletta, Victoria C; Brown, Erin L; Cho, Yoshitake; Snow, Rod J; Kralli, Anastasia; Russell, Aaron P

    2013-12-01

    The stress-responsive, tumor suppressor N-myc downstream-regulated gene 2 (Ndrg2) is highly expressed in striated muscle. In response to anabolic and catabolic signals, Ndrg2 is suppressed and induced, respectively, in mouse C2C12 myotubes. However, little is known about the mechanisms regulating Ndrg2 expression in muscle, as well as the biological role for Ndrg2 in differentiated myotubes. Here, we show that Ndrg2 is a target of a peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) and estrogen-related receptor alpha (ERRα) transcriptional program and is induced in response to endurance exercise, a physiological stress known also to increase PGC-1α/ERRα activity. Analyses of global gene and protein expression profiles in C2C12 myotubes with reduced levels of NDRG2, suggest that NDRG2 affects muscle growth, contractile properties, MAPK signaling, ion and vesicle transport and oxidative phosphorylation. Indeed, suppression of NDRG2 in myotubes increased protein synthesis and the expression of fast glycolytic myosin heavy chain isoforms, while reducing the expression of embryonic myosin Myh3, other contractile-associated genes and the MAPK p90 RSK1. Conversely, enhanced expression of NDRG2 reduced protein synthesis, and furthermore, partially blocked the increased protein synthesis rates elicited by a constitutively active form of ERRα. In contrast, suppressing or increasing levels of NDRG2 did not affect mRNA expression of genes involved in mitochondrial biogenesis that are regulated by PGC-1α or ERRα. This study shows that in C2C12 myotubes Ndrg2 is a novel PGC-1α/ERRα transcriptional target, which influences protein turnover and the regulation of genes involved in muscle contraction and function. © 2013 Elsevier B.V. All rights reserved.

  17. Epigallocatechin Gallate Reduces Slow-Twitch Muscle Fiber Formation and Mitochondrial Biosynthesis in C2C12 Cells by Repressing AMPK Activity and PGC-1α Expression.

    PubMed

    Wang, Lina; Wang, Zhen; Yang, Kelin; Shu, Gang; Wang, Songbo; Gao, Ping; Zhu, Xiaotong; Xi, Qianyun; Zhang, Yongliang; Jiang, Qingyan

    2016-08-31

    Epigallocatechin gallate (EGCG) is a major active compound in green tea polyphenols. EGCG acts as an antioxidant to prevent the cell damage caused by free radicals and their derivatives. In skeletal muscle, exercise causes the accumulation of intracellular reactive oxygen species (ROS) and promotes the formation of slow-type muscle fiber. To determine whether EGCG, as a ROS scavenger, has any effect on skeletal muscle fiber type, we applied different concentrations (0, 5, 25, and 50 μM) of EGCG in the culture medium of differentiated C2C12 cells for 2 days. The fiber-type composition, mitochondrial biogenesis-related gene expression, antioxidant and glucose metabolism enzyme activity, and ROS levels in C2C12 cells were then detected. According to our results, 5 μM EGCG significantly decreased the cellular activity of SDH, 25 μM EGCG significantly downregulated the MyHC I, PGC-1α, NRF-1, and p-AMPK levels and SDH activity while enhancing the CAT and GSH-Px activity and decreasing the intracellular ROS levels, and 50 μM EGCG significantly downregulated MyHC I, PGC-1α, and NRF-1 expression and HK and SDH activity while increasing LDH activity. Furthermore, 300 μM H2O2 and 0.5 mM AMPK agonist (AICAR) improved the expression of MyHC I, PGC-1α, and p-AMPK, which were all reversed by 25 μM EGCG. In conclusion, the effect of EGCG on C2C12 cells may occur through the reduction of the ROS level, thereby decreasing both AMPK activity and PGC-1α expression and eventually reducing slow-twitch muscle fiber formation and mitochondrial biosynthesis.

  18. Atractylenolide III Enhances Energy Metabolism by Increasing the SIRT-1 and PGC1α Expression with AMPK Phosphorylation in C2C12 Mouse Skeletal Muscle Cells.

    PubMed

    Song, Mi Young; Jung, Hyo Won; Kang, Seok Yong; Park, Yong-Ki

    2017-01-01

    Targeting energy expenditure provides a potential alternative strategy for achieving energy balance to combat obesity and the development of type 2 diabetes mellitus (T2DM). In the present study, we investigated whether atractylenolide III (AIII) regulates energy metabolism in skeletal muscle cells. Differentiated C2C12 myotubes were treated with AIII (10, 20, or 50 µM) or metformin (2.5 mM) for indicated times. The levels of glucose uptake, the expressions of key mitochondrial biogenesis-related factors and their target genes were measured in C2C12 myotubes. AIII significantly increased the glucose uptake levels, and significantly increased the expressions of peroxisome proliferator-activated receptor coactivator-1α (PGC1α) and mitochondrial biogenesis-related markers, such as, nuclear respiratory factor-1 (NRF-1), and mitochondrial transcription factor A (TFAM) and mitochondrial mass and total ATP contents. In addition, AIII significantly increased the phosphorylation of AMP-activated protein kinase (AMPK) and the expression of sirtuin1 (SIRT1). These results suggest that AIII may have beneficial effects on obesity and T2DM by improving energy metabolism in skeletal muscle.

  19. 1,25(OH)2vitamin D3 Enhances Myogenic Differentiation by Modulating the Expression of Key Angiogenic Growth factors and Angiogenic Inhibitors in C2C12 Skeletal Muscle Cells

    PubMed Central

    Garcia, Leah A.; Ferrini, Monica G.; Norris, Keith C.; Artaza, Jorge N.

    2012-01-01

    Vitamin D is mostly recognized for its regulation of calcium homeostasis in relation to the intestine, kidney, and bone. Although clinical studies have linked vitamin D with increased muscle function and strength, little is known of its underlying molecular mechanism. We recently demonstrated that 1,25-D3 exerts a direct pro-myogenic effect on skeletal muscle cells; this has provoked our investigation of 1,25-D’s effect on angiogenesis, a vital process for new capillary development and tissue repair. In this study, we examined the mechanism by which 1,25-D3 modulates key angiogenic growth factors and angiogenic inhibitors. C2C12 myoblasts were incubated with 100 nM 1,25-D3 or placebo for 1, 4 and 10 days. At the end of the respective incubation time, total RNA was isolated for PCR arrays and for qRT-PCR. Total proteins were isolated for western blots and proteome profiler arrays. The addition of 1,25-D3 to C2C12 myoblasts increased VEGFa and FGF-1: two pro-angiogenic growth factors that promote neo-vascularization and tissue regeneration, and decreased FGF-2 and TIMP-3: two myogenic and/or angiogenic inhibitors. Our previous study demonstrated that 1,25-D3 altered IGF-I/II expression, consistent with the observed changes in VEGFa and FGF-2 expression. These results extend our previous findings and demonstrate the modulation of angiogenesis which may be an additional mechanism by which 1,25-D3 promotes myogenesis. This study supports the mechanistic rationale for assessing the administration of vitamin D and/or vitamin D analogues to treat select muscle disorders and may also provide an alternative solution for therapies that directly manipulate VEGF and FGF’s to promote angiogenesis. PMID:22982629

  20. Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid on Mitochondrial DNA Replication and PGC-1α Gene Expression in C2C12 Muscle Cells

    PubMed Central

    Lee, Mak-Soon; Shin, Yoonjin; Moon, Sohee; Kim, Seunghae; Kim, Yangha

    2016-01-01

    Mitochondrial biogenesis is a complex process requiring coordinated expression of nuclear and mitochondrial genomes. The peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) is a key regulator of mitochondrial biogenesis, and it controls mitochondrial DNA (mtDNA) replication within diverse tissues, including muscle tissue. The aim of this study was to investigate the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on mtDNA copy number and PGC-1α promoter activity in C2C12 muscle cells. mtDNA copy number and mRNA levels of genes related to mitochondrial biogenesis such as PGC-1α, nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (Tfam) were assayed by quantitative real-time PCR. The PGC-1α promoter from −970 to +412 bp was subcloned into the pGL3-basic vector, which includes a luciferase reporter gene. Both EPA and DHA significantly increased mtDNA copy number, dose and time dependently, and up-regulated mRNA levels of PGC-1α, NRF1, and Tfam. Furthermore, EPA and DHA stimulated PGC-1α promoter activity in a dose-dependent manner. These results suggest that EPA and DHA may modulate mitochondrial biogenesis, which was partially associated with increased mtDNA replication and PGC-1α gene expression in C2C12 muscle cells. PMID:28078253

  1. Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid on Mitochondrial DNA Replication and PGC-1α Gene Expression in C2C12 Muscle Cells.

    PubMed

    Lee, Mak-Soon; Shin, Yoonjin; Moon, Sohee; Kim, Seunghae; Kim, Yangha

    2016-12-01

    Mitochondrial biogenesis is a complex process requiring coordinated expression of nuclear and mitochondrial genomes. The peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) is a key regulator of mitochondrial biogenesis, and it controls mitochondrial DNA (mtDNA) replication within diverse tissues, including muscle tissue. The aim of this study was to investigate the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on mtDNA copy number and PGC-1α promoter activity in C2C12 muscle cells. mtDNA copy number and mRNA levels of genes related to mitochondrial biogenesis such as PGC-1α, nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (Tfam) were assayed by quantitative real-time PCR. The PGC-1α promoter from -970 to +412 bp was subcloned into the pGL3-basic vector, which includes a luciferase reporter gene. Both EPA and DHA significantly increased mtDNA copy number, dose and time dependently, and up-regulated mRNA levels of PGC-1α, NRF1, and Tfam. Furthermore, EPA and DHA stimulated PGC-1α promoter activity in a dose-dependent manner. These results suggest that EPA and DHA may modulate mitochondrial biogenesis, which was partially associated with increased mtDNA replication and PGC-1α gene expression in C2C12 muscle cells.

  2. Glutamine, insulin and glucocorticoids regulate glutamine synthetase expression in C2C12 myotubes, Hep G2 hepatoma cells and 3T3 L1 adipocytes.

    PubMed

    Wang, Yanxin; Watford, Malcolm

    2007-04-01

    The cell-specific regulation of glutamine synthetase expression was studied in three cell lines. In C2C12 myotubes, glucocorticoids increased the abundance of both glutamine synthetase protein and mRNA. Culture in the absence of glutamine also resulted in very high glutamine synthetase protein abundance but mRNA levels were unchanged. Glucocorticoids also increased the abundance of glutamine synthetase mRNA in Hep G2 hepatoma cells but this was not reflected in changes in protein abundance. Culture of Hep G2 cells without glutamine resulted in very high levels of protein, again with no change in mRNA abundance. Insulin was without effect in both C2C12 and Hep G2 cells. In 3T3 L1 adipocytes glucocorticoids increased the abundance of both glutamine synthetase mRNA and protein, insulin added alone had no effect but in the presence of glucocorticoids resulted in lower mRNA levels than seen with glucocorticoids alone, although protein levels remained high under such conditions. In contrast to the other cell lines glutamine synthetase protein levels were relatively unchanged by culture in the absence of glutamine. The results support the hypothesis that in myocytes, and hepatomas, but not in adipocytes, glutamine acts to moderate glutamine synthetase induction by glucocorticoids.

  3. Nuclear co-repressor (NCoR) is required to maintain insulin sensitivity in C2 C12 myotubes.

    PubMed

    Choudhary, Abhijeet K; Dey, Chinmoy S

    2017-02-01

    Nuclear co-repressor (NCoR) regulates peripheral insulin sensitivity; however, its role in modulating insulin sensitivity in skeletal muscle remains elusive. Present study investigated protein expression and effect of NCoR on insulin sensitivity in murine skeletal muscle cell line C2 C12 . Myotubes as compared to myoblasts of C2 C12 cells were found to be more sensitive in response to insulin as increase in insulin-stimulated phosphorylation of AKT at serine 473 residue (pAKT(S473) ) was significantly higher in myotubes. Incidentally, reduced protein level of NCoR coincided with differentiation of myoblasts into myotubes of C2 C12 cells. However, insulin stimulation per se failed to affect protein level of NCoR either in myoblasts or myotubes of C2 C12 cells. To assess the role of NCoR on insulin sensitivity, NCoR was transiently knocked down using siRNA in myotubes of C2 C12 . In fact, transient silencing of NCoR led to significant reduction in insulin-stimulated pAKT(S473) and impaired glucose uptake. This observation is in contrast to published studies where NCoR has been reported to negatively regulate insulin signaling cascade. Furthermore, transient silencing of NCoR failed to improve insulin sensitivity in chronic hyperinsulinemia-induced insulin-resistant model of C2 C12 cells. Importantly, inhibition of lysosomal protein degradation pathway using ammonium chloride restored protein level of NCoR but failed to increase glucose uptake in serum-starved C2 C12 myotubes. Collectively, data from present study show differential protein level of NCoR under different cell state (myoblast and myotubes) of C2 C12 cells and NCoR proves to be vital for maintaining insulin sensitivity in C2 C12 myotubes.

  4. The Cytoprotective Effect of Petalonia binghamiae Methanol Extract against Oxidative Stress in C2C12 Myoblasts: Mediation by Upregulation of Heme Oxygenase-1 and Nuclear Factor-Erythroid 2 Related Factor 2.

    PubMed

    Kang, Ji Sook; Choi, Il-Whan; Han, Min Ho; Lee, Dae-Sung; Kim, Gi-Young; Hwang, Hye Jin; Kim, Byung Woo; Kim, Cheol Min; Yoo, Young Hyun; Choi, Yung Hyun

    2015-04-29

    This study was designed to examine the protective effects of the marine brown algae Petalonia binghamiae against oxidative stress-induced cellular damage and to elucidate the underlying mechanisms. P. binghamiae methanol extract (PBME) prevented hydrogen peroxide (H2O2)-induced growth inhibition and exhibited scavenging activity against intracellular reactive oxygen species (ROS) induced by H2O2 in mouse-derived C2C12 myoblasts. PBME also significantly attenuated H2O2-induced comet tail formation in a comet assay, histone γH2A.X phosphorylation, and annexin V-positive cells, suggesting that PBME prevented H2O2-induced cellular DNA damage and apoptotic cell death. Furthermore, PBME increased the levels of heme oxygenase-1 (HO-1), a potent antioxidant enzyme, associated with the induction of nuclear factor-erythroid 2 related factor 2 (Nrf2). However, zinc protoporphyrin IX, a HO-1 competitive inhibitor, significantly abolished the protective effects of PBME on H2O2-induced ROS generation, growth inhibition, and apoptosis. Collectively, these results demonstrate that PBME augments the antioxidant defense capacity through activation of the Nrf2/HO-1 pathway.

  5. Autophagic flux data in differentiated C2C12 myotubes following exposure to acetylcholine and caffeine.

    PubMed

    Bloemberg, Darin; Quadrilatero, Joe

    2016-06-01

    The C2C12 line of mouse myoblasts is a useful cell culture model in which to conduct in vitro analyses related to skeletal muscle. Here we present data regarding the autophagic response induced by two chemicals known to influence calcium release and contraction in skeletal muscles and C2C12 cells: acetylcholine and caffeine. More specifically, by concurrently administering acetylcholine or caffeine along with chloroquine to differentiated myotubes for various amounts of time and assessing the protein expression of LC3 and p62, we report data on the relative level of autophagic flux induced by these two calcium- and contraction-regulating chemicals.

  6. Effect of mitochondrial fission inhibition on C2C12 differentiation

    PubMed Central

    Bloemberg, Darin; Quadrilatero, Joe

    2016-01-01

    The differentiation of skeletal muscle is commonly examined in cell culture using the C2C12 line of mouse skeletal myoblasts. This process shares many similarities with that which occurs during embryonic development, such as the transient activation of caspases. Here, we examined the effect of inhibiting mitochondrial fission, using mdivi-1, on the ability of C2C12 cells to terminally differentiate. This was performed using immunofluorescent identification of cell morphology and myosin expression, as well as immunoblotting for markers of muscle differentiation. Furthermore, the effect of mdivi-1 administration on activation of caspase-2 and -3 was assessed using spectrofluorometric measurement of specific enzyme activity. PMID:27054170

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

    SciTech Connect

    Tsukahara, Tamotsu; Haniu, Hisao

    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.

  8. Serum Amyloid A Induces Toll-Like Receptor 2-Dependent Inflammatory Cytokine Expression and Atrophy in C2C12 Skeletal Muscle Myotubes

    PubMed Central

    Passey, Samantha L.; Bozinovski, Steven; Vlahos, Ross; Anderson, Gary P.; Hansen, Michelle J.

    2016-01-01

    Background Skeletal muscle wasting is an important comorbidity of Chronic Obstructive Pulmonary Disease (COPD) and is strongly correlated with morbidity and mortality. Patients who experience frequent acute exacerbations of COPD (AECOPD) have more severe muscle wasting and reduced recovery of muscle mass and function after each exacerbation. Serum levels of the pro-inflammatory acute phase protein Serum Amyloid A (SAA) can rise more than 1000-fold in AECOPD and are predictively correlated with exacerbation severity. The direct effects of SAA on skeletal muscle are poorly understood. Here we have examined SAA effects on pro-inflammatory cachectic cytokine expression (IL-6 and TNFα) and atrophy in C2C12 myotubes. Results SAA increased IL-6 (31-fold) and TNFα (6.5-fold) mRNA levels compared to control untreated cells after 3h of SAA treatment, and increased secreted IL-6 protein at 24h. OxPAPC, a dual TLR2 and TLR4 inhibitor, reduced the response to SAA by approximately 84% compared to SAA alone, and the TLR2 neutralising antibody T2.5 abolished SAA-induced expression of IL-6, indicating that SAA signalling in C2C12 myotubes is primarily via TLR2. SAA also reduced myotube width by 10–13% and induced a 2.5-fold increase in the expression of the muscle atrophy gene Atrogin-1, suggesting direct effects of SAA on muscle wasting. Blocking of TLR2 inhibited the SAA-induced decrease in myotube width and Atrogin-1 gene expression, indicating that SAA induces atrophy through TLR2. Conclusions These data demonstrate that SAA stimulates a robust pro-inflammatory response in skeletal muscle myotubes via the TLR2-dependent release of IL-6 and TNFα. Furthermore, the observed atrophy effects indicate that SAA could also be directly contributing to the wasting and poor recovery of muscle mass. Therapeutic strategies targeting this SAA-TLR2 axis may therefore ameliorate muscle wasting in AECOPD and a range of other inflammatory conditions associated with loss of muscle mass. PMID

  9. Characterization of porcine SKIP gene in skeletal muscle development: polymorphisms, association analysis, expression and regulation of cell growth in C2C12 cells.

    PubMed

    Xiong, Qi; Chai, Jin; Deng, Changyan; Jiang, Siwen; Liu, Yang; Huang, Tao; Suo, Xiaojun; Zhang, Nian; Li, Xiaofeng; Yang, Qianping; Chen, Mingxin; Zheng, Rong

    2012-12-01

    Skeletal muscle and kidney-enriched inositol phosphatase (SKIP) was identified as a 5'-inositol phosphatase that hydrolyzes phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3) to PI(3,4)P2 and negatively regulates insulin-induced phosphatidylinositol 3-kinase signaling in skeletal muscle. In this study, two new single nucleotide polymorphisms (SNPs) in porcine SKIP introns 1 and 6 were detected. The C1092T locus in intron 1 showed significant associations with some meat traits, whereas the A17G locus in intron 6 showed significant associations with some carcass traits. Expression analysis showed that porcine SKIP is upregulated at d 65 of gestation and Meishan fetuses have higher and prolonged expression of SKIP compared to Large White at d 100 of gestation. Ectopic expression of porcine SKIP decreased insulin-induced cell proliferation and promoted serum starvation-induced cell cycle arrest in G0/G1 phase in C2C12. Our results suggest that SKIP plays a negative regulatory role in skeletal muscle development partly by preventing cell proliferation.

  10. CRF Type 2 Receptors Mediate the Metabolic Effects of Ghrelin in C2C12 cells

    PubMed Central

    Gershon, Eran; Vale, Wylie W

    2014-01-01

    Objective Ghrelin is known to regulate appetite control and cellular metabolism. The Corticotropin-Releasing Factor (CRF) family is also known to regulate energy balance. In this study, we investigated the links between ghrelin and the CRF family in C2C12 cells, a mouse myoblast cell line. Design and methods C2C12 cells were treated with ghrelin in the presence or absence of CRF receptor antagonists and then subjected to different metabolic analyses. Results Ghrelin enhanced glucose uptake by C2C12 cells, induced GLUT4 translocation to the cell surface and decreased RBP4 expression. A CRF-R2 selective antagonist, anti-sauvagine-30, blocked ghrelin-induced glucose uptake, Ghrelin upregulated CRF-R2 but not CRF-R1 levels. Moreover, ghrelin-treated C2C12 cells displayed a cAMP and pERK activation in response to Ucn3, a CRF-R2 specific ligand, but not in response to CRF or stressin, CRF-R1 specific ligands. Ghrelin also induced UCP2 and UCP3 expression, which were blocked by anti-sauvagine-30. Ghrelin did not induce fatty acids uptake by C2C12 cells or ACC expression. Even though C2C12 cells clearly exhibited responses to ghrelin, the known ghrelin receptor, GHSR1a, was not detectable in C2C12 cells. Conclusion Our results suggest that, ghrelin plays a role in regulating muscle glucose and, raise the possibility that suppression of the CRF-R2 pathway might provide benefits in high ghrelin states. PMID:23804489

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

    PubMed

    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.

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

    PubMed Central

    Schoneich, 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 cell 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. PMID:24129924

  13. Truncated human LMP-1 triggers differentiation of C2C12 cells to an osteoblastic phenotype in vitro.

    PubMed

    Fei, Qinming; Boden, Scott D; Sangadala, Sreedhara; Viggeswarapu, Manjula; Liu, Yunshan; Titus, Louisa

    2007-09-01

    LIM mineralization protein-1 (LMP-1) is a novel intracellular osteoinductive protein that has been shown to induce bone formation both in vitro and in vivo. LMP-1 contains an N-terminal PDZ domain and three C-terminal LIM domains. In this study, we investigated whether a truncated form of human LMP-1 (hLMP-1[t]), lacking the three C-terminal LIM domains, triggers the differentiation of pluripotent myoblastic C(2)C(12) cells to the osteoblast lineage. C(2)C(12) cells were transiently transduced with Ad5-hLMP-1(t)-green fluorescent protein or viral vector control. The expression of hLMP-1(t) RNA and the truncated protein were examined. The results showed that hLMP-1(t) blocked myotube formation in C(2)C(12) cultures and significantly enhanced the alkaline phosphatase (ALP) activity. In addition, the expressions of ALP, osteocalcin, and bone morphogenetic protein (BMP)-2 and BMP-7 genes were also increased. The induction of these key osteogenic markers suggests that hLMP-1(t) can trigger the pluripotent myoblastic C2C12 cells to differentiate into osteoblastic lineage, thus extending our previous observation that LMP-1 and LMP-1(t) enhances the osteoblastic phenotype in cultures of cells already committed to the osteoblastic lineage. Therefore, C(2)C(12) cells are an appropriate model system for the examination of LMP-1 induction of the osteoblastic phenotype and the study of mechanisms of LMP-1 action.

  14. Investigation of interactions between poly-L-lysine-coated boron nitride nanotubes and C2C12 cells: up-take, cytocompatibility, and differentiation.

    PubMed

    Ciofani, G; Ricotti, L; Danti, S; Moscato, S; Nesti, C; D'Alessandro, D; Dinucci, D; Chiellini, F; Pietrabissa, A; Petrini, M; Menciassi, A

    2010-04-15

    Boron nitride nanotubes (BNNTs) have generated considerable interest within the scientific community by virtue of their unique physical properties, which can be exploited in the biomedical field. In the present in vitro study, we investigated the interactions of poly-l-lysine-coated BNNTs with C2C12 cells, as a model of muscle cells, in terms of cytocompatibility and BNNT internalization. The latter was performed using both confocal and transmission electron microscopy. Finally, we investigated myoblast differentiation in the presence of BNNTs, evaluating the protein synthesis of differentiating cells, myotube formation, and expression of some constitutive myoblastic markers, such as MyoD and Cx43, by reverse transcription - polymerase chain reaction and Western blot analysis. We demonstrated that BNNTs are highly internalized by C2C12 cells, with neither adversely affecting C2C12 myoblast viability nor significantly interfering with myotube formation.

  15. TEAD1 controls C2C12 cell proliferation and differentiation and regulates three novel target genes.

    PubMed

    Wang, Fengli; Wang, Hongyang; Wu, Hao; Qiu, Haifang; Zeng, Cuiping; Sun, Ling; Liu, Bang

    2013-03-01

    TEAD1 is a transcription factor involved in activation of muscle specific genes, such as the cardiac muscle troponin T gene, skeletal muscle actin, myosin heavy chains genes. Here, we reported that TEAD1 was expressed ubiquitously in different mouse tissues and was up-regulated in differentiation process of the mouse myoblast cell line C2C12. Functional assay revealed that overexpression of TEAD1 gene can arrest the C2C12 cell cycle and promote C2C12 cell differentiation. To understand the physiological role of TEAD1 in muscle development, three new regulated genes of TEAD1, Mrpl21, Ndufa6 and Ccne1, were identified by expression analysis, promoter activity measurement assay. The expression patterns of target genes were detected in the cell differentiation process. The Mrpl21 and Ndufa6 genes were up-regulated in cell differentiation while Ccne1 gene was significantly down-regulated. Overexpression of Mrpl21 and Ndufa6 in C2C12 can up-regulate Myh4 gene expression thus promote C2C12 differentiation, but did not affect cell cycle. Co-overexpression of Ccne1 with Ndufa6 resulted in Myh4 expression decrease and the number of S-phase cells slight increase. Together, our results suggested that TEAD1 may mediate muscle development through its target genes, Mrpl21, Ndufa6 and Ccne1. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  16. Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells.

    PubMed

    Taylor, W E; Bhasin, S; Artaza, J; Byhower, F; Azam, M; Willard, D H; Kull, F C; Gonzalez-Cadavid, N

    2001-02-01

    Myostatin mutations in mice and cattle are associated with increased muscularity, suggesting that myostatin is a negative regulator of skeletal muscle mass. To test the hypothesis that myostatin inhibits muscle cell growth, we examined the effects of recombinant myostatin in mouse skeletal muscle C2C12 cells. After verification of the expression of cDNA constructs in a cell-free system and in transfected Chinese hamster ovary cells, the human recombinant protein was expressed as the full-length (375-amino acid) myostatin in Drosophila cells (Mst375D), or the 110-amino acid carboxy-terminal protein in Escherichia coli (Mst110EC). These proteins were identified by immunoblotting and were purified. Both Mst375D and Mst110EC dose dependently inhibited cell proliferation (cell count and Formazan assay), DNA synthesis ([3H]thymidine incorporation), and protein synthesis ([1-14C]leucine incorporation) in C2C12 cells. The inhibitory effects of both proteins were greater in myotubes than in myoblasts. Neither protein had any significant effects on protein degradation or apoptosis. In conclusion, recombinant myostatin proteins inhibit cell proliferation, DNA synthesis, and protein synthesis in C2C12 muscle cells, suggesting that myostatin may control muscle mass by inhibiting muscle growth or regeneration.

  17. Secretion of adenylate kinase 1 is required for extracellular ATP synthesis in C2C12 myotubes

    PubMed Central

    Choo, Hyo-Jung; Kim, Bong-Woo; Kwon, Oh-Bong; Lee, Chang Seok; Choi, Jong-Soon

    2008-01-01

    Extracellular ATP (exATP) has been known to be a critical ligand regulating skeletal muscle differentiation and contractibility. ExATP synthesis was greatly increased with the high level of adenylate kinase 1 (AK1) and ATP synthase β during C2C12 myogenesis. The exATP synthesis was abolished by the knock-down of AK1 but not by that of ATP synthase β in C2C12 myotubes, suggesting that AK1 is required for exATP synthesis in myotubes. However, membrane-bound AK1β was not involved in exATP synthesis because its expression level was decreased during myogenesis in spite of its localization in the lipid rafts that contain various kinds of receptors and mediate cell signal transduction, cell migration, and differentiation. Interestingly, cytoplasmic AK1 was secreted from C2C12 myotubes but not from C2C12 myoblasts. Taken together all these data, we can conclude that AK1 secretion is required for the exATP generation in myotubes. PMID:18446060

  18. Leucine modulates the effect of Walker factor, a proteolysis-inducing factor-like protein from Walker tumours, on gene expression and cellular activity in C2C12 myotubes.

    PubMed

    Gonçalves, Estela Maria; Salomão, Emilianne Miguel; Gomes-Marcondes, Maria Cristina Cintra

    2013-10-01

    Cancer-cachexia causes severe weight loss, particularly from the wasting of skeletal muscle, which occurs due to increased protein catabolism and/or decreased protein synthesis. The muscle protein degradation observed in cancer patients is mediated by a specific cytokine, proteolysis-inducing factor (PIF), which is produced by the tumour. This protein increases the ubiquitin-proteasome pathway activity, and the synthesis of muscle protein in these patients can be affected by several factors, including nutrient-related signalling. Some nutrients, such as leucine, can decrease the ubiquitin-proteasome pathway activity and increase the skeletal muscle protein content in cachectic animals. In this study, we investigated the effects of leucine on cell viability, morphology, functional proteasome activity, enzymatic activity, and protein synthesis and degradation in C2C12 myotubes exposed to the proteolysis-inducing factor (PIF)-like protein purified from Walker tumour-bearing rats. Walker factor (WF) had no cytotoxic effects on myotube cells and morphological characteristics were not altered in the presence of WF and/or leucine. However, increased alkaline phosphatase activity was observed. At higher WF concentrations, chymotrypsin-like activity, cathepsin B activity and 20S proteasome gene expression increased. Treating myotubes with leucine before exposure to WF causes leads to a decrease in proteasome activity as well as the activity of chymotrypsin and cathepsin enzymes. Total protein synthesis decreased in WF-treated cells concomitantly as protein degradation increased. After leucine exposure, the observed effects of WF were minimal or even reverted in some cases. Taken together, these results suggest an important modulatory effect for leucine on the effects of WF in C2C12 myotube cells.

  19. 17β-estradiol upregulates oxytocin and the oxytocin receptor in C2C12 myotubes

    PubMed Central

    Berio, Enrica; Starvaggi Cucuzza, Laura; Biolatti, Bartolomeo; Cannizzo, Francesca Tiziana

    2017-01-01

    Background The endocrinology of skeletal muscle is highly complex and many issues about hormone action in skeletal muscle are still unresolved. Aim of the work is to improve our knowledge on the relationship between skeletal muscle and 17β-estradiol. Methods The skeletal muscle cell line C2C12 was treated with 17β-estradiol, the oxytocin peptide and a combination of the two hormones. The mRNA levels of myogenic regulatory factors, myosin heavy chain, oxytocin, oxytocin receptor and adipogenic factors were analysed in C2C12 myotubes. Results It was demonstrated that C2C12 myoblasts and myotubes express oxytocin and its receptor, in particular the receptor levels physiologically increase in differentiated myotubes. Myotubes treated with 17β-estradiol overexpressed oxytocin and oxytocin receptor genes by approximately 3- and 29-fold, respectively. A decrease in the expression of fatty acid binding protein 4 (0.62-fold), a fat metabolism-associated gene, was observed in oxytocin-treated myotubes. On the contrary, fatty acid binding protein 4 was upregulated (2.66-fold) after the administration of the combination of 17β-estradiol and oxytocin. 17β-estradiol regulates oxytocin and its receptor in skeletal muscle cells and they act in a synergic way on fatty acid metabolism. Discussion Oxytocin and its receptor are physiologically regulated along differentiation. 17β-estradiol regulates oxytocin and its receptor in skeletal muscle cells. 17β-estradiol and oxytocin act in a synergic way on fatty acid metabolism. A better understanding of the regulation of skeletal muscle homeostasis by estrogens and oxytocin peptide could contribute to increase our knowledge of muscle and its metabolism. PMID:28382233

  20. Role of PRDM16 and its PR domain in the epigenetic regulation of myogenic and adipogenic genes during transdifferentiation of C2C12 cells.

    PubMed

    Li, Xiao; Wang, Jinquan; Jiang, Zheng; Guo, Feng; Soloway, Paul D; Zhao, Ruqian

    2015-10-10

    The positive regulatory domain containing 16 (PRDM16) is commonly regarded as a "switch" controlling the transdifferentiation of myoblasts to brown adipocytes. The N-positive regulatory (PR) domain, which is highly homologous to SET domain, is a characteristic structure for the PRDM family. Many SET domain containing proteins and several PRDM members have been found to possess histone methyltransferase activity, yet the role of PRDM16 and its PR domain in the epigenetic regulation of myogenic and adipogenic genes during myoblasts/adipocytes transdifferentiation remains unexplored. In this study, we transfected C2C12 myoblasts to stably express PRDM16 and observed the repression of myogenic genes and activation of adipogenic genes at both proliferation and differentiation stages. Ectopic PRDM16-induced reprogramming of myogenic and adipogenic genes was associated with the hypermethylation on some CpG sites in the enhancer or promoter of MyoD and myogenin, but the methylation status of PPARγ promoter was not affected. C2C12 cells expressing truncated PRDM16 lacking PR domain (ΔPR-PRDM16) demonstrated attenuation of both adipogenic and myogenic potentials, indicated by PPARγ inactivation and decreased triglyceride deposition, as well as a downregulation of MyoD, MyHC and MCK genes, as compared with C2C12 cells expressing intact PRDM16. Furthermore, C2C12 cells expressing ΔPR-PRDM16 exhibited significant differences in histone modifications on the promoters of MyoD and PPARγ genes. Taken together, PRDM16-induced C2C12 transdifferentiation is associated with alterations in CpG methylation of myogenic factors, and PR domain affects both myogenesis and adipogenesis with modified histone methylation marks on MyoD and PPARγ promoters.

  1. Palmitate induces tumor necrosis factor-alpha expression in C2C12 skeletal muscle cells by a mechanism involving protein kinase C and nuclear factor-kappaB activation.

    PubMed

    Jové, Mireia; Planavila, Anna; Sánchez, Rosa M; Merlos, Manuel; Laguna, Juan Carlos; Vázquez-Carrera, Manuel

    2006-01-01

    The mechanisms responsible for increased expression of TNF-alpha in skeletal muscle cells in diabetic states are not well understood. We examined the effects of the saturated acid palmitate on TNF-alpha expression. Exposure of C2C12 skeletal muscle cells to 0.75 mm palmitate enhanced mRNA (25-fold induction, P < 0.001) and protein (2.5-fold induction) expression of the proinflammatory cytokine TNF-alpha. This induction was inversely correlated with a fall in GLUT4 mRNA levels (57% reduction, P < 0.001) and glucose uptake (34% reduction, P < 0.001). PD98059 and U0126, inhibitors of the ERK-MAPK cascade, partially prevented the palmitate-induced TNF-alpha expression. Palmitate increased nuclear factor (NF)-kappaB activation and incubation of the cells with the NF-kappaB inhibitors pyrrolidine dithiocarbamate and parthenolide partially prevented TNF-alpha expression. Incubation of palmitate-treated cells with calphostin C, a strong and specific inhibitor of protein kinase C (PKC), abolished palmitate-induced TNF-alpha expression, and restored GLUT4 mRNA levels. Palmitate treatment enhanced the expression of phospho-PKCtheta, suggesting that this PKC isoform was involved in the changes reported, and coincubation of palmitate-treated cells with the PKC inhibitor chelerythrine prevented the palmitate-induced reduction in the expression of IkappaBalpha and insulin-stimulated Akt activation. These findings suggest that enhanced TNF-alpha expression and GLUT4 down-regulation caused by palmitate are mediated through the PKC activation, confirming that this enzyme may be a target for either the prevention or the treatment of fatty acid-induced insulin resistance.

  2. Arecoline inhibits and destabilizes agrin-induced acetylcholine receptor cluster formation in C2C12 myotubes.

    PubMed

    Chang, Yung-Fu; Liu, Ting-Yuan; Liu, Shao-Tung

    2013-10-01

    Areca nut (Areca catechu) is chewed as a medical and psychoactive food by roughly 10% of the world population. Areca nut chewing may lead to low birth weight, premature delivery and impaired muscle development. Our previous study showed that arecoline, a major alkaloid in the areca nut, inhibited the myogenic differentiation of C2C12 myoblastic cells. The clustering of acetylcholine receptors (AChRs) in the postsynaptic membrane at the neuromuscular junction (NMJ) by agrin, a signaling protein released by motor neurons, is critical for the development of functional muscles. Here, we further investigate whether arecoline affects the AChR clustering using cultured C2C12 myotubes. Rhodamine-conjugated α-bungarotoxin was used to detect the presence of AChR clusters. Our results showed that arecoline inhibited the formation of agrin-induced AChR clusters and destabilized agrin-induced or spontaneous AChR cluster formation. In addition, arecoline inhibited the expression of myogenin in C2C12 myotubes. These results shed light on the important role of arecoline on the detrimental effect of areca nut to muscle development. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. A role for the Wnt3a/β-catenin signaling pathway in the myogenic program of C2C12 cells.

    PubMed

    Abraham, S Thomas

    2016-10-01

    The Wnt/β-catenin signaling pathway is a key regulator of embryonic development that was first identified in drosophila. More recent work has suggested a role for this pathway in mammalian, myogenesis but this process remains to be more completely elucidated. The current study was designed to investigate the role of the Wnt3a/β-catenin signaling module in C2C12 cell differentiation and myotube formation. Activation of the Wnt/β-catenin pathway in differentiating myoblasts, using lithium chloride supplementation, increased both the number and size of C2C12 myotubes. On the other hand, pharmacological inhibitors of Wnt/β-catenin signaling (IWR-1, XAV939) resulted in significant decreases in myotube length without affecting their numbers. In separate experiments, a neutralizing antibody to Wnt3a also reduced myotube length without affecting the number of myotubes formed during differentiation. Confocal imaging studies revealed that β-catenin was primarily localized in the cytoplasmic region of undifferentiated myoblasts and differentiation resulted in its enhanced expression in myotubes. Additionally, LiCl treatment resulted in enhanced expression of β-catenin in pre-differentiating myoblast cultures. These data suggest that Wnt3a/β-catenin signaling plays a critical role in C2C12 cell differentiation and provides an appropriate model for elucidating its functions in more detail.

  4. Myostatin inhibits myoblast differentiation by down-regulating MyoD expression.

    PubMed

    Langley, Brett; Thomas, Mark; Bishop, Amy; Sharma, Mridula; Gilmour, Stewart; Kambadur, Ravi

    2002-12-20

    Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3. In vitro, increasing concentrations of recombinant mature myostatin reversibly blocked the myogenic differentiation of myoblasts, cultured in low serum media. Western and Northern blot analysis indicated that addition of myostatin to the low serum culture media repressed the levels of MyoD, Myf5, myogenin, and p21 leading to the inhibition of myogenic differentiation. The transient transfection of C(2)C(12) myoblasts with MyoD expressing constructs did not rescue myostatin-inhibited myogenic differentiation. Myostatin signaling specifically induced Smad 3 phosphorylation and increased Smad 3.MyoD association, suggesting that Smad 3 may mediate the myostatin signal by interfering with MyoD activity and expression. Consistent with this, the expression of dominant-negative Smad3 rescued the activity of a MyoD promoter-reporter in C(2)C(12) myoblasts treated with myostatin. Taken together, these results suggest that myostatin inhibits MyoD activity and expression via Smad 3 resulting in the failure of the myoblasts to differentiate into myotubes. Thus we propose that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that lack functional myostatin is because of deregulated proliferation and differentiation of myoblasts.

  5. Low-level laser irradiation modulates cell viability and creatine kinase activity in C2C12 muscle cells during the differentiation process.

    PubMed

    Mesquita-Ferrari, Raquel Agnelli; Alves, Agnelo Neves; de Oliveira Cardoso, Vinicius; Artilheiro, Paola Pelegrineli; Bussadori, Sandra Kalil; Rocha, Lilia Alves; Nunes, Fábio Daumas; Fernandes, Kristianne Porta Santos

    2015-11-01

    Low-level laser irradiation (LLLI) is increasingly used to treat musculoskeletal disorders, with satisfactory results described in the literature. Skeletal muscle satellite cells play a key role in muscle regeneration. The aim of the present study was to evaluate the effect of LLLI on cell viability, creatine kinase (CK) activity, and the expression of myogenic regulatory factors in C2C12 myoblasts during the differentiation process. C2C12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 2% horse serum and submitted to irradiation with GaAlAs diode laser (wavelength, 780 nm; output power, 10 mW; energy density, 5 J/cm2). Cell viability and the expression of myogenic regulatory factors were assessed 24, 48, and 72 h after irradiation by 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide (MTT) assay and quantitative real-time polymerase chain reaction (RT-qPCR), respectively. CK activity was analyzed at 24 and 72 h. An increase in cell viability was found in the laser group in comparison to the control group at all evaluation times. CK activity was significantly increased in the laser group at 72 h. Myogenin messenger RNA (mRNA) demonstrated a tendency toward an increase in the laser group, but the difference in comparison to the control group was non-significant. In conclusion, LLLI was able to modulate cell viability and CK activity in C2C12 myoblasts during the differentiation process.

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

    PubMed Central

    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. 1α,25(OH)2D3-dependent modulation of Akt in proliferating and differentiating C2C12 skeletal muscle cells.

    PubMed

    Buitrago, Claudia G; Arango, Nadia S; Boland, Ricardo L

    2012-04-01

    We previously reported that 1α,25-dihydroxy-vitamin D(3) [1α,25(OH)(2)D(3)] induces non-transcriptional rapid responses through activation of Src and MAPKs in the skeletal muscle cell line C2C12. In the present study we investigated the modulation of Akt by the secosteroid hormone in C2C12 cells at proliferative stage (myoblasts) and at early differentiation stage. In proliferating cells, 1α,25(OH)(2)D(3) activates Akt by phosphorylation in Ser473 in a time-dependent manner (5-60 min). When these cells were pretreated with methyl-beta-cyclodextrin to disrupt caveolae microdomains, hormone-induced activation of Akt was suppressed. Similar results were obtained by siRNA silencing of caveolin-1 expression, further indicating that hormone effects on cell membrane caveolae are required for downstream signaling. PI3K and p38 MAPK, but not ERK1/2, participate in 1α,25(OH)(2)D(3) activation of Akt in myoblasts. The involvement of p38 MAPK in Akt phosphorylation by the hormone probably occurs through MAPK-activated protein kinase 2 (MK2), which is activated by the steroid. In addition, the participation of Src in Akt phosphorylation by 1α,25(OH)(2)D(3) was demonstrated using the inhibitor PP2 and antisense oligodeoxynucleotides that suppress Src expression. We also observed that PI3K participates in hormone-induced proliferation. During the early phase of C2C12 cell differentiation 1α,25(OH)(2)D(3) also increases Akt phosphorylation and activates Src. Of relevance, Src and PI3K are involved in Akt activation and in MHC and myogenin increased expression by 1α,25(OH)(2)D(3). Altogether, these data suggest that 1α,25(OH)(2)D(3) upregulates Akt through Src, PI(3)K, and p38 MAPK to stimulate myogenesis in C2C12 cells.

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

    PubMed

    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.

  9. Differential sensitivity of C2-C12 striated muscle cells to lovastatin and pravastatin.

    PubMed

    Gadbut, A P; Caruso, A P; Galper, J B

    1995-10-01

    One of the major side-effects of the use of HMG CoA reductase inhibitors for the treatment of hypercholesterolemia is the development of myositis and, in some patients undergoing concomitant immunosuppressive treatment, the development of rhabdomyolysis. Experiments outlined in these studies demonstrate that inhibitors of HMG-CoA reductase activity which differ primary in the substitution of a methyl group for a hydroxyl group have differential effects on both cholesterol levels and cell viability in a striated muscle cell model, the mouse C2-C12 myoblast. Thus, concentrations as high as 200 microM of pravastatin had little effect on total cholesterol level while 25 microM of lovastatin decreased cellular cholesterol by over 90%. Simvastatin and lovastatin decreased viability of C2-C12 myoblasts by nearly 50% at concentrations as low as 1 and 5 microM, respectively, and decreased viability by almost 90% at 10 and 15 microM respectively. However, 300 microM of pravastatin decreased cell viability by less than 50%. The order of potency for the effects on cell viability wassimvastatin>lovastatin>pravastatin. The possible relationship between effects on cell viability and the development of myositis is discussed.

  10. Screening of natural medicines that efficiently activate neurite outgrowth in PC12 cells in C2C12-cultured medium.

    PubMed

    Uezato, Tadayoshi; Sato, Eiji; Miura, Naoyuki

    2012-02-01

    We have studied the effects of natural medicines on neurite outgrowth in PC12D cells in a cultured medium of C2C12 cells. Derived from mouse myoblasts, the C2C12 cells secrete neurotrophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). The secretion of these neurotrophins from C2C12 cells stimulate neurite outgrowth in PC12D cells. We have screened a total of 120 samples and found five natural medicines: Trichosanthes Root, Asiasarum Root, Lycium Bark, Sinomenium Stem, and Dictamni radicis Cortex, that enhance the activity of C2C12-cultured medium to stimulate neurite outgrowth in PC12D cells. These natural medicines promoted not only neurite outgrowth but also stabilized the neurite formation in PC12D cells for several days. RT-PCR analysis showed that NGF was significantly increased with Trichosanthes and Lycium Bark. However, BDNF was slightly decreased with Lycium Bark, Sinomenium Stem, and Dictamni radicis Cortex. NT-3 was increased slightly by all of these natural medicines except Sinomenium Stem. All these five natural medicines significantly increased the number and length of neurites in PC12D cells in co-culture with C2C12 cells.

  11. GDF11 does not improve the palmitate induced insulin resistance in C2C12.

    PubMed

    Jing, Y-Y; Li, D; Wu, F; Gong, L-L; Li, R

    2017-04-01

    GDF11 (Growth Differentiation factor 11) has been reported to rejuvenate skeletal muscle, heart and brain in aged mice, and the aged skeletal muscle is closely related to insulin resistance. We wondered whether GDF11 has an effect on skeletal muscle insulin resistance. High fat diet induced obese mice with insulin resistance were established in vivo. Palmitate-induced insulin resistance in C2C12 myotubes was established in vitro. The mRNA expression of GDF11, GLUT4, IRS-1 (insulin receptor substrate-1) and PGC-1α (peroxisome proliferator-activated receptor-gamma coactivator 1) were tested by reverse transcriptase-polymerase chain reaction (RT-PCR). The protein level of GDF11 and PGC-1α were detected by Western blot. The glucose uptake was measured by 2NBDG uptake assay. In high fat diet induced obese mice, both serum level of GDF11 and the expression of GDF11 in skeletal muscle decreased. Similarly, the expression of GDF11 also reduced in palmitate-treated C2C12 myotubes. In vitro, the glucose uptake and the expression of GLUT4, IRS-1 and PGC-1α significantly decreased after palmitate intervention, but GDF11 treatment did not reverse the reduction of glucose uptake and the expression of GLUT4, IRS-1 and PGC-1α in C2C12 myotubes. We firstly confirmed that the expression of GDF11 decreased both in the skeletal muscle of obese mice and palmitate-treated myotubes, but supplementation GDF11 does not ameliorate the palmitate-induced insulin resistance in C2C12 myotubes.

  12. Palmdelphin promotes myoblast differentiation and muscle regeneration

    PubMed Central

    Nie, Yaping; Chen, Hu; Guo, Cilin; Yuan, Zhuning; Zhou, Xingyu; Zhang, Ying; Zhang, Xumeng; Mo, Delin; Chen, Yaosheng

    2017-01-01

    Differentiation of myoblasts is essential in the development and regeneration of skeletal muscles to form multinucleated, contractile muscle fibers. However, the process of myoblast differentiation in mammals is complicated and requires to be further investigated. In this study, we found Palmdelphin (Palmd), a cytosolic protein, promotes myoblast differentiation. Palmd is predominantly expressed in the cytosol of myoblasts and is gradually up-regulated after differentiation. Knockdown of Palmd by small interfering RNA (siRNA) in C2C12 markedly inhibits myogenic differentiation, suggesting a specific role of Palmd in the morphological changes of myoblast differentiation program. Overexpression of Palmd in C2C12 enhances myogenic differentiation. Remarkably, inhibition of Palmd results in impaired myotube formation during muscle regeneration after injury. These findings reveal a new cytosolic protein that promotes mammalian myoblast differentiation and provide new insights into the molecular regulation of muscle formation. PMID:28148961

  13. Tetramethylpyrazine protects palmitate-induced oxidative damage and mitochondrial dysfunction in C2C12 myotubes.

    PubMed

    Gao, Xin; Zhao, Xiao-long; Zhu, Yan-hui; Li, Xiao-mu; Xu, Qiong; Lin, Huan-dong; Wang, Ming-wei

    2011-04-25

    Tetramethylpyrazine (TMP), one of the active ingredients isolated from a Chinese herbal prescription, possesses protective effects against oxidative stress caused by high glucose in endothelial cells. In this study, the role of TMP in preventing muscle cells from palmitate-induced oxidative damage was investigated and the possible mechanisms of action elucidated. Mitochondrial reactive oxygen species (ROS) were measured in C2C12 myotubes, a palmitate-induced oxidative stress cell model, with or without TMP. Both mitochondrial membrane potential (MMP) and oxygen consumption were assessed in conjunction with quantification of mitochondrial DNA and mitochondrial biogenesis-related factors, such as peroxisome proliferator-activated receptor-γ coactivator 1 α (PGC1α), nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (Tfam), by real-time polymerase chain reaction. Expression of mitochondrial respiratory chain complex III as an index of mitochondrial function was evaluated by immunoblotting, and glucose transport into the C2C12 myotube examined by analyzing 2-deoxy-[(3)H]glucose uptake. TMP significantly alleviated palmitate-induced mitochondrial ROS production, mitigated mitochondrial dysfunction and increased D-loop mRNA expression as compared with the control. This was accompanied by a marked reversal of palmitate-induced down-regulation in the expression of mitochondrial biogenesis-related factors (PGC1α, NRF1 and Tfam) and decreased glucose uptake in C2C12 myotubes. As a result, cell respiration, as reflected by the elevated expression of mitochondrial respiratory chain complex III and oxygen consumption, was enhanced. TMP is capable of protecting C2C12 myotubes against palmitate-induced oxidative damage and mitochondrial dysfunction, and improving glucose uptake in muscle cells partially through the up-regulation of mitochondrial biogenesis. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Cobalt triggers necrotic cell death and atrophy in skeletal C2C12 myotubes

    SciTech Connect

    Rovetta, Francesca; Stacchiotti, Alessandra; Faggi, Fiorella; Catalani, Simona; Apostoli, Pietro; Fanzani, Alessandro; Aleo, Maria Francesca

    2013-09-01

    Severe poisoning has recently been diagnosed in humans having hip implants composed of cobalt–chrome alloys due to the release of particulate wear debris on polyethylene and ceramic implants which stimulates macrophagic infiltration and destroys bone and soft tissue, leading to neurological, sensorial and muscular impairments. Consistent with this premise, in this study, we focused on the mechanisms underlying the toxicity of Co(II) ions on skeletal muscle using mouse skeletal C2C12 myotubes as an in vitro model. As detected using propidium iodide incorporation, increasing CoCl{sub 2} doses (from 5 to 200 μM) affected the viability of C2C12 myotubes, mainly by cell necrosis, which was attenuated by necrostatin-1, an inhibitor of the necroptotic branch of the death domain receptor signaling pathway. On the other hand, apoptosis was hardly detectable as supported by the lack of caspase-3 and -8 activation, the latter resulting in only faint activation after exposure to higher CoCl{sub 2} doses for prolonged time points. Furthermore, CoCl{sub 2} treatment resulted in atrophy of the C2C12 myotubes which was characterized by the increased expression of HSP25 and GRP94 stress proteins and other typical 'pro-atrophic molecular hallmarks, such as early activation of the NF-kB pathway and down-regulation of AKT phosphorylation, followed by the activation of the proteasome and autophagy systems. Overall, these results suggested that cobalt may impact skeletal muscle homeostasis as an inducer of cell necrosis and myofiber atrophy. - Highlights: • The effects of cobalt on muscle myofibers in vitro were investigated. • Cobalt treatment mainly causes cell necrosis in skeletal C2C12 myotubes. • Cobalt impacts the PI3K/AKT and NFkB pathways and induces cell stress markers. • Cobalt induces atrophy of C2C12 myotubes through the activation of proteasome and autophagy systems. • Co treatment triggers NF-kB and PI3K/AKT pathways in C2C12 myotubes.

  15. Rosiglitazone, but not epigallocatechin-3-gallate, attenuates the decrease in PGC-1α protein levels in palmitate-induced insulin-resistant C2C12 cells.

    PubMed

    Karimfar, Mohammad Hassan; Haghani, Karimeh; Babakhani, Azar; Bakhtiyari, Salar

    2015-06-01

    Alteration of lipid metabolism is an important mechanism for the treatment of insulin resistance. PGC-1α, a key regulator of mitochondrial biogenesis and function, plays an important role in the improvement of insulin sensitivity by increasing fatty acids β-oxidation. In the present study, the effects of epigallocatechin-3-gallate (EGCG), an anti-obesity agent and enhancer of lipid catabolism, on PGC-1α protein expression was examined and compared with anti-diabetic drug rosiglitazone (RGZ). After differentiation of C2C12 myoblasts to myotubes, insulin resistance was induced by palmitate treatment. Then the expression of the PGC-1a gene and glucose uptake were evaluated before and after treatment with RGZ and EGCG. Palmitate treatment significantly decreased PGC-1α protein expression in C2C12 cells (P < 0.05). RGZ could restore the expression of PGC-1α in palmitate treated cells (P > 0.05), while EGCG had no significant effect on the expression of this gene (P < 0.05). RGZ and EGCG significantly improved glucose uptake (by 2- and 1.54-fold, respectively) in myotubes treated with palmitate. These data suggest that RGZ and EGCG both exert their anti-diabetic activity by increasing insulin sensitivity, but with different molecular mechanisms. This effect of RGZ, unlike EGCG, is mediated, at least partly, by increasing PGC-1α protein expression.

  16. Leucine Modulates Mitochondrial Biogenesis and SIRT1-AMPK Signaling in C2C12 Myotubes

    PubMed Central

    Liang, Chunzi; Curry, Benjamin J.; Brown, Patricia L.; Zemel, Michael B.

    2014-01-01

    Previous studies from this laboratory demonstrate that dietary leucine protects against high fat diet-induced mitochondrial impairments and stimulates mitochondrial biogenesis and energy partitioning from adipocytes to muscle cells through SIRT1-mediated mechanisms. Moreover, β-hydroxy-β-methyl butyrate (HMB), a metabolite of leucine, has been reported to activate AMPK synergistically with resveratrol in C2C12 myotubes. Therefore, we hypothesize that leucine-induced activation of SIRT1 and AMPK is the central event that links the upregulated mitochondrial biogenesis and fatty acid oxidation in skeletal muscle. Thus, C2C12 myotubes were treated with leucine (0.5 mM), alanine (0.5 mM), valine (0.5 mM), EX527 (SIRT1 inhibitor, 25 μM), and Compound C (AMPK inhibitor, 25 μM) alone or in combination to determine the roles of AMPK and SIRT1 in leucine-modulation of energy metabolism. Leucine significantly increased mitochondrial content, mitochondrial biogenesis-related genes expression, fatty acid oxidation, SIRT1 activity and gene expression, and AMPK phosphorylation in C2C12 myotubes compared to the controls, while EX527 and Compound C markedly attenuated these effects. Furthermore, leucine treatment for 24 hours resulted in time-dependent increases in cellular NAD+, SIRT1 activity, and p-AMPK level, with SIRT1 activation preceding that of AMPK, indicating that leucine activation of SIRT1, rather than AMPK, is the primary event. PMID:25400942

  17. Leptin rapidly activates PPARs in C2C12 muscle cells

    SciTech Connect

    Bendinelli, Paola; Piccoletti, Roberta . E-mail: Roberta.Piccoletti@unimi.it; Maroni, Paola

    2005-07-08

    Experimental evidence suggests that leptin operates on the tissues, including skeletal muscle, also by modulating gene expression. Using electrophoretic mobility shift assays, we have shown that physiological doses of leptin promptly increase the binding of C2C12 cell nuclear extracts to peroxisome proliferator-activated receptor (PPAR) response elements in oligonucleotide probes and that all three PPAR isoforms participate in DNA-binding complexes. We pre-treated C2C12 cells with AACOCF{sub 3}, a specific inhibitor of cytosolic phospholipase A{sub 2} (cPLA{sub 2}), an enzyme that supplies ligands to PPARs, and found that it abrogates leptin-induced PPAR DNA-binding activity. Leptin treatment significantly increased cPLA{sub 2} activity, evaluated as the release of [{sup 3}H]arachidonic acid from pre-labelled C2C12 cells, as well as phosphorylation. Further, using MEK1 inhibitor PD-98059 we showed that leptin activates cPLA{sub 2} through ERK induction. These results support a direct effect of leptin on skeletal muscle cells, and suggest that the hormone may modulate muscle transcription also by precocious activation of PPARs through ERK-cPLA{sub 2} pathway.

  18. IL-12 involvement in myogenic differentiation of C2C12 in vitro.

    PubMed

    Romanazzo, Sara; Forte, Giancarlo; Morishima, Keisuke; Taniguchi, Akiyoshi

    2015-03-01

    Recently, the extracellular microenvironment has been shown to be critical for the correct differentiation of stem cells to specific tissues. Many factors, including physical (e.g. biomaterial stiffness and topography) and biological (as growth factors, cytokines and chemokines) components, cooperate to create an ideal microenvironment for muscle stem cells, with many of these factors having been widely investigated. We previously demonstrated that the use of non-proliferating muscle-specific and unrelated cells as feeder layers for skeletal muscle progenitor cell differentiation resulted in significant differences in the ability to form myotubes, suggesting the importance of biological factors in myogenic differentiation. In this study, we investigated the biological factors involved in this process, analyzing the expression profile of 84 genes coding for cytokines and chemokines. We successfully identified a novel role for the cytokine IL-12 in the myogenic differentiation of C2C12 mouse skeletal muscle cells. Experiments involving the overexpression or silencing of the IL-12 gene in C2C12 showed that IL-12 enhanced the myogenic differentiation process. Moreover, when IL-12 was overexpressed in non-biologically related feeder cells, the new co-culture system was able to improve myogenic differentiation of C2C12 seeded on top. Although IL-12 is known to be a cytokine involved in inflammatory responses, it also appears to be involved in the myogenic differentiation process, acting as a positive regulator of this mechanism. This fact is expected to prove to be important for the development of functional biomaterials.

  19. In vitro drug testing based on contractile activity of C2C12 cells in an epigenetic drug model

    PubMed Central

    Ikeda, Kazushi; Ito, Akira; Imada, Ryusuke; Sato, Masanori; Kawabe, Yoshinori; Kamihira, Masamichi

    2017-01-01

    Skeletal muscle tissue engineering holds great promise for pharmacological studies. Herein, we demonstrated an in vitro drug testing system using tissue-engineered skeletal muscle constructs. In response to epigenetic drugs, myotube differentiation of C2C12 myoblast cells was promoted in two-dimensional cell cultures, but the levels of contractile force generation of tissue-engineered skeletal muscle constructs prepared by three-dimensional cell cultures were not correlated with the levels of myotube differentiation in two-dimensional cell cultures. In contrast, sarcomere formation and contractile activity in two-dimensional cell cultures were highly correlated with contractile force generation of tissue-engineered skeletal muscle constructs. Among the epigenetic drugs tested, trichostatin A significantly improved contractile force generation of tissue-engineered skeletal muscle constructs. Follistatin expression was also enhanced by trichostatin A treatment, suggesting the importance of follistatin in sarcomere formation of muscular tissues. These observations indicate that contractility data are indispensable for in vitro drug screening. PMID:28300163

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

    PubMed

    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.

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

    PubMed

    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.

  2. Cortisol effect on heat shock proteins in the C2C12 and 3T3-L1 cells.

    PubMed

    Ravikumar, Sambandam; Muthuraman, Pandurangan

    2014-08-01

    The present study was carried out to understand the effect of cortisol on heat shock protein system (Hsps) in the C2C12 and 3T3-L1 cells under co-culture system. Cells were co-cultured by using Transwell inserts with a 0.4-μm porous membrane to separate C2C12 and 3T3-L1 cells. Each cell type was grown independently on the Transwell plates. After cell differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates and inserts containing C2C12 cells transferred to 3T3-L1 plates. Ten micrograms per microliter of cortisol was added to the medium. Following 72 h of treatment, the cells in the lower wells were harvested for analysis. Heat shock proteins (Hsps) such as Hsp27, Hsp70, and Hsp90 were selected for the analysis. The qRT-PCR results showed the significant increase in the mRNA expression of as Hsp27, Hsp70, and Hsp90. In addition, confocal microscopical investigation showed the cortisol treatment increases Hsps expressions in the mono and co-cultured C2C12 and 3T3-L1 cells. From the results, we concluded that the cortisol increases Hsps expression in the co-cultured C2C12 and 3T3-L1 cells, which is differed from one-dimensional mono-cultured C2C12 and 3T3-L1 cells.

  3. Creatine Prevents the Structural and Functional Damage to Mitochondria in Myogenic, Oxidatively Stressed C2C12 Cells and Restores Their Differentiation Capacity

    PubMed Central

    Guescini, Michele; Calcabrini, Cinzia; Vallorani, Luciana; Diaz, Anna Rita; Canonico, Barbara; Luchetti, Francesca; Papa, Stefano; Battistelli, Michela; Falcieri, Elisabetta; Romanello, Vanina; Sandri, Marco; Stocchi, Vilberto; Ciacci, Caterina

    2016-01-01

    Creatine (Cr) is a nutritional supplement promoting a number of health benefits. Indeed Cr has been shown to be beneficial in disease-induced muscle atrophy, improve rehabilitation, and afford mild antioxidant activity. The beneficial effects are likely to derive from pleiotropic interactions. In accord with this notion, we previously demonstrated that multiple pleiotropic effects, including preservation of mitochondrial damage, account for the capacity of Cr to prevent the differentiation arrest caused by oxidative stress in C2C12 myoblasts. Given the importance of mitochondria in supporting the myogenic process, here we further explored the protective effects of Cr on the structure, function, and networking of these organelles in C2C12 cells differentiating under oxidative stressing conditions; the effects on the energy sensor AMPK, on PGC-1α, which is involved in mitochondrial biogenesis and its downstream effector Tfam were also investigated. Our results indicate that damage to mitochondria is crucial in the differentiation imbalance caused by oxidative stress and that the Cr-prevention of these injuries is invariably associated with the recovery of the normal myogenic capacity. We also found that Cr activates AMPK and induces an upregulation of PGC-1α expression, two events which are likely to contribute to the protection of mitochondrial quality and function. PMID:27610211

  4. Creatine Prevents the Structural and Functional Damage to Mitochondria in Myogenic, Oxidatively Stressed C2C12 Cells and Restores Their Differentiation Capacity.

    PubMed

    Barbieri, Elena; Guescini, Michele; Calcabrini, Cinzia; Vallorani, Luciana; Diaz, Anna Rita; Fimognari, Carmela; Canonico, Barbara; Luchetti, Francesca; Papa, Stefano; Battistelli, Michela; Falcieri, Elisabetta; Romanello, Vanina; Sandri, Marco; Stocchi, Vilberto; Ciacci, Caterina; Sestili, Piero

    2016-01-01

    Creatine (Cr) is a nutritional supplement promoting a number of health benefits. Indeed Cr has been shown to be beneficial in disease-induced muscle atrophy, improve rehabilitation, and afford mild antioxidant activity. The beneficial effects are likely to derive from pleiotropic interactions. In accord with this notion, we previously demonstrated that multiple pleiotropic effects, including preservation of mitochondrial damage, account for the capacity of Cr to prevent the differentiation arrest caused by oxidative stress in C2C12 myoblasts. Given the importance of mitochondria in supporting the myogenic process, here we further explored the protective effects of Cr on the structure, function, and networking of these organelles in C2C12 cells differentiating under oxidative stressing conditions; the effects on the energy sensor AMPK, on PGC-1α, which is involved in mitochondrial biogenesis and its downstream effector Tfam were also investigated. Our results indicate that damage to mitochondria is crucial in the differentiation imbalance caused by oxidative stress and that the Cr-prevention of these injuries is invariably associated with the recovery of the normal myogenic capacity. We also found that Cr activates AMPK and induces an upregulation of PGC-1α expression, two events which are likely to contribute to the protection of mitochondrial quality and function.

  5. Regulation of myostatin expression and myoblast differentiation by FoxO and SMAD transcription factors.

    PubMed

    Allen, David L; Unterman, Terry G

    2007-01-01

    Myostatin, a member of the transforming growth factor (TGF)-beta family, plays an important role in regulating skeletal muscle growth and differentiation. Here we examined the role of FoxO1 and SMAD transcription factors in regulating myostatin gene expression and myoblast differentiation in C(2)C(12) myotubes in vitro. Both myostatin and FoxO1 mRNA expression were greater in fast- vs. slow-twitch skeletal muscles in vivo. Moreover, expression of a constitutively active form of FoxO1 increased myostatin mRNA and increased activity of a myostatin promoter reporter construct in differentiated C(2)C(12) myotubes. Mutagenesis of highly conserved FoxO or SMAD binding sites significantly decreased myostatin promoter activity, and binding assays showed that both FoxO1 and SMADs bind to their respective sites in the myostatin promoter. Treatment with TGF-beta and/or overexpression of SMAD2, -3, or -4 also resulted in a significant increase in myostatin promoter activity. Treatment with TGF-beta along with overexpression of SMAD2 and FoxO1 resulted in the largest increase in myostatin promoter activity. Finally, TGF-beta treatment and SMAD2 overexpression greatly potentiated FoxO1-mediated suppression of myoblast differentiation. Together these data demonstrate that FoxO1 and SMAD transcription factors regulate the expression of myostatin and contribute to the control of muscle cell growth and differentiation.

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

    NASA Astrophysics Data System (ADS)

    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. Effects of type IV collagen on myogenic characteristics of IGF-I gene-engineered myoblasts.

    PubMed

    Ito, Akira; Yamamoto, Masahiro; Ikeda, Kazushi; Sato, Masanori; Kawabe, Yoshinori; Kamihira, Masamichi

    2015-05-01

    Skeletal muscle regeneration requires migration, proliferation and fusion of myoblasts to form multinucleated myotubes. In our previous study, we showed that insulin-like growth factor (IGF)-I gene delivery stimulates the proliferation and differentiation of mouse myoblast C2C12 cells and promotes the contractile force generated by tissue-engineered skeletal muscles. The aim of this study was to investigate the effects of the extracellular matrix on IGF-I gene-engineered C2C12 cells in vitro. Retroviral vectors for doxycycline (Dox)-inducible expression of the IGF-I gene were transduced into C2C12 cells. When cultured on a type IV collagen-coated surface, we observed significant increases in the migration speed and number of IGF-I gene-engineered C2C12 cells with Dox addition, designated as C2C12/IGF (+) cells. Co-culture of C2C12/IGF (+) cells and parental C2C12 cells, which had been cultured in differentiation medium for 3 days, greatly enhanced myotube formation. Moreover, type IV collagen supplementation promoted the fusion of C2C12/IGF (+) cells with differentiated C2C12 cells and increased the number of myotubes with striations. Myotubes formed by C2C12/IGF (+) cells cultured on type IV collagen showed a dynamic contractile activity in response to electrical pulse stimulation. These findings indicate that type IV collagen promotes skeletal muscle regeneration mediated by IGF-I-expressing myoblasts, which may have important clinical implications in the design of myoblast-based therapies.

  8. Independent AMP and NAD signaling regulates C2C12 differentiation and metabolic adaptation.

    PubMed

    Hsu, Chia George; Burkholder, Thomas J

    2016-12-01

    The balance of ATP production and consumption is reflected in adenosine monophosphate (AMP) and nicotinamide adenine dinucleotide (NAD) content and has been associated with phenotypic plasticity in striated muscle. Some studies have suggested that AMPK-dependent plasticity may be an indirect consequence of increased NAD synthesis and SIRT1 activity. The primary goal of this study was to assess the interaction of AMP- and NAD-dependent signaling in adaptation of C2C12 myotubes. Changes in myotube developmental and metabolic gene expression were compared following incubation with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and nicotinamide mononucleotide (NMN) to activate AMPK- and NAD-related signaling. AICAR showed no effect on NAD pool or nampt expression but significantly reduced histone H3 acetylation and GLUT1, cytochrome C oxidase subunit 2 (COX2), and MYH3 expression. In contrast, NMN supplementation for 24 h increased NAD pool by 45 % but did not reduce histone H3 acetylation nor promote mitochondrial gene expression. The combination of AMP and NAD signaling did not induce further metabolic adaptation, but NMN ameliorated AICAR-induced myotube reduction. We interpret these results as indication that AMP and NAD contribute to C2C12 differentiation and metabolic adaptation independently.

  9. TNF-α and IFN-s-Dependent Muscle Decay Is Linked to NF-κB- and STAT-1α-Stimulated Atrogin1 and MuRF1 Genes in C2C12 Myotubes

    PubMed Central

    Pijet, Barbara; Pijet, Maja; Litwiniuk, Anna; Gajewska, Małgorzata; Pająk, Beata

    2013-01-01

    TNF-α was shown to stimulate mitogenicity in C2C12 myoblasts. Selected cytokines TNF-α, IFNα, or IFNγ reduced the expression of myosin heavy chain (MyHC IIa) when given together. Molecular mechanisms of cytokine activities were controlled by NF-κB and JAK/STAT signaling pathways, as metabolic inhibitors, curcumin and AG490, inhibited some of TNF-α and IFNα/IFNγ effects. Insulin was hardly antagonistic to TNF-α- and IFNα/IFNγ-dependent decrease in MyHC IIa protein expression. Cytokines used individually or together also repressed myogenesis of C2C12 cells. Moreover, TNF-α- and IFNα/IFNγ-dependent effects on C2C12 myotubes were associated with increased activity of Atrogin1 and MuRF1 genes, which code ubiquitin ligases. MyHC IIa gene activity was unaltered by cytokines. Inhibition of NF-κB or JAK/STAT with specific metabolic inhibitors decreased activity of Atrogin1 and MuRF1 but not MyHC IIa gene. Overall, these results suggest cooperation between cytokines in the reduction of MyHC IIa protein expression level via NF-κB/JAK/STAT signaling pathways and activation of Atrogin1 and MuRF1 genes as their molecular targets. Insulin cotreatment or pretreatment does not protect against muscle decay induced by examined proinflammatory cytokines. PMID:24453411

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

    PubMed

    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.

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

    PubMed Central

    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. PMID:28123341

  12. Caffeine and nicotine decrease acetylcholine receptor clustering in C2C12 myotube culture.

    PubMed

    Kordosky-Herrera, Kaia; Grow, Wade A

    2009-02-01

    As motor neurons approach skeletal muscle during development, agrin is released and induces acetylcholine receptor (AChR) clustering. Our laboratory investigates the effect of environmental agents on skeletal muscle development by using C2C12 cell culture. For the current project, we investigated both short-term and long-term exposure to caffeine, nicotine, or both, at physiologically relevant concentrations. Short-term exposure was limited to the last 48 h of myotube formation, whereas a long-term exposure of 2 weeks allowed for several generations of myoblast proliferation followed by myotube formation. Both agrin-induced and spontaneous AChR clustering frequencies were assessed. For agrin-induced AChR clustering, agrin was added for the last 16 h of myotube formation. Caffeine, nicotine, or both significantly decreased agrin-induced AChR clustering during short-term and long-term exposure. Furthermore, caffeine, nicotine, or both significantly decreased spontaneous AChR clustering during long-term, but not short-term exposure. Surprisingly, caffeine and nicotine in combination did not decrease AChR clustering beyond the effect of either treatment alone. We conclude that physiologically relevant concentrations of caffeine or nicotine decrease AChR clustering. Moreover, we predict that fetuses exposed to caffeine or nicotine may be less likely to form appropriate neuromuscular synapses.

  13. PUGNAc induces protein ubiquitination in C2C12 myotube cells.

    PubMed

    Park, Ja-Hye; Lee, Jeong-Eun; Moon, Pyong-Gon; Baek, Moon-Chang

    2015-12-01

    O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) regulates many cellular processes including the cell cycle, cell signaling, and protein trafficking. Dysregulation of O-GlcNAcylation may be involved in the development of insulin resistance and type 2 diabetes. Therefore, it is necessary to identify cellular proteins that are induced by elevated O-GlcNAcylation. Here, using adenosine 5'-triphosphate affinity chromatography, we employed a proteomic approach in order to identify differentially expressed proteins in response to treatment with the O-GlcNAcase inhibitor, O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), in mouse C2C12 myotube cells. Among 205 selected genes, we identified 68 nucleotide-binding proteins, 14 proteins that have adenosinetriphosphatase activity, and 10 proteins with ligase activity. Upregulation of proteins, including ubiquitin-activating enzyme E1, proteasome subunit 20S, cullin-associated NEDD8-dissociated protein 1, ezrin, and downregulation of the protein nucleoside diphosphate kinase B, were confirmed by western blot analysis. In particular, we found that the protein ubiquitination level in C2C12 cells was increased by PUGNAc treatment. This is the first report of quantitative proteomic profiles of myotube cells after treatment with PUGNAc, and our results demonstrate the potential to enhance understanding of the relationship between insulin resistance, O-GlcNAc, and PUGNAc in the future. Copyright © 2015 John Wiley & Sons, Ltd.

  14. Expression of Non-acetylatable H2A.Z in Myoblast Cells Blocks Myoblast Differentiation through Disruption of MyoD Expression*

    PubMed Central

    Law, Cindy; Cheung, Peter

    2015-01-01

    H2A.Z is a histone H2A variant that is essential for viability in Tetrahymena and Drosophila and also during embryonic development of mice. Although implicated in diverse cellular processes, including transcriptional regulation, chromosome segregation, and heterochromatin formation, its essential function in cells remains unknown. Cellular differentiation is part of the developmental process of multicellular organisms. To elucidate the roles of H2A.Z and H2A.Z acetylation in cellular differentiation, we examined the effects of expressing wild type (WT) or a non-acetylatable form of H2A.Z in the growth and differentiation of the myoblast C2C12 cell line. Ectopic expression of wild type or mutant H2A.Z resulted in distinct phenotypes in the differentiation of the C2C12 cells and the formation of myotubes. Most strikingly, expression of the H2A.Z non-acetylatable mutant (H2A.Z-Ac-mut) resulted in a complete block of myoblast differentiation. We determined that this phenotype is caused by a loss of MyoD expression in the Ac-mut-expressing cells prior to and after induction of differentiation. Moreover, chromatin accessibility assays showed that the promoter region of MyoD is less accessible in the differentiation-defective cells. Altogether, these new findings show that expression of the Ac-mut form of H2A.Z resulted in a dominant phenotype that blocked differentiation due to chromatin changes at the MyoD promoter. PMID:25839232

  15. Extracellular guanosine-5'-triphosphate modulates myogenesis via intermediate Ca(2+)-activated K+ currents in C2C12 mouse cells.

    PubMed

    Pietrangelo, Tiziana; Fioretti, Bernard; Mancinelli, Rosa; Catacuzzeno, Luigi; Franciolini, Fabio; Fanò, Giorgio; Fulle, Stefania

    2006-05-01

    In this study we investigated the role of extracellular 5'-guanosine-triphosphate (GTP) on early phases of skeletal muscle differentiation using the widely used C2C12 mouse cells as a myogenic model. We show that extracellular GTP binding to specific sites activates a metabotropic cascade that leads to a transient intracellular Ca2+ mobilization, consequent activation of the intermediate Ca(2+)-activated K+ channels (IK(Ca)), and hyperpolarization of the plasma membrane. We further show that in differentiating C2C12 myoblasts GTP induces a proliferative boost, and increases the number of cells positive for the myosin heavy chain (MyHC) proteins. These effects were shown to be mediated by the IK(Ca) channel-dependent hyperpolarization, as evidenced by their disappearance when myoblasts were incubated with the IK(Ca) channel inhibitor charybdotoxin. These data give new insights into nucleotide purinergic signalling pathways, and address the role of the GTP-dependent IK(Ca) channel activation and hyperpolarization in myogenesis.

  16. Quantitative evaluation method for differentiation of C2C12 myoblasts by ultrasonic microscopy

    NASA Astrophysics Data System (ADS)

    Takanashi, Kyoichi; Washiya, Mamoru; Ota, Kazuki; Yoshida, Sachiko; Hozumi, Naohiro; Kobayashi, Kazuto

    2017-07-01

    Cell differentiation was evaluated by ultrasonic microscopy. However, there were some regions that showed a lower acoustic impedance than the culture liquid. It was considered that, in such regions, the cells were not perfectly in contact with the film substrate. Hence, a waveform analysis was performed, and compensated acoustic impedances in such regions were in a reasonable range of values. By the same analysis, the displacements of partially floated cells were also successfully calculated. The elapsed day transitions of the compensated acoustic impedances and displacements were successfully evaluated. In the process of differentiation, actin fibers comprising the cytoskeleton are supposed to loosen in order to induce cellular fusion. In addition, the progress in cell differentiation accompanied by a change into a three-dimensional structure can partially be assessed by the displacement between a cell and a cultured film. Hence, we believe that cell differentiation can be evaluated using an ultrasonic microscope.

  17. 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.

    PubMed

    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.

  18. 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

    PubMed Central

    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

    2014-01-01

    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. PMID:24550007

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

    SciTech Connect

    Okamoto, Michio; Tanaka, Hiroyuki; Okada, Kiyoshi; Kuroda, Yusuke; Nishimoto, Shunsuke; Murase, Tsuyoshi; Yoshikawa, Hideki

    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.

  20. Insulin Sensitizing Effects of Oligomannuronate-Chromium (III) Complexes in C2C12 Skeletal Muscle Cells

    PubMed Central

    Wang, Wei; Han, Zhangrun; Li, Guangsheng; Zhang, Lijuan; Zhao, Xia; Yu, Guangli

    2011-01-01

    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 insulin

  1. Systemic delivery of recombinant proteins by genetically modified myoblasts

    SciTech Connect

    Barr, E.; Leiden, J.M. )

    1991-12-06

    The ability to stably deliver recombinant proteins to the systemic circulation would facilitate the treatment of a variety of acquired and inherited diseases. To explore the feasibility of the use of genetically engineered myoblasts as a recombinant protein delivery system, stable transfectants of the murine C2C12 myoblast cell line were produced that synthesize and secrete high levels of human growth hormone (hGH) in vitro. Mice injected with hGH-transfected myoblasts had significant levels of hGH in both muscle and serum that were stable for at least 3 weeks after injection. Histological examination of muscles injected with {beta}-galactosidase-expressing C2C12 myoblasts demonstrated that many of the injected cells had fused to form multinucleated myotubes. Thus, genetically engineered myoblasts can be used for the stable delivery of recombinant proteins into the circulation.

  2. [Effects of mechanical stimulation on expression of autoantigens in myoblasts].

    PubMed

    Chen, Rong; Liu, Xinghui; Huang, Weiyi; Zeng, Huijun; Shi, Dandan; Cao, Biao; Liao, Hua

    2013-09-01

    To explore the effects of mechanical stimulation on the expression of autoantigens in myoblasts. According to different processing methods, C2C12 cells were divided into the experimental group and control group; the experimental group was divided into 4 subgroups: 2-, 4-, and 6-day and 1-day stretch groups. In 2-, 4-, and 6-day stretch groups, mechanical loading was added on the C2C12 cells at a stretching frequency of 0.25 Hz and cellular deformation amplitude of 10%, 2 hours a day for 2, 4, and 6 days respectively by Flexercell 5000 strain unit, and at a stretching frequency of 1 Hz and cellular deformation amplitude of 15% for 1 hour in 1-day stretch group. In the control group, the cells were routinely cultured for 1, 2, 4, and 6 days (1-, 2-, 4-, and 6-day control). The cells were observed by inverted phase contrast microscope. The cell proliferation was detected by flow cytometry; the expressions of autoantigens were detected by Western blot method, including the Ku/the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), U1-70 (A part of ATP-dependent DNA helicase II), histidyl tRNA synthetase (HRS), and Mi-2 (reconfigurable components deacetylase complexes of NuRD). The exfoliated cells were found in 1-day stretch group, but no exfoliated cell was seen in the control group for 1-day culture. The cells proliferated more obviously in 2-day stretch group than in the control group for 2-day culture; cell differentiation was found in 4-day stretch group, and cell fusion in 6-day stretch group, which were similar to those in the control group for 4- and 6-day culture. After single stretching, cell apoptosis was found in 1-day stretch group, showing no significant difference in the relative DNA proliferation index (DPI) when compared with DPI of control group for 1-day culture (t = 0.346, P = 0.747). After cyclic stretching, DPIs of 2- and 4- day stretch groups were significantly increased when compared with those of the control group for 2- and 4-day

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

    PubMed Central

    2011-01-01

    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. PMID:21943021

  4. Arachidonic acid activates release of calcium ions from reticulum via ryanodine receptor channels in C2C12 skeletal myotubes.

    PubMed

    Muslikhov, E R; Sukhanova, I F; Avdonin, P V

    2014-05-01

    Arachidonic acid causes an increase in free cytoplasmic calcium concentration ([Ca2+]i) in differentiated skeletal multinucleated myotubes C2C12 and does not induce calcium response in C2C12 myoblasts. The same reaction of myotubes to arachidonic acid is observed in Ca2+-free medium. This indicates that arachidonic acid induces release of calcium ions from intracellular stores. The blocker of ryanodine receptor channels of sarcoplasmic reticulum dantrolene (20 µM) inhibits this effect by 68.7 ± 6.3% (p < 0.001). The inhibitor of two-pore calcium channels of endolysosomal vesicles trans-NED19 (10 µM) decreases the response to arachidonic acid by 35.8 ± 5.4% (p < 0.05). The phospholipase C inhibitor U73122 (10 µM) has no effect. These data indicate the involvement of ryanodine receptor calcium channels of sarcoplasmic reticulum in [Ca2+]i elevation in skeletal myotubes caused by arachidonic acid and possible participation of two-pore calcium channels from endolysosomal vesicles in this process.

  5. Down-regulation of uncoupling protein-3 and -2 by thiazolidinediones in C2C12 myotubes.

    PubMed

    Cabrero, A; Alegret, M; Sánchez, R M; Adzet, T; Laguna, J C; Vázquez, M

    2000-10-27

    Uncoupling proteins (UCPs) are mitochondrial membrane proton transporters that uncouple respiration from oxidative phosphorylation by dissipating the proton gradient across the membrane. We studied the direct effect of several peroxisome proliferator-activated receptor (PPAR) ligands on UCP-3 and UCP-2 mRNA expression in C2C12 myotubes for 24 h. In the absence of exogenous fatty acids, treatment of C2C12 cells with a selective PPARalpha activator (Wy-14,643) or a non-selective PPAR activator (bezafibrate) did not affect the expression of UCP-3 mRNA levels, whereas UCP-2 expression was slightly increased. In contrast, troglitazone, a thiazolidinedione which selectively activates PPARgamma, strongly decreased UCP-3 and UCP-2 mRNA levels. Another thiazolidinedione, ciglitazone, had the same effect, but to a lower extent, suggesting that PPARgamma activation is involved. Further, the presence of 0.5 mM oleic acid strongly increased UCP-3 mRNA levels and troglitazone addition failed to block the effect of this fatty acid. The drop in UCP expression after thiazolidinedione treatment correlated well with a reduction in PPARalpha mRNA levels produced by this drug, linking the reduction in PPARalpha mRNA levels with the down-regulation of UCP mRNA in C2C12 myotubes after thiazolidinedione treatment.

  6. The Roots of Atractylodes macrocephala Koidzumi Enhanced Glucose and Lipid Metabolism in C2C12 Myotubes via Mitochondrial Regulation

    PubMed Central

    Song, Mi Young; Kang, Seok Yong; Oh, Tae Woo; Kumar, Rethineswaran Vinoth; Jung, Hyo Won; Park, Yong-Ki

    2015-01-01

    The root of Atractylodes macrocephala Koidzumi (Atractylodis Rhizoma Alba, ARA) is a Traditional Korean Medicine and has been commonly used for weight control. Mitochondrial dysfunction appears to be a key contributor to insulin resistance, and therefore mitochondrial targeting drugs represent an important potential strategy for the treatment of insulin resistance and obesity. In this study, the authors investigated the regulatory effects of ARA on mitochondrial function with respect to the stimulation of glucose and lipid metabolism in C2C12 myotubes. After differentiating C2C12 myotubes, cells were treated with or without different concentrations (0.2, 0.5, and 1.0 mg/mL) of ARA extract. ARA extract significantly increased the expression of peroxisome proliferator-activated receptor coactivator 1 alpha (PGC1α) and the downregulations of its targets, nuclear respiratory factor-1 (NRF-1), transcription factor A (TFAM), and total ATP content in C2C12 myotubes. ARA extract also increased the expressions of PGC1α activator and of the metabolic sensors, AMP-activated protein kinase (AMPK), and acetyl-CoA carboxylase and sirtuin (SIRT) 1. Furthermore, it significantly increased glucose uptake by enhancing glucose consumption and subsequently decreased FFA contents and increased carnitine palmitoyltransferase (CPT) 1b expression. Our study indicates that ARA has a potential for stimulating mitochondrial function and energy metabolism in muscle. PMID:26617661

  7. microRNA133a Targets Foxl2 and Promotes Differentiation of C2C12 into Myogenic Progenitor Cells

    PubMed Central

    Luo, Yueqiu; Wu, Xiaoxing; Ling, Zongxin; Yuan, Li; Cheng, Yiwen; Chen, Jingyang

    2015-01-01

    microRNAs are endogenous noncoding RNA molecules of ∼22 nucleotides that regulate gene function by modification of target mRNAs. Due to tissue specific of miR-133a and miR-1/206 for skeletal muscles, we investigated the role of miR-133a and miR-1/206 in promoting the differentiation of the C2C12 cells. The results show that directly transfecting mature miR-133a, miR-1/206, or combinations (miR-1 and miR-206, miR-1 and miR-133a, and miR-133a and miR-206) into C2C12 cells, respectively, for 5 days induces formation of myogenic progenitor cells. Overexpression of miR-133a and miR-206 in C2C12 cells greatly improved multinucleated myotube formation. microRNA-133a (miR-133a) is highly expressed during human muscle development. Using bioinformatics, we identified one putative miR-133a binding site within the 3′-untranslated region of the mouse Foxl2 mRNA. The expression of Foxl2 was shown to be downregulated by subsequent western blot analysis. PMID:25317675

  8. microRNA133a targets Foxl2 and promotes differentiation of C2C12 into myogenic progenitor cells.

    PubMed

    Luo, Yueqiu; Wu, Xiaoxing; Ling, Zongxin; Yuan, Li; Cheng, Yiwen; Chen, Jingyang; Xiang, Charlie

    2015-01-01

    microRNAs are endogenous noncoding RNA molecules of ∼22 nucleotides that regulate gene function by modification of target mRNAs. Due to tissue specific of miR-133a and miR-1/206 for skeletal muscles, we investigated the role of miR-133a and miR-1/206 in promoting the differentiation of the C2C12 cells. The results show that directly transfecting mature miR-133a, miR-1/206, or combinations (miR-1 and miR-206, miR-1 and miR-133a, and miR-133a and miR-206) into C2C12 cells, respectively, for 5 days induces formation of myogenic progenitor cells. Overexpression of miR-133a and miR-206 in C2C12 cells greatly improved multinucleated myotube formation. microRNA-133a (miR-133a) is highly expressed during human muscle development. Using bioinformatics, we identified one putative miR-133a binding site within the 3'-untranslated region of the mouse Foxl2 mRNA. The expression of Foxl2 was shown to be downregulated by subsequent western blot analysis.

  9. Resistin Increases Ectopic Deposition of Lipids Through miR-696 in C2C12 Cells.

    PubMed

    Wen, Fengyun; Zhang, Haiwei; Bao, Chen; Yang, Mengshi; Wang, Nan; Zhang, Jie; Hu, Yajie; Yang, Xi; Geng, Jinjing; Yang, Zaiqing

    2015-06-01

    Resistin is associated with ectopic deposition of lipids, and determining its developmental and molecular mechanisms may help in the development of novel treatments. MicroRNAs (miRNAs) are involved in many physiological and pathological processes as negative regulators. We performed mouse liver miRNA microarrays to analyze the differences in expression between resistin-treated and control mice; the results showed that miR-696 was significantly upregulated by resistin. Therefore, we aimed to study whether miR-696 played a role in the resistin-induced ectopic deposition of lipids. Quantitative RT-PCR results showed that miR-696 was upregulated both in vivo and in vitro, consistent with the microarray. We transfected C2C12 cells and used miR-696 mimics and inhibitors to assess the role of miR-696 in the resistin-induced ectopic deposition of lipids. The overexpression of miR-696 increased the TG content in C2C12 cells and decreased the mitochondrial content. Next, a study of miR-696's role in the deposition of lipids in C2C12 induced by resistin showed that inhibition of miR-696 restored the TG content by up to 80%, which suggests that, in C2C12 cells, resistin at least partially increases the deposition of lipids through miR-696. miR-696 plays a role in the ectopic deposition of lipids induced by resistin in skeletal muscle at least partially.

  10. Enhanced contractile force generation by artificial skeletal muscle tissues using IGF-I gene-engineered myoblast cells.

    PubMed

    Sato, Masanori; Ito, Akira; Kawabe, Yoshinori; Nagamori, Eiji; Kamihira, Masamichi

    2011-09-01

    The aim of this study was to investigate whether insulin-like growth factor (IGF)-I gene delivery to myoblast cells promotes the contractile force generated by hydrogel-based tissue-engineered skeletal muscles in vitro. Two retroviral vectors allowing doxycycline (Dox)-inducible expression of the IGF-I gene were transduced into mouse myoblast C2C12 cells to evaluate the effects of IGF-I gene expression on these cells. IGF-I gene expression stimulated the proliferation of C2C12 cells, and a significant increase in the growth rate was observed for IGF-I-transduced C2C12 cells with Dox addition, designated C2C12/IGF (Dox+) cells. Quantitative morphometric analyses showed that the myotubes induced from C2C12/IGF (Dox+) cells had a larger area and a greater width than control myotubes induced from normal C2C12 cells. Artificial skeletal muscle tissues were prepared from the respective cells using hydrogels composed of type I collagen and Matrigel. Western blot analyses revealed that the C2C12/IGF (Dox+) tissue constructs showed activation of a skeletal muscle hypertrophy marker (Akt) and enhanced expression of muscle-specific markers (myogenin, myosin heavy chain and tropomyosin). Moreover, the creatine kinase activity was increased in the C2C12/IGF (Dox+) tissue constructs. The C2C12/IGF (Dox+) tissue constructs contracted in response to electrical pulses, and generated a significantly higher physical force than the control C2C12 tissue constructs. These findings indicate that IGF-I gene transfer has the potential to yield functional skeletal muscle substitutes that are capable of in vivo restoration of the load-bearing function of injured muscle or acting as in vitro electrically-controlled bio-actuators. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. FOXO1 and GSK-3β Are Main Targets of Insulin-Mediated Myogenesis in C2C12 Muscle Cells

    PubMed Central

    Litwiniuk, Anna; Pijet, Barbara; Pijet-Kucicka, Maja; Gajewska, Małgorzata; Pająk, Beata; Orzechowski, Arkadiusz

    2016-01-01

    Myogenesis and muscle hypertrophy account for muscle growth and adaptation to work overload, respectively. In adults, insulin and insulin-like growth factor 1 stimulate muscle growth, although their links with cellular energy homeostasis are not fully explained. Insulin plays critical role in the control of mitochondrial activity in skeletal muscle cells, and mitochondria are essential for insulin action. The aim of this study was to elucidate molecular mechanism(s) involved in mitochondrial control of insulin-dependent myogenesis. The effects of several metabolic inhibitors (LY294002, PD98059, SB216763, LiCl, rotenone, oligomycin) on the differentiation of C2C12 myoblasts in culture were examined in the short-term (hours) and long-term (days) experiments. Muscle cell viability and mitogenicity were monitored and confronted with the activities of selected genes and proteins expression. These indices focus on the roles of insulin, glycogen synthase kinase 3 beta (GSK-3β) and forkhead box protein O1 (FOXO1) on myogenesis using a combination of treatments and inhibitors. Long-term insulin (10 nM) treatment in “normoglycemic” conditions led to increased myogenin expression and accelerated myogenesis in C2C12 cells. Insulin-dependent myogenesis was accompanied by the rise of mtTFA, MtSSB, Mfn2, and mitochondrially encoded Cox-1 gene expressions and elevated levels of proteins which control functions of mitochondria (kinase—PKB/AKT, mitofusin 2 protein—Mfn-2). Insulin, via the phosphatidylinositol 3-kinase (PI3-K)/AKT-dependent pathway reduced transcription factor FOXO1 activity and altered GSK-3β phosphorylation status. Once FOXO1 and GSK-3β activities were inhibited the rise in Cox-1 gene action and nuclear encoded cytochrome c oxidase subunit IV (COX IV) expressions were observed, even though some mRNA and protein results varied. In contrast to SB216763, LiCl markedly elevated Mfn2 and COX IV protein expression levels when given together with insulin. Thus

  12. FOXO1 and GSK-3β Are Main Targets of Insulin-Mediated Myogenesis in C2C12 Muscle Cells.

    PubMed

    Litwiniuk, Anna; Pijet, Barbara; Pijet-Kucicka, Maja; Gajewska, Małgorzata; Pająk, Beata; Orzechowski, Arkadiusz

    2016-01-01

    Myogenesis and muscle hypertrophy account for muscle growth and adaptation to work overload, respectively. In adults, insulin and insulin-like growth factor 1 stimulate muscle growth, although their links with cellular energy homeostasis are not fully explained. Insulin plays critical role in the control of mitochondrial activity in skeletal muscle cells, and mitochondria are essential for insulin action. The aim of this study was to elucidate molecular mechanism(s) involved in mitochondrial control of insulin-dependent myogenesis. The effects of several metabolic inhibitors (LY294002, PD98059, SB216763, LiCl, rotenone, oligomycin) on the differentiation of C2C12 myoblasts in culture were examined in the short-term (hours) and long-term (days) experiments. Muscle cell viability and mitogenicity were monitored and confronted with the activities of selected genes and proteins expression. These indices focus on the roles of insulin, glycogen synthase kinase 3 beta (GSK-3β) and forkhead box protein O1 (FOXO1) on myogenesis using a combination of treatments and inhibitors. Long-term insulin (10 nM) treatment in "normoglycemic" conditions led to increased myogenin expression and accelerated myogenesis in C2C12 cells. Insulin-dependent myogenesis was accompanied by the rise of mtTFA, MtSSB, Mfn2, and mitochondrially encoded Cox-1 gene expressions and elevated levels of proteins which control functions of mitochondria (kinase--PKB/AKT, mitofusin 2 protein--Mfn-2). Insulin, via the phosphatidylinositol 3-kinase (PI3-K)/AKT-dependent pathway reduced transcription factor FOXO1 activity and altered GSK-3β phosphorylation status. Once FOXO1 and GSK-3β activities were inhibited the rise in Cox-1 gene action and nuclear encoded cytochrome c oxidase subunit IV (COX IV) expressions were observed, even though some mRNA and protein results varied. In contrast to SB216763, LiCl markedly elevated Mfn2 and COX IV protein expression levels when given together with insulin. Thus

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

    PubMed Central

    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

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

    PubMed

    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 Ca(2+) concentration ([Ca(2+)]i). Putative cell membrane pathways involved in these [Ca(2+)]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 Ca(2+). 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 [Ca(2+)]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.

  15. Proliferation and skeletal myotube formation capability of C2C12 and H9c2 cells on isotropic and anisotropic electrospun nanofibrous PHB scaffolds.

    PubMed

    Ricotti, Leonardo; Polini, Alessandro; Genchi, Giada G; Ciofani, Gianni; Iandolo, Donata; Vazão, Helena; Mattoli, Virgilio; Ferreira, Lino; Menciassi, Arianna; Pisignano, Dario

    2012-06-01

    This study aims at investigating the behavior in terms of the proliferation and skeletal muscle differentiation capability of two myoblastic cell lines, C2C12 and H9c2, on both isotropic and anisotropic electrospun nanofibrous poly(hydroxybutyrate) (PHB) scaffolds, as well as on PHB films and polystyrene controls. After a careful characterization of the matrices in terms of surface morphology, surface roughness and mechanical properties, the proliferation rate and the capability of the two cell lines to form skeletal myotubes were evaluated. Genetic analyses were also performed in order to assess the differentiation level of the cells on the different substrates. We demonstrated that the aligned nanofibrous mesh decreases the proliferation activity and provides a higher differentiative stimulus. We also clarified how the nanofibrous substrate influences myotube formation, and quantified a series of myotube-related parameters for both C2C12 and H9c2 cells.

  16. Attenuation of muscle wasting in murine C2C 12 myotubes by epigallocatechin-3-gallate.

    PubMed

    Mirza, Kamran A; Pereira, Suzette L; Edens, Neile K; Tisdale, Michael J

    2014-12-01

    Loss of muscle protein is a common feature of wasting diseases where currently treatment is limited. This study investigates the potential of epigallocatechin-3-gallate (EGCg), the most abundant catechin in green tea, to reverse the increased protein degradation and rescue the decreased protein synthesis which leads to muscle atrophy. Studies were conducted in vitro using murine C2C12 myotubes. Increased protein degradation and reduced rates of protein synthesis were induced by serum starvation and tumour necrosis factor-α (TNF-α). EGCg effectively attenuated the depression of protein synthesis and increase in protein degradation in murine myotubes at concentrations as low as 10 μM. Serum starvation increased expression of the proteasome 20S and 19S subunits, as well as the proteasome 'chymotrypsin-like' enzyme activity, and these were all attenuated down to basal values in the presence of EGCg. Serum starvation did not increase expression of the ubiquitin ligases MuRF1 and MAFbx, but EGCg reduced their expression below basal levels, possibly due to an increased expression of phospho Akt (pAkt) and phospho forkhead box O3a (pFoxO3a). Attenuation of protein degradation by EGCg was increased in the presence of ZnSO4, suggesting an EGCg-Zn(2+) complex may be the active species. The ability of EGCg to attenuate depressed protein synthesis and increase protein degradation in the myotubule model system suggests that it may be effective in preserving skeletal muscle mass in catabolic conditions.

  17. Development of Insulin Resistance through Induction of miRNA-135 in C2C12 Cells

    PubMed Central

    Honardoost, Maryam; Arefian, Ehsan; Soleimani, Masoud; Soudi, Sara; Sarookhani, Mohammad Reza

    2016-01-01

    Objective Micro-RNAs (miRNAs) are a class of posttranscriptional regulators that play crucial roles in various biological processes. Emerging evidence suggests a direct link between miRNAs and development of several diseases including type 2 diabetes (T2D). In this study, we aimed to investigate the effect of predicted miRNA and target genes on insulin resistance. Materials and Methods This experimental study was conducted on the C2C12 cell line. Using bioinformatics tools miRNA-135 and two respective target genes-insulin receptor (Insr) and vesicle associated membrane protein 2 (Vamp2)were selected as potential factors involved in insulin resistance process. Levels of glucose uptake miRNA expression and respective gene targets were determined after cell transfaction by miR-135. Results It was determined that Insr gene expression was significantly down-regulated in miR-135 transfected C2C12 cell line (P≤0.05). Interestingly; these transfected cells have shown a significant difference in glucose uptake incomparision the positive control cells, while it was similar to the insulin resistant cell line (P≤0.05). In contrast, no significant alteration of Vamp2 gene expression was observed. Conclusion Our data indicated no change on the Vamp2 expression level after miRNA transfection, while expression level of Insr was reduced and miR-135 expression was contrarily increased leading to poor stimulation of glucose uptake through insulin, and development of insulin resistance phenotype in C2C12 cell line. PMID:27602317

  18. Synergistic effect of Wnt modulatory small molecules and an osteoinductive ceramic on C2C12 cell osteogenic differentiation.

    PubMed

    Chen, Sigeng; Ryan, Daniel A; Dwyer, Mary A; Cashman, John R

    2014-10-01

    Although osteoinductive ceramics can induce osteoblast differentiation in vitro and bone regeneration in vivo, their effects rely solely on the limited number of endogenous stem cells. More recently, ceramic carriers seeded with culture-expanded stem cells have been reported as implants capable of in vivo bone formation. However, effective and safe signaling agents that promote cell differentiation to the osteogenic lineage are still needed. In the present report, two osteogenic small-molecules THQ-1a and PP-9 were identified by testing a series of compounds for Runx2 and BMP2 expression in C2C12 cells. Compounds THQ-1a and PP-9 modulated Wnt signaling and enhanced the expression of molecular markers of osteoblast differentiation. To probe the utility of these compounds for use with ceramic cell implants, the effect of THQ-1a and PP-9 on C2C12 cell osteogenic differentiation was investigated in the presence of a tricalcium phosphate (TCP) ceramic. The effect of THQ-1a and PP-9 on markers such as Osteocalcin and Collagen I was significantly increased in the presence of TCP ceramic. Additionally, THQ-1a or PP-9 in the presence of TCP ceramic gave a synergistic increase in alkaline phosphatase activity in the differentiation of C2C12 cells. Taken together, the results suggest an approach to directing cell lineage commitment for bone regeneration by the application of small-molecule osteogenic agents to cells in the presence of osteoinductive ceramics. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. ERK2 is required for efficient terminal differentiation of skeletal myoblasts

    SciTech Connect

    Li Ju; Johnson, Sally E. . E-mail: sjohnson@animal.ufl.edu

    2006-07-14

    Terminal differentiation of skeletal myoblasts involves alignment of the mononucleated cells, fusion into multinucleated syncitia, and transcription of muscle-specific genes. Myogenesis in vivo is regulated partially by IGF-I initiated signaling that results in activation of an intracellular phosphatidylinositol 3 kinase (PI3K) signaling cascade. Downstream signaling through the Raf/MEK/ERK axis, a pathway initiated by IGF-I, also is implicated in the regulation of muscle formation. The involvement of ERK1 and ERK2 during myogenesis was examined in C2C12 myoblasts. C2C12 myoblasts stably expressing a small interfering RNA (siRNA) directed against ERK1 or ERK2 were created. Both of the kinases were reduced to trace levels as measured by Western for total ERK and retained the capacity to become phosphorylated. C2C12siERK2 knockdown myoblasts failed to fuse into multinucleated myofibers. By contrast, cells expressing a scrambled siRNA or ERK1 siRNA fused into large multinucleated structures. The block to muscle formation did not involve continued cell cycle progression or apoptosis. C2C12siERK1 myoblasts expressed an increased amount of ERK2 protein and formed larger myofibers in response to IGF-I treatment. Interestingly, IGF-I treatment of C2C12 ERK2 knockdown myoblasts did not reinstate the myogenic program arguing that ERK2 is required for differentiation. These results provide evidence for ERK2 as a positive regulator of myogenesis and suggest that ERK1 is dispensable for myoblast proliferation and differentiation.

  20. Effect of cortisol on caspases in the co-cultured C2C12 and 3 T3-L1 cells.

    PubMed

    Muthuraman, Pandurangan

    2014-06-01

    The present study was carried out to understand the effect of cortisol on caspase expression in the C2C12 and 3 T3-L1 cells under co-culture system. Cells were co-cultured by using transwell inserts with a 0.4-μm porous membrane to separate C2C12 and 3 T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3 T3-L1 cells were transferred to C2C12 plates and inserts containing C2C12 cells were transferred to 3 T3-L1 plates. A total of 10 μg/μl of cortisol was added to the medium. Following treatment of cortisol for 3 days, the cells in the lower well were harvested for analysis. Caspases such as caspase 3, caspase 7, and caspase 9 were selected for the analysis. qRT-PCR results indicated the significant increase in the mRNA expression of caspase 3, caspase 7, and caspase 9. Caspase 3, 7, and 9 activities were also increased in the mono- and co-cultured C2C12 and 3 T3-L1 cells. In addition, confocal microscopical investigation indicated that cortisol increases caspase expressions in the mono- and co-cultured C2C12 and 3 T3-L1 cells. Taking all these together, we concluded that the co-culture system reflects the exact effect of cortisol on caspase expression, which is quite distinct from one dimensional mono-cultured experiments.

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

    PubMed

    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.

  2. Leptin and leucine synergistically regulate protein metabolism in C2C12 myotubes and mouse skeletal muscles.

    PubMed

    Mao, Xiangbing; Zeng, Xiangfang; Huang, Zhimin; Wang, Junjun; Qiao, Shiyan

    2013-07-28

    Leucine and leptin play important roles in regulating protein synthesis and degradation in skeletal muscles in vitro and in vivo. However, the objective of the present study was to determine whether leptin and leucine function synergistically in regulating protein metabolism of skeletal muscles. In the in vitro experiment, C2C12 myotubes were cultured for 2 h in the presence of 5 mm-leucine and/or 50 ng/ml of leptin. In the in vivo experiment, C57BL/6 and ob/ob mice were randomly assigned to be fed a non-purified diet supplemented with 3 % L-leucine or 2·04 % L-alanine (isonitrogenous control) for 14 d. Ob/ob mice were injected intraperitoneally with sterile PBS or recombinant mouse leptin (0·1 μg/g body weight) for 14 d. In C57BL/6 mice, dietary leucine supplementation increased (P< 0·05) plasma leptin, leptin receptor expression and protein synthesis in skeletal muscles, but reduced (P< 0·05) plasma urea and protein degradation in skeletal muscles. Dietary leucine supplementation and leptin injection increased the relative weight of the gastrocnemius and soleus muscles in ob/ob mice. Moreover, leucine and leptin treatments stimulated (P< 0·05) protein synthesis and inhibited (P< 0·05) protein degradation in C2C12 myotubes and skeletal muscles of ob/ob mice. There were interactions (P< 0·05) between the leucine and leptin treatments with regard to protein metabolism in C2C12 myotubes and soleus muscles of ob/ob mice but not in the gastrocnemius muscles of ob/ob mice. Collectively, these results suggest that leptin and leucine synergistically regulate protein metabolism in skeletal muscles both in vitro and in vivo.

  3. Lysine suppresses protein degradation through autophagic-lysosomal system in C2C12 myotubes.

    PubMed

    Sato, Tomonori; Ito, Yoshiaki; Nedachi, Taku; Nagasawa, Takashi

    2014-06-01

    Muscle mass is determined between protein synthesis and protein degradation. Reduction of muscle mass leads to bedridden condition and attenuation of resistance to diseases. Moreover, bedridden condition leads to additional muscle loss due to disuse muscle atrophy. In our previous study (Sato et al. 2013), we showed that administered lysine (Lys), one of essential amino acid, suppressed protein degradation in skeletal muscle. In this study, we investigated that the mechanism of the suppressive effects of Lys on skeletal muscle proteolysis in C2C12 cell line. C2C12 myotubes were incubated in the serum-free medium containing 10 mM Lys or 20 mM Lys, and myofibrillar protein degradation was determined by the rates of 3-methylhistidine (MeHis) release from the cells. The mammalian target of rapamycin (mTOR) activity from the phosphorylation levels of p70-ribosormal protein S6 kinase 1 and eIF4E-binding protein 1 and the autophagic-lysosomal system activity from the ratio of LC3-II/I in C2C12 myotubes stimulated by 10 mM Lys for 0-3 h were measured. The rates of MeHis release were markedly reduced by addition of Lys. The autophagic-lysosomal system activity was inhibited upon 30 min of Lys supplementation. The activity of mTOR was significantly increased upon 30 min of Lys supplementation. The suppressive effect of Lys on the proteolysis by the autophagic-lysosomal system was maintained partially when mTOR activity was inhibited by 100 nM rapamycin, suggesting that some regulator other than mTOR signaling, for example, Akt, might also suppress the autophagic-lysosomal system. From these results, we suggested that Lys suppressed the activity of the autophagic-lysosomal system in part through activation of mTOR and reduced myofibrillar protein degradation in C2C12 myotubes.

  4. IGF-1 prevents simvastatin-induced myotoxicity in C2C12 myotubes.

    PubMed

    Bonifacio, Annalisa; Sanvee, Gerda M; Brecht, Karin; Kratschmar, Denise V; Odermatt, Alex; Bouitbir, Jamal; Krähenbühl, Stephan

    2017-05-01

    Statins are generally well tolerated, but treatment with these drugs may be associated with myopathy. The mechanisms of statin-associated myopathy are not completely understood. Statins inhibit AKT phosphorylation by an unclear mechanism, whereas insulin-like growth factor (IGF-1) activates the IGF-1/AKT signaling pathway and promotes muscle growth. The aims of the study were to investigate mechanisms of impaired AKT phosphorylation by simvastatin and to assess effects of IGF-1 on simvastatin-induced myotoxicity in C2C12 myotubes. C2C12 mouse myotubes were exposed to 10 μM simvastatin and/or 10 ng/mL IGF-1 for 18 h. Simvastatin inhibited the IGF-1/AKT signaling pathway, resulting in increased breakdown of myofibrillar proteins, impaired protein synthesis and increased apoptosis. Simvastatin inhibited AKT S473 phosphorylation, indicating reduced activity of mTORC2. In addition, simvastatin impaired stimulation of AKT T308 phosphorylation by IGF-1, indicating reduced activation of the IGF-1R/PI3K pathway by IGF-1. Nevertheless, simvastatin-induced myotoxicity could be at least partially prevented by IGF-1. The protective effects of IGF-1 were mediated by activation of the IGF-1R/AKT signaling cascade. Treatment with IGF-1 also suppressed muscle atrophy markers, restored protein synthesis and inhibited apoptosis. These results were confirmed by normalization of myotube morphology and protein content of C2C12 cells exposed to simvastatin and treated with IGF-1. In conclusion, impaired activity of AKT can be explained by reduced function of mTORC2 and of the IGF-1R/PI3K pathway. IGF-1 can prevent simvastatin-associated cytotoxicity and metabolic effects on C2C12 cells. The study gives insight into mechanisms of simvastatin-associated myotoxicity and provides potential targets for therapeutic intervention.

  5. Hydro-ethanolic extract of cashew tree (Anacardium occidentale) nut and its principal compound, anacardic acid, stimulate glucose uptake in C2C12 muscle cells.

    PubMed

    Tedong, Leonard; Madiraju, Padma; Martineau, Louis C; Vallerand, Diane; Arnason, John T; Desire, Dzeufiet D P; Lavoie, Louis; Kamtchouing, Pierre; Haddad, Pierre S

    2010-12-01

    Products of cashew tree (Anacardium occidentale) are used in traditional medicine for various ailments, including diabetes. The anti-diabetic properties of cashew plant parts were studied using differentiated C2C12 myoblasts (myotubes) and rat liver mitochondria. Hydroethanolic extract of cashew seed (CSE) and its active component, anacardic acid (AA), stimulated glucose transport into C2C12 myotubes in a concentration-dependent manner. Extracts of other parts (leaves, bark and apple) of cashew plant were inactive. Significant synergistic effect on glucose uptake with insulin was noticed at 100 μg/mL CSE. CSE and AA caused activation of adenosine monophosphate-activated protein kinase in C2C12 myotubes after 6 h of incubation. No significant effect was noticed on Akt and insulin receptor phosphorylation. Both CSE and AA exerted significant uncoupling of succinate-stimulated respiration in rat liver mitochondria. Activation of adenosine monophosphate-activated protein kinase by CSE and AA likely increases plasma membrane glucose transporters, resulting in elevated glucose uptake. In addition, the dysfunction of mitochondrial oxidative phosphorylation may enhance glycolysis and contribute to increased glucose uptake. These results collectively suggest that CSE may be a potential anti-diabetic nutraceutical. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

    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.

  7. Synergism between baltergin metalloproteinase and Ba SPII RP4 PLA2 from Bothrops alternatus venom on skeletal muscle (C2C12) cells.

    PubMed

    Bustillo, Soledad; Gay, Claudia C; García Denegri, María E; Ponce-Soto, Luis A; Bal de Kier Joffé, Elisa; Acosta, Ofelia; Leiva, Laura C

    2012-02-01

    Acute muscle damage, myonecrosis, is one of the main characteristics of envenoming by Bothrops genus. In this in vitro study we investigated the role of a metalloproteinase (baltergin) and an acidic phospholipase A2 (Ba SPII RP4) in the cytotoxicity exhibited by Bothrops alternatus venom. Baltergin metalloproteinase purified from the venom exerted a toxic effect on C2C12 myoblast cells (CC50: 583.34 μg/mL) which involved morphological alterations compatible with apoptosis/anoikis. On the contrary, the most abundant PLA2 isolated from this venom did not exhibit cytotoxicity at times and doses tested. However, when myoblasts were treated with both enzymes together, synergic activity was demonstrated. Neutralization of the venom with specific antibodies (IgG anti-baltergin and IgG anti-PLA2) confirmed this synergism. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    SciTech Connect

    Tsukahara, Tamotsu; Haniu, Hisao; Matsuda, Yoshikazu

    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.

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

    PubMed

    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.

  10. Possibility of Autocrine β-Adrenergic Signaling in C2C12 Myotubes

    PubMed Central

    SMITH, JILL L.; PATIL, PANKAJ B.; MINTEER, SHELLEY D.; LIPSITZ, JASON R.; FISHER, JONATHAN S.

    2005-01-01

    Levodopa reportedly inhibits insulin action in skeletal muscle. Here we show that C2C12 myotubes produce levodopa and that insulin-stimulated glucose transport is enhanced when endogenous levodopa is depleted. Exogenous levodopa prevented the stimulation of glucose transport by insulin (P < 0.05) and increased cAMP concentrations (P < 0.05). The decrease in insulin-stimulated glucose transport caused by levodopa was attenuated by propranolol (a β-adrenergic antagonist) and prevented by NSD-1015 (NSD), an inhibitor of DOPA decarboxylase (DDC; converts levodopa to dopamine). Propranolol and NSD both prevented levodopa-related increases in [cAMP]. However, the effects of levodopa were unlikely to be dependent on the conversion of levodopa to catecholamines because we could detect neither DDC in myotubes nor catecholamines in media after incubation of myotubes with levodopa. The data suggest the possibility of novel autocrine β-adrenergic action in C2C12 myotubes in which levodopa, produced by myotubes, could have hormone-like effects that impinge on glucose metabolism. PMID:16339749

  11. Low molecular weight guluronate prevents TNF-α-induced oxidative damage and mitochondrial dysfunction in C2C12 skeletal muscle cells.

    PubMed

    Dun, Yun-lou; Zhou, Xiao-lin; Guan, Hua-shi; Yu, Guang-li; Li, Chun-xia; Hu, Ting; Zhao, Xia; Cheng, Xiao-lei; He, Xiao-xi; Hao, Jie-jie

    2015-09-01

    Muscle wasting is associated with a variety of chronic or inflammatory disorders. Evidence suggests that inflammatory cytokines play a vital role in muscle inflammatory pathology and this may result in oxidative damage and mitochondrial dysfunction in skeletal muscle. In our study, we used microwave degradation to prepare a water-soluble low molecular weight guluronate (LMG) of 3000 Da from Fucus vesiculosus obtained from Canada, the Atlantic Ocean. We demonstrated the structural characteristics, using HPLC, FTIR and NMR of LMG and investigated its effects on oxidative damage and mitochondrial dysfunction in C2C12 skeletal muscle cells induced by tumor necrosis factor alpha (TNF-α), a cell inflammatory cytokine. The results indicated that LMG could alleviate mitochondrial reactive oxygen species (ROS) production, increase the activities of antioxidant enzymes (GSH and SOD), promote mitochondrial membrane potential (MMP) and upregulate the expression of mitochondrial respiratory chain protein in TNF-α-induced C2C12 cells. LMG supplement also increased the mitochondrial DNA copy number and mitochondrial biogenesis related genes in TNF-α-induced C2C12 cells. LMG may exert these protective effects through the nuclear factor kappa B (NF-κB) signaling pathway. These suggest that LMG is capable of protecting TNF-α-induced C2C12 cells against oxidative damage and mitochondrial dysfunction.

  12. p75NTR-mediated signaling promotes the survival of myoblasts and influences muscle strength.

    PubMed

    Reddypalli, Shailaja; Roll, Kristin; Lee, Hyung-Kook; Lundell, Martha; Barea-Rodriguez, Edwin; Wheeler, Esther F

    2005-09-01

    During muscle development, the p75(NTR) is expressed transiently on myoblasts. The temporal expression pattern of the receptor raises the possibility that the receptor is influencing muscle development. To test this hypothesis, p75(NTR)-deficient mutant mice were tested for muscle strength by using a standard wire gripe strength test and were found to have significantly decreased strength relative to that of normal mice. When normal mybolasts were examined in vivo for expression of NGF receptors, p75(NTR) was detected on myoblasts but the high affinity NGF receptor, trk A, was not co-expressed with p75(NTR). In vitro, proliferating C2C12 and primary myoblasts co-expressed the p75(NTR) and MyoD, but immunofluorescent analysis of primary myoblasts and RT-PCR analysis of C2C12 mRNA revealed that myoblasts were devoid of trk A. In contrast to the cell death functions that characterize the p75(NTR) in neurons, p75(NTR)-positive primary and C2C12 myoblasts did not differentiate or undergo apoptosis in response to neurotrophins. Rather, myoblasts survived and even proliferated when grown at subconfluent densities in the presence of the neurotrophins. Furthermore, when myoblasts treated with NGF were lysed and immunoprecipitated with antibodies against phosphorylated I-kappaB and AKT, the cells contained increased levels of both phospho-proteins, both of which promote cell survival. By contrast, neurotrophin-treated myoblasts did not induce phosphorylation of Map Kinase p42/44 or p38, indicating the survival was not mediated by the trk A receptor. Taken together, the data indicate that the p75(NTR) mediates survival of myoblasts prior to differentiation and that the activity of this receptor during myogenesis is important for developing muscle. Copyright 2005 Wiley-Liss, Inc.

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

    PubMed Central

    Matsuzaka, Yasunari; Tanihata, Jun; Komaki, Hirofumi; Ishiyama, Akihiko; Oya, Yasushi; Rüegg, Urs; Takeda, Shin-ichi; Hashido, Kazuo

    2016-01-01

    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. PMID:27977725

  14. Cdo Regulates Surface Expression of Kir2.1 K+ Channel in Myoblast Differentiation

    PubMed Central

    Koh, Jewoo; Kang, KyeongJin; Bae, Gyu-Un; Cho, Hana; Kang, Jong-Sun

    2016-01-01

    A potassium channel Kir2.1-associated membrane hyperpolarization is required for myogenic differentiation. However the molecular regulatory mechanisms modulating Kir2.1 channel activities in early stage of myogenesis are largely unknown. A cell surface protein, Cdo functions as a component of multiprotein cell surface complexes to promote myogenesis. In this study, we report that Cdo forms a complex with Kir2.1 during myogenic differentiation, and is required for the channel activity by enhancing the surface expression of Kir2.1 in the early stage of differentiation. The expression of a constitutively active form of the upstream kinase for p38MAPK, MKK6(EE) can restore Kir2.1 activities in Cdo-depleted C2C12 cells, while the treatment with a p38MAPK inhibitor, SB203580 exhibits a similar effect of Cdo depletion on Kir2.1 surface expression. Furthermore, Cdo-/- primary myoblasts, which display a defective differentiation program, exhibit a defective Kir2.1 activity. Taken together, our results suggest that a promyogenic Cdo signaling is critical for Kir2.1 activities in the induction of myogenic differentiation. PMID:27380411

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

    PubMed

    Oláh, Gábor; Szczesny, Bartosz; Brunyánszki, Attila; López-García, Isabel A; Gerö, Domokos; Radák, Zsolt; Szabo, Csaba

    2015-01-01

    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

  16. Tadalafil alters energy metabolism in C2C12 skeletal muscle cells.

    PubMed

    Sabatini, Stefania; Sgrò, Paolo; Duranti, Guglielmo; Ceci, Roberta; Di Luigi, Luigi

    2011-01-01

    Phosphodiesterases (PDEs) are a family of enzymes that hydrolyze cyclic nucleotides, thereby modulating cell functions. Three highly selective PDE5 inhibitors (PDE5i), sildenafil, vardenafil and tadalafil, have been developed for treatment of erectile dysfunction (ED). Experimental evidence showed that chronic treatment with sildenafil PDE5i in a mouse model of diet-induced obesity and insulin resistance improved insulin action and decreased circulating fatty acid levels. It has recently been shown that healthy athletes use PDE5i as performance enhancers, hence in the present study we investigated whether the long-lasting PDE5i tadalafil influences energy metabolism in C2C12 skeletal muscle cells by evaluating lactate production, glucose consumption, and citrate synthase and 3-OH acyl CoA dehydrogenase activities. Our data demonstrate that tadalafil is able to modulate energy homeostasis in mouse skeletal muscle cells, depending on the treatment length and dose.

  17. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    SciTech Connect

    Itoigawa, Yoshiaki; Kishimoto, Koshi N.; Okuno, Hiroshi; Sano, Hirotaka; Kaneko, Kazuo; Itoi, Eiji

    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.

  18. Suppression of free fatty acid-induced insulin resistance by phytopolyphenols in C2C12 mouse skeletal muscle cells.

    PubMed

    Deng, Yea-Tzy; Chang, Tsai-Wen; Lee, Ming-Shyue; Lin, Jen-Kun

    2012-02-01

    It was reported that increased plasma levels of free fatty acids (FFAs) are associated with profound insulin resistance in skeletal muscle and may also play a critical role in the insulin resistance of obesity and type 2 diabetes mellitus. Skeletal muscle is the major site for insulin-stimulated glucose uptake and is involved in energy regulation and homeostasis. In this study, we used 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C (PKC) activator, and palmitate to induce insulin resistance in C2C12 mouse skeletal muscle cells. Our data show that epigallocatechin gallate (EGCG) and curcumin treatment reduce insulin receptor substrate-1 (IRS-1) Ser307 phosphorylation, and curcumin is more potent to increase Akt phosphorylation in TPA induction. Moreover, we found that after 5 h of palmitate incubation, epicatechin gallate (ECG) can suppress IRS-1 Ser307 phosphorylation and significantly promote Akt, ERK1/2, p38 MAPK, and AMP-activated protein kinase activation. With a longer incubation with palmitate, IRS-1 exhibited a dramatic depletion, and treatment with EGCG, ECG, and curcumin could reverse IRS-1 expression, Akt phosphorylation, and MAPK signaling cascade activation and improve glucose uptake in C2C12 skeletal muscle cells, especially ECG and curcumin. In addition, treatment with these polyphenols can suppress acetyl-CoA carboxylase activation, but only EGCG could inhibit lipid accumulation in the intracellular site. These findings may suggest that curcumin shows the best capacity to improve FFA-induced insulin resistance than the other two, and ECG was more effective than EGCG in attenuating insulin resistance.

  19. Visfatin induces oxidative stress in differentiated C2C12 myotubes in an Akt- and MAPK-independent, NFkB-dependent manner.

    PubMed

    Oita, Radu C; Ferdinando, Dudley; Wilson, Steve; Bunce, Christopher; Mazzatti, Dawn J

    2010-03-01

    Adipose tissue is an important endocrine and metabolic tissue that is actively involved in cross-talk with peripheral organs such as skeletal muscle. It is likely that adipose-derived factors may underlie the development of insulin resistance in muscle. Thus, the cross-talk between adipose and muscle may be important for the propagation of obesity-related diseases. Visfatin (Pre-B-cell colony-enhancing factor 1 homolog/Nampt) is a recently discovered adipokine with pleiotropic functions. The aim of this study was to examine the effect of visfatin on cellular stress responses and signalling pathways in skeletal muscle. Visfatin treatment of differentiated C2C12 myotubes generated reactive oxygen species (ROS) comprising both superoxide and hydrogen peroxide that was dependent on de novo transcription and translation. In differentiated C2C12 myoblasts, visfatin had no effects on insulin-stimulated Akt phosphorylation nor on activation of the Akt signalling pathway. Additionally, visfatin-induced oxidative stress occurred independent of activation of the stress-activated protein kinases (MAPKs) ERK and p38. In contrast, phosphorylation of NFkB was associated with visfatin-mediated generation of ROS and blockade of this pathway via selective IKK inhibition led to a partial reduction in oxidative stress. Furthermore, the generation of ROS following visfatin treatment was highly dependent on both de novo transcription and translation. Taken together, these findings provide novel insights for the unique pathophysiological role of visfatin in skeletal muscle.

  20. TNF alpha inhibits myogenic differentiation of C2C12 cells through NF-κB activation and impairment of IGF-1 signaling pathway.

    PubMed

    Zhao, Q; Yang, S T; Wang, J J; Zhou, J; Xing, S S; Shen, C C; Wang, X X; Yue, Y X; Song, J; Chen, M; Wei, Y Y; Zhou, Q P; Dai, T; Song, Y H

    2015-03-20

    Cachexia or muscle wasting is a common condition that occurs in many chronic diseases. The wasting conditions are characterized by increased levels of TNF-α which was also known as cachectin in the past. But how TNF-α exerts its cachetic effects remains controversial. To clarify this issue, we investigated the impact of TNF-α on C2C12 cell myogenic differentiation. Our results demonstrate that myotube formation was completely inhibited by TNF-α when added to differentiating C2C12 myoblasts. The inhibitory effect of TNF-α on differentiation was accompanied by activation of NF-κB and down regulation of myogenin and Akt. Importantly, TNF-α's effect on differentiation was abolished when IGF-1 was added to the culture. IGF-1 treatment also inhibited NF-κB reporter activity and restored Akt levels. Our data suggest that TNF-α inhibits myogenic differentiation through NF-κB activation and impairment of IGF-1 signaling pathway. The reversal of TNF-α induced inhibition of myogenesis by IGF-1 may have significant therapeutic potential.

  1. Antiapoptotic effect of a novel synthetic peptide from bovine muscle and MPG peptide on H2O2-induced C2C12 cells.

    PubMed

    Sivakumar, Allur Subramaniyan; Ochirbat, Chinzorio; Cho, Soo-Hyun; Yang, Jieun; Hwang, Inho

    2014-08-01

    The objective of this study was to evaluate antioxidant and antiapoptotic effects of a novel peptide (T.peptide) isolated from bovine and commercially available MPG peptide. The amino acid sequences of the T.peptide were (Glu-Val-Pro-Glu-Val-His-Glu-Glu-Val). The antioxidant activities of these peptides were determined by measuring the 1,1-diphenyl-2-picrylhydrazyl (DPPH•) and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS+•) radical scavenging assays. The in vitro cytotoxicity of T.peptide and MPG peptide was determined against H2O2-induced C2C12 cells. H2O2-induced apoptosis in C2C12 cells were determined by mRNA expression of caspase-3. Moreover, the mRNA expression of tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NF-κB) were assayed by reverse transcription polymerase chain reaction. The findings of the study indicate that the mRNA expression of TNF-α and NF-κB are significantly (p < 0.05) increased in H2O2-induced C2C12 cells, whereas a significant decrease (p < 0.05) in these mRNA expressions are observed when pretreated with T.peptide or MPG peptide. Pretreatment with MPG or T.peptides is also found to significantly (p < 0.05) decrease the mRNA expression of caspase-3 in H2O2-induced C2C12 cells. The results of the study demonstrate that both T.peptide and MPG peptide could reduce the DPPH• and ABTS+• radical and inhibit cytotoxicity against H2O2-induced injury, resulting in prevention of free radical generation and subsequent apoptotic cell death, which indicates the potential of bovine meat as a source of antioxidant peptides.

  2. Mitochondria dysfunction in lung cancer-induced muscle wasting in C2C12 myotubes.

    PubMed

    McLean, Julie B; Moylan, Jennifer S; Andrade, Francisco H

    2014-01-01

    Cancer cachexia is a syndrome which results in severe loss of muscle mass and marked fatigue. Conditioned media from cachexia-inducing cancer cells triggers metabolic dysfunction in skeletal muscle, including decreased mitochondrial respiration, which may contribute to fatigue. We hypothesized that Lewis lung carcinoma conditioned medium (LCM) would impair the mitochondrial electron transport chain (ETC) and increase production of reactive oxygen species, ultimately leading to decreased mitochondrial respiration. We incubated C2C12 myotubes with LCM for 30 min, 2, 4, 24 or 48 h. We measured protein content by western blot; oxidant production by 2',7'-dichlorofluorescin diacetate (DCF), 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF), and MitoSox; cytochrome c oxidase activity by oxidation of cytochrome c substrate; and oxygen consumption rate (OCR) of intact myotubes by Seahorse XF Analyzer. LCM treatment for 2 or 24 h decreased basal OCR and ATP-related OCR, but did not alter the content of mitochondrial complexes I, III, IV and V. LCM treatment caused a transient rise in reactive oxygen species (ROS). In particular, mitochondrial superoxide (MitoSOX) was elevated at 2 h. 4-Hydroxynonenal, a marker of oxidative stress, was elevated in both cytosolic and mitochondrial fractions of cell lysates after LCM treatment. These data show that lung cancer-conditioned media alters electron flow in the ETC and increases mitochondrial ROS production, both of which may ultimately impair aerobic metabolism and decrease muscle endurance.

  3. Rotenone induces reductive stress and triacylglycerol deposition in C2C12 cells.

    PubMed

    He, Quan; Wang, Miao; Petucci, Christopher; Gardell, Stephen J; Han, Xianlin

    2013-12-01

    Environmental rotenone is associated with Parkinson's disease due to its inhibitory property to the complex I of mitochondrial respiration chain. Although environmental pollution has been postulated as a causal factor for the increasing prevalence of obesity, the role of rotenone in the pathogenesis of obesity has not been studied. We employed muscle-derived cell C2C12 as a model and shotgun lipidomics as a tool for lipid analysis and found that treatment with rotenone led to the profound deposition of intracellular triacylglycerol (TAG) in a time- and dose-dependent fashion. The TAG deposition resulted from complex I inhibition. Further studies revealed that rotenone induced mitochondrial stress as shown by decreased mitochondrial oxygen consumption rate, increased NADH/NAD+ ratio (i.e., reductive stress) and mitochondrial metabolites. We demonstrated that rotenone activated fatty acid de novo synthesis and TAG synthesis and ultimately resulted in intracellular TAG deposition. These studies suggested that increased mitochondrial stresses might be an underlying mechanism responsible for TAG accumulation manifest in obesity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Optogenetic approach for targeted activation of global calcium transients in differentiated C2C12 myotubes.

    PubMed

    Sebille, Stéphane; Ayad, Oualid; Chapotte-Baldacci, Charles-Albert; Cognard, Christian; Bois, Patrick; Chatelier, Aurélien

    2017-09-11

    Excitation-contraction coupling in muscle cells is initiated by a restricted membrane depolarization delimited within the neuromuscular junction. This targeted depolarization triggers an action potential that propagates and induces a global cellular calcium response and a consequent contraction. To date, numerous studies have investigated this excitation-calcium response coupling by using different techniques to depolarize muscle cells. However, none of these techniques mimic the temporal and spatial resolution of membrane depolarization observed in the neuromuscular junction. By using optogenetics in C2C12 muscle cells, we developed a technique to study the calcium response following membrane depolarization induced by photostimulations of membrane surface similar or narrower than the neuromuscular junction area. These stimulations coupled to confocal calcium imaging generate a global cellular calcium response that is the consequence of a membrane depolarization propagation. In this context, this technique provides an interesting, contactless and relatively easy way of investigation of calcium increase/release as well as calcium decrease/re-uptake triggered by a propagated membrane depolarization.

  5. Antioxidant effects of whey protein on muscle C2C12 cells.

    PubMed

    Kerasioti, Efthalia; Stagos, Dimitrios; Priftis, Alexandros; Aivazidis, Stefanos; Tsatsakis, Aristidis M; Hayes, A Wallace; Kouretas, Demetrios

    2014-07-15

    In the present study, the in vitro scavenging activity of sheep whey protein against free radicals, as well as its reducing power were determined and compared with that of beef protein, soy protein and cow whey protein. Moreover, the possible protective effects of sheep whey protein from tert-butyl hydroperoxide (tBHP)-induced oxidative stress in muscle C2C12 cells were determined by assessing oxidative stress markers by flow cytometry and spectrophotometry. The results showed that sheep whey protein scavenged DPPH, ABTS(+) and OH radicals with IC50 values of 3.1, 4.1 and 1.8 mg of protein/ml. Moreover, the reducing power activity assessed with potassium ferricyanide of sheep whey protein was 1.3mg/ml. As regards to the antioxidant effects in muscle cell line, sheep whey protein at 0.78, 1.56, 3.12 and 6.24 mg of protein/ml increased GSH levels up to 138%, lowered TBARS levels up to 25% and decreased ROS levels up to 41.4%.

  6. Electrical pulse stimulation decreases electrochemical Na(+) and K(+) gradients in C2C12 myotubes.

    PubMed

    Danilov, Kirill; Sidorenko, Svetlana; Milovanova, Kseniya; Klimanova, Elizaveta; Kapilevich, Leonid V; Orlov, Sergei N

    2017-11-18

    Electrical pulse stimulation (EPS)-treated cultured myotubes are widely employed as an in vitro model of muscle contraction. Here we examined time-dependent EPS action and dose-dependent ouabain action on [Na(+)]i and [K(+)]i in C2C12 myotubes. After 2 h of EPS (40 V, 1 Hz, 10 ms) [Na(+)]i increased by ∼150% whereas [K(+)]i declined by ∼20%. 3 μM ouabain had a negligible impact on [Na(+)]i and [K(+)]i in control cells but increased the [Na(+)]i/[K(+)]i ratio in EPS-treated myotubes by 85%. Thus, our results show for the first time that EPS results in dissipation of Na(+) and K(+) gradients in cultured myotubes and suggest that the augmented production of endogenous cardiotonic steroids may contribute to elevation of the [Na(+)]i/[K(+)]i ratio in exercising muscle. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Cyclic stretch induced miR-146a upregulation delays C2C12 myogenic differentiation through inhibition of Numb

    SciTech Connect

    Kuang Wei; Tan Jiali; Duan Yinzhong; Duan Jianmin; Wang Weijian; Jin Fang; Jin Zuolin; Yuan Xiao Liu Yanpu

    2009-01-09

    Proliferation and differentiation of muscle stem cells must be tightly regulated by intrinsic and extrinsic signals for effective regeneration and adaptive response. MicroRNAs have been implicated as potent regulators in diverse biological processes at the level of posttranscriptional repression. In this study, we found that miR-146a was significantly upregulated upon a 48-h cyclic stretch of 5% elongation/10cycles/min. Importantly, miR-146 was predicted to base-pair with sequences in the 3' UTR of Numb, which promotes satellite cell differentiation towards muscle cells by inhibiting Notch signaling. Through reporter assay and exogenous expression experiment, we confirmed Numb was inhibited by miR-146a. Inhibition of miR-146a by antago-miR-146a rescued the expression of Numb and facilitated the differentiation of C2C12 at a cost of compromised proliferation. Thus, for the first time, we propose a role of miR-146a in skewing the balance of muscle differentiation and proliferation through inhibiting the expression of Numb.

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

    SciTech Connect

    Dey, Sumit K.; Saha, Shekhar; Das, Provas; Das, Mahua R.; Jana, Siddhartha S.

    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.

  9. Biomarker-free dielectrophoretic sorting of differentiating myoblast multipotent progenitor cells and their membrane analysis by Raman spectroscopy.

    PubMed

    Muratore, Massimo; Srsen, Vlastimil; Waterfall, Martin; Downes, Andrew; Pethig, Ronald

    2012-09-01

    Myoblasts are muscle derived mesenchymal stem cell progenitors that have great potential for use in regenerative medicine, especially for cardiomyogenesis grafts and intracardiac cell transplantation. To utilise such cells for pre-clinical and clinical applications, and especially for personalized medicine, it is essential to generate a synchronised, homogenous, population of cells that display phenotypic and genotypic homogeneity within a population of cells. We demonstrate that the biomarker-free technique of dielectrophoresis (DEP) can be used to discriminate cells between stages of differentiation in the C2C12 myoblast multipotent mouse model. Terminally differentiated myotubes were separated from C2C12 myoblasts to better than 96% purity, a result validated by flow cytometry and Western blotting. To determine the extent to which cell membrane capacitance, rather than cell size, determined the DEP response of a cell, C2C12 myoblasts were co-cultured with GFP-expressing MRC-5 fibroblasts of comparable size distributions (mean diameter ∼10 μm). A DEP sorting efficiency greater than 98% was achieved for these two cell types, a result concluded to arise from the fibroblasts possessing a larger membrane capacitance than the myoblasts. It is currently assumed that differences in membrane capacitance primarily reflect differences in the extent of folding or surface features of the membrane. However, our finding by Raman spectroscopy that the fibroblast membranes contained a smaller proportion of saturated lipids than those of the myoblasts suggests that the membrane chemistry should also be taken into account.

  10. Possible role of TIEG1 as a feedback regulator of myostatin and TGF-{beta} in myoblasts

    SciTech Connect

    Miyake, Masato; Hayashi, Shinichiro; Iwasaki, Shunsuke; Chao, Guozheng; Takahashi, Hideyuki; Watanabe, Kouichi; Ohwada, Shyuichi; Aso, Hisashi; Yamaguchi, Takahiro

    2010-03-19

    Myostatin and TGF-{beta} negatively regulate skeletal muscle development and growth. Both factors signal through the Smad2/3 pathway. However, the regulatory mechanism of myostatin and TGF-{beta} signaling remains unclear. TGF-{beta} inducible early gene (TIEG) 1 is highly expressed in skeletal muscle and has been implicated in the modulation of TGF-{beta} signaling. These findings prompted us to investigate the effect of TIEG1 on myostatin and TGF-{beta} signaling using C2C12 myoblasts. Myostatin and TGF-{beta} induced the expression of TIEG1 and Smad7 mRNAs, but not TIEG2 mRNA, in proliferating C2C12 cells. When differentiating C2C12 myoblasts were stimulated by myostatin, TIEG1 mRNA was up-regulated at a late stage of differentiation. In contrast, TGF-{beta} enhanced TIEG1 expression at an early stage. Overexpression of TIEG1 prevented the transcriptional activation of Smad by myostatin and TGF-{beta} in both proliferating or differentiating C2C12 cells, but the expression of Smad2 and Smad7 mRNAs was not affected. Forced expression of TIEG1 inhibited myogenic differentiation but did not cause more inhibition than the empty vector in the presence of myostatin or TGF-{beta}. These results demonstrate that TIEG1 is one possible feedback regulator of myostatin and TGF-{beta} that prevents excess action in myoblasts.

  11. Non-classical localization of androgen receptor in the C2C12 skeletal muscle cell line.

    PubMed

    Pronsato, Lucía; Boland, Ricardo; Milanesi, Lorena

    2013-02-01

    The classical model of testosterone action has been traditionally described as being mediated by the androgen receptor (AR) localized exclusively in the nucleus. However, there is increasing functional evidence for extranuclear localization of AR. We present biochemical and immunological data supporting mitochondrial and microsomal localization of AR in the C2C12 skeletal muscle cell line. As a first approach AR was detected by immunoblotting, using specific antibodies after subcellular fractionation, not only in nucleus and cytosol, but also in mitochondria and microsomes. We then established [(3)H] testosterone binding characteristics in total homogenates and subcellular fractions. Specific and saturable [(3)H] testosterone binding sites were detected in mitochondria and microsomes. Immunolocalization of the non-classical AR was also confirmed using confocal microscopy. Sucrose gradient fractionation demonstrated the presence of the AR in lipid rafts and caveolae. Besides, the AR interacts physically with Caveolin-1, association that is lost after testosterone treatment. Accordingly, Western blot analysis revealed a decrease of AR expression in the microsomal fraction after testosterone treatment, suggesting translocation of the membrane AR to another subcellular compartment. The non-classical distribution of native pools of AR in skeletal muscle cells suggests an alternative mode of AR localization/function. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    SciTech Connect

    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

  13. Fabrication of PLGA/MWNTs composite electrospun fibrous scaffolds for improved myogenic differentiation of C2C12 cells.

    PubMed

    Xu, Jiazhu; Xie, Ya; Zhang, Hongbo; Ye, Zhaoyang; Zhang, Wenjun

    2014-11-01

    Electrically conducting scaffolds have attracted tremendous attention in skeletal muscle tissue engineering. In this paper, poly(lactic-co-glycolic acid) (PLGA)/multi-wall carbon nanotubes (MWNTs) composite fibrous scaffolds were fabricated using the electrospinning technique. The physical properties of the composite fibers were characterized and proliferation and differentiation of C2C12 cells on these scaffolds were examined. It was found that the addition of MWNTs modulated the physical properties of PLGA fibers including morphology, fiber diameter, degradation, tensile strength and electrical conductivity, depending on the amount of MWNTs. These fibrous scaffolds were cytocompatible and supported the proliferation of C2C12 cells. Importantly, C2C12 cells showed more mature myotube formation on PLGA/MWNTs composite fibrous scaffolds compared to PLGA scaffolds. These results indicate that PLGA/MWNTs composite electrospun fibers have great potential in skeletal muscle tissue engineering.

  14. Dexamethasone-Induced Skeletal Muscle Atrophy Increases O-GlcNAcylation in C2C12 Cells.

    PubMed

    Massaccesi, Luca; Goi, Giancarlo; Tringali, Cristina; Barassi, Alessandra; Venerando, Bruno; Papini, Nadia

    2016-08-01

    Skeletal muscle atrophy is a well-known adverse effect of chronic treatment with glucocorticoids and it also occurs when stress conditions such as sepsis and cachexia increase the release of endogenous glucocorticoids. Although the mechanisms of action of these hormones have been elucidated, the possible molecular mechanisms causing atrophy are not yet fully understood. The involvement of the O-GlcNAcylation process has recently been reported in disuse atrophy. O-GlcNAcylation, a regulatory post-translational modification of nuclear and cytoplasmic proteins consists in the attachment of O-GlcNAc residues on cell proteins and is regulated by two enzymes: O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays a crucial role in many cellular processes and it seems to be related to skeletal muscle physiological function. The aim of this study is to investigate the involvement of O-GlcNAcylation in glucocorticoid-induced atrophy by using an "in vitro" model, achieved by treatment of C2C12 with 10 μM dexamethasone for 48 h. In atrophic condition, we observed that O-GlcNAc levels in cell proteins increased and concomitantly protein phosphorylation on serine and threonine residues decreased. Analysis of OGA expression at mRNA and protein levels showed a reduction in this enzyme in atrophic myotubes, whereas no significant changes of OGT expression were found. Furthermore, inhibition of OGA activity by Thiamet G induced atrophy marker expression. Our current findings suggest that O-GlcNAcylation is involved in dexamethasone-induced atrophy. In particular, we propose that the decrease in OGA content causes an excessive and mostly durable level of O-GlcNAc residues on sarcomeric proteins that might modify their function and stability. J. Cell. Biochem. 117: 1833-1842, 2016. © 2016 Wiley Periodicals, Inc.

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

    SciTech Connect

    Kamli, Majid Rasool; Kim, Jihoe; Pokharel, Smritee; Jan, Arif Tasleem; Lee, Eun Ju; Choi, Inho

    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}.

  16. Transcription Factor ZBED6 Mediates IGF2 Gene Expression by Regulating Promoter Activity and DNA Methylation in Myoblasts

    NASA Astrophysics Data System (ADS)

    Huang, Yong-Zhen; Zhang, Liang-Zhi; Lai, Xin-Sheng; Li, Ming-Xun; Sun, Yu-Jia; Li, Cong-Jun; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Zhao, Xin; Chen, Hong

    2014-04-01

    Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were upregulated during C2C12 differentiation. The IGF2 expression levels were negatively associated with the methylation status in beef cattle (P < 0.05). A luciferase assay for the IGF2 intron 3 and P3 promoter showed that the mutant-type 439 A-SNP-pGL3 in driving reporter gene transcription is significantly higher than that of the wild-type 439 G-SNP-pGL3 construct (P < 0.05). An over-expression assay revealed that ZBED6 regulate IGF2 expression and promote myoblast differentiation. Furthermore, knockdown of ZBED6 led to IGF2 expression change in vitro. Taken together, these results suggest that ZBED6 inhibits IGF2 activity and expression via a G to A transition disrupts the interaction. Thus, we propose that ZBED6 plays a critical role in myogenic differentiation.

  17. In Vitro Selective Anti-Proliferative Effect of Zinc Oxide Nanoparticles Against Co-Cultured C2C12 Myoblastoma Cancer and 3T3-L1 Normal Cells.

    PubMed

    Chandrasekaran, Murugesan; Pandurangan, Muthuraman

    2016-07-01

    The zinc oxide (ZnO) nanoparticle has been widely used in biomedical applications and cancer therapy and has been reported to induce a selective cytotoxic effect on cancer cell proliferation. The present study investigated the cytotoxicity of ZnO nanoparticles against co-cultured C2C12 myoblastoma cancer cells and 3T3-L1 adipocytes. Our results showed that the ZnO nanoparticles could be cytotoxic to C2C12 myoblastoma cancer cells than 3T3-L1 cells. The messenger RNA (mRNA) expressions of p53 and bax were significantly increased 114.3 and 118.2 % in the C2C12 cells, whereas 42.5 and 40 % were increased in 3T3-L1 cells, respectively. The mRNA expression of bcl-2 was reduced 38.2 and 28.5 % in the C2C12 and 3T3-L1 cells, respectively, whereas the mRNA expression of caspase-3 was increased 80.7 and 51.6 % in the C2C12 and 3T3-L1 cells, respectively. The protein expressions of p53, bax, and caspase-3 were significantly increased 40, 81.8, and 80 % in C2C12 cells, whereas 20.3, 28.2, and 37.9 % were increased in 3T3-L1 cells, respectively. The mRNA expression of bcl-2 was significantly reduced 32.2 and 22.7 % in C2C12 and 3T3-L1 cells, respectively. Caspase-3 enzyme activity and reactive oxygen species (ROS) were increased in co-cultured C2C12 cells compared to 3T3-L1 cells. Taking all these data together, it may suggest that ZnO nanoparticles severely induce apoptosis in C2C12 myoblastoma cancer cells than 3T3-L1 cells.

  18. Pollen Typhae total flavone improves insulin-induced glucose uptake through the β-arrestin-2-mediated signaling in C2C12 myotubes.

    PubMed

    Feng, Xiao-Tao; Wang, Tian-Zhan; Chen, Yi; Liu, Ji-Bo; Liu, Yi; Wang, Wen-Jian

    2012-10-01

    Defects in insulin-stimulated glucose uptake in skeletal muscle result from the dysfunction of insulin signaling including the phosphatidylinositol-3 kinase (PI3K) pathway and a novel β-arrestin-2-mediated signaling, which leads to insulin resistance (IR). Pollen Typhae, a Chinese herb, has been used for thousands of years in traditional Chinese medicine, and has the potential to inhibit the development of IR. We have previously reported that Pollen Typhae total flavone (PTF), the extract from Pollen Typhae, ameliorates high-glucose- and high-insulin-induced impairment of glucose uptake in 3T3-L1 adipocytes, but the mechanisms remain unclear. The objective of this study was to investigate the effects of PTF on glucose uptake, and to explore the underlying mechanisms in C2C12 myotubes. PTF improved insulin-stimulated glucose uptake in a dose- and time-dependent manner in C2C12 myotubes, and prevented palmitate-induced IR. Furthermore, PTF enhanced the basal gene expression of Src and Akt2, elevated the protein expression of β-arrestin-2, Src and Akt, increased the phosphorylation of insulin receptor-β at Tyr1150/1151 and Akt at Thr308/Ser473 in an insulin-dependent manner, but had no effects on the protein expression of PI3K-p85 or the activity of PI3K. Inhibition of Src but not PI3K restrained PTF-induced phosphorylation of Akt and glucose uptake. Our findings indicate that PTF improves insulin-induced glucose uptake via the β-arrestin-2-mediated signaling in C2C12 myotubes.

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

    PubMed

    Lee, Sang-Jin; Go, Ga-Yeon; Yoo, Miran; Kim, Yong Kee; Seo, Dong-Wan; Kang, Jong-Sun; Bae, Gyu-Un

    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.

  20. Skeletal muscle myoblasts possess a stretch-responsive local angiotensin signalling system.

    PubMed

    Johnston, Adam P W; Baker, Jeff; De Lisio, Michael; Parise, Gianni

    2011-06-01

    A paucity of information exists regarding the presence of local renin-angiotensin systems (RASs) in skeletal muscle and associated muscle stem cells. Skeletal muscle and muscle stem cells were isolated from C57BL/6 mice and examined for the presence of a local RAS using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), Western blotting and liquid chromatography-mass spectrometry (LC-MS). Furthermore, the effect of mechanical stimulation on RAS member gene expression was analysed. Whole skeletal muscle, primary myoblasts and C2C12 derived myoblasts and myotubes differentially expressed members of the RAS including angiotensinogen, angiotensin-converting enzyme (ACE), angiotensin II (Ang II) type 1 (AT(1)) and type 2 (AT(2)). Renin transcripts were never detected, however, mRNA for the 'renin-like' enzyme cathepsin D was observed and Ang I and Ang II were identified in cell culture supernatants from proliferating myoblasts. AT(1) appeared to co-localise with polymerised actin filaments in proliferating myoblasts and was primarily found in the nucleus of terminally differentiated myotubes. Furthermore, mechanical stretch of proliferating and differentiating C2C12 cells differentially induced mRNA expression of angiotensinogen, AT(1) and AT(2). Proliferating and differentiated muscle stem cells possess a local stress-responsive RAS in vitro. The precise function of a local RAS in myoblasts remains unknown. However, evidence presented here suggests that Ang II may be a regulator of skeletal muscle myoblasts.

  1. MicroRNA-27a promotes myoblast proliferation by targeting myostatin

    SciTech Connect

    Huang, Zhiqing; Chen, Xiaoling; Yu, Bing; He, Jun; Chen, Daiwen

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer We identified a myogenic role for miR-27a and a new target, myostatin. Black-Right-Pointing-Pointer The miR-27a was confirmed to target myostatin 3 Prime UTR. Black-Right-Pointing-Pointer miR-27a is upregulated and myostatin is downregulated during myoblast proliferation. Black-Right-Pointing-Pointer miR-27a promotes myoblast proliferation by reducing the expression of myostatin. -- Abstract: MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. However, the role of miRNAs in myoblast proliferation remains poorly understood. Here we found that the expression of miR-27a was increased during proliferation of C2C12 myoblasts. Moreover, overexpression of miR-27a in C2C12 cells promoted myoblast proliferation by reducing the expression of myostatin, a critical inhibitor of skeletal myogenesis. In addition, the miR-27a was confirmed to target myostatin 3 Prime UTR by a luciferase reporter analysis. Together, these results suggest that miR-27a promotes myoblast proliferation through targeting myostatin.

  2. PPARγ regulates inflammatory reaction by inhibiting the MAPK/NF-κB pathway in C2C12 skeletal muscle cells.

    PubMed

    Kim, Jeong-Seok; Lee, Young-Hee; Chang, Yong-Uoo; Yi, Ho-Keun

    2017-02-01

    Excessive exercise induces an inflammatory response caused by oxidative stress, which delays recovery of damaged muscle fibers. The reduction of inflammatory response is important for skeletal muscle homeostasis. Peroxisome proliferator-activated receptor gamma (PPARγ) is an anti-inflammatory molecule, but the role of PPARγ in skeletal muscle as anti-inflammatory activity is not clear. Thus, this study examined the anti-inflammatory role of PPARγ against H2O2-induced oxidative stress in skeletal muscle. Sprague Dawley (SD) rats were exercised on a treadmill to induce oxidative stress. In vitro oxidative stress was evaluated in differentiated C2C12 cells stimulated using 200 μM H2O2. Inflammation-related molecules were determined by immunohistochemistry and Western blot analysis. Expressions of the inflammatory molecules tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), cyclooxygenase-2 (COX-2), and matrix metalloproteinase-2 (MMP-2) in muscles of the acute exercise group were highly increased. PPARγ was also highly expressed in these muscles. These inflammatory molecules were also markedly increased in C2C12 cells with H2O2 stimulation. However, PPARγ overexpression in C2C12 transfected by Ad/PPARγ dramatically reduced the inflammatory molecules. PPARγ also enhanced the anti-oxidants molecules like Cu/Zn-SOD, Mn-SOD, and hemeoxygenase-1 by reducing the generation of ROS, even in the presence of H2O2. PPARγ displayed dual anti-inflammatory and anti-oxidant roles by inhibiting the mitogen-activated protein kinase (MAPK) pathway and translocation of nuclear transcriptional factor-κB (NF-κB) from the cytosol to the nucleus. These results demonstrate a potential role of PPARγ in protecting muscle fibers against oxidative stress caused by excessive acute exercise due to its anti-inflammatory and anti-oxidant activity exerted by inhibition of the MAPK/NF-κB pathway.

  3. Vascular Endothelial Growth Factor Modulates Skeletal Myoblast Function

    PubMed Central

    Germani, Antonia; Di Carlo, Anna; Mangoni, Antonella; Straino, Stefania; Giacinti, Cristina; Turrini, Paolo; Biglioli, Paolo; Capogrossi, Maurizio C.

    2003-01-01

    Vascular endothelial growth factor (VEGF) expression is enhanced in ischemic skeletal muscle and is thought to play a key role in the angiogenic response to ischemia. However, it is still unknown whether, in addition to new blood vessel growth, VEGF modulates skeletal muscle cell function. In the present study immunohistochemical analysis showed that, in normoperfused mouse hindlimb, VEGF and its receptors Flk-1 and Flt-1 were expressed mostly in quiescent satellite cells. Unilateral hindlimb ischemia was induced by left femoral artery ligation. At day 3 and day 7 after the induction of ischemia, Flk-1 and Flt-1 were expressed in regenerating muscle fibers and VEGF expression by these fibers was markedly enhanced. Additional in vitro experiments showed that in growing medium both cultured satellite cells and myoblast cell line C2C12 expressed VEGF and its receptors. Under these conditions, Flk-1 receptor exhibited constitutive tyrosine phosphorylation that was increased by VEGF treatment. During myogenic differentiation Flk-1 and Flt-1 were down-regulated. In a modified Boyden Chamber assay, VEGF enhanced C2C12 myoblasts migration approximately fivefold. Moreover, VEGF administration to differentiating C2C12 myoblasts prevented apoptosis, while inhibition of VEGF signaling either with selective VEGF receptor inhibitors (SU1498 and CB676475) or a neutralizing Flk-1 antibody, enhanced cell death approximately 3.5-fold. Finally, adenovirus-mediated VEGF165 gene transfer inhibited ischemia-induced apoptosis in skeletal muscle. These results support a role for VEGF in myoblast migration and survival, and suggest a novel autocrine role of VEGF in skeletal muscle repair during ischemia. PMID:14507649

  4. DHA inhibits protein degradation more efficiently than EPA by regulating the PPARγ/NFκB pathway in C2C12 myotubes.

    PubMed

    Wang, Yue; Lin, Qiao-wei; Zheng, Pei-pei; Zhang, Jian-song; Huang, Fei-ruo

    2013-01-01

    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 (P < 0.01). However, the total protein degradation was decreased when the concentrations of DHA ranged from 300 µM to 700 µM (P < 0.01). DHA (400 µM, 24 h) more efficiently decreased the I κ B α phosphorylation and increased in the I κ B α protein level than 400 µM EPA (P < 0.01). Compared with BSA, 400 µM EPA and DHA resulted in a 47% or 68% induction of the NF κ B DNA binding activity, respectively (P < 0.01). Meanwhile, 400 µM EPA and DHA resulted in a 1.3-fold and 2.0-fold induction of the PPAR γ expression, respectively (P < 0.01). In C2C12 myotubes for PPAR γ knockdown, neither 400 µM EPA nor DHA affected the levels of p-I κ B α , total I κ B α or NF κ B DNA binding activity compared with BSA (P > 0.05). Interestingly, EPA and DHA both still decreased the total protein degradation, although PPAR γ 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 PPAR γ /NF- κ B pathway in C2C12 myotubes.

  5. Increased reactive oxygen species production down-regulates peroxisome proliferator-activated alpha pathway in C2C12 skeletal muscle cells.

    PubMed

    Cabrero, Agatha; Alegret, Marta; Sanchez, Rosa M; Adzet, Tomas; Laguna, Juan C; Carrera, Manuel Vazquez

    2002-03-22

    Generation of reactive oxygen species may contribute to the pathogenesis of diseases involving intracellular lipid accumulation. To explore the mechanisms leading to these pathologies we tested the effects of etomoxir, an inhibitor of carnitine palmitoyltransferase I which contains a fatty acid-derived structure, in C2C12 skeletal muscle cells. Etomoxir treatment for 24 h resulted in a down-regulation of peroxisome proliferator-activated receptor alpha (PPARalpha) mRNA expression, achieving an 87% reduction at 80 microm etomoxir. The mRNA levels of most of the PPARalpha target genes studied were reduced at 100 microm etomoxir. By using several inhibitors of de novo ceramide synthesis and C(2)-ceramide we showed that they were not involved in the effects of etomoxir. Interestingly, the addition of triacsin C, a potent inhibitor of acyl-CoA synthetase, to etomoxir-treated C2C12 skeletal muscle cells did not prevent the down-regulation in PPARalpha mRNA levels, suggesting that the active form of the drug, etomoxir-CoA, was not involved. Given that saturated fatty acids may generate reactive oxygen species (ROS), we determined whether the addition of etomoxir resulted in ROS generation. Etomoxir increased ROS production and the activity of the well known redox transcription factor NF-kappaB. In the presence of the pyrrolidine dithiocarbamate, a potent antioxidant and inhibitor of NF-kappaB activity, etomoxir did not down-regulate PPARalpha mRNA in C2C12 skeletal muscle cells. These results indicate that ROS generation and NF-kappaB activation are responsible for the down-regulation of PPARalpha and may provide a new mechanism by which intracellular lipid accumulation occurs in skeletal muscle cells.

  6. Metabolic Fate of Branched-Chain Amino Acids During Adipogenesis, in Adipocytes From Obese Mice and C2C12 Myotubes.

    PubMed

    Estrada-Alcalde, Isabela; Tenorio-Guzman, Miriam R; Tovar, Armando R; Salinas-Rubio, Daniela; Torre-Villalvazo, Ivan; Torres, Nimbe; Noriega, Lilia G

    2017-04-01

    Branched-chain amino acid (BCAA) catabolism is regulated by the branched-chain aminotransferase (BCAT2) and the branched-chain α-keto acid dehydrogenase complex (BCKDH). BCAT2 and BCKDH expression and activity are modified during adipogenesis and altered in adipose tissues of mice with genetic or diet-induced obesity. However, little is known about how these modifications and alterations affect the intracellular metabolic fate of BCAAs during adipogenesis, in adipocytes from mice fed a control or high-fat diet or in C2C12 myotubes. Here, we demonstrate that BCAAs are mainly incorporated into proteins during the early stages of adipocyte differentiation. However, they are oxidized and incorporated into lipids during the late days of differentiation. Conversely, 92% and 97% of BCAA were oxidized, 1.6% and 6% were used for protein synthesis and 1.2% and 1.5% were incorporated into lipids in adipocytes from epididymal and subcutaneous adipose tissue, respectively. All three pathways were decreased in adipocytes from mice fed a high-fat diet. In C2C12 myotubes, leucine is mainly used for protein synthesis and palmitate is incorporated into lipids. Interestingly, leucine decreased both palmitate oxidation and its incorporation to lipids and proteins; and palmitate increased leucine oxidation and decreased its incorporation to lipids and proteins in a dose-dependent manner. These results demonstrate that BCAA metabolic fate differs between the early and late stages of adipocyte differentiation and in adipocytes from mice fed a control or high-fat diet; and that leucine affects the metabolic fate of palmitate and vice versa in C2C12 myotubes. J. Cell. Biochem. 118: 808-818, 2017. © 2016 Wiley Periodicals, Inc.

  7. FOXO1 delays skeletal muscle regeneration and suppresses myoblast proliferation.

    PubMed

    Yamashita, Atsushi; Hatazawa, Yukino; Hirose, Yuma; Ono, Yusuke; Kamei, Yasutomi

    2016-08-01

    Unloading stress, such as bed rest, inhibits the regenerative potential of skeletal muscles; however, the underlying mechanisms remain largely unknown. FOXO1 expression, which induces the upregulated expression of the cell cycle inhibitors p57 and Gadd45α, is known to be increased in the skeletal muscle under unloading conditions. However, there is no report addressing FOXO1-induced inhibition of myoblast proliferation. Therefore, we induced muscle injury by cardiotoxin in transgenic mice overexpressing FOXO1 in the skeletal muscle (FOXO1-Tg mice) and observed regeneration delay in skeletal muscle mass and cross-sectional area in FOXO1-Tg mice. Increased p57 and Gadd45α mRNA levels, and decreased proliferation capacity were observed in C2C12 myoblasts expressing a tamoxifen-inducible active form of FOXO1. These results suggest that decreased proliferation capacity of myoblasts by FOXO1 disrupts skeletal muscle regeneration under FOXO1-increased conditions, such as unloading.

  8. Mitochondrial membrane potential is reduced in copper-deficient C2C12 cells in the absence of apoptosis.

    PubMed

    Chen, Xiulian; Medeiros, Denis M; Jennings, Dianne

    2005-07-01

    Mitochondrial membrane potential is reduced in copper-deficient rat hearts, but it is uncertain if this will lead to the onset of apoptosis. To determine if copper deficiency per se leads to apoptosis, C2C12 cells were made copper deficient by treatment with the copper chelator tetraethylenepentamine (TEPA). In TEPA-treated cells, the activity of Cu, Zn-superoxide dismutase and cytochrome-c oxidase decreased dramatically. The protein levels of nuclear-encoded subunits of the cytochromie-c oxidase decreased, but the mitochondrial-encoded subunits remained unchanged. Decreased mitochondrial membrane potential was indicated in TEPA-treated cells, but further investigation of the potential induction of apoptosis by measuring caspase-3 activity, protein concentrations of Bcl-2 and Bax, and DNA fragmentation suggested that apoptosis is not induced in TEPA-treated C2C12 cells. Cells with decreased mitochondrial membrane potential were not destined to apoptosis as a result of copper deficiency.

  9. EPO-receptor is present in mouse C2C12 and human primary skeletal muscle cells but EPO does not influence myogenesis.

    PubMed

    Lamon, Séverine; Zacharewicz, Evelyn; Stephens, Andrew N; Russell, Aaron P

    2014-01-01

    Abstract The role and regulation of the pleiotropic cytokine erythropoietin (EPO) in skeletal muscle are controversial. EPO exerts its effects by binding its specific receptor (EPO-R), which activates intracellular signaling and gene transcription in response to internal and external stress signals. EPO is suggested to play a direct role in myogenesis via the EPO-R, but several studies have questioned the effect of EPO treatment in muscle in vitro and in vivo. The lack of certainty surrounding the use of nonspecific EPO-R antibodies contributes to the ambiguity of the field. Our study demonstrates that the EPO-R gene and protein are expressed at each stage of mouse C2C12 and human skeletal muscle cell proliferation and differentiation and validates a specific antibody for the detection of the EPO-R protein. However, in our experimental conditions, EPO treatment had no effect on mouse C2C12 and human muscle cell proliferation, differentiation, protein synthesis or EPO-R expression. While an increase in Akt and MAPK phosphorylation was observed, we demonstrate that this effect resulted from the stress caused by changing medium and not from EPO treatment. We therefore suggest that skeletal muscle EPO-R might be present in a nonfunctional form, or too lowly expressed to play a role in muscle cell function.

  10. Chronic reactive oxygen species exposure inhibits glucose uptake and causes insulin resistance in C2C12 myotubes.

    PubMed

    Ding, Hongwen; Heng, Baoli; He, Wenfang; Shi, Liping; Lai, Caiyong; Xiao, Long; Ren, Haolin; Mo, Shijie; Su, Zexuan

    2016-09-16

    Reactive oxygen species (ROS) is an important regulator in cellular signaling transduction, and many previous studies have indicated that acute ROS stimulation improves insulin sensitivity in skeletal muscle. In the study, we found that chronic ROS treatment caused serious insulin resistance in C2C12 myotubes. Glucose uptake and consumption assay indicated that pretreatment with 80 μM H2O2 for 2 h inhibited insulin-stimulated glucose uptake in C2C12 myotubes, and the reason for it, is that chronic H2O2 treatment decreased insulin-induced glucose transporter 4 (GLUT4) translocation from cell plasma to cell membrane. Moreover, Akt2 phosphorylation depended on insulin was reduced in C2C12 myotubes of chronic H2O2 treatment. Together, this study provides further demonstration that chronic ROS stress is associated with insulin resistance of skeletal muscle in the progression of type 2 diabetes. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. α-Syntrophin Modulates Myogenin Expression in Differentiating Myoblasts

    PubMed Central

    Kim, Min Jeong; Hwang, Sung Ho; Lim, Jeong A.; Froehner, Stanley C.; Adams, Marvin E.; Kim, Hye Sun

    2010-01-01

    Background α-Syntrophin is a scaffolding protein linking signaling proteins to the sarcolemmal dystrophin complex in mature muscle. However, α-syntrophin is also expressed in differentiating myoblasts during the early stages of muscle differentiation. In this study, we examined the relationship between the expression of α-syntrophin and myogenin, a key muscle regulatory factor. Methods and Findings The absence of α-syntrophin leads to reduced and delayed myogenin expression. This conclusion is based on experiments using muscle cells isolated from α-syntrophin null mice, muscle regeneration studies in α-syntrophin null mice, experiments in Sol8 cells (a cell line that expresses only low levels of α-syntrophin) and siRNA studies in differentiating C2 cells. In primary cultured myocytes isolated from α-syntrophin null mice, the level of myogenin was less than 50% that from wild type myocytes (p<0.005) 40 h after differentiation induction. In regenerating muscle, the expression of myogenin in the α-syntrophin null muscle was reduced to approximately 25% that of wild type muscle (p<0.005). Conversely, myogenin expression is enhanced in primary cultures of myoblasts isolated from a transgenic mouse over-expressing α-syntrophin and in Sol8 cells transfected with a vector to over-express α-syntrophin. Moreover, we find that myogenin mRNA is reduced in the absence of α-syntrophin and increased by α-syntrophin over-expression. Immunofluorescence microscopy shows that α-syntrophin is localized to the nuclei of differentiating myoblasts. Finally, immunoprecipitation experiments demonstrate that α-syntrophin associates with Mixed-Lineage Leukemia 5, a regulator of myogenin expression. Conclusions We conclude that α-syntrophin plays an important role in regulating myogenesis by modulating myogenin expression. PMID:21179410

  12. Spatial Geometries of Self-Assembled Chitohexaose Monolayers Regulate Myoblast Fusion

    PubMed Central

    Poosala, Pornthida; Ichinose, Hirofumi; Kitaoka, Takuya

    2016-01-01

    Myoblast fusion into functionally-distinct myotubes to form in vitro skeletal muscle constructs under differentiation serum-free conditions still remains a challenge. Herein, we report that our microtopographical carbohydrate substrates composed of bioactive hexa-N-acetyl-d-glucosamine (GlcNAc6) modulated the efficiency of myoblast fusion without requiring horse serum or any differentiation medium during cell culture. Promotion of the differentiation of dissociated mononucleated skeletal myoblasts (C2C12; a mouse myoblast cell line) into robust myotubes was found only on GlcNAc6 micropatterns, whereas the myoblasts on control, non-patterned GlcNAc6 substrates or GlcNAc6-free patterns exhibited an undifferentiated form. We also examined the possible role of GlcNAc6 micropatterns with various widths in the behavior of C2C12 cells in early and late stages of myogenesis through mRNA expression of myosin heavy chain (MyHC) isoforms. The spontaneous contraction of myotubes was investigated via the regulation of glucose transporter type 4 (GLUT4), which is involved in stimulating glucose uptake during cellular contraction. Narrow patterns demonstrated enhanced glucose uptake rate and generated a fast-twitch muscle fiber type, whereas the slow-twitch muscle fiber type was dominant on wider patterns. Our findings indicated that GlcNAc6-mediated integrin interactions are responsible for guiding myoblast fusion forward along with myotube formation. PMID:27164094

  13. Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro

    PubMed Central

    Zhong, Xia; Wang, Qian-Qian; Li, Jian-Wei; Zhang, Yu-Mei; An, Xiao-Rong; Hou, Jian

    2017-01-01

    Muscle cell differentiation is a complex process that is principally governed by related myogenic regulatory factors (MRFs). DNA methylation is considered to play an important role on the expression of MRF genes and on muscle cell differentiation. However, the roles of enzymes specifically in myogenesis are not fully understood. Here, we demonstrate that Tet2, a ten-eleven translocation (Tet) methylcytosine dioxygenase, exerts a role during skeletal myoblast differentiation. By using an immunostaining method, we found that the levels of 5-hydroxymethylcytosine (5-hmC) were much higher in differentiated myotubes than in undifferentiated C2C12 myoblasts. Both Tet1 and Tet2 expression were upregulated after differentiation induction of C2C12 myoblasts. Knockdown of Tet2, but not Tet1, significantly reduced the expression of myogenin as well as Myf6 and myomaker, and impaired myoblast differentiation. DNA demethylation of myogenin and myomaker promoters was negatively influenced by Tet2 knockdown as detected by bisulfite sequencing analysis. Furthermore, although vitamin C could promote genomic 5hmC generation, myogenic gene expression and myoblast differentiation, its effect was significantly attenuated by Tet2 knockdown. Taken together, these results indicate that Tet2 is involved in myoblast differentiation through promoting DNA demethylation and myogenic gene expression. PMID:28272491

  14. Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro.

    PubMed

    Zhong, Xia; Wang, Qian-Qian; Li, Jian-Wei; Zhang, Yu-Mei; An, Xiao-Rong; Hou, Jian

    2017-03-08

    Muscle cell differentiation is a complex process that is principally governed by related myogenic regulatory factors (MRFs). DNA methylation is considered to play an important role on the expression of MRF genes and on muscle cell differentiation. However, the roles of enzymes specifically in myogenesis are not fully understood. Here, we demonstrate that Tet2, a ten-eleven translocation (Tet) methylcytosine dioxygenase, exerts a role during skeletal myoblast differentiation. By using an immunostaining method, we found that the levels of 5-hydroxymethylcytosine (5-hmC) were much higher in differentiated myotubes than in undifferentiated C2C12 myoblasts. Both Tet1 and Tet2 expression were upregulated after differentiation induction of C2C12 myoblasts. Knockdown of Tet2, but not Tet1, significantly reduced the expression of myogenin as well as Myf6 and myomaker, and impaired myoblast differentiation. DNA demethylation of myogenin and myomaker promoters was negatively influenced by Tet2 knockdown as detected by bisulfite sequencing analysis. Furthermore, although vitamin C could promote genomic 5hmC generation, myogenic gene expression and myoblast differentiation, its effect was significantly attenuated by Tet2 knockdown. Taken together, these results indicate that Tet2 is involved in myoblast differentiation through promoting DNA demethylation and myogenic gene expression.

  15. Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

    NASA Astrophysics Data System (ADS)

    Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2016-07-01

    Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm-2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

  16. Activation of AMPK/TSC2/PLD by alcohol regulates mTORC1 and mTORC2 assembly in C2C12 myocytes.

    PubMed

    Hong-Brown, Ly Q; Brown, C Randell; Navaratnarajah, Maithili; Lang, Charles H

    2013-11-01

    Ethanol (EtOH) decreases muscle protein synthesis, and this is associated with reduced mammalian target of rapamycin complex (mTORC)1 and increased mTORC2 activities. In contrast, phospholipase D (PLD) and its metabolite phosphatidic acid (PA) positively regulate mTORC1 signaling, whereas their role in mTORC2 function is less well defined. Herein, we examine the role that PLD and PA play in EtOH-mediated mTOR signaling. C2C12 myoblasts were incubated with EtOH for 18 to 24 hours. For PA experiments, cells were pretreated with the drug for 25 minutes followed by 50-minute incubation with PA in the presence or absence of EtOH. The phosphorylation state of various proteins was assessed by immunoblotting. Protein-protein interactions were determined by immunoprecipitation and immunoblotting. PLD activity was measured using the Amplex Red PLD assay kit. PA concentrations were determined with a total PA assay kit. PA levels and PLD activity increased in C2C12 myocytes exposed to EtOH (100 mM). Increased PLD activity was blocked by inhibitors of AMP-activated protein kinase (AMPK) (compound C) and phosphoinositide 3-kinase (PI3K) (wortmannin). Likewise, suppression of PLD activity with CAY10594 prevented EtOH-induced Akt (S473) phosphorylation. PLD inhibition also enhanced the binding of Rictor to mSin1 and the negative regulatory proteins Deptor and 14-3-3. Addition of PA to myocytes decreased Akt phosphorylation, but changes in mTORC2 activity were not associated with altered binding of complex members and 14-3-3. PA increased S6K1 phosphorylation, with the associated increase in mTORC1 activity being regulated by reduced phosphorylation of AMPKα (T172) and its target tuberous sclerosis protein complex (TSC)2 (S1387). This resulted in increased Rheb and RagA/RagC GTPase interactions with mTOR, as well as suppression of mTORC2. EtOH-induced increases in PLD activity and PA may partially counterbalance the adverse effects of this agent. EtOH and PA regulate mTORC1 via

  17. Altered expression of ganglioside GM3 molecular species and a potential regulatory role during myoblast differentiation.

    PubMed

    Go, Shinji; Go, Shiori; Veillon, Lucas; Ciampa, Maria Grazia; Mauri, Laura; Sato, Chihiro; Kitajima, Ken; Prinetti, Alessandro; Sonnino, Sandro; Inokuchi, Jin-Ichi

    2017-04-28

    Gangliosides (sialic acid-containing glycosphingolipids) help regulate many important biological processes, including cell proliferation, signal transduction, and differentiation, via formation of functional microdomains in plasma membranes. The structural diversity of gangliosides arises from both the ceramide moiety and glycan portion. Recently, differing molecular species of a given ganglioside are suggested to have distinct biological properties and regulate specific and distinct biological events. Elucidation of the function of each molecular species is important and will provide new insights into ganglioside biology. Gangliosides are also suggested to be involved in skeletal muscle differentiation; however, the differential roles of ganglioside molecular species remain unclear. Here we describe striking changes in quantity and quality of gangliosides (particularly GM3) during differentiation of mouse C2C12 myoblast cells and key roles played by distinct GM3 molecular species at each step of the process. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Involvement of Ras and Ral in Chemotactic Migration of Skeletal Myoblasts

    PubMed Central

    Suzuki, Jotaro; Yamazaki, Yuji; Guang, Li; Kaziro, Yoshito; Koide, Hiroshi

    2000-01-01

    In skeletal myoblasts, Ras has been considered to be a strong inhibitor of myogenesis. Here, we demonstrate that Ras is involved also in the chemotactic response of skeletal myoblasts. Expression of a dominant-negative mutant of Ras inhibited chemotaxis of C2C12 myoblasts in response to basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1), key regulators of limb muscle development and skeletal muscle regeneration. A dominant-negative Ral also decreased chemotactic migration by these growth factors, while inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase (MEK) showed no effect. Activation of the Ras-Ral pathway by expression of an activated mutant of either Ras, the guanine-nucleotide dissociation stimulator for Ral, or Ral resulted in increased motility of myoblasts. The ability of Ral to stimulate motility was reduced by introduction of a mutation which prevents binding to Ral-binding protein 1 or phospholipase D. These results suggest that the Ras-Ral pathway is essential for the migration of myoblasts. Furthermore, we found that Ras and Ral are activated in C2C12 cells by bFGF, HGF and IGF-1 and that the Ral activation is regulated by the Ras- and the intracellular Ca2+-mediated pathways. Taken together, our data indicate that Ras and Ral regulate the chemotactic migration of skeletal muscle progenitors. PMID:10848592

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

    PubMed Central

    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

    Background: 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. Results: 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. Conclusion: These findings position STARS as an important regulator of skeletal muscle growth and regeneration. PMID:26903873

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

    PubMed

    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.

  1. Activation of Mechanosensitive Transcription Factors in Murine C2C12 Mesenchymal Precursors by Focused Low-Intensity Pulsed Ultrasound (FLIPUS).

    PubMed

    Puts, Regina; Rikeit, Paul; Ruschke, Karen; Kadow-Romacker, Anke; Hwang, Soyoung; Jenderka, Klaus-Vitold; Knaus, Petra; Raum, Kay

    2016-10-01

    In this paper, we investigated the mechanoresponse of C2C12 mesenchymal precursor cells to focused low-intensity pulsed ultrasound (FLIPUS). The setup has been developed for in vitro stimulation of adherent cells in the defocused far field of the ultrasound propagating through the bottom of the well plate. Twenty-four-well tissue culture plates, carrying the cell monolayers, were incubated in a temperature-controlled water tank. The ultrasound was applied at 3.6-MHz frequency, pulsed at 100-Hz repetition frequency with a 27.8% duty cycle, and calibrated at an output intensity of ISATA = 44.5 ±7.1 mW/cm(2). Numerical sound propagation simulations showed no generation of standing waves in the well plate. The response of murine C2C12 cells to FLIPUS was evaluated by measuring activation of mechanosensitive transcription factors, i.e., activator protein-1 (AP-1), specificity protein 1 (Sp1), and transcriptional enhancer factor (TEAD), and expression of mechanosensitive genes, i.e., c-fos, c-jun, heparin binding growth associated molecule (HB-GAM), and Cyr-61. FLIPUS induced 50% ( p ≤ 0.05 ) and 70% ( p ≤ 0.05 ) increases in AP-1 and TEAD promoter activities, respectively, when stimulated for 5 min. The Sp1 activity was enhanced by about 20% ( p ≤ 0.05 ) after 5-min FLIPUS exposure and the trend persisted for 30-min ( p ≤ 0.05 ) and 1-h ( p ≤ 0.05 ) stimulation times. Expressions of mechanosensitive genes c-fos ( p ≤ 0.05 ), c-jun ( p ≤ 0.05 ), HB-GAM ( p ≤ 0.05 ), and cystein-rich protein 61 ( p ≤ 0.05 ) were enhanced in response to 5-min FLIPUS stimulation. The increase in proliferation of C2C12s occurred after the FLIPUS stimulation ( p ≤ 0.05 ), with AP-1, Sp1, and TEAD possibly regulating the observed cellular activities.

  2. Hydro-Alcoholic Cinnamon Extract, Enhances Glucose Transporter Isotype-4 Translocation from Intracellular Compartments into the Cytoplasmic Membrane of C2C12 Myotubes.

    PubMed

    Absalan, Abdorrahim; Mohiti-Ardakani, Javad; Hadinedoushan, Hossein; Khalili, Mohammad Ali

    2012-10-01

    Cinnamon has been used as an anti-diabetic agent for centuries but only in recent few years its mechanism of action has been under investigation. Previous studies showed that cinnamon might exert its anti-diabetic effect via increasing glucose transporter isotype-4 (GLUT4) gene and glycoprotein contents in fat cells. To study if hydro-alcoholic cinnamon extract (HACE) enhances GLUT4 translocation from intracellular compartments of nuclear or endoplasmic reticulum membranes (N/ER) into the cytoplasmic membrane (CM). C2C12 myoblastic cell line were seeded in DMEM plus 20 % FBS and differentiated to myotubes using 2 % horse serum. After myotubes formation, 100 or 1,000 μg/ml HACE, as intervention, and as control 1 % DMSO were added for 3 h. Cells were washed and homogenized followed by ultracentrifuge fractionation, protein separation by SDS-PAGE and GLUT4 detection using semi-quantitative Western blotting. Data analysis was done by two-independent samples t test for comparison of mean ± SD of GLUT4 percent in categories. GLUT4 contents were higher in CM of groups 100 and 1,000 μg/ml HACE and lower in 1 % DMSO treated myotubes (CI = 0.95, P < 0.05). For N/ER reverse results were obtained (CI = 0.95, P < 0.05). As our results have shown HACE induces GLUT4 translocation from intra-cell into cell surface. We conclude that cinnamon maybe a choice of type-2 diabetes mellitus treatment because its extract enhances GLUT4 contents in CM where it facilitates glucose entrance into the cell. However it is necessary to trace the signaling pathways which are activated by HACE in muscular tissue.

  3. Therapeutic angiogenesis by a myoblast layer harvested by tissue transfer printing from cell-adhesive, thermosensitive hydrogels.

    PubMed

    Kim, Dong Wan; Jun, Indong; Lee, Tae-Jin; Lee, Ji Hye; Lee, Young Jun; Jang, Hyeon-Ki; Kang, Seokyung; Park, Ki Dong; Cho, Seung-Woo; Kim, Byung-Soo; Shin, Heungsoo

    2013-11-01

    Peripheral arterial disease (PAD) is characterized by the altered structure and function of arteries caused by accumulated plaque. There have been many studies on treating this disease by the direct injection of various types of therapeutic cells, however, the low cell engraftment efficiency and diffusion of the transplanted cells have been major problems. In this study, we developed an approach (transfer printing) to deliver monolayer of cells to the hindlimb ischemic tissue using thermosensitive hydrogels, and investigated its efficacy in long term retention upon transplantation and therapeutic angiogenesis. We first investigated the in vitro maintenance of robust cell-cell contacts and stable expression of the ECM proteins in myoblast layer following transfer printing process. In order to confirm the therapeutic effect of the myoblasts in vivo, we cultured a monolayer of C2C12 myoblasts on thermosensitive hydrogels, which was then transferred to the hindlimb ischemia tissue of athymic mice directly from the hydrogel by conformal contact. The transferred myoblast layer was retained for a longer period of time than an intramuscularly injected cell suspension. In addition, the morphology of the mice and laser Doppler perfusion (28 days after treatment) supported that the myoblast layer enhanced the therapeutic effects on the ischemic tissue. In summary, the transplantation of the C2C12 myoblast layer using a tissue transfer printing method could represent a new approach for the treatment of PAD by therapeutic angiogenesis.

  4. The brain expressed x-linked gene 1 (Bex1) regulates myoblast fusion

    PubMed Central

    Yue, Feng; Kuang, Shihuan

    2015-01-01

    Skeletal muscle development (myogenesis) is a complex but precisely orchestrated process involving spatiotemporal regulation of the proliferation, differentiation and fusion of myogenic progenitor cells (myoblasts). Here we identify brain expressed x-linked gene 1 (Bex1) as a transient, developmentally regulated gene involved in myoblast fusion. Bex1 expression is undetectable in adult muscles or in quiescent muscle stem cells (satellite cells). During embryonic myogenesis, however, Bex1 is robustly expressed by myogenin+ differentiating myoblasts, but not by Pax7+ proliferating myoblasts. Interestingly, Bex1 is initially localized in the cytoplasm and then translocates into the nucleus. During adult muscle regeneration, Bex1 is highly expressed in newly regenerated myofibers and the expression is rapidly downregulated during maturation. Consistently, in cultured myoblasts, Bex1 is not expressed at the proliferation stage but transiently expressed upon induction of myogenic differentiation, following a similar cytoplasm to nucleus translocation pattern as seen in vivo. Using gain- and loss-of-function studies, we found that overexpression of Bex1 promotes the fusion of primary myoblasts without affecting myogenic differentiation and myogenin expression. Conversely, Bex1 knockout myoblasts exhibit obvious fusion defects, even though they express normal levels of myogenin and differentiate normally. These results elucidate a novel role of Bex1 in myogenesis through regulating myoblast fusion. PMID:26586200

  5. Hydroxylamine enhances glucose uptake in C2C12 skeletal muscle cells through the activation of insulin receptor substrate 1.

    PubMed

    Kimura, Taro; Kato, Eisuke; Machikawa, Tsukasa; Kimura, Shunsuke; Katayama, Shinji; Kawabata, Jun

    2014-02-28

    Diabetes mellitus is a global disease, and the number of patients with it is increasing. Of various agents for treatment, those that directly act on muscle are currently attracting attention because muscle is one of the main tissues in the human body, and its metabolism is decreased in type II diabetes. In this study, we found that hydroxylamine (HA) enhances glucose uptake in C2C12 myotubes. Analysis of HA's mechanism revealed the involvement of IRS1, PI3K and Akt that is related to the insulin signaling pathway. Further investigation about the activation mechanism of insulin receptor or IRS1 by HA may provide a way to develop a novel anti-diabetic agent alternating to insulin. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    SciTech Connect

    Kamolrat, Torkamol; Gray, Stuart R.

    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.

  7. Melatonin protects against uric acid-induced mitochondrial dysfunction, oxidative stress, and triglyceride accumulation in C2C12 myotubes.

    PubMed

    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 C2C12 myotubes and assess how these are modulated by the antioxidant molecule melatonin. Differentiated C2C12 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.

  8. Leucine-induced activation of translational initiation is partly regulated by the branched-chain {alpha}-keto acid dehydrogenase complex in C2C12 cells

    SciTech Connect

    Nakai, Naoya . E-mail: nakai@hss.osaka-u.ac.jp; Shimomura, Yoshiharu; Tamura, Tomohiro; Tamura, Noriko; Hamada, Koichiro; Kawano, Fuminori; Ohira, Yoshinobu

    2006-05-19

    Branched-chain amino acid leucine has been shown to activate the translational regulators through the mammalian target of rapamycin. However, the leucine's effects are self-limiting because leucine promotes its own disposal by an oxidative pathway. The irreversible and rate-limiting step in the leucine oxidation pathway is catalyzed by the branched-chain {alpha}-keto acid dehydrogenase (BCKDH) complex. The complex contains E1 ({alpha}2{beta}2), E2, and E3 subunits, and its activity is abolished by phosphorylation of the E1{alpha} subunit by BCKDH kinase. The relationship between the activity of BCKDH complex and leucine-mediated activation of the protein translation was investigated using the technique of RNA interference. The activity of BCKDH complex in C2C12 cell was modulated by transfection of small interfering RNA (siRNA) for BCKDH E2 subunit or BCKDH kinase. Transfection of siRNAs decreased the mRNA expression and protein amount of corresponding gene. Suppression of either E2 subunit or kinase produced opposite effects on the cell proliferation and the activation of translational regulators by leucine. Suppression of BCKDH kinase for 48 h resulted in decreasing cell proliferation. In contrast, E2 suppression led to increased amount of total cellular protein. The phosphorylation of p70 S6 kinase by leucine was increased in E2-siRNA transfected C2C12 cells, whereas the leucine's effect was diminished in kinase-siRNA transfected cells. These results suggest that the activation of the translational regulators by leucine was partly regulated by the activity of BCKDH complex.

  9. Ductile electroactive biodegradable hyperbranched polylactide copolymers enhancing myoblast differentiation

    PubMed Central

    Xie, Meihua; Wang, Ling; Guo, Baolin; Wang, Zhong; Chen, Y. Eugene; Ma, Peter X.

    2015-01-01

    Myotube formation is crucial to restoring muscular functions, and biomaterials that enhance the myoblast differentiation into myotubes are highly desirable for muscular repair. Here, we report the synthesis of electroactive, ductile, and degradable copolymers and their application in enhancing the differentiation of myoblasts to myotubes. A hyperbranched ductile polylactide (HPLA) was synthesized and then copolymerized with aniline tetramer (AT) to produce a series of electroactive, ductile and degradable copolymers (HPLAAT). The HPLA and HPLAAT showed excellent ductility with strain to failure from 158.9% to 42.7% and modulus from 265.2 to 758.2 MPa. The high electroactivity of the HPLAAT was confirmed by UV spectrometer and cyclic voltammogram measurements. These HPLAAT polymers also showed improved thermal stability and controlled biodegradation rate compared to HPLA. Importantly, when applying these polymers for myotube formation, the HPLAAT significantly improved the proliferation of C2C12 myoblasts in vitro compared to HPLA. Furthermore, these polymers greatly promoted myogenic differentiation of C2C12 cells as measured by quantitative analysis of myotube number, length, diameter, maturation index, and gene expression of MyoD and TNNT. Together, our study shows that these electroactive, ductile and degradable HPLAAT copolymers represent significantly improved biomaterials for muscle tissue engineering compared to HPLA. PMID:26335860

  10. Ductile electroactive biodegradable hyperbranched polylactide copolymers enhancing myoblast differentiation.

    PubMed

    Xie, Meihua; Wang, Ling; Guo, Baolin; Wang, Zhong; Chen, Y Eugene; Ma, Peter X

    2015-12-01

    Myotube formation is crucial to restoring muscular functions, and biomaterials that enhance the myoblast differentiation into myotubes are highly desirable for muscular repair. Here, we report the synthesis of electroactive, ductile, and degradable copolymers and their application in enhancing the differentiation of myoblasts to myotubes. A hyperbranched ductile polylactide (HPLA) was synthesized and then copolymerized with aniline tetramer (AT) to produce a series of electroactive, ductile and degradable copolymers (HPLAAT). The HPLA and HPLAAT showed excellent ductility with strain to failure from 158.9% to 42.7% and modulus from 265.2 to 758.2 MPa. The high electroactivity of the HPLAAT was confirmed by UV spectrometer and cyclic voltammogram measurements. These HPLAAT polymers also showed improved thermal stability and controlled biodegradation rate compared to HPLA. Importantly, when applying these polymers for myotube formation, the HPLAAT significantly improved the proliferation of C2C12 myoblasts in vitro compared to HPLA. Furthermore, these polymers greatly promoted myogenic differentiation of C2C12 cells as measured by quantitative analysis of myotube number, length, diameter, maturation index, and gene expression of MyoD and TNNT. Together, our study shows that these electroactive, ductile and degradable HPLAAT copolymers represent significantly improved biomaterials for muscle tissue engineering compared to HPLA. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Transcription factor ZBED6 mediates IGF2 gene expression by regulating promoter activity and DNA methylation in myoblasts

    USDA-ARS?s Scientific Manuscript database

    Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were up regulated during C2C12 differentiation. The IGF2 expression levels wer...

  12. Decreased proliferation kinetics of mouse myoblasts overexpressing FRG1.

    PubMed

    Chen, Steven C; Frett, Ellie; Marx, Joseph; Bosnakovski, Darko; Reed, Xylena; Kyba, Michael; Kennedy, Brian K

    2011-01-01

    Although recent publications have linked the molecular events driving facioscapulohumeral muscular dystrophy (FSHD) to expression of the double homeobox transcription factor DUX4, overexpression of FRG1 has been proposed as one alternative causal agent as mice overexpressing FRG1 present with muscular dystrophy. Here, we characterize proliferative defects in two independent myoblast lines overexpressing FRG1. Myoblasts isolated from thigh muscle of FRG1 transgenic mice, an affected dystrophic muscle, exhibit delayed proliferation as measured by decreased clone size, whereas myoblasts isolated from the unaffected diaphragm muscle proliferated normally. To confirm the observation that overexpression of FRG1 could impair myoblast proliferation, we examined C2C12 myoblasts with inducible overexpression of FRG1, finding increased doubling time and G1-phase cells in mass culture after induction of FRG1 and decreased levels of pRb phosphorylation. We propose that depressed myoblast proliferation may contribute to the pathology of mice overexpressing FRG1 and may play a part in FSHD.

  13. SMAD3 and SP1/SP3 Transcription Factors Collaborate to Regulate Connective Tissue Growth Factor Gene Expression in Myoblasts in Response to Transforming Growth Factor β.

    PubMed

    Córdova, Gonzalo; Rochard, Alice; Riquelme-Guzmán, Camilo; Cofré, Catalina; Scherman, Daniel; Bigey, Pascal; Brandan, Enrique

    2015-09-01

    Fibrotic disorders are characterized by an increase in extracellular matrix protein expression and deposition, Duchene Muscular Dystrophy being one of them. Among the factors that induce fibrosis are Transforming Growth Factor type β (TGF-β) and the matricellular protein Connective Tissue Growth Factor (CTGF/CCN2), the latter being a target of the TGF-β/SMAD signaling pathway and is the responsible for the profibrotic effects of TGF-β. Both CTGF and TGF are increased in tissues affected by fibrosis but little is known about the regulation of the expression of CTGF mediated by TGF-β in muscle cells. By using luciferase reporter assays, site directed mutagenesis and specific inhibitors in C2C12 cells; we described a novel SMAD Binding Element (SBE) located in the 5' UTR region of the CTGF gene important for the TGF-β-mediated expression of CTGF in myoblasts. In addition, our results suggest that additional transcription factor binding sites (TFBS) present in the 5' UTR of the CTGF gene are important for this expression and that SP1/SP3 factors are involved in TGF-β-mediated CTGF expression.

  14. Dorsomorphin and LDN-193189 inhibit BMP-mediated Smad, p38 and Akt signalling in C2C12 cells.

    PubMed

    Boergermann, J H; Kopf, J; Yu, P B; Knaus, P

    2010-11-01

    Bone morphogenetic proteins (BMPs) are key regulators of cell fate decisions during embryogenesis and tissue homeostasis. BMPs signal through a coordinated assembly of two types of transmembrane serine/threonine kinase receptors to induce Smad1/5/8 plus non-Smad pathways, such as MAPK and Akt. The recent discovery of BMP receptor inhibitors opened new avenues to study specific BMP signalling and to delineate this effect from TGF-β and Activin signalling. Here we present comprehensive and quantitative analyses on both canonical and non-Smad mediated BMP signalling under Dorsomorphin (DM) and LDN-193189 (LDN) treatment conditions. We demonstrate for the first time, that both compounds affect not only the Smad but also the non-Smad signalling pathways induced by either BMP2, BMP6 or GDF5. The activation of p38, ERK1/2 and Akt in C2C12 cells was inhibited by DM and LDN. In addition "off-target" effects on all branches of BMP non-Smad signalling are presented. From this we conclude that the inhibition of BMP receptors by DM and more efficiently by LDN-193189 affects all known BMP induced signalling cascades.

  15. The Effects of Propionate and Valerate on Insulin Responsiveness for Glucose Uptake in 3T3-L1 Adipocytes and C2C12 Myotubes via G Protein-Coupled Receptor 41

    PubMed Central

    Han, Joo-Hui; Kim, In-Su; Jung, Sang-Hyuk; Lee, Sang-Gil; Son, Hwa-Young; Myung, Chang-Seon

    2014-01-01

    Since insulin resistance can lead to hyperglycemia, improving glucose uptake into target tissues is critical for regulating blood glucose levels. Among the free fatty acid receptor (FFAR) family of G protein-coupled receptors, GPR41 is known to be the Gαi/o-coupled receptor for short-chain fatty acids (SCFAs) such as propionic acid (C3) and valeric acid (C5). This study aimed to investigate the role of GPR41 in modulating basal and insulin-stimulated glucose uptake in insulin-sensitive cells including adipocytes and skeletal muscle cells. Expression of GPR41 mRNA and protein was increased with maximal expression at differentiation day 8 for 3T3-L1 adipocytes and day 6 for C2C12 myotubes. GPR41 protein was also expressed in adipose tissues and skeletal muscle. After analyzing dose-response relationship, 300 µM propionic acid or 500 µM valeric acid for 30 min incubation was used for the measurement of glucose uptake. Both propionic acid and valeric acid increased insulin-stimulated glucose uptake in 3T3-L1 adipocyte, which did not occur in cells transfected with siRNA for GPR41 (siGPR41). In C2C12 myotubes, these SCFAs increased basal glucose uptake, but did not potentiate insulin-stimulated glucose uptake, and siGPR41 treatment reduced valerate-stimulated basal glucose uptake. Therefore, these findings indicate that GPR41 plays a role in insulin responsiveness enhanced by both propionic and valeric acids on glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes, and in valerate-induced increase in basal glucose uptake in C2C12 myotubes. PMID:24748202

  16. 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

    SciTech Connect

    Yuasa, Katsutoshi; Aoki, Natsumi; Hijikata, Takao

    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.

  17. Increasing leucine concentration stimulates mechanistic target of rapamycin signaling and cell growth in C2C12 skeletal muscle cells.

    PubMed

    Areta, José L; Hawley, John A; Ye, Ji-Ming; Chan, M H Stanley; Coffey, Vernon G

    2014-11-01

    Leucine is a key amino acid for initiating translation in muscle cells, but the dose-dependent effects of leucine on intracellular signaling are poorly characterized. This study examined the effect that increasing doses of leucine would have on changes in mechanistic target of rapamycin (mTOR)-mediated signaling, rates of protein synthesis, and cell size in C2C12 cells. We hypothesized that a leucine "threshold" exists, which represents the minimum stimulus required to initiate mTOR signaling in muscle cells. Acute exposure to 1.5, 3.2, 5.0, and 16.1 mM leucine increased phosphorylation of mTOR(Ser2448) (~1.4-fold; P < .04), 4E-BP1 (Thr37/46) (~1.9-fold; P < .001), and rpS6(Ser235/6) (~2.3-fold; P < .001). However, only p70S6k(Thr389) exhibited a dose-dependent response to leucine with all treatments higher than control (~4-fold; P < .001) and at least 5 mM higher than the 1.5-mM concentration (1.2-fold; P < .02). Rates of protein synthesis were not altered by any treatment. Seven days of exposure to 0.5, 1.5, 5.0, and 16.5 mM leucine resulted in an increase in cell size in at least 5 mM treatments (~1.6-fold, P < .001 vs control). Our findings indicate that even at low leucine concentrations, phosphorylation of proteins regulating translation initiation signaling is enhanced. The phosphorylation of p70S6k(Thr389) follows a leucine dose-response relationship, although this was not reflected by the acute protein synthetic response. Nevertheless, under the conditions of the present study, it appears that leucine concentrations of at least 5 mM are necessary to enhance cell growth.

  18. RUNX1 Plays an Important Role in Mediating BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells Line C3H10T1/2, Murine Multi-Lineage Cells Lines C2C12 and MEFs.

    PubMed

    Ji, Caixia; Liu, Xiaohua; Xu, Li; Yu, Tingting; Dong, Chaoqun; Luo, Jinyong

    2017-06-23

    As one of the least studied bone morphogenetic proteins (BMPs), BMP9 is highly capable of promoting osteogenic differentiation. However, the underlying mechanism involved remains largely unknown. Recent studies have demonstrated that RUNX1 (runt-related transcription factor 1) is essential in osteoblast/chondrocyte maturation. In this study, we investigated the function of RUNX1 in BMP9-induced osteogenic of murine mesenchymal stem cell line (C3H10T1/2) and murine multi-lineage cell lines (C2C12 and MEFs). Our data showed that BMP9 promoted the endogenous expression of RUNX1 in C3H10T1/2, C2C12 and MEFs. Moreover, RUNX1 was probably a direct target of BMP9/Smad signaling. BMP9-induced osteogenic differentiation was enhanced by overexpression of RUNX1, whereas inhibited by knockdown RUNX1 in C3H10T1/2, C2C12 and MEFs. Further mechanism studies demonstrated that RUNX1 might affect BMP9-induced phosphorylation of Smad1/5/8, but not the phosphorylation of p38 and ERK1/2.Our results suggest that RUNX1 may be an essential modulator in BMP9- induced osteogenic differentiation of MSCs (Mesenchymal stem cells).

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

    USDA-ARS?s Scientific Manuscript database

    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...

  20. The nuclear protein-coding gene ANKRD23 negatively regulates myoblast differentiation.

    PubMed

    Wang, Xiaojing; Zeng, Rui; Xu, Haiyang; Xu, Zaiyan; Zuo, Bo

    2017-09-20

    Muscle fiber formation is a complex process and subject to fine regulation of a variety of protein-coding genes and non-coding RNA. In this study, we identified a nuclear protein-coding gene ANKRD23 which was highly expressed in muscle. Quantitative real-time PCR, western blotting and immunofluorescence were used to detect the expression change of myoblast differentiation marker genes after knockdown and overexpression of ANKRD23. The results showed that the expression of myoblast differentiation marker genes were increased by interference and reduced by ANKRD23 overexpression, indicating that ANKRD23 played a negative role in the myoblast differentiation. Interestingly, we discovered a long non-coding RNA-AK004293 which was overlapped with the 3'UTR of ANKRD23 gene. Then we detected the effect of AK004293 on the expression of ANKRD23 and myoblast differentiation marker genes in C2C12 myoblasts. The results showed that AK004293 had no significant effect on the expression of myoblast differentiation maker genes and ANKRD23. In conclusion, our results established the foundation for further studies about the regulation mechanism of ANKRD23 in muscle development. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Simvastatin inhibits glucose uptake activity and GLUT4 translocation through suppression of the IR/IRS-1/Akt signaling in C2C12 myotubes.

    PubMed

    Li, Weihua; Liang, Xiaojing; Zeng, Zhipeng; Yu, Kaizhen; Zhan, Shaopeng; Su, Qiang; Yan, Yinzhi; Mansai, Huseen; Qiao, Weitong; Yang, Qi; Qi, Zhongquan; Huang, Zhengrong

    2016-10-01

    Simvastatin,a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, is clinically used in the prevention and treatment of cardiovascular diseases. Numerous studies demonstrate that statins increase the risk of new-onset diabetes in long-term therapy, but mechanisms underpinning this effect are still unclear. Here, we investigated whether simvastatin inhibited the glucose uptake activity and the underlying mechanisms in C2C12 myotubes. Our studies showed that simvastatin significantly inhibited glucose uptake activity and GLUT4 translocation, whereas the effect was reversible with mevalonolactone (ML), which acts as an intermediate of cholesterol synthesis pathway. Mechanistically, the inhibition of glucose uptake and GLUT4 translocation elicited by simvastatin were associated with the suppression of the insulin receptor (IR)/IR substrate (IRS)/Akt signaling cascade. Simvastatin suppressed the phosphorylation of IR, IRS-1 and Akt, and total expression of IR or IRS-1, but did not affect Akt. Furthermore, simvastatin decreased Rac1 GTP binding. In conclusion, our findings indicate that simvastatin suppresses glucose uptake activity and GLUT4 translocation via IR-dependent IRS-1/PI3K/Akt pathway. These results provide an important new insight into the mechanism of statins on insulin sensitivity which may be associated with new-onset diabetes.

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

    PubMed

    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.

  3. CaMKK2 Suppresses Muscle Regeneration through the Inhibition of Myoblast Proliferation and Differentiation

    PubMed Central

    Ye, Cheng; Zhang, Duo; Zhao, Lei; Li, Yan; Yao, Xiaohan; Wang, Hui; Zhang, Shengjie; Liu, Wei; Cao, Hongchao; Yu, Shuxian; Wang, Yucheng; Jiang, Jingjing; Wang, Hui; Li, Xihua; Ying, Hao

    2016-01-01

    Skeletal muscle has a major role in locomotion and muscle disorders are associated with poor regenerative efficiency. Therefore, a deeper understanding of muscle regeneration is needed to provide a new insight for new therapies. CaMKK2 plays a role in the calcium/calmodulin-dependent kinase cascade; however, its role in skeletal muscle remains unknown. Here, we found that CaMKK2 expression levels were altered under physiological and pathological conditions including postnatal myogensis, freeze or cardiotoxin-induced muscle regeneration, and Duchenne muscular dystrophy. Overexpression of CaMKK2 suppressed C2C12 myoblast proliferation and differentiation, while inhibition of CaMKK2 had opposite effect. We also found that CaMKK2 is able to activate AMPK in C2C12 myocytes. Inhibition of AMPK could attenuate the effect of CaMKK2 overexpression, while AMPK agonist could abrogate the effect of CaMKK2 knockdown on C2C12 cell differentiation and proliferation. These results suggest that CaMKK2 functions as an AMPK kinase in muscle cells and AMPK mediates the effect of CaMKK2 on myoblast proliferation and differentiation. Our data also indicate that CaMKK2 might inhibit myoblast proliferation through AMPK-mediated cell cycle arrest by inducing cdc2-Tyr15 phosphorylation and repress differentiation through affecting PGC1α transcription. Lastly, we show that overexpressing CaMKK2 in the muscle of mice via electroporation impaired the muscle regeneration during freeze-induced injury, indicating that CaMKK2 could serve as a potential target to treat patients with muscle injury or myopathies. Together, our study reveals a new role for CaMKK2 as a negative regulator of myoblast differentiation and proliferation and sheds new light on the molecular regulation of muscle regeneration. PMID:27783047

  4. Effect of biologically active substances present in water extracts of white mustard and coriander on antioxidant status and lipid peroxidation of mouse C2C12 skeletal muscle cells.

    PubMed

    Zielniok, K; Szkoda, K; Gajewska, M; Wilczak, J

    2016-10-01

    Coriander and white mustard, an annual plants originated in the Mediterranean region, have been cultivated and used as spices for a long time. Recent studies have shown that they may constitute a potential source of phenolic compounds. The aim of this study was to evaluate the content of polyphenols in coriander and white mustard water extracts and to investigate their antioxidant activity in C2C12 mouse skeletal muscle cells, which serve as a good model of cells with intensive metabolism. HPLC analysis showed that polyphenols were able to permeate from the water extracts of studied plants into the undifferentiated myoblasts as well as myocytes undergoing differentiation, increasing the concentration of reduced glutathione and upregulating glutathione reductase and peroxidase activity. White mustard and coriander extracts also decreased the levels of oxysterols and sum of tiobarbituric acid reactive substances (TBARS) in both: myoblasts and differentiating myocytes, demonstrating protective effect on cell membranes. The obtained results indicate that polyphenols synthesized by both herbs may have beneficial effects on muscle tissue. Journal of Animal Physiology and Animal Nutrition © 2015 Blackwell Verlag GmbH.

  5. Induction of angiogenesis by implantation of encapsulated primary myoblasts expressing vascular endothelial growth factor.

    PubMed

    Springer, M L; Hortelano, G; Bouley, D M; Wong, J; Kraft, P E; Blau, H M

    2000-01-01

    We previously demonstrated that intramuscular implantation of primary myoblasts engineered to express vascular endothelial growth factor (VEGF) constitutively resulted in hemangioma formation and the appearance of VEGF in the circulation. To investigate the potential for using allogeneic myoblasts and the effects of delivery of VEGF-expressing myoblasts to non-muscle sites, we have enclosed them in microcapsules that protect allogeneic cells from rejection, yet allow the secretion of proteins produced by the cells. Encapsulated mouse primary myoblasts that constitutively expressed murine VEGF164, or encapsulated negative control cells, were implanted either subcutaneously or intraperitoneally into mice. Upon subcutaneous implantation, capsules containing VEGF-expressing myoblasts gave rise to large tissue masses at the implantation site that continued to grow and were composed primarily of endothelial and smooth muscle cells directly surrounding the capsules, and macrophages and capillaries further away from the capsules. Similarly, when injected intraperitoneally, VEGF-producing capsules caused significant localized inflammation and angiogenesis within the peritoneum, and ultimately led to fatal intraperitoneal hemorrhage. Notably, however, VEGF was not detected in the plasma of any mice. We conclude that encapsulated primary myoblasts persist and continue to secrete VEGF subcutaneously and intraperitoneally, but that the heparin-binding isoform VEGF164 exerts localized effects at the site of production. VEGF secreted from the capsules attracts endothelial and smooth muscle cells in a macrophage-independent manner. These results, along with our previous results, show that the mode and site of delivery of the same factor by the same engineered myoblasts can lead to markedly different outcomes. Moreover, the results confirm that constitutive delivery of high levels of VEGF is not desirable. In contrast, regulatable expression may lead to efficacious, safe, and

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

    SciTech Connect

    Lee, Sang-Jin; Go, Ga-Yeon; Yoo, Miran; Kim, Yong Kee; Seo, Dong-Wan; Kang, Jong-Sun; Bae, Gyu-Un

    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.

  7. AICAR-induced activation of AMPK negatively regulates myotube hypertrophy through the HSP72-mediated pathway in C2C12 skeletal muscle cells.

    PubMed

    Egawa, Tatsuro; Ohno, Yoshitaka; Goto, Ayumi; Ikuta, Akihiro; Suzuki, Miho; Ohira, Tomotaka; Yokoyama, Shingo; Sugiura, Takao; Ohira, Yoshinobu; Yoshioka, Toshitada; Goto, Katsumasa

    2014-02-01

    5'-AMP-activated protein kinase (AMPK) plays an important role as a negative regulator of skeletal muscle mass. However, the precise mechanism of AMPK-mediated regulation of muscle mass is not fully clarified. Heat shock proteins (HSPs), stress-induced molecular chaperones, are related with skeletal muscle adaptation, but the association between AMPK and HSPs in skeletal muscle hypertrophy is unknown. Thus, we investigated whether AMPK regulates hypertrophy by mediating HSPs in C2C12 cells. The treatment with AICAR, a potent stimulator of AMPK, decreased 72-kDa HSP (HSP72) expression, whereas there were no changes in the expressions of 25-kDa HSP, 70-kDa heat shock cognate, and heat shock transcription factor 1 in myotubes. Protein content and diameter were less in the AICAR-treated myotubes in those without treatment. AICAR-induced suppression of myotube hypertrophy and HSP72 expression was attenuated in the siRNA-mediated AMPKα knockdown myotubes. AICAR increased microRNA (miR)-1, a modulator of HSP72, and the increase of miR-1 was not induced in AMPKα knockdown condition. Furthermore, siRNA-mediated HSP72 knockdown blocked AICAR-induced inhibition of myotube hypertrophy. AICAR upregulated the gene expression of muscle Ring-finger 1, and this alteration was suppressed in either AMPKα or HSP72 knockdown myotubes. The phosphorylation of p70 S6 kinase Thr(389) was downregulated by AICAR, whereas this was attenuated in AMPKα, but not in HSP72, knockdown myotubes. These results suggest that AMPK inhibits hypertrophy through, in part, an HSP72-associated mechanism via miR-1 and protein degradation pathways in skeletal muscle cells.

  8. GH-Releasing Hormone Promotes Survival and Prevents TNF-α-Induced Apoptosis and Atrophy in C2C12 Myotubes.

    PubMed

    Gallo, Davide; Gesmundo, Iacopo; Trovato, Letizia; Pera, Giulia; Gargantini, Eleonora; Minetto, Marco Alessandro; Ghigo, Ezio; Granata, Riccarda

    2015-09-01

    Skeletal muscle atrophy is a consequence of different chronic diseases, including cancer, heart failure, and diabetes, and also occurs in aging and genetic myopathies. It results from an imbalance between anabolic and catabolic processes, and inflammatory cytokines, such as TNF-α, have been found elevated in muscle atrophy and implicated in its pathogenesis. GHRH, in addition to stimulating GH secretion from the pituitary, exerts survival and antiapoptotic effects in different cell types. Moreover, we and others have recently shown that GHRH displays antiapoptotic effects in isolated cardiac myocytes and protects the isolated heart from ischemia/reperfusion injury and myocardial infarction in vivo. On these bases, we investigated the effects of GHRH on survival and apoptosis of TNF-α-treated C2C12 myotubes along with the underlying mechanisms. GHRH increased myotube survival and prevented TNF-α-induced apoptosis through GHRH receptor-mediated mechanisms. These effects involved activation of phosphoinositide 3-kinase/Akt pathway and inactivation of glycogen synthase kinase-3β, whereas mammalian target of rapamycin was unaffected. GHRH also increased the expression of myosin heavy chain and the myogenic transcription factor myogenin, which were both reduced by the cytokine. Furthermore, GHRH inhibited TNF-α-induced expression of nuclear factor-κB, calpain, and muscle ring finger1, which are all involved in muscle protein degradation. In summary, these results indicate that GHRH exerts survival and antiapoptotic effects in skeletal muscle cells through the activation of anabolic pathways and the inhibition of proteolytic routes. Overall, our findings suggest a novel therapeutic role for GHRH in the treatment of muscle atrophy-associated diseases.

  9. CD36 is required for myoblast fusion during myogenic differentiation

    SciTech Connect

    Park, Seung-Yoon; Yun, Youngeun; Kim, In-San

    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.

  10. 1,25(OH)2-vitamin D3 enhances the stimulating effect of leucine and insulin on protein synthesis rate through Akt/PKB and mTOR mediated pathways in murine C2C12 skeletal myotubes.

    PubMed

    Salles, Jérôme; Chanet, Audrey; Giraudet, Christophe; Patrac, Véronique; Pierre, Philippe; Jourdan, Marion; Luiking, Yvette C; Verlaan, Sjors; Migné, Carole; Boirie, Yves; Walrand, Stéphane

    2013-12-01

    In recent years, there has been a growing body of evidence pointing to an effect of vitamin D on muscle mass and function. Our aim was to investigate the combined effect of 1,25(OH)2-vitamin D3 (1,25(OH)2D3) with anabolic factors insulin and leucine on protein fractional synthesis rate (FSR) and regulation in the mouse C2C12 myotube. After differentiation, myotubes were cultured in 1,25(OH)2D3 solutions at 0, 1, or 10 nM for 72 h. Cells were treated by L-[1-(13) C]valine and puromycin in presence or not of leucine and insulin, and protein FSR was determined by measuring tracer enrichments and puromycin incorporation in proteins, respectively. Protein expression and phosphorylation state of insulin receptor (IR), Akt, GSK3, mTOR, p70 S6 kinase, rpS6, and 4EBP1 were measured by Western blot. Transcript levels of IR and 1,25(OH)2D3 receptor (VDR) were determined by qPCR. 1,25(OH)2D3 (10 nM) with leucine and insulin increased protein FSR in C2C12 myotubes (14-16%). IR and VDR mRNA expression was increased with 1,25(OH)2D3 treatment. The Akt/mTOR-dependent pathway was activated by insulin and leucine and further enhanced by 1,25(OH)2D3. 1,25(OH)2D3 sensitizes the Akt/mTOR-dependant pathway to the stimulating effect of leucine and insulin, resulting in a further activation of protein synthesis in murine C2C12 skeletal myotubes. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. 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

    PubMed Central

    Kim, Sung Hee; Sung, Mi Jeong; Park, Jae Ho; Yang, Hye Jeong; Hwang, Jin-Taek

    2013-01-01

    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-yl)amino]-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. PMID:23690860

  12. Idesolide, an isolate of Idesia polycarpa, inhibits apoptosis through induction of intracellular heat shock protein 70 in C2C12 muscle cells.

    PubMed

    Jung, Min-Ho; Yoo, Jung-Min; Kang, Yeo-Jin; Lee, Hyoung Woo; Kim, Seung Hyun; Sung, Sang Hyun; Lee, Yong-Jin; Choi, Inho; Kim, Tack-Joong

    2010-01-01

    Muscle disorders, such as muscular dystrophy, are associated with an increase in oxidative stress. Proposed treatments for muscular dystrophy, some in clinical trials, include gene therapy and muscle cell transplantation. In this study, we investigated the effects of idesolide, isolated from the fruits of Idesia polycarpa, on changes that occur in muscle disuse atrophy. We noted protective effects on oxidative stress response and HSP70 regulation. Pre-treatment with idesolide for 24 h maintained cell viability and decreased apoptosis in H(2)O(2)-treated C(2)C(12) muscle cells. The idesolide pretreatment also increased intracellular HSP70 protein. Our results suggest that idesolide inhibits cell death through induction of HSP70 in C(2)C(12) muscle cells. This work is the first to report that idesolide can regulate the decrease in HSP70 that occurs during skeletal muscle atrophy.

  13. AAV-2-mediated expression of IGF-1 in skeletal myoblasts stimulates angiogenesis and cell survival.

    PubMed

    Subramanian, Indira V; Fernandes, Brian C A; Robinson, Timothy; Koening, Jennifer; Lapara, Kelly S; Ramakrishnan, S

    2009-03-01

    The transplantation of skeletal myoblasts is being tested in various organ systems to facilitate tissue repair and regeneration. Previous studies have indicated that transplanted cells for varied reasons were not surviving in sufficient numbers following transplantation, thus negatively affecting overall therapeutic efficacy of the approach. We hypothesize that the genetic modification of myoblasts to express insulin-like growth factor 1 (IGF-1) locally may enhance the survival of transplanted cells by stimulating neo-vascularization, decreasing apoptosis, and promoting cell proliferation. Using an adeno-associated virus (adeno-associated virus type 2) vector system, the IGF-1 gene was introduced into canine skeletal myoblasts. As a negative control, myoblasts transduced with the green fluorescence protein (GFP) was used. Relative angiogenic response induced by IGF-1 myoblast was compared to VEGF165-induced neo-vascularization using Matrigel plugs under similar conditions. In vitro evaluation and characterization revealed that the secreted IGF-1 protein was biologically and functionally active in promoting endothelial cell proliferation, migration and assembly into vessel-like structures. Matrigel plugs containing the three test groups were implanted subcutaneously in nude mice (n = 5). After 3 weeks, analysis of explanted samples revealed an enhanced neo-vascularization with an average microvessel density per field for IGF-1 at 55.9 versus 33.4 for vascular endothelial growth factor and 24 for GFP. Additionally, apoptosis was significantly reduced (p myoblasts. We conclude that the genetic modification of skeletal myoblasts with the IGF-1 gene offers a potential means for enhanced cell survival following transplantation.

  14. Cisplatin triggers atrophy of skeletal C2C12 myotubes via impairment of Akt signalling pathway and subsequent increment activity of proteasome and autophagy systems

    SciTech Connect

    Fanzani, Alessandro Zanola, Alessandra; Rovetta, Francesca; Rossi, Stefania; Aleo, Maria Francesca

    2011-02-01

    Cisplatin (cisPt) is an antineoplastic drug which causes an array of adverse effects on different organs and tissues, including skeletal muscle. In this work we show that cisPt behaves as a potent trigger to activate protein hypercatabolism in skeletal C2C12 myotubes. Within 24 h of 50 {mu}M cisPt administration, C2C12 myotubes displayed unchanged cell viability but showed a subset of hallmark signs typically recognized during atrophy, including severe reduction in body size, repression of Akt phosphorylation, transcriptional up-regulation of atrophy-related genes, such as atrogin-1, gabarap, beclin-1 and bnip-3, and loss of myogenic markers. As a consequence, proteasomal activity and formation of autophagosomes were remarkably increased in cisPt-treated myotubes, but forced stimulation of Akt pathway, as obtained through insulin administration or delivery of a constitutively activated Akt form, was sufficient to counter the cisPt-induced protein breakdown, leading to rescue of atrophic size. Overall, these results indicate that cisPt induces atrophy of C2C12 myotubes via activation of proteasome and autophagy systems, suggesting that the Akt pathway represents one sensitive target of cisPt molecular action in skeletal muscle.

  15. Effect of simvastatin on cholesterol metabolism in C2C12 myotubes and HepG2 cells, and consequences for statin-induced myopathy.

    PubMed

    Mullen, Peter James; Lüscher, Barbara; Scharnagl, Hubert; Krähenbühl, Stephan; Brecht, Karin

    2010-04-15

    The mechanism of statin-induced skeletal muscle myopathy is poorly understood. We investigated how simvastatin affects cholesterol metabolism, ubiquinone levels, and the prenylation and N-linked glycosylation of proteins in C2C12 myotubes. We used liver HepG2 cells for comparison, as their responses to statins are well-characterized in terms of their cholesterol metabolism (in contrast to muscle cells), and statins are well-tolerated in the liver. Differences between the two cell lines could indicate the mechanism behind statin-induced myopathy. Simvastatin reduced de novo cholesterol production in C2C12 myotubes by 95% after 18h treatment. The reduction was 82% in the HepG2 cells. Total cholesterol pools, however, remained constant in both cell lines. Simvastatin treatment similarly did not affect total ubiquinone levels in the myotubes, unlike in HepG2 cells (22% reduction in CoQ10). Statin treatment reduced levels of Ras and Rap1 prenylation in both cell lines, whereas N-linked glycosylation was only affected in C2C12 myotubes (21% reduction in rate). From these observations, we conclude that total cholesterol and ubiquinone levels are unlikely to be involved in statin-mediated myopathy, but reductions in protein prenylation and especially N-linked glycosylation may play a role. This first comparison of the responses to simvastatin between liver and skeletal muscle cell lines may be important for future research directions concerning statin-induced myopathy.

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

    PubMed

    Tsukahara, Tamotsu; Haniu, Hisao; Matsuda, Yoshikazu

    2013-04-12

    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.

  17. p130/p107 expression distinguishes adipogenic potential in primary myoblasts based on age.

    PubMed

    Guan, Yu; Taylor-Jones, Jane M; Peterson, Charlotte A; McGehee, Robert E

    2002-09-06

    Recent investigations have provided significant evidence that many mesodermally derived tissues contain stem cell-like precursors capable of being stimulated to undergo differentiation into a variety of cellular lineages. We have recently reported that primary myoblasts isolated from 23-month-old mice have an increased adipogenic potential when compared to their 8-month-old counterparts. To further characterize the degree of adipocyte differentiation in these myoblasts, we examined early and late markers of adipocyte differentiation. Within the first 24h of adipocyte differentiation, expression of p130 and p107, two members of the retinoblastoma tumor suppressor gene family, are regulated and this event is an important one early in adipogenesis. Consistent with the increased adipogenic potential of the older myoblasts and in contrast to the younger cells, the p130:p107 pattern of expression is very similar to that observed in adipogenesis where there is a transient increase in p107 expression accompanied by a decrease in p130 expression. Interestingly, while these older cells accumulated lipid and expressed genes associated with lipid metabolism, they failed to express adipsin and leptin, two well-established markers of terminal adipocyte differentiation. These results suggest that older myoblasts are capable of initiating and progressing through the adipogenic program to a point where they express genes associated with lipid metabolism, but do not reach a terminally differentiated state. This finding may have important metabolic implications in the aging population.

  18. Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest.

    PubMed

    Subramaniam, Sindhu; Sreenivas, Prethish; Cheedipudi, Sirisha; Reddy, Vatrapu Rami; Shashidhara, Lingadahalli Subrahmanya; Chilukoti, Ravi Kumar; Mylavarapu, Madhavi; Dhawan, Jyotsna

    2014-01-01

    Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological

  19. Role of pulsed electromagnetic fields (PEMF) on tenocytes and myoblasts-potential application for treating rotator cuff tears.

    PubMed

    Liu, Mengyao; Lee, Carlin; Laron, Dominique; Zhang, Nianli; Waldorff, Erik I; Ryaby, James T; Feeley, Brian; Liu, Xuhui

    2017-05-01

    The post-surgery integrity of the tendons and muscle quality are the two major factors in success of rotator cuff (RC) repair. Though surgical techniques for rotator cuff repair have significantly improved in the past two decades, there are no effective treatments to improve tendon-to-bone healing and muscle quality after repair at this point in time. Pulsed electromagnetic fields (PEMF) have previously been used for promoting fracture healing. Previous studies have shown that PEMF has a positive role in promoting osteoblast precursors proliferation and differentiation. However, PEMFs effect on tenocytes and muscle cells has not been determined fully yet. The purpose of this study is to define the role of a commercially available PEMF on tenocytes and myoblasts growth and differentiation in vitro. Human rotator cuff tenocytes and C2C12 murine myoblasts were cultured and treated with PEMF for 2 weeks under regular and inflammatory conditions. Our results showed that 2 weeks treatment of PEMF enhanced gene expressions of growth factors in human rotator cuff tenocytes under inflammatory conditions. PEMF significantly enhanced C2C12 myotube formation under normal and inflammatory conditions. Results from this study suggest that PEMF has a positive role in promoting tenocyte gene expression and myoblast differentiation. Therefore, PEMF may potentially serve as a non-operative treatment to improve clinical incomes rotator cuff tendon repairs. Results © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:956-964, 2017. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  20. AlphaB-crystallin is involved in oxidative stress protection determined by VEGF in skeletal myoblasts.

    PubMed

    Mercatelli, Neri; Dimauro, Ivan; Ciafré, Silvia Anna; Farace, Maria Giulia; Caporossi, Daniela

    2010-08-01

    Recent studies suggest that the effects of VEGF-A, the prototype VEGF ligand, may extend to a variety of cell types other than endothelial cells. The expression of VEGF-A and its main receptors, Flt-1/VEGFR-1 and KDR/Flk-1/VEGFR-2, was indeed detected in several cell types, including cardiac myocytes and regenerating myotubes. In addition to its proangiogenic activity, evidence indicates that VEGF-A can sustain skeletal muscle regeneration by enhancing the survival and migration of myogenic cells and by promoting the growth of myogenic fibers. In this study, our aim was to investigate whether VEGF could protect skeletal muscle satellite cells from apoptotic cell death triggered by reactive oxygen species and to identify the main molecular mechanisms. C2C12 mouse myoblasts, cultured in vitro in the presence of exogenous VEGF or stably transfected with a plasmid vector expressing VEGF-A, were subjected to oxidative stress and analyzed for cell growth and survival, induction of apoptosis, and molecular signaling. The results of our study demonstrated that VEGF protects C2C12 myoblasts from apoptosis induced by oxidative or hypoxic-like stress. This protection did not correlate with the modulation of the expression of VEGF receptors, but is clearly linked to the phosphorylation of the KDR/Flk-1 receptor, the activation of NF-kappaB, and/or the overexpression of the antiapoptotic protein alphaB-crystallin. Copyright 2010 Elsevier Inc. All rights reserved.

  1. Celastrus paniculatus Willd. mitigates t-BHP induced oxidative and apoptotic damage in C2C12 murine muscle cells.

    PubMed

    Kumar, Kandikattu Hemanth; Venuprasad, M P; Jayashree, G V; Rachitha, P; Krupashree, K; Pal, Ajay; Khanum, Farhath

    2015-12-01

    Identification, exploration and scientific validation of antioxidant rich herbal extracts to mitigate the radical induced cell damage provide new insights in the field of ayurvedic research/therapies. In the present study, we evaluated the anti-oxidant and anti-apoptotic potential of Celastrus paniculatus seed extract (CPSE) against tertiary butyl hydroperoxide (t-BHP) induced mice muscle cell damage. The extract at a dose of 50 µg/ml protected the cells up to 70 % as evidenced by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival assay and also prevented LDH leakage against t-BHP induced cytotoxicity. CPSE showed potential antioxidant activity by restoring mitochondrial membrane potential and inhibited reactive oxygen species generation and lipid peroxidation. CPSE pretreatment also regulated the antioxidant markers such as superoxide dismutase and catalase enzymes content and proteins expression. Further CPSE showed anti-apoptotic effects by regulating cytochrome-C and heat shock protein-70 expression and also showed 43 % muscle cell DNA damage inhibitory activity against t-BHP challenge as observed by single cell gel electrophoresis assay. Overall the extract inhibits the muscle cell damage, thus explaining the possible anti-oxidant/anti-apoptotic defense status of the C. paniculatus seed extract.

  2. Protein Kinase D2 Is an Essential Regulator of Murine Myoblast Differentiation

    PubMed Central

    Pusapati, Ganesh V.; Armacki, Milena; Müller, Martin; Tümpel, Stefan; Illing, Anett; Hartmann, Daniel; Brunner, Cornelia; Liebau, Stefan; Rudolph, Karl L.; Adler, Guido; Seufferlein, Thomas

    2011-01-01

    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. PMID:21298052

  3. Apoptosis induced by copper oxide quantum dots in cultured C2C12 cells via caspase 3 and caspase 7: a study on cytotoxicity assessment.

    PubMed

    Amna, Touseef; Van Ba, Hoa; Vaseem, M; Hassan, M Shamshi; Khil, Myung-Seob; Hahn, Y B; Lee, Hak-Kyo; Hwang, I H

    2013-06-01

    We report herein the synthesis and characterization of copper oxide quantum dots and their cytotoxic impact on mouse C2C12 cells. The utilized CuO quantum dots were prepared by the one-pot wet chemical method using copper acetate and hexamethylenetetramine as precursors. The physicochemical characterization of the synthesized CuO quantum dots was carried out using X-ray diffraction, energy-dispersive X-ray analysis, and transmission electron microscopy. To examine the in vitro cytotoxicity, C2C12 cell lines were treated with different concentrations of as-prepared quantum dots and the viability of cells was analyzed using Cell Counting Kit-8 assay at regular time intervals. The morphology of the treated C2C12 cells was observed under a phase-contrast microscope, whereas the quantification of cell viability was carried out via confocal laser scanning microscopy. To gain insight into the mechanism of cell death, we examined the effect of CuO quantum dots on the candidate genes such as caspases 3 and 7, which are key mediators of apoptotic events. In vitro investigations of the biological effect of CuO quantum dots have shown that it binds genomic DNA, decreases significantly the viability of cells in culture in a concentration (10-20 μg/mL) dependent manner, and inhibits mitochondrial caspases 3 and 7. To sum up, the elucidation of the pathways is to help in understanding CuO quantum dot-induced effects and evaluating CuO quantum dot-related hazards to human health.

  4. The Activity of Differentiation Factors Induces Apoptosis in Polyomavirus Large T-Expressing Myoblasts

    PubMed Central

    Fimia, Gian Maria; Gottifredi, Vanesa; Bellei, Barbara; Ricciardi, Maria Rosaria; Tafuri, Agostino; Amati, Paolo; Maione, Rossella

    1998-01-01

    It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor β or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G00 arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis. PMID:9614186

  5. Influence of the PDE5 inhibitor tadalafil on redox status and antioxidant defense system in C2C12 skeletal muscle cells.

    PubMed

    Duranti, Guglielmo; Ceci, Roberta; Sgrò, Paolo; Sabatini, Stefania; Di Luigi, Luigi

    2017-05-01

    Phosphodiesterase type 5 inhibitors (PDE5Is), widely known for their beneficial effects onto male erectile dysfunction, seem to exert favorable effects onto metabolism as well. Tadalafil exposure increases oxidative metabolism of C2C12 skeletal muscle cells. A rise in fatty acid (FA) metabolism, requiring more oxygen, could induce a larger reactive oxygen species (ROS) release as a byproduct thus leading to a redox imbalance. The aim of this study was to determine how PDE5I tadalafil influences redox status in skeletal muscle cells to match the increasing oxidative metabolism. To this purpose, differentiated C2C12 skeletal muscle cells were treated with tadalafil and analyzed for total antioxidant capacity (TAC) and glutathione levels as marker of redox status; enzyme activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) engaged in antioxidant defense; and lipid peroxidation (TBARS) and protein carbonyls (PrCar) as markers of oxidative damage. Tadalafil increased total intracellular glutathione (tGSH), CAT, SOD, and GPx enzymatic activities while no changes were found in TAC. A perturbation of redox status, as showed by the decrease in the ratio between reduced/oxidized glutathione (GSH/GSSG), was observed. Nevertheless, it did not cause any change in TBARS and PrCar levels probably due to the enhancement in the antioxidant enzymatic network. Taken together, these data indicate that tadalafil, besides improving oxidative metabolism, may be beneficial to skeletal muscle cells by enhancing the enzymatic antioxidant system capacity.

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

    PubMed

    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.

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

    PubMed

    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.

  8. Differential use of the regulatory elements of the alpha B-crystallin enhancer in cultured murine lung (MLg), lens (alpha TN4-1) and muscle (C2C12) cells.

    PubMed

    Haynes, J I; Gopal-Srivastava, R; Frederikse, P H; Piatigorsky, J

    1995-04-03

    The mouse alpha B-crystallin-encoding gene (alpha B-cry) is highly expressed in the lens and expressed to lesser extents in other tissues. Here, we investigated alpha B-cry expression in mouse-lung-derived MLg cells. Two sizes of MLg alpha B-cry transcripts comigrated with alpha B-cry transcripts contained in total and poly(A)+RNA from mouse lung, with preference for the larger species in the MLg cells. Expression of both alpha B-cry promoter/cat reporter gene constructs and alpha B-cry enhancer (nt -427/-259)/herpes simplex virus (HSV) thymidine kinase promoter (ptk)/human growth hormone reporter gene (hGH) constructs was studied in transfected MLg cells and the results compared with those obtained from alpha TN4-1 lens and C2C12 muscle cells. The alpha B-cry enhancer increased activity of the endogenous and tk promoters approx. 2-fold in the MLg cells, in contrast to its 3-7-fold effect in alpha TN4-1 cells and 17-20-fold effect in C2C12 myotubes. Site-specific mutagenesis of the previously identified enhancer control elements, alpha B-E-1 (nt -407 to -397), alpha BE-2 (-360 to -327) and MRF (-300 to -288), decreased enhancer strength in transfected MLg cells. DNase I footprinting showed that MLg nuclear proteins occupy only alpha BE-1 and alpha BE-2. Previous data have shown that lens cells use alpha BE-1, alpha BE-2 and alpha BE-3, while muscle cells use, in addition, the muscle regulatory factor-binding site (MRF). Thus, the present experiments correlate tissue-specific enhancer strength and the number of control elements utilized.

  9. Three-dimensional Myoblast Aggregates--Effects of Modeled Microgravity

    NASA Technical Reports Server (NTRS)

    Byerly, Diane; Sognier, M. A.; Marquette, M. L.

    2006-01-01

    The overall objective of these studies is to elucidate the molecular and cellular alterations that contribute to muscle atrophy in astronauts caused by exposure to microgravity conditions in space. To accomplish this, a three-dimensional model test system was developed using mouse myoblast cells (C2C12). Myoblast cells were grown as three-dimensional aggregates (without scaffolding or other solid support structures) in both modeled microgravity (Rotary Cell Culture System, Synthecon, Inc.) and at unit gravity in coated Petri dishes. Evaluation of H&E stained thin sections of the aggregates revealed the absence of any necrosis. Confocal microscopy evaluations of cells stained with the Live/Dead assay (Molecular Probes) confirmed that viable cells were present throughout the aggregates with an average of only three dead cells observed per aggregate. Preliminary results from gene array analysis (Affymetrix chip U74Av2) showed that approximately 14% of the genes were down regulated (decreased more than 3 fold) and 4% were upregulated in cells exposed to modeled microgravity for 12 hours compared to unit gravity controls. Additional studies using fluorescent phallacidin revealed a decrease in F-actin in the cells exposed to modeled microgravity compared to unit gravity. Myoblast cells grown as aggregates in modeled microgravity exhibited spontaneous differentiation into syncitia while no differentiation was seen in the unit gravity controls. These studies show that 1)the model test system developed is suitable for assessing cellular and molecular alterations in myoblasts; 2) gene expression alterations occur rapidly (within 12 hours) following exposure to modeled microgravity; and 3) modeled microgravity conditions stimulated myoblast cell differentiation. Achieving a greater understanding of the molecular alterations leading to muscle atrophy will eventually enable the development of cell-based countermeasures, which may be valuable for treatment of muscle diseases on

  10. Inhibiting c-Jun N-terminal kinase partially attenuates caffeine-dependent cell death without alleviating the caffeine-induced reduction in mitochondrial respiration in C2C12 skeletal myotubes.

    PubMed

    Downs, R M; Hughes, M A; Kinsey, S T; Johnson, M C; Baumgarner, B L

    2016-11-04

    Caffeine is a widely consumed stimulant that has previously been shown to promote cytotoxic stress and even cell death in numerous mammalian cell lines. Thus far there is little information available regarding the toxicity of caffeine in skeletal muscle cells. Our preliminary data revealed that treating C2C12 myotubes with 5 mM caffeine for 6 h increased nuclear fragmentation and reduced basal and maximal oxygen consumption rate (OCR) in skeletal myotubes. The purpose of this study was to further elucidate the pathways by which caffeine increased cell death and reduced mitochondrial respiration. We specifically examined the role of c-Jun N-terminal kinase (JNK), which has previously been shown to simultaneously increase caspase-dependent cell death and reduce mitochondrial respiration in other mammalian cell lines. We found that caffeine promoted a dose-dependent increase in cell death in multinucleated myotubes but did not in mononucleated myoblasts. The addition of 10 μM Z-DEVD-FMK, a specific inhibitor of executioner caspases, completely inhibited caffeine-dependent cell death. Further, the addition of 400 μM dantrolene, a specific ryanodine receptor (RYR) inhibitor, prevented the caffeine-dependent increase in cell death and the reduction in basal and maximal OCR. We also discovered that caffeine treatment significantly increased the phosphorylation of JNK and that the addition of 30 μM SP600125 (JNKi), a specific JNK inhibitor, partially attenuated caffeine-induced cell death without preventing the caffeine-dependent reduction in basal and maximal OCR. Our results suggest that JNK partially mediates the increase in caspase-dependent cell death but does not contribute to reduced mitochondrial respiration in caffeine-treated skeletal muscle cells. We conclude that caffeine increased cell death and reduced mitochondrial respiration in a calcium-dependent manner by activating the RYR and promoting reticular calcium release. Copyright © 2016 Elsevier Inc

  11. Inhibiting c-Jun N-terminal kinase partially attenuates caffeine-dependent cell death without alleviating the caffeine-induced reduction in mitochondrial respiration in C2C12 skeletal myotubes

    PubMed Central

    Downs, R.M.; Hughes, M.A.; Kinsey, S.T.; Johnson, M.C.; Baumgarner, B.L.

    2017-01-01

    Caffeine is a widely consumed stimulant that has previously been shown to promote cytotoxic stress and even cell death in numerous mammalian cell lines. Thus far there is little information available regarding the toxicity of caffeine in skeletal muscle cells. Our preliminary data revealed that treating C2C12 myotubes with 5 mM caffeine for 6 h increased nuclear fragmentation and reduced basal and maximal oxygen consumption rate (OCR) in skeletal myotubes. The purpose of this study was to further elucidate the pathways by which caffeine increased cell death and reduced mitochondrial respiration. We specifically examined the role of c-Jun N-terminal kinase (JNK), which has previously been shown to simultaneously increase caspase-dependent cell death and reduce mitochondrial respiration in other mammalian cell lines. We found that caffeine promoted a dose-dependent increase in cell death in multinucleated myotubes but did not in mononucleated myoblasts. The addition of 10 μM Z-DEVD-FMK, a specific inhibitor of executioner caspases, completely inhibited caffeine-dependent cell death. Further, the addition of 400 μM dantrolene, a specific ryanodine receptor (RYR) inhibitor, prevented the caffeine-dependent increase in cell death and the reduction in basal and maximal OCR. We also discovered that caffeine treatment significantly increased the phosphorylation of JNK and that the addition of 30 μM SP600125 (JNKi), a specific JNK inhibitor, partially attenuated caffeine-induced cell death without preventing the caffeine-dependent reduction in basal and maximal OCR. Our results suggest that JNK partially mediates the increase in caspase-dependent cell death but does not contribute to reduced mitochondrial respiration in caffeine-treated skeletal muscle cells. We conclude that caffeine increased cell death and reduced mitochondrial respiration in a calcium-dependent manner by activating the RYR and promoting reticular calcium release. PMID:27717822

  12. TEAD transcription factors are required for normal primary myoblast differentiation in vitro and muscle regeneration in vivo.

    PubMed

    Joshi, Shilpy; Davidson, Guillaume; Le Gras, Stéphanie; Watanabe, Shuichi; Braun, Thomas; Mengus, Gabrielle; Davidson, Irwin

    2017-02-01

    The TEAD family of transcription factors (TEAD1-4) bind the MCAT element in the regulatory elements of both growth promoting and myogenic differentiation genes. Defining TEAD transcription factor function in myogenesis has proved elusive due to overlapping expression of family members and their functional redundancy. We show that silencing of either Tead1, Tead2 or Tead4 did not effect primary myoblast (PM) differentiation, but that their simultaneous knockdown strongly impaired differentiation. In contrast, Tead1 or Tead4 silencing impaired C2C12 differentiation showing their different contributions in PMs and C2C12 cells. Chromatin immunoprecipitation identified enhancers associated with myogenic genes bound by combinations of Tead4, Myod1 or Myog. Tead4 regulated distinct gene sets in C2C12 cells and PMs involving both activation of the myogenic program and repression of growth and signaling pathways. ChIP-seq from mature mouse muscle fibres in vivo identified a set of highly transcribed muscle cell-identity genes and sites bound by Tead1 and Tead4. Although inactivation of Tead4 in mature muscle fibres caused no obvious phenotype under normal conditions, notexin-induced muscle regeneration was delayed in Tead4 mutants suggesting an important role in myogenic differentiation in vivo. By combining knockdown in cell models in vitro with Tead4 inactivation in muscle in vivo, we provide the first comprehensive description of the specific and redundant roles of Tead factors in myogenic differentiation.

  13. TEAD transcription factors are required for normal primary myoblast differentiation in vitro and muscle regeneration in vivo

    PubMed Central

    Joshi, Shilpy; Le Gras, Stéphanie; Watanabe, Shuichi; Braun, Thomas

    2017-01-01

    The TEAD family of transcription factors (TEAD1-4) bind the MCAT element in the regulatory elements of both growth promoting and myogenic differentiation genes. Defining TEAD transcription factor function in myogenesis has proved elusive due to overlapping expression of family members and their functional redundancy. We show that silencing of either Tead1, Tead2 or Tead4 did not effect primary myoblast (PM) differentiation, but that their simultaneous knockdown strongly impaired differentiation. In contrast, Tead1 or Tead4 silencing impaired C2C12 differentiation showing their different contributions in PMs and C2C12 cells. Chromatin immunoprecipitation identified enhancers associated with myogenic genes bound by combinations of Tead4, Myod1 or Myog. Tead4 regulated distinct gene sets in C2C12 cells and PMs involving both activation of the myogenic program and repression of growth and signaling pathways. ChIP-seq from mature mouse muscle fibres in vivo identified a set of highly transcribed muscle cell-identity genes and sites bound by Tead1 and Tead4. Although inactivation of Tead4 in mature muscle fibres caused no obvious phenotype under normal conditions, notexin-induced muscle regeneration was delayed in Tead4 mutants suggesting an important role in myogenic differentiation in vivo. By combining knockdown in cell models in vitro with Tead4 inactivation in muscle in vivo, we provide the first comprehensive description of the specific and redundant roles of Tead factors in myogenic differentiation. PMID:28178271

  14. In vitro protective effects of resveratrol and stilbene alkanoic derivatives on induced oxidative stress on C2C12 and MCF7 cells.

    PubMed

    Leporini, L; Giampietro, L; Amororso, R; Ammazzalorso, A; Fantacuzzi, M; Menghini, L; Maccallini, C; Ferrante, C; Orlando, G; De Filippis, B

    2017-09-11

    Resveratrol (3,4’,5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine, which has been extensively studied for a wide range of biological effects. A large number of stilbene-containing derivatives have displayed antioxidant and antiproliferative activities on various cancer cell lines. In this study, a series of stilbene hybrids 1-9, previously reported as peroxisome proliferator-activated receptor (PPAR) agonists, were assessed at micromolar concentrations using MTT cell viability assay in C2C12 and MCF7 cell lines. The modulation of oxidative stress was also evaluated by measuring the protective effects on reactive oxygen species (ROS) production induced or not by oxidative stimulus. Among these, compounds 2 and 8 showed significant radical scavenging activity.

  15. Electron probe X-ray microanalysis of cultured myogenic C2C12 cells with scanning and scanning transmission electron microscopy.

    PubMed

    Tylko, G; Karasiński, J; Wróblewski, R; Roomans, G M; Kilarski, W M

    2000-01-01

    Heterogeneity of the elemental content of myogenic C2C12 cultured cells was studied by electron probe X-ray microanalysis (EPXMA) with scanning (SEM EPXMA) and scanning transmission electron microscopy (STEM EPXMA). The best plastic substrate for growing cells was Thermanox. For STEM EPXMA, a Formvar film coated with carbon was found to be suitable substrate. The cells examined by scanning transmission electron microscopy showed great heterogeneity in their elemental content in comparison with the cells examined in the scanning electron microscope despite of an almost identical preparation procedure for EPXMA. Nevertheless the K/Na ratios obtained from both methods of EPXMA were very close (4.1 and 4.3). We conclude that the observed discrepancy in the elemental content obtained by the two methods may be due to differences in instrumentation and this must be taken into account when planning a comparative study.

  16. Mitochondrial Hyperfusion during Oxidative Stress Is Coupled to a Dysregulation in Calcium Handling within a C2C12 Cell Model

    PubMed Central

    Drozdzal, Gregory; Radisic, Milica; McBride, Heidi M.

    2013-01-01

    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 conclude that the

  17. Structure–function analysis of myomaker domains required for myoblast fusion

    PubMed Central

    Millay, Douglas P.; Gamage, Dilani G.; Quinn, Malgorzata E.; Min, Yi-Li; Mitani, Yasuyuki; Bassel-Duby, Rhonda; Olson, Eric N.

    2016-01-01

    During skeletal muscle development, myoblasts fuse to form multinucleated myofibers. Myomaker [Transmembrane protein 8c (TMEM8c)] is a muscle-specific protein that is essential for myoblast fusion and sufficient to promote fusion of fibroblasts with muscle cells; however, the structure and biochemical properties of this membrane protein have not been explored. Here, we used CRISPR/Cas9 mutagenesis to disrupt myomaker expression in the C2C12 muscle cell line, which resulted in complete blockade to fusion. To define the functional domains of myomaker required to direct fusion, we established a heterologous cell–cell fusion system, in which fibroblasts expressing mutant versions of myomaker were mixed with WT myoblasts. Our data indicate that the majority of myomaker is embedded in the plasma membrane with seven membrane-spanning regions and a required intracellular C-terminal tail. We show that myomaker function is conserved in other mammalian orthologs; however, related family members (TMEM8a and TMEM8b) do not exhibit fusogenic activity. These findings represent an important step toward deciphering the cellular components and mechanisms that control myoblast fusion and muscle formation. PMID:26858401

  18. Structure-function analysis of myomaker domains required for myoblast fusion.

    PubMed

    Millay, Douglas P; Gamage, Dilani G; Quinn, Malgorzata E; Min, Yi-Li; Mitani, Yasuyuki; Bassel-Duby, Rhonda; Olson, Eric N

    2016-02-23

    During skeletal muscle development, myoblasts fuse to form multinucleated myofibers. Myomaker [Transmembrane protein 8c (TMEM8c)] is a muscle-specific protein that is essential for myoblast fusion and sufficient to promote fusion of fibroblasts with muscle cells; however, the structure and biochemical properties of this membrane protein have not been explored. Here, we used CRISPR/Cas9 mutagenesis to disrupt myomaker expression in the C2C12 muscle cell line, which resulted in complete blockade to fusion. To define the functional domains of myomaker required to direct fusion, we established a heterologous cell-cell fusion system, in which fibroblasts expressing mutant versions of myomaker were mixed with WT myoblasts. Our data indicate that the majority of myomaker is embedded in the plasma membrane with seven membrane-spanning regions and a required intracellular C-terminal tail. We show that myomaker function is conserved in other mammalian orthologs; however, related family members (TMEM8a and TMEM8b) do not exhibit fusogenic activity. These findings represent an important step toward deciphering the cellular components and mechanisms that control myoblast fusion and muscle formation.

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

    PubMed

    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.

  20. Gene expression of selenoproteins can be regulated by selenoprotein K silencing in chicken myoblasts.

    PubMed

    Fan, Ruifeng; Yao, Haidong; Zhao, Xia; Cao, Changyu; Yang, Tianshu; Luan, Yilin; Zhang, Ziwei; Xu, Shiwen

    2016-08-01

    The aim of the present study was to clarify the effect of Selenoprotein K (Selk) silencing on the mRNA expression of 25 selenoproteins in chicken myoblasts. The specific small interfering RNA (siRNA) for Selk gene was designed and transfected into chicken myoblasts. Post-transfection mRNA expression of 25 selenoproteins was determined at various time periods i.e., 24, 48 and 72 h. Moreover, based on the results of expression of 25 selenoproteins, correlation analysis and principal component analysis (PCA) were used for further analysis. The results showed that the designed siRNA effectively inhibited Selk expression (decreased by 20, 29 and 43 % on 24, 48 and 72 h, respectively) and the mRNA expression levels of the 23 selenoproteins were influenced by silencing Selk differently (P < 0.05). Time-dependent pattern of mRNA expression after siRNA treatment in three groups were found similar: one group including Gpx1, Gpx2, Gpx3, Gpx4, Txnrd1, Txnrd2, Txnrd3, Sepw1, Selh, Sepp1, Selo and Sepx1, another group including Sepn1, Sels, Selt, Selm and Sep15 and other group including Dio2 and Dio3. The results of correlation analysis showed that Gpx1, Gpx2, Gpx3, Gpx4, Dio1, Dio3, Sepn1, Sels, Sepw1, Selt, Selh, Sep15, Seli and Selu had a positive correlation with Selk, while Dio2 and Sepp1 had a negative correlation with Selk. PCA data also indicated that Txnrd1, Txnrd2, Dio2, Selpb, Sepp1and Selo may play special roles in response to Selk silencing. In summary, these results indicated that different selenoproteins possess and exhibits distinct responses to silencing of Selk in chicken myoblasts.

  1. Low-level infrared laser modulates muscle repair and chromosome stabilization genes in myoblasts.

    PubMed

    da Silva Neto Trajano, Larissa Alexsandra; Stumbo, Ana Carolina; da Silva, Camila Luna; Mencalha, Andre Luiz; Fonseca, Adenilson S

    2016-08-01

    Infrared laser therapy is used for skeletal muscle repair based on its biostimulative effect on satellite cells. However, shortening of telomere length limits regenerative potential in satellite cells, which occurs after each cell division cycle. Also, laser therapy could be more effective on non-physiologic tissues. This study evaluated low-level infrared laser exposure effects on mRNA expression from muscle injury repair and telomere stabilization genes in myoblasts in normal and stressful conditions. Laser fluences were those used in clinical protocols. C2C12 myoblast cultures were exposed to low-level infrared laser (10, 35, and 70 J/cm(2)) in standard or normal (10 %) and reduced (2 %) fetal bovine serum concentrations; total RNA was extracted for mRNA expression evaluation from muscle injury repair (MyoD and Pax7) and chromosome stabilization (TRF1 and TRF2) genes by real time quantitative polymerization chain reaction. Data show that low-level infrared laser increases the expression of MyoD and Pax7 in 10 J/cm(2) fluence, TRF1 expression in all fluences, and TRF2 expression in 70 J/cm(2) fluence in both 10 and 2 % fetal bovine serum. Low-level infrared laser increases mRNA expression from genes related to muscle repair and telomere stabilization in myoblasts in standard or normal and stressful conditions.

  2. Analysis of adhesive binding forces between laminin-1 and C2C12 muscle cell membranes measured via high resolution force spectroscopy

    NASA Astrophysics Data System (ADS)

    Gluck, George; Gilbert, Richard; Ortiz, Christine

    2002-03-01

    Laminins are a family of glycoproteins that regulate cell differentiation, shape, and motility through interactions with various cell surface receptors. Here, we have directly measured the biomolecular adhesive binding forces between a cantilever / probe tip that was covalently attached with laminin-1 and membrane receptors on C2C12 muscle cells using the technique of high-resolution force spectroscopy (HRFS). On retraction of the probe tip away from the membrane surface, discrete, long-range adhesive unbinding events were always observed. Statistical analysis of the data revealed an initial broad distribution of heterogeneous unbinding events (occurring at separation distances, D=0-2µm from the point of maximum compression) of magnitude 92.23±37.87pN followed by a narrow distribution of homogeneous unbinding events (occurring at D > 2µm) of magnitude 38.16±9.10pN, which is suggestive of an individual biomolecular adhesive interaction. On-going studies include loading rate dependence and effect of dystroglycan mutation.

  3. Cortisone induces insulin resistance in C2C12 myotubes through activation of 11beta-hydroxysteroid dehydrogenase 1 and autocrinal regulation.

    PubMed

    Park, Seung Yeon; Bae, Ji Hyun; Cho, Young Sik

    2014-04-01

    The enzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) is known to catalyse inactive glucocorticoids into active forms, and its dysregulation in adipose and muscle tissues has been implicated in the development of metabolic syndrome. To delineate the molecular mechanism by which active cortisol has an antagonizing effect against insulin, we optimized the metabolic production of cortisol and its biological functions in myotubes (C2C12). Myotubes supplemented with cortisone actively catalysed its conversion into cortisol, which in turn abolished phosphorylation of Akt in response to insulin treatment. This led to diminished uptake of insulin-induced glucose. This was corroborated by the application of 11β-HSD1 inhibitor glycyrrhetinic acid and a glucocorticoid receptor antagonist RU-486, which reversed completely the antagonizing effects of cortisol on insulin action. Therefore, development of specific inhibitors targeting 11β-HSD1 might be a promising way to improve impaired insulin-stimulated glucose uptake. Copyright © 2013 John Wiley & Sons, Ltd.

  4. Cinnamon Extract Enhances Glucose Uptake in 3T3-L1 Adipocytes and C2C12 Myocytes by Inducing LKB1-AMP-Activated Protein Kinase Signaling

    PubMed Central

    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. PMID:24551069

  5. CLA reduces inflammatory mediators from A427 human lung cancer cells and A427 conditioned medium promotes differentiation of C2C12 murine muscle cells.

    PubMed

    Oraldi, Manuela; Maggiora, Marina; Paiuzzi, Elena; Canuto, Rosa A; Muzio, Giuliana

    2013-01-01

    Conjugated linoleic acid (CLA) is thought to have anti-proliferative and anti-inflammatory properties, but its effect on cancer cachexia is unknown. Two effects were here investigated: that of CLA on inflammatory mediator production in human lung cancer cells, and that of reduced mediators on the myogenic differentiation of murine muscle C2C12 cells. The latter cells were grown in medium conditioned by human lung cancer A427 cells, with or without CLA, to mimic only the effect of molecules released from the tumor "in vivo", excluding the effect of host-produced cachectic factors. The results obtained show that CLA was found to reduce the production of tumor necrosis factor-α, interleukin (IL)-1β and prostaglandin E2 (PGE2), but had no effect on IL-6 production. The mechanisms underlying the effect of CLA on cytokine or PGE2 release in A427 cells are probably mediated by activation of peroxisome proliferator-activated receptor (PPAR)α, which increased at 24 h CLA treatment. In turn, the reduced content of inflammatory mediators in medium conditioned by A427 cells, in the presence of CLA, allowed muscle cells to proliferate, again by inducing PPAR. The involvement of PPARα was demonstrated by treatment with the antagonist MK-886. The findings demonstrate the anti-inflammatory and myogenic action of CLA and point to its possible application as a novel dietary supplement and therapeutic agent in inflammatory disease states, such as cachexia.

  6. Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest.

    PubMed

    Sellathurai, Jeeva; Nielsen, Joachim; Hejbøl, Eva Kildall; Jørgensen, Louise Helskov; Dhawan, Jyotsna; Nielsen, Michael Friberg Bruun; Schrøder, Henrik Daa

    2016-01-01

    Most cell culture studies have been performed at atmospheric oxygen tension of 21%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G0 arrested myoblasts in 21% O2 and in 1% O2 in order to see how oxygen tension affects activation and proliferation of human myoblasts. Human myoblasts were isolated from skeletal muscle tissue and G0 arrested in vitro followed by reactivation at 21% O2 and 1% O2. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot. We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch 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-catenin indicated that notch signaling may be induced in 21% O2, while the canonical Wnt signaling may be induced in 1% O2 during activation and proliferation of myoblasts.

  7. Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest

    PubMed Central

    Sellathurai, Jeeva; Nielsen, Joachim; Hejbøl, Eva Kildall; Jørgensen, Louise Helskov; Dhawan, Jyotsna; Nielsen, Michael Friberg Bruun; Schrøder, Henrik Daa

    2016-01-01

    Objectives Most cell culture studies have been performed at atmospheric oxygen tension of 21%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G0 arrested myoblasts in 21% O2 and in 1% O2 in order to see how oxygen tension affects activation and proliferation of human myoblasts. Materials and Methods Human myoblasts were isolated from skeletal muscle tissue and G0 arrested in vitro followed by reactivation at 21% O2 and 1% O2. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot. Results and Conclusions We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch 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-catenin indicated that notch signaling may be induced in 21% O2, while the canonical Wnt signaling may be induced in 1% O2 during activation and proliferation of myoblasts. PMID:27442119

  8. Evidence for myoblast-extrinsic regulation of slow myosin heavy chain expression during muscle fiber formation in embryonic development.

    PubMed

    Cho, M; Webster, S G; Blau, H M

    1993-05-01

    Vertebrate muscles are composed of an array of diverse fast and slow fiber types with different contractile properties. Differences among fibers in fast and slow MyHC expression could be due to extrinsic factors that act on the differentiated myofibers. Alternatively, the mononucleate myoblasts that fuse to form multinucleated muscle fibers could differ intrinsically due to lineage. To distinguish between these possibilities, we determined whether the changes in proportion of slow fibers were attributable to inherent differences in myoblasts. The proportion of fibers expressing slow myosin heavy chain (MyHC) was found to change markedly with time during embryonic and fetal human limb development. During the first trimester, a maximum of 75% of fibers expressed slow MyHC. Thereafter, new fibers formed which did not express this MyHC, so that the proportion of fibers expressing slow MyHC dropped to approximately 3% of the total by midgestation. Several weeks later, a subset of the new fibers began to express slow MyHC and from week 30 of gestation through adulthood, approximately 50% of fibers were slow. However, each myoblast clone (n = 2,119) derived from muscle tissues at six stages of human development (weeks 7, 9, 16, and 22 of gestation, 2 mo after birth and adult) expressed slow MyHC upon differentiation. We conclude from these results that the control of slow MyHC expression in vivo during muscle fiber formation in embryonic development is largely extrinsic to the myoblast. By contrast, human myoblast clones from the same samples differed in their expression of embryonic and neonatal MyHCs, in agreement with studies in other species, and this difference was shown to be stably heritable. Even after 25 population doublings in tissue culture, embryonic stage myoblasts did not give rise to myoblasts capable of expressing MyHCs typical of neonatal stages, indicating that stage-specific differences are not under the control of a division dependent mechanism, or

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

    PubMed

    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.

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

    PubMed

    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.

  11. Muscle acellular scaffold as a biomaterial: effects on C2C12 cell differentiation and interaction with the murine host environment

    PubMed Central

    Perniconi, Barbara; Coletti, Dario; Aulino, Paola; Costa, Alessandra; Aprile, Paola; Santacroce, Luigi; Chiaravalloti, Ernesto; Coquelin, Laura; Chevallier, Nathalie; Teodori, Laura; Adamo, Sergio; Marrelli, Massimo; Tatullo, Marco

    2014-01-01

    The extracellular matrix (ECM) of decellularized organs possesses the characteristics of the ideal tissue-engineering scaffold (i.e., histocompatibility, porosity, degradability, non-toxicity). We previously observed that the muscle acellular scaffold (MAS) is a pro-myogenic environment in vivo. In order to determine whether MAS, which is basically muscle ECM, behaves as a myogenic environment, regardless of its location, we analyzed MAS interaction with both muscle and non-muscle cells and tissues, to assess the effects of MAS on cell differentiation. Bone morphogenetic protein treatment of C2C12 cells cultured within MAS induced osteogenic differentiation in vitro, thus suggesting that MAS does not irreversibly commit cells to myogenesis. In vivo MAS supported formation of nascent muscle fibers when replacing a muscle (orthotopic position). However, heterotopically grafted MAS did not give rise to muscle fibers when transplanted within the renal capsule. Also, no muscle formation was observed when MAS was transplanted under the xiphoid process, in spite of the abundant presence of cells migrating along the laminin-based MAS structure. Taken together, our results suggest that MAS itself is not sufficient to induce myogenic differentiation. It is likely that the pro-myogenic environment of MAS is not strictly related to the intrinsic properties of the muscle scaffold (e.g., specific muscle ECM proteins). Indeed, it is more likely that myogenic stem cells colonizing MAS recognize a muscle environment that ultimately allows terminal myogenic differentiation. In conclusion, MAS may represent a suitable environment for muscle and non-muscle 3D constructs characterized by a highly organized structure whose relative stability promotes integration with the surrounding tissues. Our work highlights the plasticity of MAS, suggesting that it may be possible to consider MAS for a wider range of tissue engineering applications than the mere replacement of volumetric muscle loss

  12. Class III phosphoinositide 3-kinase--Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12 myotubes.

    PubMed Central

    Tassa, Amina; Roux, Marie Paule; Attaix, Didier; Bechet, Daniel M

    2003-01-01

    Increased proteolysis contributes to muscle atrophy that prevails in many diseases. Elucidating the signalling pathways responsible for this activation is of obvious clinical importance. Autophagy is a ubiquitous degradation process, induced by amino acid starvation, that delivers cytoplasmic components to lysosomes. Starvation markedly stimulates autophagy in myotubes, and the present studies investigate the mechanisms of this regulation. In C(2)C(12) myotubes incubated with serum growth factors, amino acid starvation stimulated autophagic proteolysis independently of p38 and p42/p44 mitogen-activated protein kinases, but in a PI3K (phosphoinositide 3-kinase)-dependent manner. Starvation, however, did not alter activities of class I and class II PI3Ks, and was not sufficient to affect major signalling proteins downstream from class I PI3K (glycogen synthase kinase, Akt/protein kinase B and protein S6). In contrast, starvation increased class III PI3K activity in whole-myotube extracts. In fact, this increase was most pronounced for a population of class III PI3K that coimmunoprecipitated with Beclin1/Apg6 protein, a major determinant in the initiation of autophagy. Stimulation of proteolysis was reproduced by feeding myotubes with synthetic dipalmitoyl-PtdIns3 P, the class III PI3K product. Conversely, protein transfection of anti-class III PI3K inhibitory antibody into starved myotubes inverted the induction of proteolysis. Therefore, independently of class I PI3K/Akt, protein S6 and mitogen-activated protein kinase pathways, amino acid starvation stimulates proteolysis in myotubes by regulating class III PI3K-Beclin1 autophagic complexes. PMID:12967324

  13. Ethanol extract of the Prunus mume fruits stimulates glucose uptake by regulating PPAR-γ in C2C12 myotubes and ameliorates glucose intolerance and fat accumulation in mice fed a high-fat diet.

    PubMed

    Shin, Eun Ju; Hur, Haeng Jeon; Sung, Mi Jeong; Park, Jae Ho; Yang, Hye Jeong; Kim, Myung Sunny; Kwon, Dae Young; Hwang, Jin-Taek

    2013-12-15

    In this study, we performed in vitro and in vivo studies to examine whether a 70% ethanol extract of Prunus mume fruits (EMS) exhibits anti-diabetic effects. Treatment with EMS increased glucose uptake in C2C12 myotubes, and also increased PPAR-γ activity or PPAR-γ mRNA expression. To confirm these in vitro results, we next conducted an animal experiment. A high-fat diet significantly increased the body weight, fat accumulation, and glucose levels in mice. Under the same conditions, 5% EMS attenuated the high-fat diet-induced increase in body weight and fat accumulation and improved the impaired fasting glucose level and glucose tolerance. High performance liquid chromatography analysis demonstrated that EMS contained chlorogenic acid, caffeic acid, rutin, luteolin-7-glucoside, naringin, apigenin-7-glucoside, and hesperidin. Taken together, these findings suggest that EMS exerts an anti-diabetic effect both in vitro and in vivo, which is mediated, at least in part, by the activation of PPAR-γ.

  14. Ursolic acid induces mitochondrial biogenesis through the activation of AMPK and PGC-1 in C2C12 myotubes: a possible mechanism underlying its beneficial effect on exercise endurance.

    PubMed

    Chen, Jihang; Wong, Hoi Shan; Leong, Pou Kuan; Leung, Hoi Yan; Chan, Wing Man; Ko, Kam Ming

    2017-07-19

    Mitochondrial biogenesis, which involves an increase in mitochondrial number and the overall capacity of oxidative phosphorylation, is a critical determinant of skeletal muscle function. Recent findings have shown that some natural products can enhance mitochondrial adaptation to aerobic exercise, which in turn improves exercise performance, presumably by delaying muscle fatigue. Ursolic acid (UA), a natural triterpene, is commonly found in various vegetables and fruits. In the current study, UA was shown to increase mitochondrial mass and ATP generation capacity, with a concomitant production of a low level of mitochondrial reactive oxygen species (ROS) in C2C12 myotubes. Mitochondrial ROS, in turn, activated the redox sensitive adenosine monophosphate-dependent protein kinase (AMPK)/peroxisome proliferator-activated receptor γ coactivator-1(PGC-1) pathway. The activation of AMPK/PGC-1 further increased the expression of cytochrome c oxidase (COX) and uncoupling protein 3. Animal studies showed that UA can also dose-dependently increase the endurance exercise capacity in mice, as assessed by a weight-loaded swimming test and a hanging wire test. Our findings suggest that UA may induce mitochondrial biogenesis through the activation of AMPK and PGC-1 pathways in skeletal muscle, thereby offering a promising prospect for its use to enhance exercise endurance and alleviating fatigue in humans.

  15. Long-chain acylcarnitines activate cell stress and myokine release in C2C12 myotubes: calcium-dependent and -independent effects.

    PubMed

    McCoin, Colin S; Knotts, Trina A; Ono-Moore, Kikumi D; Oort, Pieter J; Adams, Sean H

    2015-06-01

    Acylcarnitines, important lipid biomarkers reflective of acyl-CoA status, are metabolites that possess bioactive and inflammatory properties. This study examined the potential for long-chain acylcarnitines to activate cellular inflammatory, stress, and death pathways in a skeletal muscle model. Differentiated C2C12 myotubes treated with l-C14, C16, C18, and C18:1 carnitine displayed dose-dependent increases in IL-6 production with a concomitant rise in markers of cell permeability and death, which was not observed for shorter chain lengths. l-C16 carnitine, used as a representative long-chain acylcarnitine at initial extracellular concentrations ≥25 μM, increased IL-6 production 4.1-, 14.9-, and 31.4-fold over vehicle at 25, 50, and 100 μM. Additionally, l-C16 carnitine activated c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase between 2.5- and 11-fold and induced cell injury and death within 6 h with modest activation of the apoptotic caspase-3 protein. l-C16 carnitine rapidly increased intracellular calcium, most clearly by 10 μM, implicating calcium as a potential mechanism for some activities of long-chain acylcarnitines. The intracellular calcium chelator BAPTA-AM blunted l-C16 carnitine-mediated IL-6 production by >65%. However, BAPTA-AM did not attenuate cell permeability and death responses, indicating that these outcomes are calcium independent. The 16-carbon zwitterionic compound amidosulfobetaine-16 qualitatively mimicked the l-C16 carnitine-associated cell stress outcomes, suggesting that the effects of high experimental concentrations of long-chain acylcarnitines are through membrane disruption. Herein, a model is proposed in which acylcarnitine cell membrane interactions take place along a spectrum of cellular concentrations encountered in physiological-to-pathophysiological conditions, thus regulating function of membrane-based systems and impacting cell biology.

  16. Long-chain acylcarnitines activate cell stress and myokine release in C2C12 myotubes: calcium-dependent and -independent effects

    PubMed Central

    McCoin, Colin S.; Knotts, Trina A.; Ono-Moore, Kikumi D.; Oort, Pieter J.

    2015-01-01

    Acylcarnitines, important lipid biomarkers reflective of acyl-CoA status, are metabolites that possess bioactive and inflammatory properties. This study examined the potential for long-chain acylcarnitines to activate cellular inflammatory, stress, and death pathways in a skeletal muscle model. Differentiated C2C12 myotubes treated with l-C14, C16, C18, and C18:1 carnitine displayed dose-dependent increases in IL-6 production with a concomitant rise in markers of cell permeability and death, which was not observed for shorter chain lengths. l-C16 carnitine, used as a representative long-chain acylcarnitine at initial extracellular concentrations ≥25 μM, increased IL-6 production 4.1-, 14.9-, and 31.4-fold over vehicle at 25, 50, and 100 μM. Additionally, l-C16 carnitine activated c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase between 2.5- and 11-fold and induced cell injury and death within 6 h with modest activation of the apoptotic caspase-3 protein. l-C16 carnitine rapidly increased intracellular calcium, most clearly by 10 μM, implicating calcium as a potential mechanism for some activities of long-chain acylcarnitines. The intracellular calcium chelator BAPTA-AM blunted l-C16 carnitine-mediated IL-6 production by >65%. However, BAPTA-AM did not attenuate cell permeability and death responses, indicating that these outcomes are calcium independent. The 16-carbon zwitterionic compound amidosulfobetaine-16 qualitatively mimicked the l-C16 carnitine-associated cell stress outcomes, suggesting that the effects of high experimental concentrations of long-chain acylcarnitines are through membrane disruption. Herein, a model is proposed in which acylcarnitine cell membrane interactions take place along a spectrum of cellular concentrations encountered in physiological-to-pathophysiological conditions, thus regulating function of membrane-based systems and impacting cell biology. PMID:25852008

  17. Gene Expression Profiling of H9c2 Myoblast Differentiation towards a Cardiac-Like Phenotype

    PubMed Central

    Branco, Ana F.; Pereira, Susana P.; Gonzalez, Susana; Gusev, Oleg; Rizvanov, Albert A.; Oliveira, Paulo J.

    2015-01-01

    H9c2 myoblasts are a cell model used as an alternative for cardiomyocytes. H9c2 cells have the ability to differentiate towards a cardiac phenotype when the media serum is reduced in the presence of all-trans-retinoic acid (RA), creating multinucleated cells with low proliferative capacity. In the present study, we performed for the first time a transcriptional analysis of the H9c2 cell line in two differentiation states, i.e. embryonic cells and differentiated cardiac-like cells. The results show that RA-induced H9c2 differentiation increased the expression of genes encoding for cardiac sarcomeric proteins such as troponin T, or calcium transporters and associated machinery, including SERCA2, ryanodine receptor and phospholamban as well as genes associated with mitochondrial energy production including respiratory chain complexes subunits, mitochondrial creatine kinase, carnitine palmitoyltransferase I and uncoupling proteins. Undifferentiated myoblasts showed increased gene expression of pro-survival proteins such as Bcl-2 as well as cell cycle-regulating proteins. The results indicate that the differentiation of H9c2 cells lead to an increase of transcripts and protein levels involved in calcium handling, glycolytic and mitochondrial metabolism, confirming that H9c2 cell differentiation induced by RA towards a more cardiac-like phenotype involves remodeled mitochondrial function. PI3K, PDK1 and p-CREB also appear to be involved on H9c2 differentiation. Furthermore, complex analysis of differently expressed transcripts revealed significant up-regulation of gene expression related to cardiac muscle contraction, dilated cardiomyopathy and other pathways specific for the cardiac tissue. Metabolic and gene expression remodeling impacts cell responses to different stimuli and determine how these cells are used for biochemical assays. PMID:26121149

  18. YB1/p32, a nuclear Y-box binding protein 1, is a novel regulator of myoblast differentiation that interacts with Msx1 homeoprotein

    SciTech Connect

    Song, Young Joon; Lee, Hansol

    2010-02-15

    Precisely controlled cellular differentiation is essential for the proper development of vertebrate embryo and deregulated differentiation is a major cause of many human congenital diseases as well as cancer. Msx1 is a member of the homeoprotein family implicated in these processes, which inhibits the differentiation of skeletal muscle and other cell types, presumably by regulating transcription of target genes through interaction with other cellular factors. We presently show that YB1/p32, a nuclear Y-box binding protein 1, interacts with Msx1 homeoprotein and functions as a regulator of C2C12 myoblast differentiation. We demonstrate that YB1/p32 functionally interacts with Msx1 through its N-terminal region and colocalizes with Msx1 at the nuclear periphery. Moreover, we find that YB1/p32 is competent for inhibition of C2C12 myoblast differentiation, which is correlated with its activity as a negative regulator of MyoD gene expression and binding to the MyoD core enhancer region (CER). Furthermore, YB1/p32 cooperates with Msx1 in transcriptional repression and knocking down the expression of endogenous YB1 attenuates the effects of Msx1. Taken together, our study has uncovered a new function of YB1/p32, a regulator of skeletal muscle differentiation.

  19. Role of Phosphatidylinositol 3,4,5-Trisphosphate (PIP3) 5-Phosphatase Skeletal Muscle- and Kidney-enriched Inositol Polyphosphate Phosphatase (SKIP) in Myoblast Differentiation*

    PubMed Central

    Ijuin, Takeshi; Takenawa, Tadaomi

    2012-01-01

    Insulin-like growth factors (IGFs) are essential for the development, regeneration, and hypertrophy of skeletal muscles. IGF-II promotes myoblast differentiation through phosphatidylinositol 3-kinase (PI 3-kinase), Akt, and mTOR signaling. Here, we report that skeletal muscle- and kidney-enriched inositol polyphosphate phosphatase (SKIP) negatively regulates myogenesis through inhibition of IGF-II production and attenuation of the IGF-II-Akt-mTOR signaling pathway. We also demonstrate that SKIP expression, which was markedly elevated during differentiation, was controlled by MyoD in C2C12 cells. Expression of SKIP inhibited IGF-II at the transcription level. These results indicate that SKIP regulates MyoD-mediated muscle differentiation. Silencing of SKIP increased IGF-II transcription and myoblast differentiation. Furthermore, knockdown of SKIP resulted in thick myotubes with a larger number of nuclei than that in control C2C12 cells. Taken together, these data indicate that SKIP controls the IGF-II-PI 3-kinase-Akt-mTOR auto-regulation loop during myogenesis. Our findings identify SKIP as a key regulator of muscle cell differentiation. PMID:22815484

  20. Histone methyltransferase Setd2 is critical for the proliferation and differentiation of myoblasts.

    PubMed

    Yi, Xin; Tao, Ye; Lin, Xi; Dai, Yuan; Yang, Tingli; Yue, Xiaojing; Jiang, Xuejun; Li, Xiaoyan; Jiang, Ding-Sheng; Andrade, Kelsey C; Chang, Jiang

    2017-04-01

    Skeletal muscle cell proliferation and differentiation are tightly regulated. Epigenetic regulation is a major component of the regulatory mechanism governing these processes. Histone modification is part of the epigenetic code used for transcriptional regulation of chromatin through the establishment of an active or repressive state for genes involved in myogenesis in a temporal manner. Here, we uncovered the function of SET domain containing 2 (Setd2), an essential histone 3 lysine 36 trimethyltransferase, in regulating the proliferation and differentiation of myoblasts. Setd2 was silenced in the skeletal muscle myoblast cell line, C2C12, using the CRISPR/CAS9 system. The mutant cells exhibited defect in myotube formation. The myotube formation marker, myosin heavy chain (MHC), was downregulated earlier in Setd2 silenced cells compared to wild-type myoblasts during differentiation. The deficiency in Setd2 also resulted in repression of Myogenin (MyoG) expression, a key myogenic regulator during differentiation. In addition to the myoblast differentiation defect, decreased proliferation rate with significantly reduced levels of histone 3 phosphorylation, indicative of cell proliferation defect, were observed in the Setd2 silenced cells; suggesting an impaired proliferation phenotype. Furthermore, compromised G1/S- and G2/M-phase transition and decreased expression levels of major regulators of cell cycle G1/S checkpoints, cyclin D1, CDK4, CDK6, and cyclin E2 were detected in Setd2 silenced cells. Consistent with the cell cycle arrested phenotype, cyclin-dependent kinase inhibitor p21 was upregulated in Setd2 silenced cells. Together, this study demonstrates an essential role of Setd2 in myoblast proliferation and differentiation, and uncovers Setd2-mediated molecular mechanism through regulating MyoG and p21.

  1. Segregation of myoblast fusion and muscle-specific gene expression by distinct ligand-dependent inactivation of GSK-3β

    PubMed Central

    Pansters, N. A. M.; van der Velden, J. L. J.; Kelders, M. C. J. M.; Laeremans, H.; Schols, A. M. W. J.

    2010-01-01

    Myogenic differentiation involves myoblast fusion and induction of muscle-specific gene expression, which are both stimulated by pharmacological (LiCl), genetic, or IGF-I-mediated GSK-3β inactivation. To assess whether stimulation of myogenic differentiation is common to ligand-mediated GSK-3β inactivation, myoblast fusion and muscle-specific gene expression were investigated in response to Wnt-3a. Moreover, crosstalk between IGF-I/GSK-3β/NFATc3 and Wnt/GSK-3β/β-catenin signaling was assessed. While both Wnt-3a and LiCl promoted myoblast fusion, muscle-specific gene expression was increased by LiCl, but not by Wnt-3a or β-catenin over-expression. Furthermore, LiCl and IGF-I, but not Wnt-3a, increased NFATc3 transcriptional activity. In contrast, β-catenin-dependent transcriptional activity was increased by Wnt-3a and LiCl, but not IGF-I. These results for the first time reveal a segregated regulation of myoblast fusion and muscle-specific gene expression following stimulation of myogenic differentiation in response to distinct ligand-specific signaling routes of GSK-3β inactivation. Electronic supplementary material The online version of this article (doi:10.1007/s00018-010-0467-7) contains supplementary material, which is available to authorized users. PMID:20694829

  2. Rab35 regulates cadherin-mediated adherens junction formation and myoblast fusion

    PubMed Central

    Charrasse, Sophie; Comunale, Franck; De Rossi, Sylvain; Echard, Arnaud; Gauthier-Rouvière, Cécile

    2013-01-01

    Cadherins are homophilic cell–cell adhesion molecules implicated in many fundamental processes, such as morphogenesis, cell growth, and differentiation. They accumulate at cell–cell contact sites and assemble into large macromolecular complexes named adherens junctions (AJs). Cadherin targeting and function are regulated by various cellular processes, many players of which remain to be uncovered. Here we identify the small GTPase Rab35 as a new regulator of cadherin trafficking and stabilization at cell–cell contacts in C2C12 myoblasts and HeLa cells. We find that Rab35 accumulates at cell–cell contacts in a cadherin-dependent manner. Knockdown of Rab35 or expression of a dominant-negative form of Rab35 impaired N- and M-cadherin recruitment to cell–cell contacts, their stabilization at the plasma membrane, and association with p120 catenin and led to their accumulation in transferrin-, clathrin-, and AP-2–positive intracellular vesicles. We also find that Rab35 function is required for PIP5KIγ accumulation at cell–cell contacts and phosphatidyl inositol 4,5-bisphosphate production, which is involved in cadherin stabilization at contact sites. Finally, we show that Rab35 regulates myoblast fusion, a major cellular process under the control of cadherin-dependent signaling. Taken together, these results reveal that Rab35 regulates cadherin-dependent AJ formation and myoblast fusion. PMID:23197472

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

    PubMed Central

    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

  4. The cytoprotective effects of ethanol extract of Ecklonia cava against oxidative stress are associated with upregulation of Nrf2-mediated HO-1 and NQO-1 expression through activation of the MAPK pathway.

    PubMed

    Choi, Yung Hyun

    2016-01-01

    The aim of the present study was to examine the cytoprotective effect of Ecklonia cava against oxidative stress in C2C12 myoblasts. The ethanol extract of E. cava (EEEC) prevented hydrogen peroxide (H₂O₂)-induced inhibition of the growth of C2C12 myoblasts and exhibited scavenging activity against intracellular reactive oxygen species (ROS) induced by H₂O₂. EEEC treatment attenuated H2O2-induced comet tail formation and phospho-histone γH2A.X expression. Furthermore, EEEC treatment enhanced the level of the phosphorylated form of nuclear factor erythroid 2- related factor 2 (Nrf2) and its nuclear translocation, which was associated with the induction of heme oxygenase-1 (HO-1) and NADPH-quinone oxidoreductase 1 (NQO-1). Zinc protoporphyrin IX, a HO-1 competitive inhibitor, significantly abolished the protective effects of EEEC against H₂O₂-induced ROS generation and growth inhibition in C2C12 myoblasts. Transient transfection with Nrf2-specific small interfering RNA restored the elevated HO-1 and NQO-1 expression and the phosphorylation of Nrf2 to near normal levels. The EEEC treatment also induced the activation of mitogen-activated protein kinases (MAPKs), and specific inhibitors of MAPKs abolished upregulated HO-1 and NQO-1, as well as the phosphorylation of Nrf2. Taken together, these data suggest that EEEC attenuates oxidative stress by activating Nrf2-mediated HO-1 and inducing NQO-1 via the activation of MAPK signaling pathways.

  5. Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR

    PubMed Central

    Wang, Haohan; Li, Xinxin; Liu, Hehe; Sun, Lingli; Zhang, Rongping; Li, Liang; Wangding, Mincheng; Wang, Jiwen

    2016-01-01

    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. PMID:27007909

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

    PubMed

    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.

  7. miR-29 targets Akt3 to reduce proliferation and facilitate differentiation of myoblasts in skeletal muscle development.

    PubMed

    Wei, W; He, H-B; Zhang, W-Y; Zhang, H-X; Bai, J-B; Liu, H-Z; Cao, J-H; Chang, K-C; Li, X-Y; Zhao, S-H

    2013-06-13

    MicroRNAs (miRNAs) are a type of endogenous noncoding small RNAs involved in the regulation of multiple biological processes. Recently, miR-29 was found to participate in myogenesis. However, the underlying mechanisms by which miR-29 promotes myogenesis have not been identified. We found here that miR-29 was significantly upregulated with age in postnatal mouse skeletal muscle and during muscle differentiation. Overexpression of miR-29 inhibited mouse C2C12 myoblast proliferation and promoted myotube formation. miR-29 specifically targeted Akt3, a member of the serine/threonine protein kinase family responsive to growth factor cell signaling, to result in its post-transcriptional downregulation. Furthermore, knockdown of Akt3 by siRNA significantly inhibited the proliferation of C2C12 cells, and conversely, overexpression of Akt3 suppressed their differentiation. Collectively and given the inverse endogenous expression pattern of rising miR-29 levels and decreasing Akt3 protein levels with age in mouse skeletal muscle, we propose a novel mechanism in which miR-29 modulates growth and promotes differentiation of skeletal muscle through the post-transcriptional downregulation of Akt3.

  8. Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells

    PubMed Central

    2014-01-01

    Background Via the hepatocyte growth factor receptor (Met), hepatocyte growth factor (HGF) exerts key roles involving skeletal muscle development and regeneration. Heparan sulfate proteoglycans (HSPGs) are critical modulators of HGF activity, but the role of specific HSPGs in HGF regulation is poorly understood. Glypican-1 is the only HSPG expressed in myoblasts that localize in lipid raft membrane domains, controlling cell responses to extracellular stimuli. We determined if glypican-1 in these domains is necessary to stabilize the HGF-Met signaling complex and myoblast response to HGF. Methods C2C12 myoblasts and a derived clone (C6) with low glypican-1 expression were used as an experimental model. The activation of Met, ERK1/2 and AKT in response to HGF was evaluated. The distribution of Met and its activated form in lipid raft domains, as well as its dependence on glypican-1, were characterized by sucrose density gradient fractionation in both cell types. Rescue experiments reexpressing glypican-1 or a chimeric glypican-1 fused to the transmembrane and cytoplasmic domains of mouse syndecan-1 or myoblast pretreatment with MβCD were conducted. In vitro and in vivo myoblast migration assays in response to HGF were also performed. Results Glypican-1 localization in membrane raft domains was required for a maximum cell response to HGF. It stabilized Met and HGF in lipid raft domains, forming a signaling complex where the active phospho-Met receptor was concentrated. Glypican-1 also stabilized CD44 in a HGF-dependent manner. In addition, glypican-1 was required for in vitro and in vivo HGF-dependent myoblast migration. Conclusions Glypican-1 is a regulator of HGF-dependent signaling via Met in lipid raft domains. PMID:24517345

  9. Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells.

    PubMed

    Gutiérrez, Jaime; Cabrera, Daniel; Brandan, Enrique

    2014-02-12

    Via the hepatocyte growth factor receptor (Met), hepatocyte growth factor (HGF) exerts key roles involving skeletal muscle development and regeneration. Heparan sulfate proteoglycans (HSPGs) are critical modulators of HGF activity, but the role of specific HSPGs in HGF regulation is poorly understood. Glypican-1 is the only HSPG expressed in myoblasts that localize in lipid raft membrane domains, controlling cell responses to extracellular stimuli. We determined if glypican-1 in these domains is necessary to stabilize the HGF-Met signaling complex and myoblast response to HGF. C2C12 myoblasts and a derived clone (C6) with low glypican-1 expression were used as an experimental model. The activation of Met, ERK1/2 and AKT in response to HGF was evaluated. The distribution of Met and its activated form in lipid raft domains, as well as its dependence on glypican-1, were characterized by sucrose density gradient fractionation in both cell types. Rescue experiments reexpressing glypican-1 or a chimeric glypican-1 fused to the transmembrane and cytoplasmic domains of mouse syndecan-1 or myoblast pretreatment with MβCD were conducted. In vitro and in vivo myoblast migration assays in response to HGF were also performed. Glypican-1 localization in membrane raft domains was required for a maximum cell response to HGF. It stabilized Met and HGF in lipid raft domains, forming a signaling complex where the active phospho-Met receptor was concentrated. Glypican-1 also stabilized CD44 in a HGF-dependent manner. In addition, glypican-1 was required for in vitro and in vivo HGF-dependent myoblast migration. Glypican-1 is a regulator of HGF-dependent signaling via Met in lipid raft domains.

  10. Conjugated linoleic acid (CLA) stimulates mitochondrial biogenesis signaling by the upregulation of PPARγ coactivator 1α (PGC-1α) in C2C12 cells.

    PubMed

    Kim, Yoo; Park, Yeonhwa

    2015-04-01

    Along with its effect on body fat reduction, dietary conjugated linoleic acid (CLA) has been reported to improve physical activity and endurance capacity in mice. It has been suggested these effects may in part be due to physiological changes in skeletal muscle, however, the mode of action is not completely understood. Thus, the purpose of this study was to determine the relevant mechanisms of CLA isomers for mitochondrial biogenesis, one of the most important adaptive responses in skeletal muscle. Both cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12) CLA isomers increased the expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), however, only the t10,c12 isomer, but not c9,t11, increased phosphorylation of AMP-activated protein kinase (AMPK) compared to the control. Among downstream biomarkers of PGC-1α, the CLA mixed isomer enhanced the expression of peroxisome proliferator-activated receptor-δ (PPARδ). Both c9,t11 and t10,c12 CLA isomers increased expression of nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (Tfam), while the c9,t11 increased expression of cytochrome c (Cyt C) and t10,c12 CLA increased expression of voltage-dependent anion channel (VDAC), respectively. Both CLA isomers significantly increased mitochondrial DNA copy number compared to that of control. These findings suggest that the individual CLA isomers potentiate mitochondrial biogenesis via PGC-1α-NRF-1-Tfam signaling cascade, although downstream regulation may be isomer dependent.

  11. Alignment of skeletal muscle myoblasts and myotubes using linear micropatterned surfaces ground with abrasives.

    PubMed

    Shimizu, Kazunori; Fujita, Hideaki; Nagamori, Eiji

    2009-06-15

    Alignment of cells plays a significant key role in skeletal muscle tissue engineering because skeletal muscle tissue in vivo has a highly organized structure consisting of long parallel multinucleated myotubes formed through differentiation and fusion of myoblasts. In the present study, we developed an easy, simple, and low-cost method for aligning skeletal muscle cells by using surfaces with linear microscale features fabricated by grinding. Iron blocks were ground in one direction with three kinds of abrasives (9 microm diamond suspension, #400 sandpaper, and #150 sandpaper) and then used as molds to make micropatterned polydimethylsiloxane (PDMS) substrates (type I, type II, and type III). Observation of the surface topography revealed that the PDMS substrates exhibited different degree of mean roughness (Ra), 0.03 microm for type I, 0.16 microm for type II, and 0.56 microm for type III, respectively. Murine skeletal muscle cell line C2C12 myoblasts were cultured and differentiated on the patterned PDMS substrates, and it was examined whether the alignment of C2C12 myoblasts and myotubes was possible. Although the cell growth and differentiation on the three types of patterned substrates were similar to those on the flat PDMS substrate as a control, the alignment of both C2C12 myoblasts and myotubes was obviously observed on types II and III, but not on type I or the control substrate. These results indicate that surfaces ground with abrasives will be useful for fabricating aligned skeletal muscle tissues.

  12. A synthetic compound that potentiates bone morphogenetic protein-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype.

    PubMed

    Kato, Satoshi; Sangadala, Sreedhara; Tomita, Katsuro; Titus, Louisa; Boden, Scott D

    2011-03-01

    There is an urgent need to develop methods that lower costs of using recombinant human bone morphogenetic proteins (BMPs) to promote bone induction. In this study, we demonstrate the osteogenic effect of a low-molecular weight compound, SVAK-12, that potentiated the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. Here, we report a specific compound, SVAK-12, which was selected based on in silico screenings of small-molecule databases using the homology modeled interaction motif of Smurf1-WW2 domain. The enhancement of BMP-2 activity by SVAK-12 was characterized by evaluating a BMP-specific reporter activity and by monitoring the BMP-2-induced expression of mRNA for osteocalcin and alkaline phosphatase (ALP), which are widely accepted marker genes of osteoblast differentiation. Finally, we confirmed these results by also measuring the enhancement of BMP-2-induced activity of ALP. Smurf1 is an E3 ligase that targets osteogenic Smads for ubiquitin-mediated proteasomal degradation. Smurf1 is an interesting potential target to enhance bone formation based on the positive effects on bone of proteins that block Smurf1-binding to Smad targets or in Smurf1-/- knockout mice. Since Smads bind Smurf1 via its WW2 domain, we performed in silico screening to identify compounds that might interact with the Smurf1-WW2 domain. We recently reported the activity of a compound, SVAK-3. However, SVAK-3, while exhibiting BMP-potentiating activity, was not stable and thus warranted a new search for a more stable and efficacious compound among a selected group of candidates. In addition to being more stable, SVAK-12 exhibited a dose-dependent activity in inducing osteoblastic differentiation of myoblastic C2C12 cells even when multiple markers of the osteoblastic phenotype were parallelly monitored.

  13. A synthetic compound that potentiates bone morphogenetic protein-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype

    PubMed Central

    Kato, Satoshi; Tomita, Katsuro; Titus, Louisa; Boden, Scott D.

    2011-01-01

    There is an urgent need to develop methods that lower costs of using recombinant human bone morphogenetic proteins (BMPs) to promote bone induction. In this study, we demonstrate the osteogenic effect of a low-molecular weight compound, SVAK-12, that potentiated the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. Here, we report a specific compound, SVAK-12, which was selected based on in silico screenings of small-molecule databases using the homology modeled interaction motif of Smurf1-WW2 domain. The enhancement of BMP-2 activity by SVAK-12 was characterized by evaluating a BMP-specific reporter activity and by monitoring the BMP-2-induced expression of mRNA for osteocalcin and alkaline phosphatase (ALP), which are widely accepted marker genes of osteoblast differentiation. Finally, we confirmed these results by also measuring the enhancement of BMP-2-induced activity of ALP. Smurf1 is an E3 ligase that targets osteogenic Smads for ubiquitin-mediated proteasomal degradation. Smurf1 is an interesting potential target to enhance bone formation based on the positive effects on bone of proteins that block Smurf1-binding to Smad targets or in Smurf1−/− knockout mice. Since Smads bind Smurf1 via its WW2 domain, we performed in silico screening to identify compounds that might interact with the Smurf1-WW2 domain. We recently reported the activity of a compound, SVAK-3. However, SVAK-3, while exhibiting BMP-potentiating activity, was not stable and thus warranted a new search for a more stable and efficacious compound among a selected group of candidates. In addition to being more stable, SVAK-12 exhibited a dose-dependent activity in inducing osteoblastic differentiation of myoblastic C2C12 cells even when multiple markers of the osteoblastic phenotype were parallelly monitored. PMID:21110071

  14. Transient and stable transfections of mouse myoblasts with genes coding for pro-angiogenic factors.

    PubMed

    Bialas, M; Krupka, M; Janeczek, A; Rozwadowska, N; Fraczek, M; Kotlinowski, J; Kucharzewska, P; Lackowska, B; Kurpisz, M

    2011-04-01

    Cardiomyocyte loss in the ischaemic heart can be the reason of many complications, eventually being even the cause of patient's death. Despite many promises, cell therapy with the use of skeletal muscle stem cells (SMSC) still remains to be modified and improved. Combined cell and gene therapy seems to be a promising strategy to heal damaged myocardium. In the present study we have investigated the influence of a simultaneous overexpression of two potent pro-angiogenic genes encoding the fibroblast growth factor-4 (FGF-4) and the vascular endothelial growth factor-A (VEGF-A) on a myogenic murine C2C12 cell line. We have demonstrated in in vitro conditions that myoblasts which overexpressed these factors exhibited significant changes in the cell cycle and pro-angiogenic potential with only slight differences in the expression of the myogenic genes. There was not observed the influence of transient or stable overexpression of FGF-4 and VEGF on cell apoptosis/necrosis in standard or oxidative stress conditions comparing to non transfected controls. Overall, our results suggest that the possible transplantation of myoblasts overexpressing pro-angiogenic factors may potentially improve the functionality of the injured myocardium although the definite proof must originate from in situ conducted pre-clinical studies.

  15. Macrophage colony-stimulating factor-induced macrophage differentiation promotes regrowth in atrophied skeletal muscles and C2C12 myotubes.

    PubMed

    Dumont, Nicolas A; Frenette, Jérôme

    2013-02-01

    Skeletal muscle injury and regeneration are closely associated with an inflammatory reaction that is usually characterized by sequential recruitment of neutrophils and monocytes or macrophages. Selective macrophage depletion models have shown that macrophages are essential for complete regeneration of muscle fibers after freeze injuries, toxin injuries, ischemia-reperfusion, and hindlimb unloading and reloading. Although there is growing evidence that macrophages possess major myogenic capacities, it is not known whether the positive effects of macrophages can be optimized to stimulate muscle regrowth. We used in vivo and in vitro mouse models of atrophy to investigate the effects of stimulating macrophages with macrophage colony-stimulating factor (M-CSF) on muscle regrowth. When atrophied soleus muscles were injected intramuscularly with M-CSF, we observed a 1.6-fold increase in macrophage density and a faster recovery in muscle force (20%), combined with an increase in muscle fiber diameter (10%), after 7 days of reloading, compared with PBS-injected soleus muscles. Furthermore, coculture of atrophied myotubes with or without bone marrow-derived macrophages (BMDM) and/or M-CSF revealed that the combination of BMDMs and M-CSF was required to promote myotube growth (15%). More specifically, M-CSF promoted the anti-inflammatory macrophage phenotype, which in turn decreased protein degradation and MuRF-1 expression by 25% in growing myotubes. These results indicate that specific macrophage subsets can be stimulated to promote muscle cell regrowth after atrophy.

  16. ADP-Ribosylation Factor 6 Regulates Mammalian Myoblast Fusion through Phospholipase D1 and Phosphatidylinositol 4,5-Bisphosphate Signaling Pathways

    PubMed Central

    Bach, Anne-Sophie; Enjalbert, Sandrine; Comunale, Franck; Bodin, Stéphane; Vitale, Nicolas; Charrasse, Sophie

    2010-01-01

    Myoblast fusion is an essential step during myoblast differentiation that remains poorly understood. M-cadherin–dependent pathways that signal through Rac1 GTPase activation via the Rho-guanine nucleotide exchange factor (GEF) Trio are important for myoblast fusion. The ADP-ribosylation factor (ARF)6 GTPase has been shown to bind to Trio and to regulate Rac1 activity. Moreover, Loner/GEP100/BRAG2, a GEF of ARF6, has been involved in mammalian and Drosophila myoblast fusion, but the specific role of ARF6 has been not fully analyzed. Here, we show that ARF6 activity is increased at the time of myoblast fusion and is required for its implementation in mouse C2C12 myoblasts. Specifically, at the onset of myoblast fusion, ARF6 is associated with the multiproteic complex that contains M-cadherin, Trio, and Rac1 and accumulates at sites of myoblast fusion. ARF6 silencing inhibits the association of Trio and Rac1 with M-cadherin. Moreover, we demonstrate that ARF6 regulates myoblast fusion through phospholipase D (PLD) activation and phosphatidylinositol 4,5-bis-phosphate production. Together, these data indicate that ARF6 is a critical regulator of C2C12 myoblast fusion and participates in the regulation of PLD activities that trigger both phospholipids production and actin cytoskeleton reorganization at fusion sites. PMID:20505075

  17. Let-7b Regulates Myoblast Proliferation by Inhibiting IGF2BP3 Expression in Dwarf and Normal Chicken

    PubMed Central

    Lin, Shumao; Luo, Wen; Ye, Yaqiong; Bekele, Endashaw J.; Nie, Qinghua; Li, Yugu; Zhang, Xiquan

    2017-01-01

    The sex-linked dwarf chicken is caused by the mutation of growth hormone receptor (GHR) gene and characterized by shorter shanks, lower body weight, smaller muscle fiber diameter and fewer muscle fiber number. However, the precise regulatory pathways that lead to the inhibition of skeletal muscle growth in dwarf chickens still remain unclear. Here we found a let-7b mediated pathway might play important role in the regulation of dwarf chicken skeletal muscle growth. Let-7b has higher expression in the skeletal muscle of dwarf chicken than in normal chicken, and the expression of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which is a translational activator of IGF2, showed opposite expression trend to let-7b. In vitro cellular assays validated that let-7b directly inhibits IGF2BP3 expression through binding to its 3′UTR region, and the protein level but not mRNA level of IGF2 would be reduced in let-7b overexpressed chicken myoblast. Let-7b can inhibit cell proliferation and induce cell cycle arrest in chicken myoblast through let-7b-IGF2BP3-IGF2 signaling pathway. Additionally, let-7b can also regulate skeletal muscle growth through let-7b-GHR-GHR downstream genes pathway, but this pathway is non-existent in dwarf chicken because of the deletion mutation of GHR 3′UTR. Notably, as the loss binding site of GHR for let-7b, let-7b has enhanced its binding and inhibition on IGF2BP3 in dwarf myoblast, suggesting that the miRNA can balance its inhibiting effect through dynamic regulate its binding to target genes. Collectively, these results not only indicate that let-7b can inhibit skeletal muscle growth through let-7b-IGF2BP3-IGF2 signaling pathway, but also show that let-7b regulates myoblast proliferation by inhibiting IGF2BP3 expression in dwarf and normal chickens. PMID:28736533

  18. ZBED6, a Novel Transcription Factor Derived from a Domesticated DNA Transposon Regulates IGF2 Expression and Muscle Growth

    PubMed Central

    Jaffe, Jacob D.; Mikkelsen, Tarjei S.; Wallerman, Ola; Larhammar, Martin; Zhang, Xiaolan; Wang, Li; Saenz-Vash, Veronica; Gnirke, Andreas; Lindroth, Anders M.; Barrés, Romain; Yan, Jie; Strömberg, Sara; De, Sachinandan; Pontén, Fredrik; Lander, Eric S.; Carr, Steven A.; Zierath, Juleen R.; Kullander, Klas; Wadelius, Claes; Lindblad-Toh, Kerstin; Andersson, Göran; Hjälm, Göran; Andersson, Leif

    2009-01-01

    A single nucleotide substitution in intron 3 of IGF2 in pigs abrogates a binding site for a repressor and leads to a 3-fold up-regulation of IGF2 in skeletal muscle. The mutation has major effects on muscle growth, size of the heart, and fat deposition. Here, we have identified the repressor and find that the protein, named ZBED6, is previously unknown, specific for placental mammals, and derived from an exapted DNA transposon. Silencing of Zbed6 in mouse C2C12 myoblasts affected Igf2 expression, cell proliferation, wound healing, and myotube formation. Chromatin immunoprecipitation (ChIP) sequencing using C2C12 cells identified about 2,500 ZBED6 binding sites in the genome, and the deduced consensus motif gave a perfect match with the established binding site in Igf2. Genes associated with ZBED6 binding sites showed a highly significant enrichment for certain Gene Ontology classifications, including development and transcriptional regulation. The phenotypic effects in mutant pigs and ZBED6-silenced C2C12 myoblasts, the extreme sequence conservation, its nucleolar localization, the broad tissue distribution, and the many target genes with essential biological functions suggest that ZBED6 is an important transcription factor in placental mammals, affecting development, cell proliferation, and growth. PMID:20016685

  19. Stem cell antigen-1 regulates the tempo of muscle repair through effects on proliferation of {alpha}7 integrin-expressing myoblasts

    SciTech Connect

    Epting, Conrad L.; Lopez, Javier E.; Pedersen, Anissa; Brown, Courtney; Spitz, Paul; Ursell, Philip C.; Bernstein, Harold S.

    2008-03-10

    Skeletal muscle repair occurs through a programmed series of events including myogenic precursor activation, myoblast proliferation, and differentiation into new myofibers. We previously identified a role for Stem cell antigen-1 (Sca-1) in myoblast proliferation and differentiation in vitro. We demonstrated that blocking Sca-1 expression resulted in sustained myoblast cell division. Others have since demonstrated that Sca-1-null myoblasts display a similar phenotype when cultured ex vivo. To test the importance of Sca-1 during myogenesis in vivo, we employed a myonecrotic injury model in Sca-1{sup -/-} and Sca-1{sup +/+} mice. Our results demonstrate that Sca-1{sup -/-} myoblasts exhibit a hyperproliferative response consisting of prolonged and accelerated cell division in response to injury. This leads to delayed myogenic differentiation and muscle repair. These data provide the first in vivo evidence for Sca-1 as a regulator of myoblast proliferation during muscle regeneration. These studies also suggest that the balance between myogenic precursor proliferation and differentiation is critical to normal muscle repair.

  20. Inulin increases glucose transport in C2C12 myotubes and HepG2 cells via activation of AMP-activated protein kinase and phosphatidylinositol 3-kinase pathways.

    PubMed

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

    2009-10-01

    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.

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

    PubMed Central

    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

  2. MAP kinases p38 and JNK are activated by the steroid hormone 1alpha,25(OH)2-vitamin D3 in the C2C12 muscle cell line.

    PubMed

    Buitrago, Claudia G; Ronda, Ana C; de Boland, Ana Russo; Boland, Ricardo

    2006-03-01

    In chick skeletal muscle cell primary cultures, we previously demonstrated that 1alpha,25(OH)2-vitamin D3 [1alpha,25(OH)2D3], the hormonally active form of vitamin D, increases the phosphorylation and activity of the extracellular signal-regulated mitogen-activated protein (MAP) kinase isoforms ERK1 and ERK2, their subsequent translocation to the nucleus and involvement in DNA synthesis stimulation. In this study, we show that other members of the MAP kinase superfamily are also activated by the hormone. Using the muscle cell line C2C12 we found that 1alpha,25(OH)2D3 within 1 min phosphorylates and increases the activity of p38 MAPK. The immediately upstream mitogen-activated protein kinase kinases 3/6 (MKK3/MKK6) were also phosphorylated by the hormone suggesting their participation in p38 activation. 1Alpha,25(OH)2D3 was able to dephosphorylate/activate the ubiquitous cytosolic tyrosine kinase c-Src in C2C12 cells and studies with specific inhibitors imply that Src participates in hormone induced-p38 activation. Of relevance, 1alpha,25(OH)2D3 induced in the C2C12 line the stimulation of mitogen-activated protein kinase activating protein kinase 2 (MAPKAP-kinase 2) and subsequent phosphorylation of heat shock protein 27 (HSP27) in a p38 kinase activation-dependent manner. Treatment with the p38 inhibitor, SB203580, blocked p38 phosphorylation caused by the hormone and inhibited the phosphorylation of its downstrean substrates. 1Alpha,25(OH)2D3 also promotes the phosphorylation of c-jun N-terminal protein kinases (JNK 1/2), the response is fast (0.5-1 min) and maximal phosphorylation of the enzyme is observed at physiological doses of 1alpha,25(OH)2D3 (1 nM). The relative contribution of ERK-1/2, p38, and JNK-1/2 and their interrelationships in hormonal regulation of muscle cell proliferation and differentiation remain to be established.

  3. Tumor necrosis factor-α alters integrins and metalloprotease ADAM12 levels and signaling in differentiating myoblasts.

    PubMed

    Grzelkowska-Kowalczyk, K; Tokarska, J; Grabiec, K; Gajewska, M; Milewska, M; Błaszczyk, M

    2016-01-01

    The extracellular matrix (ECM) is important in the regulation of myogenesis. We hypothesized that tumor necrosis factor-α (TNF-α) modifies ECM during differentiation of mouse C2C12 myoblasts. Exogenous TNF-α (1 ng/ml) stimulated myoblast fusion on the 3rd day (by 160% vs control) but not on the 5th day of myogenesis. The level of integrin α5 was significantly augmented by TNF-α during 5 day-differentiation; however, integrin β1 was higher than control only on the 3rd day of cytokine treatment. Both the abundance of integrin α5 bound to actin and the level of integrin β1 complexed with integrin α5 increased in the presence of TNF-α, especially on the 3rd day of differentiation. Similarly, the stimulatory effects of TNF-α on integrin α3, metalloprotease ADAM12 and kinases related to integrins, FAK and ILK, were limited to the 3rd day of differentiation. We concluded that TNF-α-induced changes in ECM components in differentiating myogenic cells, i.e. i) increased expression of integrin α5, β1, α3, and metalloprotease ADAM12, ii) enhanced formation of α5β1 integrin receptors and interaction of integrin α5-cytoskeleton, and iii) increased expression of kinases associated with integrin signaling, FAK and ILK, were temporarily associated with the onset of myocyte fusion.

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

    PubMed

    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.

  5. Elastase levels and activity are increased in dystrophic muscle and impair myoblast cell survival, proliferation and differentiation

    PubMed Central

    Arecco, N.; Clarke, C. J.; Jones, F. K.; Simpson, D. M.; Mason, D.; Beynon, R. J.; Pisconti, A.

    2016-01-01

    In Duchenne muscular dystrophy, progressive loss of muscle tissue is accompanied by fibrosis, chronic inflammation and reduced muscle regenerative capacity. Although much is known about the development of fibrosis and chronic inflammation in muscular dystrophy, less is known about how they are mechanistically linked to loss of muscle regenerative capacity. We have developed a proteomics method to discover dystrophy-associated changes in the muscle progenitor cell niche, which identified serine proteases, and especially neutrophil elastase, as candidates. We show that elastase activity is increased in dystrophic (mdx4cv) muscle and impairs myoblast survival in culture. While the effect of elastase on C2C12 cell survival correlates with the kinetics of elastase-mediated degradation of the substrate to which the cells adhere, the effect of elastase on satellite cell-derived primary myoblast growth and differentiation is substrate-independent and even more dramatic than the effect on C2C12 cells, suggesting a detrimental role for elastase on myogenesis in vivo. Additionally, elastase impairs differentiation of both primary and C2C12 myoblasts into myotubes. Our findings evidence the importance of neutrophil-mediated inflammation in muscular dystrophy and indicate elastase-mediated regulation of myoblast behaviour as a potential mechanism underlying loss of regenerative capacity in dystrophic muscle. PMID:27241590

  6. Co-transplantation of plasmid-transfected myoblasts and myotubes into rat brains enables high levels of gene expression long-term

    NASA Technical Reports Server (NTRS)

    Jiao, S.; Williams, P.; Safda, N.; Schultz, E.; Wolff, J. A.

    1993-01-01

    We have previously proposed the use of primary muscle cells as a "platform," or "vehicle" for intracerebral transgene expression. Brain grafts of minced muscle, or cultured muscle cells persisted in rat brains for at least 6 mo without any decrease in graft size, or tumor formation. Stable, but moderate levels of intracerebral transgene expression were obtained by transplanting plasmid-transfected myotubes in culture. In the present study, high and stable levels of intracerebral transgene expression were achieved by the co-transplantation of plasmid-transfected myoblasts and myotubes in culture. Approximately 5 X 10(5) myoblasts and myotubes were transfected with 10 micrograms pRSVL plasmid DNA, and 30 micrograms Lipofectin (BRL), respectively. They were mixed together (total cell number was 1 million), and stereotactically injected into the caudate nucleus of an adult rat brain. The activity of luciferase, the product of transgene expression, was stable for at least 4 mo, and much higher than the levels in myotube grafts, or co-grafts of myoblasts and minced muscle. Presumably, the myotubes served as a framework on which the myoblasts can form myotubes. The sections of brains transplanted with co-graft of myoblasts, and myotubes transfected with pRSVLac-Z were stained immunofluorescently for beta-galactosidase activity. The muscle grafts contained beta-galactosidase positive myofibers 4 mo after transplantation. Such high and stable levels of in vivo expression after postnatal gene transfer have rarely been achieved. Primary muscle cells are useful vehicle for transgene expression in brains, and potentially valuable for gene therapy of degenerative neurological disorders.

  7. 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

    SciTech Connect

    Zuloaga, R.; Fuentes, E.N.; Molina, A.; Valdés, J.A.

    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.

  8. Co-transplantation of plasmid-transfected myoblasts and myotubes into rat brains enables high levels of gene expression long-term

    NASA Technical Reports Server (NTRS)

    Jiao, S.; Williams, P.; Safda, N.; Schultz, E.; Wolff, J. A.

    1993-01-01

    We have previously proposed the use of primary muscle cells as a "platform," or "vehicle" for intracerebral transgene expression. Brain grafts of minced muscle, or cultured muscle cells persisted in rat brains for at least 6 mo without any decrease in graft size, or tumor formation. Stable, but moderate levels of intracerebral transgene expression were obtained by transplanting plasmid-transfected myotubes in culture. In the present study, high and stable levels of intracerebral transgene expression were achieved by the co-transplantation of plasmid-transfected myoblasts and myotubes in culture. Approximately 5 X 10(5) myoblasts and myotubes were transfected with 10 micrograms pRSVL plasmid DNA, and 30 micrograms Lipofectin (BRL), respectively. They were mixed together (total cell number was 1 million), and stereotactically injected into the caudate nucleus of an adult rat brain. The activity of luciferase, the product of transgene expression, was stable for at least 4 mo, and much higher than the levels in myotube grafts, or co-grafts of myoblasts and minced muscle. Presumably, the myotubes served as a framework on which the myoblasts can form myotubes. The sections of brains transplanted with co-graft of myoblasts, and myotubes transfected with pRSVLac-Z were stained immunofluorescently for beta-galactosidase activity. The muscle grafts contained beta-galactosidase positive myofibers 4 mo after transplantation. Such high and stable levels of in vivo expression after postnatal gene transfer have rarely been achieved. Primary muscle cells are useful vehicle for transgene expression in brains, and potentially valuable for gene therapy of degenerative neurological disorders.

  9. The NF-κB-modulated microRNAs miR-195 and miR-497 inhibit myoblast proliferation by targeting Igf1r, Insr and cyclin genes.

    PubMed

    Wei, Wei; Zhang, Wei-Ya; Bai, Jian-Bo; Zhang, Hai-Xin; Zhao, Yuan-Yuan; Li, Xin-Yun; Zhao, Shu-Hong

    2016-01-01

    MicroRNAs (miRNAs) play important roles in the development of skeletal muscle. In our previous study, expression of miR-195 and miR-497 were shown to be upregulated during muscle development in pigs. In this study, we investigated the roles of these two miRNAs in myogenesis and analyzed their transcriptional regulation. Our results showed that miR-195 and miR-497 were upregulated during muscle development and myoblast differentiation. Moreover, miR-195 and miR-497 inhibited proliferation but not differentiation in C2C12 cells. Further investigation revealed that Igf1r, Insr, Ccnd2 and Ccne1 were directly targeted by miR-195 and miR-497 in myoblasts. In addition, we confirmed that miR-195 and miR-497, which shared the similar expression profiling, were negatively regulated by nuclear factor κB (NF-κB) in both myoblasts and skeletal muscle tissue. Our data illustrate that the signaling pathway NF-κB-miR-195/497-Igf1r/Insr-Ccnd2/Ccne1 plays important roles in myogenesis. Our study provides novel evidence for the roles of miR-195 and miR-497 in muscle development.

  10. A gene-trap strategy identifies quiescence-induced genes in synchronized myoblasts.

    PubMed

    Sambasivan, Ramkumar; Pavlath, Grace K; Dhawan, Jyotsna

    2008-03-01

    Cellular quiescence is characterized not only by reduced mitotic and metabolic activity but also by altered gene expression. Growing evidence suggests that quiescence is not merely a basal state but is regulated by active mechanisms. To understand the molecular programme that governs reversible cell cycle exit, we focused on quiescence-related gene expression in a culture model of myogenic cell arrest and activation. Here we report the identification of quiescence-induced genes using a gene-trap strategy. Using a retroviral vector, we generated a library of gene traps in C2C12 myoblasts that were screened for arrest-induced insertions by live cell sorting (FACS-gal). Several independent gene- trap lines revealed arrest-dependent induction of betagal activity, confirming the efficacy of the FACS screen. The locus of integration was identified in 15 lines. In three lines,insertion occurred in genes previously implicated in the control of quiescence, i.e. EMSY - a BRCA2--interacting protein, p8/com1 - a p300HAT -- binding protein and MLL5 - a SET domain protein. Our results demonstrate that expression of chromatin modulatory genes is induced in G0, providing support to the notion that this reversibly arrested state is actively regulated.

  11. Gravitational force modulates G2/M phase exit in mechanically unloaded myoblasts

    PubMed Central

    Benavides Damm, Tatiana; Franco-Obregón, Alfredo; Egli, Marcel

    2013-01-01

    Prolonged spaceflight gives rise to muscle loss and reduced strength, a condition commonly referred to as space atrophy. During exposure to microgravity, skeletal muscle myoblasts are mechanically unloaded and respond with attenuated cell proliferation, slowed cell cycle progression, and modified protein expression. To elucidate the underlying mechanisms by which muscle mass declines in response to prolonged microgravity exposure, we grew C2C12 mouse muscle cells under conditions of simulated microgravity (SM) and analyzed their proliferative capacity, cell cycle progression, and cyclin B and D expression. We demonstrated that the retarded cell growth observed in SM was correlated with an approximate 16 h delay in G2/M phase progression, where cells accumulated specifically between the G2 checkpoint and the onset of anaphase, concomitantly with a positive expression for cyclin B. The effect was specific for gravitational mechanical unloading as cells grown under conditions of hypergravity (HG, 4 g) for similar durations of time exhibited normal proliferation and normal cell cycle progression. Our results show that SM and HG exert phenomenological distinct responses over cell cycle progression. The deficits of SM can be restored by terrestrial gravitational force, whereas the effects of HG are indistinguishable from the 1 g control. This suggests that the mechanotransduction apparatus of cells responds differently to mechanical unloading and loading. PMID:23974110

  12. Signaling Mechanisms in Mammalian Myoblast Fusion

    PubMed Central

    Hindi, Sajedah M.; Tajrishi, Marjan M.; Kumar, Ashok

    2013-01-01

    Myoblast fusion is a critical process that contributes to the growth of muscle during development and to the regeneration of myofibers upon injury. Myoblasts fuse with each other as well as with multinucleated myotubes to enlarge the myofiber. Initial studies demonstrated that myoblast fusion requires extracellular calcium and changes in cell membrane topography and cytoskeletal organization. More recent studies have identified several cell-surface and intracellular proteins that mediate myoblast fusion. Furthermore, emerging evidence suggests that myoblast fusion is also regulated by the activation of specific cell-signaling pathways that lead to the expression of genes whose products are essential for the fusion process and for modulating the activity of molecules that are involved in cytoskeletal rearrangement. Here, we review the roles of the major signaling pathways in mammalian myoblast fusion. PMID:23612709

  13. Signaling mechanisms in mammalian myoblast fusion.

    PubMed

    Hindi, Sajedah M; Tajrishi, Marjan M; Kumar, Ashok

    2013-04-23

    Myoblast fusion is a critical process that contributes to the growth of muscle during development and to the regeneration of myofibers upon injury. Myoblasts fuse with each other as well as with multinucleated myotubes to enlarge the myofiber. Initial studies demonstrated that myoblast fusion requires extracellular calcium and changes in cell membrane topography and cytoskeletal organization. More recent studies have identified several cell-surface and intracellular proteins that mediate myoblast fusion. Furthermore, emerging evidence suggests that myoblast fusion is also regulated by the activation of specific cell-signaling pathways that lead to the expression of genes whose products are essential for the fusion process and for modulating the activity of molecules that are involved in cytoskeletal rearrangement. Here, we review the roles of the major signaling pathways in mammalian myoblast fusion.

  14. Differential thiol oxidation of the signaling proteins Akt, PTEN or PP2A determines whether Akt phosphorylation is enhanced or inhibited by oxidative stress in C2C12 myotubes derived from skeletal muscle.

    PubMed

    Tan, Pearl Lin; Shavlakadze, Tea; Grounds, Miranda D; Arthur, Peter G

    2015-05-01

    Oxidative stress, caused by excess reactive oxygen species (ROS), has been hypothesized to cause or exacerbate skeletal muscle wasting in a number of diseases and chronic conditions. ROS, such as hydrogen peroxide, have the potential to affect signal transduction pathways such as the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3 K)/Akt pathway that regulates protein synthesis. Previous studies have found contradictory outcomes for the effect of ROS on the PI3K/Akt signaling pathway, where oxidative stress can either enhance or inhibit Akt phosphorylation. The apparent contradictions could reflect differences in experimental cell types or types of ROS treatments. We replicate both effects in myotubes of cultured skeletal muscle C2C12 cells, and show that increased oxidative stress can either inhibit or enhance Akt phosphorylation. This differential response could be explained: thiol oxidation of Akt, but not the phosphatases PTEN or PP2A, caused a decline in Akt phosphorylation; whereas the thiol oxidation of Akt, PTEN and PP2A increased Akt phosphorylation. These observations indicate that a more complete understanding of the effects of oxidative stress on a signal transduction pathway comes not only from identifying the proteins susceptible to thiol oxidation, but also their relative sensitivity to ROS.

  15. DNA fragmentation induced by all-trans retinoic acid and its steroidal analogue EA-4 in C2 C12 mouse and HL-60 human leukemic cells in vitro.

    PubMed

    Alakhras, Raghda S; Stephanou, Georgia; Demopoulos, Nikos A; Grintzalis, Konstantinos; Georgiou, Christos D; Nikolaropoulos, Sotirios S

    2014-08-01

    We have recently shown that retinoic acid induces micronucleation mainly via chromosome breakage (Alakhras et al. Cancer Lett 2011; 306: 15-26). To further study retinoic acid clastogenicity and evaluate DNA damaging potential we investigated the ability of (a) all-trans retinoic acid and its steroidal analogue EA-4 to induce DNA fragmentation by using Comet assay under alkaline unwinding and neutral condition electrophoresis, and (b) the retinoids under study to induce small (0-1 kb) DNA fragments. Two cell lines, C2C12 mouse cells and HL-60 human leukemic cells were used in this study. We found that all-trans retinoic acid and its steroidal analogue EA-4 (a) provoke DNA migration due to DNA fragmentation as it is shown by the increased values of Comet parameters, and (b) induce significantly small-size fragmented genomic DNA as indicated by the quantification of necrotic/apoptotic small DNA segments in both cell systems. A different response between the two cell lines was observed in relation to retinoid ability to increase the percentage of DNA in the tail as well as break DNA in to small fragments. Our findings confirm the ability of retinoic acid to provoke micronucleation by disrupting DNA into fragments, among which small pieces of double-stranded DNA up to 1 kb are identified. Copyright © 2013 John Wiley & Sons, Ltd.

  16. Synthesis, biological activity and structure-activity relationships of new benzoic acid-based protein tyrosine phosphatase inhibitors endowed with insulinomimetic effects in mouse C2C12 skeletal muscle cells.

    PubMed

    Ottanà, Rosaria; Maccari, Rosanna; Mortier, Jérémie; Caselli, Anna; Amuso, Simona; Camici, Guido; Rotondo, Archimede; Wolber, Gerhard; Paoli, Paolo

    2014-01-01

    Insulin resistance is a complex altered metabolic condition characterized by impaired insulin signaling and implicated in the pathogenesis of serious human diseases, such as diabetes, obesity, neurodegenerative pathologies. In pursuing our aim to identify new agents able to improve cellular insulin sensitivity, we have synthesized new 4-[(5-arylidene-4-oxo-2-phenylimino/oxothiazolidin-3-yl)methyl]benzoic acids (5, 8) and evaluated their inhibitory activity towards human protein tyrosine phosphatases PTP1B, LMW-PTP and TCPTP, enzymes which are involved in the development of insulin resistance. Compounds 5 and 8 showed from moderate to significant selectivity toward PTP1B over both the highly homologous TCPTP and the two isoforms of human LMW-PTP. In addition, most of the tested compounds selectively inhibited LMW-PTP IF1 over the isoform IF2. Docking studies into the active sites of PTP1B and LMW-PTP aided the rationalization of the observed PTP inhibitory profile. Moreover, most tested compounds were capable to induce the insulin metabolic pathway in mouse C2C12 skeletal muscle cells by remarkably stimulating both IRβ phosphorylation and 2-deoxyglucose cellular uptake. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  17. Peptide separations by on-line MudPIT compared to isoelectric focusing in an off-gel format: application to a membrane-enriched fraction from C2C12 mouse skeletal muscle cells.

    PubMed

    Elschenbroich, Sarah; Ignatchenko, Vladimir; Sharma, Parveen; Schmitt-Ulms, Gerold; Gramolini, Anthony O; Kislinger, Thomas

    2009-10-01

    High-resolution peptide separation is pivotal for successful shotgun proteomics. The need for capable techniques propels invention and improvement of ever more sophisticated approaches. Recently, Agilent Technologies has introduced the OFFGEL fractionator, which conducts peptide separation by isoelectric focusing in an off-gel setup. This platform has been shown to accomplish high resolution of peptides for diverse sample types, yielding valuable advantages over comparable separation techniques. In this study, we deliver the first comparison of the newly emerging OFFGEL approach to the well-established on-line MudPIT platform. Samples from a membrane-enriched fraction isolated from murine C2C12 cells were subjected to replicate analysis by OFFGEL (12 fractions, pH 3-10) followed by RP-LC-MS/MS or 12-step on-line MudPIT. OFFGEL analyses yielded 1398 proteins (identified by 10,269 peptides), while 1428 proteins (11,078 peptides) were detected with the MudPIT approach. Thus, our data shows that both platforms produce highly comparable results in terms of protein/peptide identifications and reproducibility for the sample type analyzed. We achieve more accurate peptide focusing after OFFGEL fractionation with 88% of all peptides binned to a single fraction, as compared to 61% of peptides detected in only one step in MudPIT analyses. Our study suggests that both platforms are equally capable of high quality peptide separation of a sample with medium complexity, rendering them comparably valuable for comprehensive proteomic analyses.

  18. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts

    PubMed Central

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

  19. Overexpression of calpastatin inhibits L8 myoblast fusion

    SciTech Connect

    Barnoy, Sivia; E-mail: sivia@post.tau.ac.il; Maki, Masatoshi; Kosower, Nechama S.

    2005-07-08

    The formation of skeletal muscle fibers involves cessation of myoblast division, myoblast alignment, and fusion to multinucleated myofibers. Calpain is one of the factors shown to be involved in myoblast fusion. Using L8 rat myoblasts, we found that calpain levels did not change significantly during myoblast differentiation, whereas calpastatin diminished prior to myoblast fusion and reappeared after fusion. The transient diminution in calpastatin allows the Ca{sup 2+}-promoted activation of calpain and calpain-induced membrane proteolysis, which is required for myoblast fusion. Here we show that calpastatin overexpression in L8 myoblasts does not inhibit cell proliferation and alignment, but prevents myoblast fusion and fusion-associated protein degradation. In addition, calpastatin appears to modulate myogenic gene expression, as indicated by the lack of myogenin (a transcription factor expressed in differentiating myoblasts) in myoblasts overexpressing calpastatin. These results suggest that, in addition to the role in membrane disorganization in the fusing myoblasts, the calpain-calpastatin system may also modulate the levels of factors required for myoblast differentiation.

  20. Thyroid Hormone Receptor α Plays an Essential Role in Male Skeletal Muscle Myoblast Proliferation, Differentiation, and Response to Injury

    PubMed Central

    Lee, Jang-Won; Kim, Nam-Ho; Liu, Yan-Yun; Yang, An; Sedrakyan, Sargis; Kahng, Andrew; Cervantes, Vanessa; Tripuraneni, Nikita; Cheng, Sheue-yann; Perin, Laura

    2016-01-01

    Thyroid hormone plays an essential role in myogenesis, the process required for skeletal muscle development and repair, although the mechanisms have not been established. Skeletal muscle develops from the fusion of precursor myoblasts into myofibers. We have used the C2C12 skeletal muscle myoblast cell line, primary myoblasts, and mouse models of resistance to thyroid hormone (RTH) α and β, to determine the role of thyroid hormone in the regulation of myoblast differentiation. T3, which activates thyroid hormone receptor (TR) α and β, increased myoblast differentiation whereas GC1, a selective TRβ agonist, was minimally effective. Genetic approaches confirmed that TRα plays an important role in normal myoblast proliferation and differentiation and acts through the Wnt/β-catenin signaling pathway. Myoblasts with TRα knockdown, or derived from RTH-TRα PV (a frame-shift mutation) mice, displayed reduced proliferation and myogenic differentiation. Moreover, skeletal muscle from the TRα1PV mutant mouse had impaired in vivo regeneration after injury. RTH-TRβ PV mutant mouse model skeletal muscle and derived primary myoblasts did not have altered proliferation, myogenic differentiation, or response to injury when compared with control. In conclusion, TRα plays an essential role in myoblast homeostasis and provides a potential therapeutic target to enhance skeletal muscle regeneration. PMID:26451739

  1. Myogenic factors that regulate expression of muscle-specific microRNAs.

    PubMed

    Rao, Prakash K; Kumar, Roshan M; Farkhondeh, Mina; Baskerville, Scott; Lodish, Harvey F

    2006-06-06

    Since their discovery as key regulators of early animal development, microRNAs now are recognized as widespread regulators of gene expression. Despite their abundance, little is known regarding the regulation of microRNA biogenesis. We show that three highly conserved muscle-specific microRNAs, miR-1, miR-133 and miR-206, are robustly induced during the myoblast-myotube transition, both in primary human myoblasts and in the mouse mesenchymal C2C12 stem cell line. These microRNAs were not induced during osteogenic conversion of C2C12 cells. Moreover, both loci encoding miR-1, miR-1-1, and miR-1-2, and two of the three encoding miR-133, miR-133a-1 and miR-133a-2, are strongly induced during myogenesis. Some of the induced microRNAs are in intergenic regions, whereas two are transcribed in the opposite direction to the nonmuscle-specific gene in which they are embedded. By using CHIP analysis, we demonstrate that the myogenic factors Myogenin and MyoD bind to regions upstream of these microRNAs and, therefore, are likely to regulate their expression. Because miR-1 and miR-206 are predicted to repress similar mRNA targets, our work suggests that induction of these microRNAs is important in regulating the expression of muscle-specific proteins.

  2. NDRG2 promotes myoblast proliferation and caspase 3/7 activities during differentiation, and attenuates hydrogen peroxide - But not palmitate-induced toxicity.

    PubMed

    Anderson, Kimberley J; Russell, Aaron P; Foletta, Victoria C

    2015-01-01

    The function of the stress-responsive N-myc downstream-regulated gene 2 (NDRG2) in the control of myoblast growth, and the amino acids contributing to its function, are not well characterized. Here, we investigated the effect of increased NDRG2 levels on the proliferation, differentiation and apoptosis in skeletal muscle cells under basal and stress conditions. NDRG2 overexpression increased C2C12 myoblast proliferation and the expression of positive cell cycle regulators, cdk2, cyclin B and cyclin D, and phosphorylation of Rb, while the serine/threonine-deficient NDRG2, 3A-NDRG2, had less effect. The onset of differentiation was enhanced by NDRG2 as determined through the myogenic regulatory factor expression profiles and myocyte fusion index. However, the overall level of differentiation in myotubes was not different. While NDRG2 up-regulated caspase 3/7 activities during differentiation, no increase in apoptosis was measured by TUNEL assay or through cleavage of caspase 3 and PARP proteins. During H2O2 treatment to induce oxidative stress, NDRG2 helped protect against the loss of proliferation and ER stress as measured by GRP78 expression with 3A-NDRG2 displaying less protection. NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels. In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity. Our findings show that NDRG2 overexpression increases myoblast proliferation and caspase 3/7 activities without increasing overall differentiation. Furthermore, NDRG2 attenuates H2O2-induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells.

  3. Stiffness- and wettability-dependent myoblast cell compatibility of transparent poly(vinyl alcohol) hydrogels.

    PubMed

    Gupta, Siddhi; T, Greeshma; Basu, Bikramjit; Goswami, Sudipta; Sinha, Arvind

    2013-02-01

    This study reports the in vitro compatibility of muscle cells (C2C12 mouse myoblast cell line) with the transparent poly(vinyl alcohol) (PVA) hydrogels and the results are explained on the basis of surface wettability, crystallinity, and nanoscale elastic stiffness property. Nanoindentation was carried out with a maximum load of 100 μN for all the hydrogel compositions and the properties such as elastic stiffness, hardness and total work done during indentation were computed. The difference in cell viability as well as adhesion of cultured myoblast cells on the investigated hydrogel substrates were discussed in reference to the difference in the nanoscale elastic properties, crystallinity, and surface wettability. An important result has been that both elastic stiffness and surface wettability synergistically influence myoblast viability/adhesion on PVA hydrogels. Copyright © 2012 Wiley Periodicals, Inc.

  4. [Differences in the Ca2+ signaling in proliferating and differentiating myoblasts in mice].

    PubMed

    Krasnyĭ, A M; Ozerniuk, N D

    2010-01-01

    Specific features of Ca2+ -signaling in proliferating and differentiated C2C12 myoblasts have been studied. It was shown that the system of Ca2+ -signaling is reduced in proliferating myoblasts: the intracellular ATP-regulated stock is insignificant, the buffer protein is absent or present in minimum quantities in endoplasmic reticulum, and the entry of Ca2+ is not registered when its endocellular stocks are exhausted. The formation of the Ca -signaling system occurs during the initial stages of differentiation (within eight to ten hours after transfer of cell to differentiation medium). During this period, the buffer protein is accumulated, and the entry of Ca begins. During the initial stages of myoblast differentiation, the voltage-dependent entry of Ca2+ also appears. It was also shown that the stock of in mitochondria makes an insignificant contribution to increase in Ca2+ concentration in the cytoplasm.

  5. Transforming Growth Factor-β1 Induces Transdifferentiation of Myoblasts into Myofibroblasts via Up-Regulation of Sphingosine Kinase-1/S1P3 Axis

    PubMed Central

    Cencetti, Francesca; Bernacchioni, Caterina; Nincheri, Paola; Donati, Chiara

    2010-01-01

    The pleiotropic cytokine transforming growth factor (TGF)-β1 is a key player in the onset of skeletal muscle fibrosis, which hampers tissue repair. However, the molecular mechanisms implicated in TGFβ1-dependent transdifferentiation of myoblasts into myofibroblasts are presently unknown. Here, we show that TGFβ1 up-regulates sphingosine kinase (SK)-1 in C2C12 myoblasts in a Smad-dependent manner, and concomitantly modifies the expression of sphingosine 1-phosphate (S1P) receptors (S1PRs). Notably, pharmacological or short interfering RNA-mediated inhibition of SK1 prevented the induction of fibrotic markers by TGFβ1. Moreover, inhibition of S1P3, which became the highest expressed S1PR after TGFβ1 challenge, strongly attenuated the profibrotic response to TGFβ1. Furthermore, downstream of S1P3, Rho/Rho kinase signaling was found critically implicated in the profibrotic action of TGFβ1. Importantly, we demonstrate that SK/S1P axis, known to play a key role in myogenesis via S1P2, consequently to TGFβ1-dependent S1PR pattern remodeling, becomes responsible for transmitting a profibrotic, antidifferentiating action. This study provides new compelling information on the mechanism by which TGFβ1 gives rise to fibrosis in skeletal muscle, opening new perspectives for its pharmacological treatment. Moreover, it highlights the pleiotropic role of SK/S1P axis in skeletal myoblasts that, depending on the expressed S1PR pattern, seems capable of eliciting multiple, even contrasting biological responses. PMID:20089836

  6. Preliminary Quantitative Profile of Differential Expression between Rat L6 Myoblasts and Myotubes by Stable Isotope Labeling by Amino acids in Cell Culture

    PubMed Central

    Cui, Ziyou; Chen, Xiulan; Lu, Bingwen; Park, Sung Kyu; Xu, Tao; Xie, Zhensheng; Xue, Peng; Hou, Junjie; Hang, Haiying; Yates, John R.; Yang, Fuquan

    2010-01-01

    Defining the mechanisms governing myogenesis has advanced in recent years. Skeletal-muscle differentiation is a multi-step process controlled spatially and temporally by various factors at the transcription level. To explore those factors involved in myogenesis, stable isotope labeling with amino acids in cell culture (SILAC), coupled with high accuracy mass spectrometry (LTQ-Orbitrap), was applied successfully. Rat L6 cell line is an excellent model system for studying muslce myogenesis in vitro. When mononucleate L6 myoblast cells reach confluent in culture plate, they could transform into multinucleate myotubes by serum starvation. By comparing protein expression of L6 myoblasts and terminally differentiated multinucleated myotubes, 1170 proteins were quantified and 379 proteins changed significantly in fully differentiated myotubes in contrast to myoblasts. These differentially expressed proteins are mainly involved in inter-or intracellular signaling, protein synthesis and degradation, protein folding, cell adhesion and extracelluar matrix, cell structure and motility, metabolism, substance transportation, etc. These findings were supported by many previous studies on myogenic differentiation, of which many up-regulated proteins were found to be involved in promoting skeletal muscle differentiation for the first time in our study. In sum, our results provide new clues for understanding the mechanism of myogenesis. PMID:19253283

  7. Intercellular Adhesion Molecule-1 Expression by Skeletal Muscle Cells Augments Myogenesis

    PubMed Central

    Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T.; Pierre, Philippe; Chadee, Deborah N.; Pizza, Francis X.

    2014-01-01

    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. PMID:25281303

  8. Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis.

    PubMed

    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.

  9. IGF-1 induces IP3 -dependent calcium signal involved in the regulation of myostatin gene expression mediated by NFAT during myoblast differentiation.

    PubMed

    Valdés, Juan A; Flores, Sylvia; Fuentes, Eduardo N; Osorio-Fuentealba, Cesar; Jaimovich, Enrique; Molina, Alfredo

    2013-07-01

    Skeletal muscle differentiation is a complex and highly regulated process characterized by cell cycle arrest, which is associated with morphological changes including myoblast alignment, elongation, and fusion into multinucleated myotubes. This is a balanced process dynamically coordinated by positive and negative signals such as the insulin-like growth factor I (IGF-1) and myostatin (MSTN), respectively. In this study, we report that the stimulation of skeletal myoblasts during differentiation with IGF-1 induces a rapid and transient calcium increase from intracellular stores, which are principally mediated through the phospholipase C gamma (PLC γ)/inositol 1,4,5-triphosphate (IP3 )-dependent signaling pathways. This response was completely blocked when myoblasts were incubated with LY294002 or transfected with the dominant-negative p110 gamma, suggesting a fundamental role of phosphatidylinositol 3-kinase (PI3K) in PLCγ activation. Additionally, we show that calcium released via IP3 and induced by IGF-1 stimulates NFAT-dependent gene transcription and nuclear translocation of the GFP-labeled NFATc3 isoform. This activation was independent of extracellular calcium influx and calcium release mediated by ryanodine receptor (RyR). Finally, we examined mstn mRNA levels and mstn promoter activity in myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents and in reporter activity, which was inhibited by cyclosporin A, 11R-VIVIT, and by inhibitors of the PI3Kγ, PLCγ, and IP3 receptor. Our results strongly suggest that IGF-1 regulates myostatin transcription through the activation of the NFAT transcription factor in an IP3 /calcium-dependent manner. This is the first study to demonstrate a role of calcium-dependent signaling pathways in the mRNA expression of myostatin.

  10. Transmembrane proteoglycans syndecan-2, 4, receptor candidates for the impact of HGF and FGF2 on semaphorin 3A expression in early-differentiated myoblasts

    PubMed Central

    Do, Mai-Khoi Q; Shimizu, Naomi; Suzuki, Takahiro; Ohtsubo, Hideaki; Mizunoya, Wataru; Nakamura, Mako; Sawano, Shoko; Furuse, Mitsuhiro; Ikeuchi, Yoshihide; Anderson, Judy E; Tatsumi, Ryuichi

    2015-01-01

    Regenerative mechanisms that regulate intramuscular motor innervation are thought to reside in the spatiotemporal expression of axon-guidance molecules. Our previous studies proposed an unexplored role of resident myogenic stem cell (satellite cell)-derived myoblasts as a key presenter of a secreted neural chemorepellent semaphorin 3A (Sema3A); hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2) triggered its expression exclusively at the early differentiation phase. In order to advance this concept, the present study described that transmembrane heparan/chondroitin sulfate proteoglycans syndecan-2, 4 may be the plausible receptor candidates for HGF and FGF2 to signal Sema3A expression. Results showed that mRNA expression of syndecan-2, 4 was abundant (two magnitudes higher than syndecan-1, 3) in early-differentiated myoblasts and their in vitro knockdown diminished the HGF/FGF2-induced expression of Sema3A down to a baseline level. Pretreatment with heparitinase and chondroitinase ABC decreased the HGF and FGF2 responses, respectively, in non–knockdown cultures, supporting a possible model that HGF and FGF2 may bind to heparan and chondroitin sulfate chains of syndecan-2, 4 to signal Sema3A expression. The findings, therefore, extend our understanding that HGF/FGF2-syndecan-2, 4 association may stimulate a burst of Sema3A secretion by myoblasts recruited to the site of muscle injury; this would ensure a coordinated delay in the attachment of motoneuron terminals onto fibers early in muscle regeneration, and thus synchronize the recovery of muscle fiber integrity and the early resolution of inflammation after injury with reinnervation toward functional recovery. PMID:26381016

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

    PubMed Central

    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. Increasing α7β1-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis without changing gene expression

    PubMed Central

    Liu, Jianming; Burkin, Dean J.; Kaufman, Stephen J.

    2008-01-01

    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 α7β1-integrin also binds laminin and links the extracellular matrix with the cytoskeleton. Enhancement of α7-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 α7-integrin levels affects transcription and cellular functions, we generated α7-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 α7-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. PMID:18045857

  13. Comparative Proteomic Study of Fatty Acid-treated Myoblasts Reveals Role of Cox-2 in Palmitate-induced Insulin Resistance.

    PubMed

    Chen, Xiulan; Xu, Shimeng; Wei, Shasha; Deng, Yaqin; Li, Yiran; Yang, Fuquan; Liu, Pingsheng

    2016-02-22

    Accumulated studies demonstrate that saturated fatty acids (FAs) such as palmitic acid (PA) inhibit insulin signaling in skeletal muscle cells and monounsaturated fatty acids such as oleic acid (OA) reverse the effect of PA on insulin signaling. The detailed molecular mechanism of these opposite effects remains elusive. Here we provide a comparative proteomic study of skeletal myoblast cell line C2C12 that were untreated or treated with PA, and PA plus OA. A total of 3437 proteins were quantified using SILAC in this study and 29 proteins fall into the pattern that OA reverses PA effect. Expression of some these proteins were verified using qRT-PCR and Western blot. The most significant change was cyclooxygenase-2 (Cox-2). In addition to whole cell comparative proteomic study, we also compared lipid droplet (LD)-associated proteins and identified that Cox-2 was one of three major altered proteins under the FA treatment. This finding was then confirmed using immunofluorescence. Finally, Cox-2 selective inhibitor, celecoxib protected cells from PA-reduced insulin signaling Akt phosphorylation. Together, these results not only provide a dataset of protein expression change in FA treatment but also suggest that Cox-2 and lipid droplets (LDs) are potential players in PA- and OA-mediated cellular processes.

  14. Electrically conductive graphene/polyacrylamide hydrogels produced by mild chemical reduction for enhanced myoblast growth and differentiation.

    PubMed

    Jo, Hyerim; Sim, Myeongbu; Kim, Semin; Yang, Sumi; Yoo, Youngjae; Park, Jin-Ho; Yoon, Tae Ho; Kim, Min-Gon; Lee, Jae Young

    2017-01-15

    Graphene and graphene derivatives, such as graphene oxide (GO) and reduced GO (rGO), have been extensively employed as novel components of biomaterials because of their unique electrical and mechanical properties. These materials have also been used to fabricate electrically conductive biomaterials that can effectively deliver electrical signals to biological systems. Recently, increasing attention has been paid to electrically conductive hydrogels that have both electrical activity and a tissue-like softness. In this study, we synthesized conductive graphene hydrogels by mild chemical reduction of graphene oxide/polyacrylamide (GO/PAAm) composite hydrogels to obtain conductive hydrogels. The reduced hydrogel, r(GO/PAAm), exhibited muscle tissue-like stiffness with a Young's modulus of approximately 50kPa. The electrochemical impedance of r(GO/PAAm) could be decreased by more than ten times compared to that of PAAm and unreduced GO/PAAm. In vitro studies with C2C12 myoblasts revealed that r(GO/PAAm) significantly enhanced proliferation and myogenic differentiation compared with unreduced GO/PAAm and PAAm. Moreover, electrical stimulation of myoblasts growing on r(GO/PAAm) graphene hydrogels for 7days significantly enhanced the myogenic gene expression compared to unstimulated controls. As results, our graphene-based conductive and soft hydrogels will be useful as skeletal muscle tissue scaffolds and can serve as a multifunctional platform that can simultaneously deliver electrical and mechanical cues to biological systems. Graphene-based conductive hydrogels presenting electrical conductance and a soft tissue-like modulus were successfully fabricated via mild reduction of graphene oxide/polyacrylamide composite hydrogels to study their potential to skeletal tissue scaffold applications. Significantly promoted myoblast proliferation and differentiation were obtained on our hydrogels. Additionally, electrical stimulation of myoblasts via the graphene hydrogels could

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

    PubMed Central

    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

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

    PubMed Central

    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. PMID:26030912

  17. MicroRNAs regulate the expression of the alternative splicing factor nPTB during muscle development

    PubMed Central

    Boutz, Paul L.; Chawla, Geetanjali; Stoilov, Peter; Black, Douglas L.

    2007-01-01

    Alternative pre-mRNA splicing determines many changes in gene expression during development. Two regulators known to control splicing patterns during neuron and muscle differentiation are the polypyrimidine tract-binding protein (PTB) and its neuronal homolog nPTB. These proteins repress certain exons in early myoblasts, but upon differentiation of mature myotubes PTB/nPTB expression is reduced, leading to increased inclusion of their target exons. We show here that the repression of nPTB expression during myoblast differentiation results from its targeting by the muscle-restricted microRNA miR-133. During differentiation of C2C12 myoblasts, nPTB protein but not mRNA expression is strongly reduced, concurrent with the up-regulation of miR-133 and the induction of splicing for several PTB-repressed exons. Introduction of synthetic miR-133 into undifferentiated C2C12 cells leads to a decrease in endogenous nPTB expression. Both the miR-133 and the coexpressed miR-1/206 microRNAs are extremely conserved across animal species, and PTB proteins are predicted targets for these miRNAs in Drosophila, mice, and humans. There are two potential miR-133-responsive elements (MRE) within the nPTB 3′ untranslated region (UTR), and a luciferase reporter carrying this 3′ UTR is repressed by miR-133 in an MRE-dependent manner. Transfection of locked nucleic acid (LNA) oligonucleotides designed to block the function of miR-133 and miR-1/206 increases expression of nPTB and decreases the inclusion of PTB dependent exons. These results indicate that miR-133 directly down-regulates a key splicing factor during muscle development and establishes a role for microRNAs in the control of a developmentally dynamic splicing program. PMID:17210790

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

    PubMed

    Muratore, Massimo; Mitchell, Steve; Waterfall, Martin

    2013-09-06

    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.

  19. VITO-1, a novel vestigial related protein is predominantly expressed in the skeletal muscle lineage.

    PubMed

    Mielcarek, Michal; Günther, Stefan; Krüger, Marcus; Braun, Thomas

    2002-12-01

    In order to identify novel genes expressed in skeletal muscle we performed a subtractive hybridization for genes expressed in human skeletal muscle but not in other tissues. We identified a novel scalloped interaction domain (SID) containing protein in humans and in the mouse, which we named VITO-1. Highest homology of VITO-1 was found with the Drosophila vestigial and the human TONDU proteins in the SID (54 and 40%, respectively). Using whole-mount hybridzation and Northern blot analysis, we showed that VITO-1 is expressed in the somitic myotome from E8.75 mouse embryos onwards and later on in skeletal muscle but not in the heart. Additional expression domains during development were detected in the pharyngeal pouches and clefts starting at E8.0 as well as in the cranial pharynx and in Rathkes pouch. By Northern blot analysis, we found VITO-1 to be up-regulated in C2C12 myotubes although some expression can be detected in proliferating C2C12 myoblasts. No expression was spotted in other adult mouse tissues. Likewise, expression of human Vito-1 during fetal and adult human development was found exclusively in skeletal muscle preferentially in fast skeletal muscles. These data suggest a role of VITO-1 for the development of skeletal muscles and of pharyngeal clefts/Rathkes' pouch derived structures.

  20. Skeletal Muscle Cells Express ICAM-1 after Muscle Overload and ICAM-1 Contributes to the Ensuing Hypertrophic Response

    PubMed Central

    Dearth, Christopher L.; Goh, Qingnian; Marino, Joseph S.; Cicinelli, Peter A.; Torres-Palsa, Maria J.; Pierre, Philippe; Worth, Randall G.; Pizza, Francis X.

    2013-01-01

    We previously reported that leukocyte specific β2 integrins contribute to hypertrophy after muscle overload in mice. Because intercellular adhesion molecule-1 (ICAM-1) is an important ligand for β2 integrins, we examined ICAM-1 expression by murine skeletal muscle cells after muscle overload and its contribution to the ensuing hypertrophic response. Myofibers in control muscles of wild type mice and cultures of skeletal muscle cells (primary and C2C12) did not express ICAM-1. Overload of wild type plantaris muscles caused myofibers and satellite cells/myoblasts to express ICAM-1. Increased expression of ICAM-1 after muscle overload occurred via a β2 integrin independent mechanism as indicated by similar gene and protein expression of ICAM-1 between wild type and β2 integrin deficient (CD18-/-) mice. ICAM-1 contributed to muscle hypertrophy as demonstrated by greater (p<0.05) overload-induced elevations in muscle protein synthesis, mass, total protein, and myofiber size in wild type compared to ICAM-1-/- mice. Furthermore, expression of ICAM-1 altered (p<0.05) the temporal pattern of Pax7 expression, a marker of satellite cells/myoblasts, and regenerating myofiber formation in overloaded muscles. In conclusion, ICAM-1 expression by myofibers and satellite cells/myoblasts after muscle overload could serve as a mechanism by which ICAM-1 promotes hypertrophy by providing a means for cell-to-cell communication with β2 integrin expressing myeloid cells. PMID:23505517

  1. Skeletal muscle cells express ICAM-1 after muscle overload and ICAM-1 contributes to the ensuing hypertrophic response.

    PubMed

    Dearth, Christopher L; Goh, Qingnian; Marino, Joseph S; Cicinelli, Peter A; Torres-Palsa, Maria J; Pierre, Philippe; Worth, Randall G; Pizza, Francis X

    2013-01-01

    We previously reported that leukocyte specific β2 integrins contribute to hypertrophy after muscle overload in mice. Because intercellular adhesion molecule-1 (ICAM-1) is an important ligand for β2 integrins, we examined ICAM-1 expression by murine skeletal muscle cells after muscle overload and its contribution to the ensuing hypertrophic response. Myofibers in control muscles of wild type mice and cultures of skeletal muscle cells (primary and C2C12) did not express ICAM-1. Overload of wild type plantaris muscles caused myofibers and satellite cells/myoblasts to express ICAM-1. Increased expression of ICAM-1 after muscle overload occurred via a β2 integrin independent mechanism as indicated by similar gene and protein expression of ICAM-1 between wild type and β2 integrin deficient (CD18-/-) mice. ICAM-1 contributed to muscle hypertrophy as demonstrated by greater (p<0.05) overload-induced elevations in muscle protein synthesis, mass, total protein, and myofiber size in wild type compared to ICAM-1-/- mice. Furthermore, expression of ICAM-1 altered (p<0.05) the temporal pattern of Pax7 expression, a marker of satellite cells/myoblasts, and regenerating myofiber formation in overloaded muscles. In conclusion, ICAM-1 expression by myofibers and satellite cells/myoblasts after muscle overload could serve as a mechanism by which ICAM-1 promotes hypertrophy by providing a means for cell-to-cell communication with β2 integrin expressing myeloid cells.

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

    SciTech Connect

    Muratore, Massimo; Mitchell, Steve; Waterfall, Martin

    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.

  3. Skeletal muscle PI3K p110β regulates expression of AMP-activated protein kinase.

    PubMed

    Matheny, Ronald W; Abdalla, Mary N; Geddis, Alyssa V; Leandry, Luis A; Lynch, Christine M

    2017-01-22

    Skeletal muscle metabolic homeostasis is maintained through numerous biochemical and physiological processes. Two principal molecular regulators of skeletal muscle metabolism include AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3K); however, PI3K exists as multiple isoforms, and specific metabolic actions of each isoform have not yet been fully elucidated in skeletal muscle. Given this lack of knowledge, we performed a series of experiments to define the extent to which PI3K p110β mediated expression and (or) activation of AMPK in skeletal muscle. To determine the effect of p110β inhibition on AMPK expression and phosphorylation in cultured cells, C2C12 myoblasts were treated with a pharmacological inhibitor of p110β (TGX-221), siRNA against p110β, or overexpression of kinase-dead p110β. Expression and phosphorylation of AMPK were unaffected in myoblasts treated with TGX-221 or expressing kinase-dead p110β. Howev