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Sample records for c2c12 skeletal muscle

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

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    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.

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

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    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.

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

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    Jung Hun Ohn

    2013-06-01

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

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

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    Cui Hao

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

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

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    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.

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

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

    2015-01-01

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

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

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    Jasmer, Douglas P; Kwak, Dongmi

    2006-02-01

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

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

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

    2017-01-01

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

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

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    Pronsato, Lucía; Boland, Ricardo; Milanesi, Lorena

    2012-03-01

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

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

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

    2015-01-01

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

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

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

    2017-02-01

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

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

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

    2012-12-01

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

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

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

    2017-01-01

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

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

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

    2014-01-01

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

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

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    Zhenguo Dong

    2015-01-01

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

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

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

    2017-04-01

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

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

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

    2018-01-24

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

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

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    Yasunari Matsuzaka

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

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

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

    2017-10-01

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

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

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

    2017-06-01

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

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

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

    2017-01-01

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

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

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    Paola Pelegrineli Artilheiro

    2010-06-01

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

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

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    Jun Myoung Park

    2015-03-01

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

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

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

    2008-01-01

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

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

    Science.gov (United States)

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

    2008-10-01

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

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

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

    2016-11-01

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

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

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    Roger D. Madison

    2014-02-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

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

    2016-07-15

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

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

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

    2014-09-26

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

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

    Science.gov (United States)

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

    2014-05-01

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

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

    Science.gov (United States)

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

    2017-05-04

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

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

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

    2011-03-25

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

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

    Science.gov (United States)

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

    2017-11-01

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

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

    Science.gov (United States)

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

    2010-03-01

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Qiangling Zhang

    2015-08-01

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

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

    Science.gov (United States)

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

    2017-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Masaki Horie

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

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

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-04-12

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

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

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

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Science.gov (United States)

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

    2017-04-01

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

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

    Science.gov (United States)

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

    2013-06-07

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

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

    Directory of Open Access Journals (Sweden)

    James M Dugan

    2014-03-01

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

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

    Science.gov (United States)

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

    2017-08-22

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

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

    Science.gov (United States)

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

    2014-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Felicia Carotenuto

    2016-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Jeffrey eKim

    2014-03-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Kohei Washio

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Long Jia

    2013-12-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-03-22

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

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

    Science.gov (United States)

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

    2009-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-04-29

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

  2. Glucose deprivation attenuates sortilin levels in skeletal muscle cells.

    Science.gov (United States)

    Ariga, Miyako; Yoneyama, Yosuke; Fukushima, Toshiaki; Ishiuchi, Yuri; Ishii, Takayuki; Sato, Hitoshi; Hakuno, Fumihiko; Nedachi, Taku; Takahashi, Shin-Ichiro

    2017-03-31

    In skeletal muscle, sortilin plays a predominant role in the sorting of glucose transporter 4 (Glut4), thereby controlling glucose uptake. Moreover, our previous study suggested that the sortilin expression levels are also implicated in myogenesis. Despite the importance of sortilin in skeletal muscle, however, the regulation of sortilin expression has not been completely understood. In the present study, we analyzed if the sortilin expression is regulated by glucose in C2C12 myocytes and rat skeletal muscles in vivo. Sortilin protein expression was elevated upon C2C12 cell differentiation and was further enhanced in the presence of a high concentration of glucose. The gene expression and protein degradation of sortilin were not affected by glucose. On the other hand, rapamycin partially reduced sortilin induction by a high concentration of glucose, which suggested that sortilin translation could be regulated by glucose, at least in part. We also examined if the sortilin regulation by glucose was also observed in skeletal muscles that were obtained from fed or fasted rats. Sortilin expression in both gastrocnemius and extensor digitorum longus (EDL) muscle was significantly decreased by 17-18h of starvation. On the other hand, pathological levels of high blood glucose did not alter the sortilin expression in rat skeletal muscle. Overall, the present study suggests that sortilin protein levels are reduced under hypoglycemic conditions by post-transcriptional control in skeletal muscles.

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

    Directory of Open Access Journals (Sweden)

    Caterina Morabito

    2017-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ji Sook Kang

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

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

    Science.gov (United States)

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

    2013-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Christo J. Botha

    2017-01-01

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

  8. Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways and transcription factors

    DEFF Research Database (Denmark)

    Deshmukh, Atul S; Murgia, Marta; Nagaraja, Nagarjuna

    2015-01-01

    spectrometric (MS) workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins...

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

    Science.gov (United States)

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

    2008-11-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-01-17

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

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

    Science.gov (United States)

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

    2009-03-01

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

  12. Tissue engineering skeletal muscle for orthopaedic applications

    Science.gov (United States)

    Payumo, Francis C.; Kim, Hyun D.; Sherling, Michael A.; Smith, Lee P.; Powell, Courtney; Wang, Xiao; Keeping, Hugh S.; Valentini, Robert F.; Vandenburgh, Herman H.

    2002-01-01

    With current technology, tissue-engineered skeletal muscle analogues (bioartificial muscles) generate too little active force to be clinically useful in orthopaedic applications. They have been engineered genetically with numerous transgenes (growth hormone, insulinlike growth factor-1, erythropoietin, vascular endothelial growth factor), and have been shown to deliver these therapeutic proteins either locally or systemically for months in vivo. Bone morphogenetic proteins belonging to the transforming growth factor-beta superfamily are osteoinductive molecules that drive the differentiation pathway of mesenchymal cells toward the chondroblastic or osteoblastic lineage, and stimulate bone formation in vivo. To determine whether skeletal muscle cells endogenously expressing bone morphogenetic proteins might serve as a vehicle for systemic bone morphogenetic protein delivery in vivo, proliferating skeletal myoblasts (C2C12) were transduced with a replication defective retrovirus containing the gene for recombinant human bone morphogenetic protein-6 (C2BMP-6). The C2BMP-6 cells constitutively expressed recombinant human bone morphogenetic protein-6 and synthesized bioactive recombinant human bone morphogenetic protein-6, based on increased alkaline phosphatase activity in coincubated mesenchymal cells. C2BMP-6 cells did not secrete soluble, bioactive recombinant human bone morphogenetic protein-6, but retained the bioactivity in the cell layer. Therefore, genetically-engineered skeletal muscle cells might serve as a platform for long-term delivery of osteoinductive bone morphogenetic proteins locally.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-03-25

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

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

    Science.gov (United States)

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

    2009-11-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-07-01

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

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

    Science.gov (United States)

    Pronsato, Lucía; Milanesi, Lorena

    2016-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Dupuy Fabrice

    2009-10-01

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

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

    Science.gov (United States)

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

    2009-06-01

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

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

    Science.gov (United States)

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

    2015-07-01

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

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

    Science.gov (United States)

    Yang, Chuanbin; Li, Xuechen; Rong, Jianhui

    2014-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-08-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-15

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

  3. Proteomics of Skeletal Muscle

    DEFF Research Database (Denmark)

    Deshmukh, Atul S

    2016-01-01

    Skeletal muscle is the largest tissue in the human body and plays an important role in locomotion and whole body metabolism. It accounts for ~80% of insulin stimulated glucose disposal. Skeletal muscle insulin resistance, a primary feature of Type 2 diabetes, is caused by a decreased ability...... of muscle to respond to circulating insulin. Physical exercise improves insulin sensitivity and whole body metabolism and remains one of the most promising interventions for the prevention of Type 2 diabetes. Insulin resistance and exercise adaptations in skeletal muscle might be a cause, or consequence......, of altered protein expressions profiles and/or their posttranslational modifications (PTMs). Mass spectrometry (MS)-based proteomics offer enormous promise for investigating the molecular mechanisms underlying skeletal muscle insulin resistance and exercise-induced adaptation; however, skeletal muscle...

  4. Peripheral endocannabinoids regulate skeletal muscle development and maintenance

    Directory of Open Access Journals (Sweden)

    Dongjiao Zhao

    2010-12-01

    Full Text Available As a principal tissue responsible for insulin-mediated glucose uptake, skeletal muscle is important for whole-body health. The role of peripheral endocannabinoids as regulators of skeletal muscle metabolism has recently gained a lot of interest, as endocannabinoid system disorders could cause peripheral insulin resistance. We investigated the role of the peripheral endocannabinoid system in skeletal muscle development and maintenance. Cultures of C2C12 cells, primary satellite cells and mouse skeletal muscle single fibers were used as model systems for our studies. We found an increase in cannabinoid receptor type 1 (CB1 mRNA and endocannabinoid synthetic enzyme mRNA skeletal muscle cells during differentiation. We also found that activation of CB1 inhibited myoblast differentiation, expanded the number of satellite cells, and stimulated the fast-muscle oxidative phenotype. Our findings contribute to understanding of the role of the endocannabinoid system in skeletal muscle metabolism and muscle oxygen consumption, and also help to explain the effects of the peripheral endocannabinoid system on whole-body energy balance.

  5. Astragalus Polysaccharide Suppresses Skeletal Muscle Myostatin Expression in Diabetes: Involvement of ROS-ERK and NF-κB Pathways

    Directory of Open Access Journals (Sweden)

    Min Liu

    2013-01-01

    Full Text Available Objective. The antidiabetes drug astragalus polysaccharide (APS is capable of increasing insulin sensitivity in skeletal muscle and improving whole-body glucose homeostasis. Recent studies suggest that skeletal muscle secreted growth factor myostatin plays an important role in regulating insulin signaling and insulin resistance. We hypothesized that regulation of skeletal muscle myostatin expression may be involved in the improvement of insulin sensitivity by APS. Methods. APS was administered to 13-week-old diabetic KKAy and nondiabetic C57BL/6J mice for 8 weeks. Complementary studies examined APS effects on the saturated acid palmitate-induced insulin resistance and myostatin expression in C2C12 cells. Results. APS treatment ameliorated hyperglycemia, hyperlipidemia, and insulin resistance and decreased the elevation of myostatin expression and malondialdehyde production in skeletal muscle of noninsulin-dependent diabetic KKAy mice. In C2C12 cells in vitro, saturated acid palmitate-induced impaired glucose uptake, overproduction of ROS, activation of extracellular regulated protein kinases (ERK, and NF-κB were partially restored by APS treatment. The protective effects of APS were mimicked by ERK and NF-κB inhibitors, respectively. Conclusion. Our study demonstrates elevated myostatin expression in skeletal muscle of type 2 diabetic KKAy mice and in cultured C2C12 cells exposed to palmitate. APS is capable of improving insulin sensitivity and decreasing myostatin expression in skeletal muscle through downregulating ROS-ERK-NF-κB pathway.

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

    Directory of Open Access Journals (Sweden)

    Ya-Ju eChang

    2016-03-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-11-27

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

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

    Directory of Open Access Journals (Sweden)

    Hyunju Kim

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

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

    Science.gov (United States)

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

    2016-01-01

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

  10. Skeletal muscle tissue engineering

    National Research Council Canada - National Science Library

    Bach, A. D; Beier, J. P; Stern‐Staeter, J; Horch, R. E

    2004-01-01

    The reconstruction of skeletal muscle tissue either lost by traumatic injury or tumor ablation or functional damage due to myopathies is hampered by the lack of availability of functional substitution...

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

    Science.gov (United States)

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

    2013-10-01

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

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

    OpenAIRE

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

    2009-01-01

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

  13. CLP-1 associates with MyoD and HDAC to restore skeletal muscle cell regeneration.

    Science.gov (United States)

    Galatioto, Josephine; Mascareno, Eduardo; Siddiqui, M A Q

    2010-11-01

    Emerging evidence suggests that eukaryotic gene transcription is regulated primarily at the elongation stage by association and dissociation of the inhibitory protein cardiac lineage protein 1 (CLP-1/HEXIM1) from the positive transcription elongation factor b (P-TEFb) complex. It was reported recently that P-TEFb interacts with skeletal muscle-specific regulatory factor, MyoD, suggesting a linkage between CLP-1-mediated control of transcription and skeletal myogenesis. To examine this, we produced CLP-1 knockdown skeletal muscle C2C12 cells by homologous recombination, and demonstrated that the C2C12 CLP-1 +/- cells failed to differentiate when challenged by low serum in the medium. We also showed that CLP-1 interacts with both MyoD and histone deacetylases (HDACs) maximally at the early stage of differentiation of C2C12 cells. This led us to hypothesize that the association might be crucial to inhibition of MyoD-target proliferative genes. Chromatin immunoprecipitation analysis revealed that the CLP-1/MyoD/HDAC complex binds to the promoter of the cyclin D1 gene, which is downregulated in differentiated muscle cells. These findings suggest a novel transcriptional paradigm whereby CLP-1, in conjunction with MyoD and HDAC, acts to inhibit growth-related gene expression, a requirement for myoblasts to exit the cell cycle and transit to myotubes.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    S. E. Hurst

    2012-01-01

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

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

    Science.gov (United States)

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

    2013-12-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-07-17

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

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  19. in Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Espen E. Spangenburg

    2011-01-01

    Full Text Available Triglyceride storage is altered across various chronic health conditions necessitating various techniques to visualize and quantify lipid droplets (LDs. Here, we describe the utilization of the BODIPY (493/503 dye in skeletal muscle as a means to analyze LDs. We found that the dye was a convenient and simple approach to visualize LDs in both sectioned skeletal muscle and cultured adult single fibers. Furthermore, the dye was effective in both fixed and nonfixed cells, and the staining seemed unaffected by permeabilization. We believe that the use of the BODIPY (493/503 dye is an acceptable alternative and, under certain conditions, a simpler method for visualizing LDs stored within skeletal muscle.

  20. Noncontrast skeletal muscle oximetry.

    Science.gov (United States)

    Zheng, Jie; An, Hongyu; Coggan, Andrew R; Zhang, Xiaodong; Bashir, Adil; Muccigrosso, David; Peterson, Linda R; Gropler, Robert J

    2014-01-01

    The objective of this study was to develop a new noncontrast method to directly quantify regional skeletal muscle oxygenation. The feasibility of the method was examined in five healthy volunteers using a 3 T clinical MRI scanner, at rest and during a sustained isometric contraction. The perfusion of skeletal muscle of the calf was measured using an arterial spin labeling method, whereas the oxygen extraction fraction of the muscle was measured using a susceptibility-based MRI technique. In all volunteers, the perfusion in soleus muscle increased significantly from 6.5 ± 2.0 mL (100 g min)(-1) at rest to 47.9 ± 7.7 mL (100 g min)(-1) during exercise (P oxygen extraction fraction did not change significantly, the rate of oxygen consumption increased from 0.43 ± 0.13 to 4.2 ± 1.5 mL (100 g min)(-1) (P muscle but with greater oxygen extraction fraction increase than the soleus muscle. This is the first MR oximetry developed for quantification of regional skeletal muscle oxygenation. A broad range of medical conditions could benefit from these techniques, including cardiology, gerontology, kinesiology, and physical therapy. Copyright © 2013 Wiley Periodicals, Inc.

  1. Potential Therapeutic Role of L-Carnitine in Skeletal Muscle Oxidative Stress and Atrophy Conditions

    Directory of Open Access Journals (Sweden)

    Anna Montesano

    2015-01-01

    Full Text Available The targeting of nutraceutical treatment to skeletal muscle damage is an emerging area of research, driven by the need for new therapies for a range of muscle-associated diseases. L-Carnitine (CARN is an essential nutrient and plays a key role in mitochondrial β-oxidation and in the ubiquitin-proteasome system regulation. As a dietary supplement to improve athletic performance, CARN has been studied for its potential to enhance β-oxidation. However, CARN effects on myogenesis, mitochondrial activity, and hypertrophy process are not completely elucidated. This in vitro study aims to investigate CARN role on skeletal muscle remodeling, differentiation process, and myotubes formation. We analyzed muscle differentiation and morphological features in C2C12 myoblasts exposed to 5 mM CARN. Our results showed that CARN was able to accelerate C2C12 myotubes formation and induce morphological changes, characterizing the start of hypertrophy process. In addition, CARN improved AKT activation and downstream cellular signaling pathways involved in skeletal muscle atrophy process prevention. Also, CARN positively regulated the pathways involved in oxidative stress defense. In this work, we provide an interesting novel mechanism of the potential therapeutic use of CARN to treat pathological conditions characterized by skeletal muscle morphological and functional impairment, oxidative stress production, and atrophy process in aging.

  2. Chiral Orientation of Skeletal Muscle Cells Requires Rigid Substrate

    Directory of Open Access Journals (Sweden)

    Ninghao Zhu

    2017-06-01

    Full Text Available Reconstitution of tissue morphology with inherent left–right (LR asymmetry is essential for tissue/organ functions. For skeletal muscle, the largest tissue in mammalian organisms, successful myogenesis requires the regulation of the LR asymmetry to form the appropriate muscle alignment. However, the key factor for reproducing the LR asymmetry of skeletal tissues in a controllable, engineering context remains largely unknown. Recent reports indicate that cell chirality may underlie the LR development in tissue morphogenesis. Here, we report that a rigid substrate is required for the chirality of skeletal muscle cells. By using alternating micropatterned cell-adherent and cell-repellent stripes on a rigid substrate, we found that C2C12 skeletal muscle myoblasts exhibited a unidirectional tilted orientation with respect to the stripe boundary. Importantly, such chiral orientation was reduced when soft substrates were used instead. In addition, we demonstrated the key role of actin stress fibers in the formation of the chiral orientation. This study reveals that a rigid substrate is required for the chiral pattern of myoblasts, paving the way for reconstructing damaged muscle tissue with inherent LR asymmetry in the future.

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

    Science.gov (United States)

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

    2015-01-01

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

  4. Extra-nuclear telomerase reverse transcriptase (TERT) regulates glucose transport in skeletal muscle cells.

    Science.gov (United States)

    Shaheen, Fozia; Grammatopoulos, Dimitris K; Müller, Jürgen; Zammit, Victor A; Lehnert, Hendrik

    2014-09-01

    Telomerase reverse transcriptase (TERT) is a key component of the telomerase complex. By lengthening telomeres in DNA strands, TERT increases senescent cell lifespan. Mice that lack TERT age much faster and exhibit age-related conditions such as osteoporosis, diabetes and neurodegeneration. Accelerated telomere shortening in both human and animal models has been documented in conditions associated with insulin resistance, including T2DM. We investigated the role of TERT, in regulating cellular glucose utilisation by using the myoblastoma cell line C2C12, as well as primary mouse and human skeletal muscle cells. Inhibition of TERT expression or activity by using siRNA (100nM) or specific inhibitors (100nM) reduced basal 2-deoxyglucose uptake by ~50%, in all cell types, without altering insulin responsiveness. In contrast, TERT over-expression increased glucose uptake by 3.25-fold. In C2C12 cells TERT protein was mostly localised intracellularly and stimulation of cells with insulin induced translocation to the plasma membrane. Furthermore, co-immunoprecipitation experiments in C2C12 cells showed that TERT was constitutively associated with glucose transporters (GLUTs) 1, 4 and 12 via an insulin insensitive interaction that also did not require intact PI3-K and mTOR pathways. Collectively, these findings identified a novel extra-nuclear function of TERT that regulates an insulin-insensitive pathway involved in glucose uptake in human and mouse skeletal muscle cells. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-14

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-01-15

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

  7. Lipolysis in Skeletal Muscle

    DEFF Research Database (Denmark)

    Serup, Annette Karen Lundbeck

    Lipid is stored as triacylglycerol (TG) in lipid droplets and is in skeletal muscle stored as intra muscular triacylglycerol (IMTG). IMTG is considered an energy pool that is utilized by lipolysis during situations with low cellular energy availability, such as exercise. Lipolysis is in skeletal ......, is not an important signaling molecule in the mechanism behind insulin resistance and type 2 diabetes The findings of this PhD thesis are presented in one manuscript and in one published paper. In addition, the thesis comprises unpublished work....

  8. Nutritional regulation and role of peroxisome proliferator-activated receptor delta in fatty acid catabolism in skeletal muscle

    DEFF Research Database (Denmark)

    Holst, Dorte; Luquet, Serge; Nogueira, Véronique

    2003-01-01

    Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors primarily involved in lipid homeostasis. PPARdelta displays strong expression in tissues with high lipid metabolism, such as adipose, intestine and muscle. Its role in skeletal muscle remains largely unknown. After a 24-h...... starvation period, PPARdelta mRNA levels are dramatically up-regulated in gastrocnemius muscle of mice and restored to control level upon refeeding. The rise of PPARdelta is accompanied by parallel up-regulations of fatty acid translocase/CD36 (FAT/CD36) and heart fatty acid binding protein (H-FABP), while...... refeeding promotes down-regulation of both genes. To directly access the role of PPARdelta in muscle cells, we forced its expression and that of a dominant-negative PPARdelta mutant in C2C12 myogenic cells. Differentiated C2C12 cells responds to 2-bromopalmitate or synthetic PPARdelta agonist by induction...

  9. PDH regulation in skeletal muscle

    DEFF Research Database (Denmark)

    Kiilerich, Kristian

    regulation in human skeletal muscle. 2: Effect of muscle glycogen on PDH regulation in human skeletal muscle at rest and during exercise. 3: The impact of physical inactivity on PDH regulation in human skeletal muscle at rest and during exercise. 4: Elucidating the importance of PGC-1? in PDH regulation...... in mouse skeletal muscle at rest and in response to fasting and during recovery from exercise. The studies indicate that the content of PDH-E1? in human muscle follows the metabolic profile of the muscle, rather than the myosin heavy chain fiber distribution of the muscle. The larger lactate accumulation...... in human skeletal muscle. It may be noted that the increased PDK4 protein associated with elevated plasma FFA occurs already 2 hours after different dietary intake. A week of physical inactivity (bed rest), leading to whole body glucose intolerance, does not affect muscle PDH-E1? content, or the exercise...

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

    Science.gov (United States)

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

    2015-09-30

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

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

    Science.gov (United States)

    2014-01-01

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

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

    Science.gov (United States)

    Cheon, Hyae Gyeong; Cho, Young Sik

    2014-02-12

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

  13. A vertebrate slow skeletal muscle actin isoform

    National Research Council Canada - National Science Library

    Mudalige, Wasana A. K. A; Jackman, Donna M; Waddleton, Deena M; Heeley, David H

    2007-01-01

    Salmonids utilize a unique, class II isoactin in slow skeletal muscle. This actin contains 12 replacements when compared with those from salmonid fast skeletal muscle, salmonid cardiac muscle and rabbit skeletal muscle...

  14. Skeletal muscle connective tissue

    DEFF Research Database (Denmark)

    Brüggemann, Dagmar Adeline

      The connective tissue content of skeletal muscle is believed to be the major factor responsible for defining the eating quality of different meat cuts, although attempts to correlate quantifications based on traditional histological methods have not as yet been able to prove this relation...... composition, the organizational structure of connective tissue, the role of connective tissue in muscle contraction and the generation of force, metabolic regulation of arterial structure focusing on associated collagen changes, and a new highly-specific technique for following in three-dimensions changes...... in the structure of fibrous collagen and myofibers at high-resolution. The results demonstrate that the collagen composition in the extra cellular matrix of Gadus morhua fish muscle is much more complex than previously anticipated, as it contains type III, IV, V  and VI collagen in addition to type I. The vascular...

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

    Directory of Open Access Journals (Sweden)

    Yang Shen

    2015-01-01

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

  16. DHA at nutritional doses restores insulin sensitivity in skeletal muscle by preventing lipotoxicity and inflammation.

    Science.gov (United States)

    Capel, Frédéric; Acquaviva, Cécile; Pitois, Elodie; Laillet, Brigitte; Rigaudière, Jean-Paul; Jouve, Chrystèle; Pouyet, Corinne; Gladine, Cècile; Comte, Blandine; Vianey Saban, Christine; Morio, Bèatrice

    2015-09-01

    Skeletal muscle plays a major role in the control of whole body glucose disposal in response to insulin stimulus. Excessive supply of fatty acids to this tissue triggers cellular and molecular disturbances leading to lipotoxicity, inflammation, mitochondrial dysfunctions, impaired insulin response and decreased glucose uptake. This study was conducted to analyze the preventive effect of docosahexaenoic acid (DHA), a long-chain polyunsaturated n-3 fatty acid, against insulin resistance, lipotoxicity and inflammation in skeletal muscle at doses compatible with nutritional supplementation. DHA (30 μM) prevented insulin resistance in C2C12 myotubes exposed to palmitate (500 μM) by decreasing protein kinase C (PKC)-θ activation and restoring cellular acylcarnitine profile, insulin-dependent AKT phosphorylation and glucose uptake. Furthermore, DHA protected C2C12 myotubes from palmitate- or lipopolysaccharide-induced increase in Ptgs2, interleukin 6 and tumor necrosis factor-α mRNA level, probably through the inhibition of p38 MAP kinase and c-Jun amino-terminal kinase. In LDLR -/- mice fed a high-cholesterol-high-sucrose diet, supplementation with DHA reaching up to 2% of daily energy intake enhanced the insulin-dependent AKT phosphorylation and reduced the PKC-θ activation in skeletal muscle. Therefore, DHA used at physiological doses participates in the regulation of muscle lipid and glucose metabolisms by preventing lipotoxicity and inflammation. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Nrf2-Mediated Regulation of Skeletal Muscle Glycogen Metabolism.

    Science.gov (United States)

    Uruno, Akira; Yagishita, Yoko; Katsuoka, Fumiki; Kitajima, Yasuo; Nunomiya, Aki; Nagatomi, Ryoichi; Pi, Jingbo; Biswal, Shyam S; Yamamoto, Masayuki

    2016-06-01

    Nrf2 (NF-E2-related factor 2) contributes to the maintenance of glucose homeostasis in vivo Nrf2 suppresses blood glucose levels by protecting pancreatic β cells from oxidative stress and improving peripheral tissue glucose utilization. To elucidate the molecular mechanisms by which Nrf2 contributes to the maintenance of glucose homeostasis, we generated skeletal muscle (SkM)-specific Keap1 knockout (Keap1MuKO) mice that express abundant Nrf2 in their SkM and then examined Nrf2 target gene expression in that tissue. In Keap1MuKO mice, blood glucose levels were significantly downregulated and the levels of the glycogen branching enzyme (Gbe1) and muscle-type PhKα subunit (Phka1) mRNAs, along with those of the glycogen branching enzyme (GBE) and the phosphorylase b kinase α subunit (PhKα) protein, were significantly upregulated in mouse SkM. Consistent with this result, chemical Nrf2 inducers promoted Gbe1 and Phka1 mRNA expression in both mouse SkM and C2C12 myotubes. Chromatin immunoprecipitation analysis demonstrated that Nrf2 binds the Gbe1 and Phka1 upstream promoter regions. In Keap1MuKO mice, muscle glycogen content was strongly reduced and forced GBE expression in C2C12 myotubes promoted glucose uptake. Therefore, our results demonstrate that Nrf2 induction in SkM increases GBE and PhKα expression and reduces muscle glycogen content, resulting in improved glucose tolerance. Our results also indicate that Nrf2 differentially regulates glycogen metabolism in SkM and the liver. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. Paraplegia increases skeletal muscle autophagy.

    Science.gov (United States)

    Fry, Christopher S; Drummond, Micah J; Lujan, Heidi L; DiCarlo, Stephen E; Rasmussen, Blake B

    2012-11-01

    Paraplegia results in significant skeletal muscle atrophy through increases in skeletal muscle protein breakdown. Recent work has identified a novel SIRT1-p53 pathway that is capable of regulating autophagy and protein breakdown. Soleus muscle was collected from 6 male Sprague-Dawley rats 10 weeks after complete T4-5 spinal cord transection (paraplegia group) and 6 male sham-operated rats (control group). We utilized immunoblotting methods to measure intracellular proteins and quantitative real-time polymerase chain reaction to measure the expression of skeletal muscle microRNAs. SIRT1 protein expression was 37% lower, and p53 acetylation (LYS379) was increased in the paraplegic rats (P paraplegia group compared with controls (P paraplegia appears to increase skeletal muscle autophagy independent of SIRT1 signaling. We conclude that chronic paraplegia may cause an increase in autophagic cell death and negatively impact skeletal muscle protein balance. Copyright © 2012 Wiley Periodicals, Inc.

  19. Diabetes-Related Ankyrin Repeat Protein (DARP/Ankrd23 Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle.

    Directory of Open Access Journals (Sweden)

    Yoshiaki Shimoda

    Full Text Available Skeletal muscle is the major site for glucose disposal, the impairment of which closely associates with the glucose intolerance in diabetic patients. Diabetes-related ankyrin repeat protein (DARP/Ankrd23 is a member of muscle ankyrin repeat proteins, whose expression is enhanced in the skeletal muscle under diabetic conditions; however, its role in energy metabolism remains poorly understood. Here we report a novel role of DARP in the regulation of glucose homeostasis through modulating AMP-activated protein kinase (AMPK activity. DARP is highly preferentially expressed in skeletal muscle, and its expression was substantially upregulated during myotube differentiation of C2C12 myoblasts. Interestingly, DARP-/- mice demonstrated better glucose tolerance despite similar body weight, while their insulin sensitivity did not differ from that in wildtype mice. We found that phosphorylation of AMPK, which mediates insulin-independent glucose uptake, in skeletal muscle was significantly enhanced in DARP-/- mice compared to that in wildtype mice. Gene silencing of DARP in C2C12 myotubes enhanced AMPK phosphorylation, whereas overexpression of DARP in C2C12 myoblasts reduced it. Moreover, DARP-silencing increased glucose uptake and oxidation in myotubes, which was abrogated by the treatment with AICAR, an AMPK activator. Of note, improved glucose tolerance in DARP-/- mice was abolished when mice were treated with AICAR. Mechanistically, gene silencing of DARP enhanced protein expression of LKB1 that is a major upstream kinase for AMPK in myotubes in vitro and the skeletal muscle in vivo. Together with the altered expression under diabetic conditions, our data strongly suggest that DARP plays an important role in the regulation of glucose homeostasis under physiological and pathological conditions, and thus DARP is a new therapeutic target for the treatment of diabetes mellitus.

  20. Metformin suppresses lipid accumulation in skeletal muscle by promoting fatty acid oxidation.

    Science.gov (United States)

    Wang, Chao; Liu, Fang; Yuan, Yuan; Wu, Jie; Wang, Hui; Zhang, Lijun; Hu, Peizhen; Li, Zengshan; Li, Qing; Ye, Jing

    2014-01-01

    Obesity is a major risk factor for metabolic syndrome, including insulin resistance (IR), type 2 diabetes mellitus (T2DM), and cardiovascular disease; ectopic fat deposition plays a key role in the development of these conditions. In insulin-resistant and/or T2DM patients, lipid accumulation is increased in skeletal muscle; the intramuscular accumulation of fatty acid metabolites is recognized to play a critical role in metabolic syndrome. Besides improving insulin sensitivity, the anti-diabetic drug metformin can reduce lipid accumulation in skeletal muscle; however, its mechanism of action remains unclear. Ob/ob mice and C2C12 cells were used to explore the effects of metformin on the morphological and physiological changes of lipid droplets. To clarify the mechanism by which metformin regulates fatty acid metabolism, a cDNA microarray and quantitative real-time PCR were used to examine the effects of metformin on the transcriptome of C2C12 cells treated with 200 micromol/L oleic acid. Metformin could retard body weight gain, improve insulin sensitivity and reduce intramyocellular lipid accumulation in ob/ob mice. In C2C12 cells, metformin inhibited lipid accumulation, stimulated fatty acid oxidation, and decreased triglyceride synthesis. Twenty-seven differentially expressed genes, including 12 upregulated and 15 downregulated genes, were involved in fatty acid metabolism. Interestingly, several genes involved in acyl-CoA synthesis and fatty acid oxidation were also upregulated, such as Ppard, Acsbg1, Ascl3, and Mlycd. However, several genes related to lipolysis were downregulated, such as Ces1d and Cel. Moreover, several important genes related to lipid metabolism were also downregulated, such as Fabp4, Adipoq, and Apoc2. Metformin retards body weight gain, improves insulin sensitivity, and suppresses lipid accumulation in skeletal muscle by promoting fatty acid oxidation.

  1. Designing of a Si-MEMS device with an integrated skeletal muscle cell-based bio-actuator.

    Science.gov (United States)

    Fujita, Hideaki; Van Dau, Thanh; Shimizu, Kazunori; Hatsuda, Ranko; Sugiyama, Susumu; Nagamori, Eiji

    2011-02-01

    With the aim of designing a mechanical drug delivery system involving a bio-actuator, we fabricated a Micro Electro Mechanical Systems (MEMS) device that can be driven through contraction of skeletal muscle cells. The device is composed of a Si-MEMS with springs and ratchets, UV-crosslinked collagen film for cell attachment, and C2C12 muscle cells. The Si-MEMS device is 600 μm x 1000 μm in size and the width of the collagen film is 250 ~ 350 μm, which may allow the device to go through small blood vessels. To position the collagen film on the MEMS device, a thermo-sensitive polymer was used as the sacrifice-layer which was selectively removed with O₂ plasma at the positions where the collagen film was glued. The C2C12 myoblasts were seeded on the collagen film, where they proliferated and formed myotubes after induction of differentiation. When C2C12 myotubes were stimulated with electric pulses, contraction of the collagen film-C2C12 myotube complex was observed. When the edge of the Si-MEMS device was observed, displacement of ~8 μm was observed, demonstrating the possibility of locomotive movement when the device is placed on a track of adequate width. Here, we propose that the C2C12-collagen film complex is a new generation actuator for MEMS devices that utilize glucose as fuel, which will be useful in environments in which glucose is abundant such as inside a blood vessel.

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

    Directory of Open Access Journals (Sweden)

    Xinyi Liu

    2017-01-01

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

  3. Attenuation of skeletal muscle wasting with recombinant human growth hormone secreted from a tissue-engineered bioartificial muscle

    Science.gov (United States)

    Vandenburgh, H.; Del Tatto, M.; Shansky, J.; Goldstein, L.; Russell, K.; Genes, N.; Chromiak, J.; Yamada, S.

    1998-01-01

    Skeletal muscle wasting is a significant problem in elderly and debilitated patients. Growth hormone (GH) is an anabolic growth factor for skeletal muscle but is difficult to deliver in a therapeutic manner by injection owing to its in vivo instability. A novel method is presented for the sustained secretion of recombinant human GH (rhGH) from genetically modified skeletal muscle implants, which reduces host muscle wasting. Proliferating murine C2C12 skeletal myoblasts stably transduced with the rhGH gene were tissue engineered in vitro into bioartificial muscles (C2-BAMs) containing organized postmitotic myofibers secreting 3-5 microg of rhGH/day in vitro. When implanted subcutaneously into syngeneic mice, C2-BAMs delivered a sustained physiologic dose of 2.5 to 11.3 ng of rhGH per milliliter of serum. rhGH synthesized and secreted by the myofibers was in the 22-kDa monomeric form and was biologically active, based on downregulation of a GH-sensitive protein synthesized in the liver. Skeletal muscle disuse atrophy was induced in mice by hindlimb unloading, causing the fast plantaris and slow soleus muscles to atrophy by 21 to 35% ( muscle-wasting disorders.

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

    Directory of Open Access Journals (Sweden)

    Tisdale Michael J

    2008-01-01

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

  5. Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis.

    Science.gov (United States)

    Goh, Qingnian; Dearth, Christopher L; Corbett, Jacob T; Pierre, Philippe; Chadee, Deborah N; Pizza, Francis X

    2015-02-15

    We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast-myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube-myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube-myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    DEFF Research Database (Denmark)

    Grossi, Alberto; Lawson, Moira Ann

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

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

    Science.gov (United States)

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

    2012-11-01

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

  8. Skeletal Muscle Na+ Channel Disorders

    Directory of Open Access Journals (Sweden)

    Dina eSimkin

    2011-10-01

    Full Text Available Five inherited human disorders affecting skeletal muscle contraction have been traced to mutations in the gene encoding the voltage-gated sodium channel Nav1.4. The main symptoms of these disorders are myotonia or periodic paralysis caused by changes in skeletal muscle fiber excitability. Symptoms of these disorders vary from mild or latent disease to incapacitating or even death in severe cases. As new human sodium channel mutations corresponding to disease states become discovered, the importance of understanding the role of the sodium channel in skeletal muscle function and disease state grows.

  9. Metformin ameliorates high uric acid-induced insulin resistance in skeletal muscle cells.

    Science.gov (United States)

    Yuan, Huier; Hu, Yaqiu; Zhu, Yuzhang; Zhang, Yongneng; Luo, Chaohuan; Li, Zhi; Wen, Tengfei; Zhuang, Wanling; Zou, Jinfang; Hong, Liangli; Zhang, Xin; Hisatome, Ichiro; Yamamoto, Tetsuya; Cheng, Jidong

    2017-03-05

    Hyperuricemia occurs together with abnormal glucose metabolism and insulin resistance. Skeletal muscle is an important organ of glucose uptake, disposal, and storage. Metformin activates adenosine monophosphate-activated protein kinase (AMPK) to regulate insulin signaling and promote the translocation of glucose transporter type 4 (GLUT4), thereby stimulating glucose uptake to maintain energy balance. Our previous study showed that high uric acid (HUA) induced insulin resistance in skeletal muscle tissue. However, the mechanism of metformin ameliorating UA-induced insulin resistance in muscle cells is unknown and we aimed to determine it. In this study, differentiated C2C12 cells were exposed to UA (15 mg/dl), then reactive oxygen species (ROS) was detected with DCFH-DA and glucose uptake with 2-NBDG. The levels of phospho-insulin receptor substrate 1 (IRS1; Ser307), phospho-AKT (Ser473) and membrane GLUT4 were examined by western blot analysis. The impact of metformin on UA-induced insulin resistance was monitored by adding Compound C, an AMPK inhibitor, and LY294002, a PI3K/AKT inhibitor. Our data indicate that UA can increase ROS production, inhibit IRS1-AKT signaling and insulin-stimulated glucose uptake, and induce insulin resistance in C2C12 cells. Metformin can reverse this process by increasing intracellular glucose uptake and ameliorating UA-induced insulin resistance. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  10. Troponin T3 regulates nuclear localization of the calcium channel Cavβ1a subunit in skeletal muscle.

    Science.gov (United States)

    Zhang, Tan; Taylor, Jackson; Jiang, Yang; Pereyra, Andrea S; Messi, Maria Laura; Wang, Zhong-Min; Hereñú, Claudia; Delbono, Osvaldo

    2015-08-15

    The voltage-gated calcium channel (Cav) β1a subunit (Cavβ1a) plays an important role in excitation-contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Cavβ1a subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160-244 aa) and Cavβ1a NH2-terminus (1-99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Cavβ1a/YFP shows that TnT3 facilitates Cavβ1a nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Skeletal muscle performance and ageing.

    Science.gov (United States)

    Tieland, Michael; Trouwborst, Inez; Clark, Brian C

    2017-11-19

    The world population is ageing rapidly. As society ages, the incidence of physical limitations is dramatically increasing, which reduces the quality of life and increases healthcare expenditures. In western society, ~30% of the population over 55 years is confronted with moderate or severe physical limitations. These physical limitations increase the risk of falls, institutionalization, co-morbidity, and premature death. An important cause of physical limitations is the age-related loss of skeletal muscle mass, also referred to as sarcopenia. Emerging evidence, however, clearly shows that the decline in skeletal muscle mass is not the sole contributor to the decline in physical performance. For instance, the loss of muscle strength is also a strong contributor to reduced physical performance in the elderly. In addition, there is ample data to suggest that motor coordination, excitation-contraction coupling, skeletal integrity, and other factors related to the nervous, muscular, and skeletal systems are critically important for physical performance in the elderly. To better understand the loss of skeletal muscle performance with ageing, we aim to provide a broad overview on the underlying mechanisms associated with elderly skeletal muscle performance. We start with a system level discussion and continue with a discussion on the influence of lifestyle, biological, and psychosocial factors on elderly skeletal muscle performance. Developing a broad understanding of the many factors affecting elderly skeletal muscle performance has major implications for scientists, clinicians, and health professionals who are developing therapeutic interventions aiming to enhance muscle function and/or prevent mobility and physical limitations and, as such, support healthy ageing. © 2017 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.

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

    OpenAIRE

    Pandurangan, Muthuraman; Kim, Doo Hwan

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Wei Li

    2013-01-01

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

  14. Mechanisms of Hyperhomocysteinemia Induced Skeletal Muscle Myopathy after Ischemia in the CBS−/+ Mouse Model

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    Sudhakar Veeranki

    2015-01-01

    Full Text Available Although hyperhomocysteinemia (HHcy elicits lower than normal body weights and skeletal muscle weakness, the mechanisms remain unclear. Despite the fact that HHcy-mediated enhancement in ROS and consequent damage to regulators of different cellular processes is relatively well established in other organs, the nature of such events is unknown in skeletal muscles. Previously, we reported that HHcy attenuation of PGC-1α and HIF-1α levels enhanced the likelihood of muscle atrophy and declined function after ischemia. In the current study, we examined muscle levels of homocysteine (Hcy metabolizing enzymes, anti-oxidant capacity and focused on protein modifications that might compromise PGC-1α function during ischemic angiogenesis. Although skeletal muscles express the key enzyme (MTHFR that participates in re-methylation of Hcy into methionine, lack of trans-sulfuration enzymes (CBS and CSE make skeletal muscles more susceptible to the HHcy-induced myopathy. Our study indicates that elevated Hcy levels in the CBS−/+ mouse skeletal muscles caused diminished anti-oxidant capacity and contributed to enhanced total protein as well as PGC-1α specific nitrotyrosylation after ischemia. Furthermore, in the presence of NO donor SNP, either homocysteine (Hcy or its cyclized version, Hcy thiolactone, not only increased PGC-1α specific protein nitrotyrosylation but also reduced its association with PPARγ in C2C12 cells. Altogether these results suggest that HHcy exerts its myopathic effects via reduction of the PGC-1/PPARγ axis after ischemia.

  15. Simvastatin effects on skeletal muscle

    DEFF Research Database (Denmark)

    Larsen, Steen; Stride, Nis; Hey-Mogensen, Martin

    2013-01-01

    Glucose tolerance and skeletal muscle coenzyme Q(10) (Q(10)) content, mitochondrial density, and mitochondrial oxidative phosphorylation (OXPHOS) capacity were measured in simvastatin-treated patients (n = 10) and in well-matched control subjects (n = 9)....

  16. Skeletal Muscle Na+ Channel Disorders

    OpenAIRE

    Dina eSimkin; Saïd eBendahhou

    2011-01-01

    Five inherited human disorders affecting skeletal muscle contraction have been traced to mutations in the gene encoding the voltage-gated sodium channel Nav1.4. The main symptoms of these disorders are myotonia or periodic paralysis caused by changes in skeletal muscle fiber excitability. Symptoms of these disorders vary from mild or latent disease to incapacitating or even death in severe cases. As new human sodium channel mutations corresponding to disease states become discovered, the impo...

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

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    Allen Herbst

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

  18. Analysis of MicroRNA Expression Profiles in Weaned Pig Skeletal Muscle after Lipopolysaccharide Challenge

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    Jing Zhang

    2015-09-01

    Full Text Available MicroRNAs (miRNAs constitute a class of non-coding RNAs that play a crucial regulatory role in skeletal muscle development and disease. Several acute inflammation conditions including sepsis and cancer are characterized by a loss of skeletal muscle due primarily to excessive muscle catabolism. As a well-known inducer of acute inflammation, a lipopolysaccharide (LPS challenge can cause serious skeletal muscle wasting. However, knowledge of the role of miRNAs in the course of inflammatory muscle catabolism is still very limited. In this study, RNA extracted from the skeletal muscle of pigs injected with LPS or saline was subjected to small RNA deep sequencing. We identified 304 conserved and 114 novel candidate miRNAs in the pig. Of these, four were significantly increased in the LPS-challenged samples and five were decreased. The expression of five miRNAs (ssc-miR-146a-5p, ssc-miR-221-5p, ssc-miR-148b-3p, ssc-miR-215 and ssc-miR-192 were selected for validation by quantitative polymerase chain reaction (qPCR, which found that ssc-miR-146a-5p and ssc-miR-221-5p were significantly upregulated in LPS-challenged pig skeletal muscle. Moreover, we treated mouse C2C12 myotubes with 1000 ng/mL LPS as an acute inflammation cell model. Expression of TNF-α, IL-6, muscle atrophy F-box (MAFbx and muscle RING finger 1 (MuRF1 mRNA was strongly induced by LPS. Importantly, miR-146a-5p and miR-221-5p also showed markedly increased expression in LPS-treated C2C12 myotubes, suggesting the two miRNAs may be involved in muscle catabolism systems in response to acute inflammation caused by a LPS challenge. To our knowledge, this study is the first to examine miRNA expression profiles in weaned pig skeletal muscle challenged with LPS, and furthers our understanding of miRNA function in the regulation of inflammatory muscle catabolism.

  19. Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T. [Department of Kinesiology, The University of Toledo, Toledo, OH (United States); Pierre, Philippe [Centre d’Immunologie de Marseille-Luminy U2M, Aix-Marseille Université, Marseille (France); INSERM U631, Institut National de la Santé et Recherche Médicale, Marseille (France); CNRS UMR6102, Centre National de la Recherche Scientifique, Marseille (France); Chadee, Deborah N. [Department of Biological Sciences, The University of Toledo, Toledo, OH (United States); Pizza, Francis X., E-mail: Francis.Pizza@utoledo.edu [Department of Kinesiology, The University of Toledo, Toledo, OH (United States)

    2015-02-15

    We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast–myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube–myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube–myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. - Highlights: • We examined mechanisms through which skeletal muscle cell expression of ICAM-1 facilitates events of in vitro myogenesis. • Expression of ICAM-1 by cultured myoblasts did not influence their ability to proliferate or differentiate. • Skeletal muscle cell expression of ICAM-1 augmented myoblast fusion, myotube alignment, myotube–myotube fusion, and myotube size. • ICAM-1 augmented myogenic processes through

  20. Urotensin II inhibits skeletal muscle glucose transport signaling pathways via the NADPH oxidase pathway.

    Directory of Open Access Journals (Sweden)

    Hong-Xia Wang

    Full Text Available Our previous studies have demonstrated that the urotensin (UII and its receptor are up-regulated in the skeletal muscle of mice with type II diabetes mellitus (T2DM, but the significance of UII in skeletal muscle insulin resistance remains unknown. The purpose of this study was to investigate the effect of UII on NADPH oxidase and glucose transport signaling pathways in the skeletal muscle of mice with T2DM and in C2C12 mouse myotube cells. KK/upj-AY/J mice (KK mice were divided into the following groups: KK group, with saline treatment for 2 weeks; KK+ urantide group, with daily 30 µg/kg body weight injections over the same time period of urantide, a potent urotensin II antagonist peptide; Non-diabetic C57BL/6J mice were used as normal controls. After urantide treatment, mice were subjected to an intraperitoneal glucose tolerance test, in addition to measurements of the levels of ROS, NADPH oxidase and the phosphorylated AKT, PKC and ERK. C2C12 cells were incubated with serum-free DMEM for 24 hours before conducting the experiments, and then administrated with 100 nM UII for 2 hours or 24 hours. Urantide treatment improved glucose tolerance, decreased the translocation of the NADPH subunits p40-phox and p47-phox, and increased levels of the phosphorylated PKC, AKT and ERK. In contrast, UII treatment increased ROS production and p47-phox and p67-phox translocation, and decreased the phosphorylated AKT, ERK1/2 and p38MAPK; Apocynin abrogated this effect. In conclusion, UII increased ROS production by NADPH oxidase, leading to the inhibition of signaling pathways involving glucose transport, such as AKT/PKC/ERK. Our data imply a role for UII at the molecular level in glucose homeostasis, and possibly in skeletal muscle insulin resistance in T2DM.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  2. Skeletal muscle ultrasound.

    NARCIS (Netherlands)

    Pillen, S.; Alfen, N. van

    2011-01-01

    Muscle ultrasound is a convenient technique to visualize normal and pathological muscle tissue as it is non-invasive and real-time. Neuromuscular disorders give rise to structural muscle changes that can be visualized with ultrasound: atrophy can be objectified by measuring muscle thickness, while

  3. Skeletal muscle ultrasound.

    Science.gov (United States)

    Pillen, Sigrid; van Alfen, Nens

    2011-12-01

    Muscle ultrasound is a convenient technique to visualize normal and pathological muscle tissue as it is non-invasive and real-time. Neuromuscular disorders give rise to structural muscle changes that can be visualized with ultrasound: atrophy can be objectified by measuring muscle thickness, while infiltration of fat and fibrous tissue increases muscle echo intensity, i.e. the muscles become whiter on the ultrasound image. Muscle echo intensity needs to be quantified to correct for age-related increase in echo intensity and differences between individual muscles. This can be done by gray scale analysis, a method that can be easily applied in daily clinical practice. Using this technique, it is possible to detect neuromuscular disorders with predictive values of 90%. Only in young children and metabolic myopathies the sensitivity is lower. Ultrasound is a dynamic technique and therefore capable of visualizing normal and pathological muscle movements. Fasciculations can easily be differentiated from other muscle movements. Ultrasound appeared to be even more sensitive in detecting fasciculations compared to Electromyography (EMG) and clinical observations, because it can visualize a large muscle area and deeper located muscles. With improving resolution and frame rate it has recently become clear that also smaller scale spontaneous muscle activity such as fibrillations can be detected by ultrasound. This opens the way to a broader use of muscle ultrasound in the diagnosis of peripheral nerve and muscle disorders.

  4. Skeletal muscle ultrasound

    Directory of Open Access Journals (Sweden)

    Sigrid Pillen

    2010-12-01

    Full Text Available Muscle ultrasound is a convenient technique to visualize normal and pathological muscle tissue as it is non-invasive and real-time. Neuromuscular disorders give rise to structural muscle changes that can be visualized with ultrasound: atrophy can be objectified by measuring muscle thickness, while infiltration of fat and fibrous tissue increase muscle echo intensity, i.e. the muscles become whiter on the ultrasound image. Muscle echo intensity need to be quantified to correct for age-related increase in echo intensity and differences between individual muscles. This can be done by gray scale analysis, a method that can be easily applied in daily clinical practice. Using this technique it is possible to detect neuromuscular disorders with predictive values of 90 percent. Only in young children and metabolic myopathies the sensitivity is lower. Ultrasound is a dynamic technique and therefore capable of visualizing normal and pathological muscle movements. Fasciculations can easily be differentiated from other muscle movements. Ultrasound appeared to be even more sensitive in detecting fasciculations compared to EMG and clinical observations, because it can visualize a large muscle area and deeper located muscles. With improving resolution and frame rate it has recently become clear that also smaller scale spontaneous muscle activity such as fibrillations can be detected by ultrasound. This opens the way to a broader use of muscle ultrasound in the diagnosis of peripheral nerve and muscle disorders.

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

    Science.gov (United States)

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

    2009-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Michelle I Smith

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

  7. Angiotensin II Evokes Angiogenic Signals within Skeletal Muscle through Co-ordinated Effects on Skeletal Myocytes and Endothelial Cells

    Science.gov (United States)

    Gorman, Jennifer L.; Liu, Sammy T. K.; Slopack, Dara; Shariati, Khashayar; Hasanee, Adam; Olenich, Sara; Olfert, I. Mark; Haas, Tara L.

    2014-01-01

    Skeletal muscle overload induces the expression of angiogenic factors such as vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-2, leading to new capillary growth. We found that the overload-induced increase in angiogenesis, as well as increases in VEGF, MMP-2 and MT1-MMP transcripts were abrogated in muscle VEGF KO mice, highlighting the critical role of myocyte-derived VEGF in controlling this process. The upstream mediators that contribute to overload-induced expression of VEGF have yet to be ascertained. We found that muscle overload increased angiotensinogen expression, a precursor of angiotensin (Ang) II, and that Ang II signaling played an important role in basal VEGF production in C2C12 cells. Furthermore, matrix-bound VEGF released from myoblasts induced the activation of endothelial cells, as evidenced by elevated endothelial cell phospho-p38 levels. We also found that exogenous Ang II elevates VEGF expression, as well as MMP-2 transcript levels in C2C12 myotubes. Interestingly, these responses also were observed in skeletal muscle endothelial cells in response to Ang II treatment, indicating that these cells also can respond directly to the stimulus. The involvement of Ang II in muscle overload-induced angiogenesis was assessed. We found that blockade of AT1R-dependent Ang II signaling using losartan did not attenuate capillary growth. Surprisingly, increased levels of VEGF protein were detected in overloaded muscle from losartan-treated rats. Similarly, we observed elevated VEGF production in cultured endothelial cells treated with losartan alone or in combination with Ang II. These studies conclusively establish the requirement for muscle derived VEGF in overload-induced angiogenesis and highlight a role for Ang II in basal VEGF production in skeletal muscle. However, while Ang II signaling is activated following overload and plays a role in muscle VEGF production, inhibition of this pathway is not sufficient to halt overload

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

    Science.gov (United States)

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

    2015-01-01

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

  9. Skeletal Muscle Loss is Associated with TNF Mediated Insufficient Skeletal Myogenic Activation After Burn.

    Science.gov (United States)

    Song, Juquan; Saeman, Melody R; De Libero, Jana; Wolf, Steven E

    2015-11-01

    Muscle loss accompanies severe burn; in this hyper-catabolic state, muscle undergoes atrophy through protein degradation and disuse. Muscle volume is related to the relative rates of cellular degradation and myogenesis. We hypothesize that muscle atrophy after injury is in part because of insufficient myogenesis associated with the hyper-inflammatory response. The aim of this study was to investigate the role of skeletal myogenesis and muscle cell homeostasis in response to severe burn. Twenty-eight male C57BL6 mice received 25% TBSA scald. Gluteus muscle from these animals was analyzed at days 1, 3, 7, and 14 after injury. Six additional animals without burn served as controls. We showed muscle wet weight and protein content decreased at days 3 and 7 after burn, with elevated tumor necrosis factor (TNF) mRNA expression (P < 0.05). Increased cell death was observed through TUNEL staining, and cleaved caspase-3 levels reached a peak in muscle lysate at day 3 (P < 0.05). The cell proliferation marker proliferating cell nuclear antigen (PCNA) significantly increased after burn, associated with increased gene and protein expression of myogenesis markers Pax7 and myogenin. Desmin mRNA expression and the ratio of desmin to PCNA protein expression, however, significantly decreased at day 7 (P < 0.05). In vitro, the ratio of desmin to PCNA protein expression significantly decreased in C2C12 murine myoblasts after TNF-α stimulation for 24 h. We showed that severe burn induces both increased cell death and proliferation. Myogenesis, however, does not counterbalance increased cell death after burn. Data suggest insufficient myogenesis might be associated with pro-inflammatory mediator TNF activity.

  10. Hyperammonaemia‐induced skeletal muscle mitochondrial dysfunction results in cataplerosis and oxidative stress

    Science.gov (United States)

    Davuluri, Gangarao; Allawy, Allawy; Thapaliya, Samjhana; Rennison, Julie H.; Singh, Dharmvir; Kumar, Avinash; Sandlers, Yana; Van Wagoner, David R.; Flask, Chris A.; Hoppel, Charles; Kasumov, Takhar

    2016-01-01

    Key points Hyperammonaemia occurs in hepatic, cardiac and pulmonary diseases with increased muscle concentration of ammonia.We found that ammonia results in reduced skeletal muscle mitochondrial respiration, electron transport chain complex I dysfunction, as well as lower NAD+/NADH ratio and ATP content.During hyperammonaemia, leak of electrons from complex III results in oxidative modification of proteins and lipids.Tricarboxylic acid cycle intermediates are decreased during hyperammonaemia, and providing a cell‐permeable ester of αKG reversed the lower TCA cycle intermediate concentrations and increased ATP content.Our observations have high clinical relevance given the potential for novel approaches to reverse skeletal muscle ammonia toxicity by targeting the TCA cycle intermediates and mitochondrial ROS. Abstract Ammonia is a cytotoxic metabolite that is removed primarily by hepatic ureagenesis in humans. Hyperammonaemia occurs in advanced hepatic, cardiac and pulmonary disease, and in urea cycle enzyme deficiencies. Increased skeletal muscle ammonia uptake and metabolism are the major mechanism of non‐hepatic ammonia disposal. Non‐hepatic ammonia disposal occurs in the mitochondria via glutamate synthesis from α‐ketoglutarate resulting in cataplerosis. We show skeletal muscle mitochondrial dysfunction during hyperammonaemia in a comprehensive array of human, rodent and cellular models. ATP synthesis, oxygen consumption, generation of reactive oxygen species with oxidative stress, and tricarboxylic acid (TCA) cycle intermediates were quantified. ATP content was lower in the skeletal muscle from cirrhotic patients, hyperammonaemic portacaval anastomosis rat, and C2C12 myotubes compared to appropriate controls. Hyperammonaemia in C2C12 myotubes resulted in impaired intact cell respiration, reduced complex I/NADH oxidase activity and electron leak occurring at complex III of the electron transport chain. Consistently, lower NAD+/NADH ratio was observed

  11. The Root Extract of Pueraria lobata and Its Main Compound, Puerarin, Prevent Obesity by Increasing the Energy Metabolism in Skeletal Muscle.

    Science.gov (United States)

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

    2017-01-04

    Radix Pueraria lobata (RP) has been reported to prevent obesity and improve glucose metabolism; however, the mechanism responsible for these effects has not been elucidated. The mechanism underlying anti-obesity effect of RP was investigated in high-fat diet (HFD) induced obese mice and skeletal muscle cells (C2C12). Five-week-old C5BL/6 mice were fed a HFD containing or not containing RP (100 or 300 mg/kg) or metformin (250 mg/kg) for 16 weeks. RP reduced body weight gain, lipid accumulation in liver, and adipocyte and blood lipid levels. In addition, RP dose-dependently improved hyperglycemia, insulinemia, and glucose tolerance, and prevented the skeletal muscle atrophy induced by HFD. Furthermore, RP increased the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) expression and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle tissues. RP and its main component, puerarin, increased mitochondrial biogenesis and myotube hypertrophy in C2C12 cells. The present study demonstrates that RP can prevent diet-induced obesity, glucose tolerance, and skeletal muscle atrophy in mouse models of obesity. The mechanism responsible for the effect of RP appears to be related to the upregulation of energy metabolism in skeletal muscle, which at the molecular level may be associated with PGC-1α and AMPK activation.

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

    Science.gov (United States)

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

    2017-05-17

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

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

    Science.gov (United States)

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

    2014-02-01

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

  14. High-Density Lipoprotein Maintains Skeletal Muscle Function by Modulating Cellular Respiration in Mice

    National Research Council Canada - National Science Library

    Lehti, Maarit; Donelan, Elizabeth; Abplanalp, William; Al-Massadi, Omar; Habegger, Kirk M; Weber, Jon; Ress, Chandler; Mansfeld, Johannes; Somvanshi, Sonal; Trivedi, Chitrang; Keuper, Michaela; Ograjsek, Teja; Striese, Cynthia; Cucuruz, Sebastian; Pfluger, Paul T; Krishna, Radhakrishna; Gordon, Scott M; Silva, R.A. Gangani D; Luquet, Serge; Castel, Julien; Martinez, Sarah; D’Alessio, David; Davidson, W Sean; Hofmann, Susanna M

    2013-01-01

    .... Endurance capacity during exercise exhaustion test was impaired in apoA-I ko mice. HDL directly enhanced glucose oxidation by increasing glycolysis and mitochondrial respiration rate in C2C12 muscle...

  15. High-Density Lipoprotein Maintains Skeletal Muscle Function by Modulating Cellular Respiration in Mice

    National Research Council Canada - National Science Library

    Lehti, Maarit; Donelan, Elizabeth; Abplanalp, William; Al-Massadi, Omar; Habegger, Kirk M; Weber, Jon; Ress, Chandler; Mansfeld, Johannes; Somvanshi, Sonal; Trivedi, Chitrang; Keuper, Michaela; Ograjsek, Teja; Striese, Cynthia; Cucuruz, Sebastian; Pfluger, Paul T; Krishna, Radhakrishna; Gordon, Scott M; Silva, R.A. Gangani D; Luquet, Serge; Castel, Julien; Martinez, Sarah; D’Alessio, David; Davidson, W Sean; Hofmann, Susanna M

    2013-01-01

    .... Endurance capacity during exercise exhaustion test was impaired in apoA-I ko mice. HDL directly enhanced glucose oxidation by increasing glycolysis and mitochondrial respiration rate in C2C12 muscle cells...

  16. PGC-1α-mediated branched-chain amino acid metabolism in the skeletal muscle.

    Science.gov (United States)

    Hatazawa, Yukino; Tadaishi, Miki; Nagaike, Yuta; Morita, Akihito; Ogawa, Yoshihiro; Ezaki, Osamu; Takai-Igarashi, Takako; Kitaura, Yasuyuki; Shimomura, Yoshiharu; Kamei, Yasutomi; Miura, Shinji

    2014-01-01

    Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (PGC-1α) is a coactivator of various nuclear receptors and other transcription factors, which is involved in the regulation of energy metabolism, thermogenesis, and other biological processes that control phenotypic characteristics of various organ systems including skeletal muscle. PGC-1α in skeletal muscle is considered to be involved in contractile protein function, mitochondrial function, metabolic regulation, intracellular signaling, and transcriptional responses. Branched-chain amino acid (BCAA) metabolism mainly occurs in skeletal muscle mitochondria, and enzymes related to BCAA metabolism are increased by exercise. Using murine skeletal muscle overexpressing PGC-1α and cultured cells, we investigated whether PGC-1α stimulates BCAA metabolism by increasing the expression of enzymes involved in BCAA metabolism. Transgenic mice overexpressing PGC-1α specifically in the skeletal muscle had increased the expression of branched-chain aminotransferase (BCAT) 2, branched-chain α-keto acid dehydrogenase (BCKDH), which catabolize BCAA. The expression of BCKDH kinase (BCKDK), which phosphorylates BCKDH and suppresses its enzymatic activity, was unchanged. The amount of BCAA in the skeletal muscle was significantly decreased in the transgenic mice compared with that in the wild-type mice. The amount of glutamic acid, a metabolite of BCAA catabolism, was increased in the transgenic mice, suggesting the activation of muscle BCAA metabolism by PGC-1α. In C2C12 cells, the overexpression of PGC-1α significantly increased the expression of BCAT2 and BCKDH but not BCKDK. Thus, PGC-1α in the skeletal muscle is considered to significantly contribute to BCAA metabolism.

  17. PGC-1α-mediated branched-chain amino acid metabolism in the skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Yukino Hatazawa

    Full Text Available Peroxisome proliferator-activated receptor (PPAR γ coactivator 1α (PGC-1α is a coactivator of various nuclear receptors and other transcription factors, which is involved in the regulation of energy metabolism, thermogenesis, and other biological processes that control phenotypic characteristics of various organ systems including skeletal muscle. PGC-1α in skeletal muscle is considered to be involved in contractile protein function, mitochondrial function, metabolic regulation, intracellular signaling, and transcriptional responses. Branched-chain amino acid (BCAA metabolism mainly occurs in skeletal muscle mitochondria, and enzymes related to BCAA metabolism are increased by exercise. Using murine skeletal muscle overexpressing PGC-1α and cultured cells, we investigated whether PGC-1α stimulates BCAA metabolism by increasing the expression of enzymes involved in BCAA metabolism. Transgenic mice overexpressing PGC-1α specifically in the skeletal muscle had increased the expression of branched-chain aminotransferase (BCAT 2, branched-chain α-keto acid dehydrogenase (BCKDH, which catabolize BCAA. The expression of BCKDH kinase (BCKDK, which phosphorylates BCKDH and suppresses its enzymatic activity, was unchanged. The amount of BCAA in the skeletal muscle was significantly decreased in the transgenic mice compared with that in the wild-type mice. The amount of glutamic acid, a metabolite of BCAA catabolism, was increased in the transgenic mice, suggesting the activation of muscle BCAA metabolism by PGC-1α. In C2C12 cells, the overexpression of PGC-1α significantly increased the expression of BCAT2 and BCKDH but not BCKDK. Thus, PGC-1α in the skeletal muscle is considered to significantly contribute to BCAA metabolism.

  18. Biocompatible, Biodegradable, and Electroactive Polyurethane-Urea Elastomers with Tunable Hydrophilicity for Skeletal Muscle Tissue Engineering.

    Science.gov (United States)

    Chen, Jing; Dong, Ruonan; Ge, Juan; Guo, Baolin; Ma, Peter X

    2015-12-30

    It remains a challenge to develop electroactive and elastic biomaterials to mimic the elasticity of soft tissue and to regulate the cell behavior during tissue regeneration. We designed and synthesized a series of novel electroactive and biodegradable polyurethane-urea (PUU) copolymers with elastomeric property by combining the properties of polyurethanes and conducting polymers. The electroactive PUU copolymers were synthesized from amine capped aniline trimer (ACAT), dimethylol propionic acid (DMPA), polylactide, and hexamethylene diisocyanate. The electroactivity of the PUU copolymers were studied by UV-vis spectroscopy and cyclic voltammetry. Elasticity and Young's modulus were tailored by the polylactide segment length and ACAT content. Hydrophilicity of the copolymer films was tuned by changing DMPA content and doping of the copolymer. Cytotoxicity of the PUU copolymers was evaluated by mouse C2C12 myoblast cells. The myogenic differentiation of C2C12 myoblasts on copolymer films was also studied by analyzing the morphology of myotubes and relative gene expression during myogenic differentiation. The chemical structure, thermal properties, surface morphology, and processability of the PUU copolymers were characterized by NMR, FT-IR, gel permeation chromatography (GPC), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), scanning electron microscopy (SEM), atomic force microscopy (AFM), and solubility testing, respectively. Those biodegradable electroactive elastic PUU copolymers are promising materials for repair of soft tissues such as skeletal muscle, cardiac muscle, and nerve.

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

    Science.gov (United States)

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

    2014-09-01

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

  20. Functional evaluation of artificial skeletal muscle tissue constructs fabricated by a magnetic force-based tissue engineering technique.

    Science.gov (United States)

    Yamamoto, Yasunori; Ito, Akira; Fujita, Hideaki; Nagamori, Eiji; Kawabe, Yoshinori; Kamihira, Masamichi

    2011-01-01

    Skeletal muscle tissue engineering is currently applied in a variety of research fields, including regenerative medicine, drug screening, and bioactuator development, all of which require the fabrication of biomimic and functional skeletal muscle tissues. In the present study, magnetite cationic liposomes were used to magnetically label C2C12 myoblast cells for the construction of three-dimensional artificial skeletal muscle tissues by an applied magnetic force. Skeletal muscle functions, such as biochemical and contractile properties, were evaluated for the artificial tissue constructs. Histological studies revealed that elongated and multinucleated myotubes were observed within the tissue. Expression of muscle-specific markers, such as myogenin, myosin heavy chain and tropomyosin, were detected in the tissue constructs by western blot analysis. Further, creatine kinase activity increased during differentiation. In response to electric pulses, the artificial tissue constructs contracted to generate a physical force (the maximum twitch force, 33.2 μN [1.06 mN/mm2]). Rheobase and chronaxie of the tissue were determined as 4.45 V and 0.72 ms, respectively. These results indicate that the artificial skeletal muscle tissue constructs fabricated in this study were physiologically functional and the data obtained for the evaluation of their functional properties may provide useful information for future skeletal muscle tissue engineering studies.

  1. Delta-like 1 homolog (dlk1: a marker for rhabdomyosarcomas implicated in skeletal muscle regeneration.

    Directory of Open Access Journals (Sweden)

    Louise H Jørgensen

    Full Text Available Dlk1, a member of the Epidermal Growth Factor family, is expressed in multiple tissues during development, and has been detected in carcinomas and neuroendocrine tumors. Dlk1 is paternally expressed and belongs to a group of imprinted genes associated with rhabdomyosarcomas but not with other primitive childhood tumors to date. Here, we investigate the possible roles of Dlk1 in skeletal muscle tumor formation. We analyzed tumors of different mesenchymal origin for expression of Dlk1 and various myogenic markers and found that Dlk1 was present consistently in myogenic tumors. The coincident observation of Dlk1 with a highly proliferative state in myogenic tumors led us to subsequently investigate the involvement of Dlk1 in the control of the adult myogenic programme. We performed an injury study in Dlk1 transgenic mice, ectopically expressing ovine Dlk1 (membrane bound C2 variant under control of the myosin light chain promotor, and detected an early, enhanced formation of myotubes in Dlk1 transgenic mice. We then stably transfected the mouse myoblast cell line, C2C12, with full-length Dlk1 (soluble A variant and detected an inhibition of myotube formation, which could be reversed by adding Dlk1 antibody to the culture supernatant. These results suggest that Dlk1 is involved in controlling the myogenic programme and that the various splice forms may exert different effects. Interestingly, both in the Dlk1 transgenic mice and the DLK1-C2C12 cells, we detected reduced myostatin expression, suggesting that the effect of Dlk1 on the myogenic programme might involve the myostatin signaling pathway. In support of a relationship between Dlk1 and myostatin we detected reciprocal expression of these two transcripts during different cell cycle stages of human myoblasts. Together our results suggest that Dlk1 is a candidate marker for skeletal muscle tumors and might be involved directly in skeletal muscle tumor formation through a modulatory effect on the

  2. Pomegranate extract prevents skeletal muscle of mice against wasting induced by acute TNF-α injection.

    Science.gov (United States)

    Rodriguez, Julie; Caille, Olivier; Ferreira, Daneel; Francaux, Marc

    2017-04-01

    We investigated whether punicalagin-rich pomegranate extract (PE) protects skeletal muscle of mice against inflammation induced by an acute injection of TNF-α. Mice fed with PE or standard chow during 6 wk were injected with TNF-α (100 ng/g) or vehicle and sacrificed 6 h later. Prior supplementation with PE prevented the loss of tibialis anterior mass induced by TNF-α. In skeletal muscle, the activation of the NF-κB signaling and the induction of cytokines mRNA were reduced in mice having received PE. In those mice, the activity of the Akt/mTORC1 pathway and the protein synthesis were maintained after TNF-α injection whereas markers involved in the ubiquitin proteasome pathway were less activated. As urolithin A was the only punicalagin metabolite detectable in plasma of mice supplemented with PE, we performed in vitro experiments using a murine cell line (C2C12) to provide evidence that urolithin A is likely the active compound protecting skeletal muscle against TNF-α-induced inflammation. These results suggest that supplementation with a punicalagin-rich PE may protect skeletal muscle against an acute inflammation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Loss of HIF-1α impairs GLUT4 translocation and glucose uptake by the skeletal muscle cells.

    Science.gov (United States)

    Sakagami, Hidemitsu; Makino, Yuichi; Mizumoto, Katsutoshi; Isoe, Tsubasa; Takeda, Yasutaka; Watanabe, Jun; Fujita, Yukihiro; Takiyama, Yumi; Abiko, Atsuko; Haneda, Masakazu

    2014-05-01

    Defects in glucose uptake by the skeletal muscle cause diseases linked to metabolic disturbance such as type 2 diabetes. The molecular mechanism determining glucose disposal in the skeletal muscle in response to cellular stimuli including insulin, however, remains largely unknown. The hypoxia-inducible factor-1α (HIF-1α) is a transcription factor operating in the cellular adaptive response to hypoxic conditions. Recent studies have uncovered pleiotropic actions of HIF-1α in the homeostatic response to various cellular stimuli, including insulin under normoxic conditions. Thus we hypothesized HIF-1α is involved in the regulation of glucose metabolism stimulated by insulin in the skeletal muscle. To this end, we generated C2C12 myocytes in which HIF-1α is knocked down by short-hairpin RNA and examined the intracellular signaling cascade and glucose uptake subsequent to insulin stimulation. Knockdown of HIF-1α expression in the skeletal muscle cells resulted in abrogation of insulin-stimulated glucose uptake associated with impaired mobilization of glucose transporter 4 (GLUT4) to the plasma membrane. Such defect seemed to be caused by reduced phosphorylation of the protein kinase B substrate of 160 kDa (AS160). AS160 phosphorylation and GLUT4 translocation by AMP-activated protein kinase activation were abrogated as well. In addition, expression of the constitutively active mutant of HIF-1α (CA-HIF-1α) or upregulation of endogenous HIF-1α in C2C12 cells shows AS160 phosphorylation comparable to the insulin-stimulated level even in the absence of insulin. Accordingly GLUT4 translocation was increased in the cells expressing CA-HIF1α. Taken together, HIF-1α is a determinant for GLUT4-mediated glucose uptake in the skeletal muscle cells thus as a possible target to alleviate impaired glucose metabolism in, e.g., type 2 diabetes.

  4. Skeletal muscle satellite cells

    Science.gov (United States)

    Schultz, E.; McCormick, K. M.

    1994-01-01

    Evidence now suggests that satellite cells constitute a class of myogenic cells that differ distinctly from other embryonic myoblasts. Satellite cells arise from somites and first appear as a distinct myoblast type well before birth. Satellite cells from different muscles cannot be functionally distinguished from one another and are able to provide nuclei to all fibers without regard to phenotype. Thus, it is difficult to ascribe any significant function to establishing or stabilizing fiber type, even during regeneration. Within a muscle, satellite cells exhibit marked heterogeneity with respect to their proliferative behavior. The satellite cell population on a fiber can be partitioned into those that function as stem cells and those which are readily available for fusion. Recent studies have shown that the cells are not simply spindle shaped, but are very diverse in their morphology and have multiple branches emanating from the poles of the cells. This finding is consistent with other studies indicating that the cells have the capacity for extensive migration within, and perhaps between, muscles. Complexity of cell shape usually reflects increased cytoplasmic volume and organelles including a well developed Golgi, and is usually associated with growing postnatal muscle or muscles undergoing some form of induced adaptive change or repair. The appearance of activated satellite cells suggests some function of the cells in the adaptive process through elaboration and secretion of a product. Significant advances have been made in determining the potential secretion products that satellite cells make. The manner in which satellite cell proliferative and fusion behavior is controlled has also been studied. There seems to be little doubt that cellcell coupling is not how satellite cells and myofibers communicate. Rather satellite cell regulation is through a number of potential growth factors that arise from a number of sources. Critical to the understanding of this form

  5. The Skeletal Muscle Satellite Cell

    Science.gov (United States)

    2011-01-01

    The skeletal muscle satellite cell was first described and named based on its anatomic location between the myofiber plasma and basement membranes. In 1961, two independent studies by Alexander Mauro and Bernard Katz provided the first electron microscopic descriptions of satellite cells in frog and rat muscles. These cells were soon detected in other vertebrates and acquired candidacy as the source of myogenic cells needed for myofiber growth and repair throughout life. Cultures of isolated myofibers and, subsequently, transplantation of single myofibers demonstrated that satellite cells were myogenic progenitors. More recently, satellite cells were redefined as myogenic stem cells given their ability to self-renew in addition to producing differentiated progeny. Identification of distinctively expressed molecular markers, in particular Pax7, has facilitated detection of satellite cells using light microscopy. Notwithstanding the remarkable progress made since the discovery of satellite cells, researchers have looked for alternative cells with myogenic capacity that can potentially be used for whole body cell-based therapy of skeletal muscle. Yet, new studies show that inducible ablation of satellite cells in adult muscle impairs myofiber regeneration. Thus, on the 50th anniversary since its discovery, the satellite cell’s indispensable role in muscle repair has been reaffirmed. PMID:22147605

  6. 4,4'-Dichlorodiphenyltrichloroethane (DDT) and 4,4'-dichlorodiphenyldichloroethylene (DDE) inhibit myogenesis in C2C12 myoblasts.

    Science.gov (United States)

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

    2017-12-01

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

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

    Science.gov (United States)

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

    2010-10-01

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

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

    Science.gov (United States)

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

    2017-03-06

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

  9. Muscle Bioenergetic Considerations for Intrinsic Laryngeal Skeletal Muscle Physiology

    Science.gov (United States)

    Sandage, Mary J.; Smith, Audrey G.

    2017-01-01

    Purpose: Intrinsic laryngeal skeletal muscle bioenergetics, the means by which muscles produce fuel for muscle metabolism, is an understudied aspect of laryngeal physiology with direct implications for voice habilitation and rehabilitation. The purpose of this review is to describe bioenergetic pathways identified in limb skeletal muscle and…

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

    Directory of Open Access Journals (Sweden)

    Yan Shen

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

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

    Science.gov (United States)

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

    2014-01-01

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

  12. Myostatin promotes distinct responses on protein metabolism of skeletal and cardiac muscle fibers of rodents

    Directory of Open Access Journals (Sweden)

    L.H. Manfredi

    2017-10-01

    Full Text Available Myostatin is a novel negative regulator of skeletal muscle mass. Myostatin expression is also found in heart in a much less extent, but it can be upregulated in pathological conditions, such as heart failure. Myostatin may be involved in inhibiting protein synthesis and/or increasing protein degradation in skeletal and cardiac muscles. Herein, we used cell cultures and isolated muscles from rats to determine protein degradation and synthesis. Muscles incubated with myostatin exhibited an increase in proteolysis with an increase of Atrogin-1, MuRF1 and LC3 genes. Extensor digitorum longus muscles and C2C12 myotubes exhibited a reduction in protein turnover. Cardiomyocytes showed an increase in proteolysis by activating autophagy and the ubiquitin proteasome system, and a decrease in protein synthesis by decreasing P70S6K. The effect of myostatin on protein metabolism is related to fiber type composition, which may be associated to the extent of atrophy mediated effect of myostatin on muscle.

  13. Mechanical modeling of skeletal muscle functioning.

    NARCIS (Netherlands)

    van der Linden, B.J.J.J.

    1998-01-01

    For movement of body or body segments is combined effort needed of the central nervous system and the muscular-skeletal system. This thesis deals with the mechanical functioning of skeletal muscle. That muscles come in a large variety of geometries, suggest the existence of a relation between muscle

  14. AMPK in skeletal muscle function and metabolism

    DEFF Research Database (Denmark)

    Kjøbsted, Rasmus; Hingst, Janne R; Fentz, Joachim

    2017-01-01

    Skeletal muscle possesses a remarkable ability to adapt to various physiologic conditions. AMPK is a sensor of intracellular energy status that maintains energy stores by fine-tuning anabolic and catabolic pathways. AMPK's role as an energy sensor is particularly critical in tissues displaying...... highly changeable energy turnover. Due to the drastic changes in energy demand that occur between the resting and exercising state, skeletal muscle is one such tissue. Here, we review the complex regulation of AMPK in skeletal muscle and its consequences on metabolism (e.g., substrate uptake, oxidation......, and storage as well as mitochondrial function of skeletal muscle fibers). We focus on the role of AMPK in skeletal muscle during exercise and in exercise recovery. We also address adaptations to exercise training, including skeletal muscle plasticity, highlighting novel concepts and future perspectives...

  15. Exercise Promotes Healthy Aging of Skeletal Muscle

    DEFF Research Database (Denmark)

    Cartee, Gregory D; Hepple, Russell T; Bamman, Marcas M

    2016-01-01

    Primary aging is the progressive and inevitable process of bodily deterioration during adulthood. In skeletal muscle, primary aging causes defective mitochondrial energetics and reduced muscle mass. Secondary aging refers to additional deleterious structural and functional age-related changes...... caused by diseases and lifestyle factors. Secondary aging can exacerbate deficits in mitochondrial function and muscle mass, concomitant with the development of skeletal muscle insulin resistance. Exercise opposes deleterious effects of secondary aging by preventing the decline in mitochondrial...... respiration, mitigating aging-related loss of muscle mass and enhancing insulin sensitivity. This review focuses on mechanisms by which exercise promotes "healthy aging" by inducing modifications in skeletal muscle....

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-08-08

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

  17. The omega-3 fatty acid, eicosapentaenoic acid (EPA, prevents the damaging effects of tumour necrosis factor (TNF-alpha during murine skeletal muscle cell differentiation

    Directory of Open Access Journals (Sweden)

    Pearson Stephen

    2008-07-01

    Full Text Available Abstract Background Eicosapentaenoic acid (EPA is a ώ-3 polyunsaturated fatty acid with anti-inflammatory and anti-cachetic properties that may have potential benefits with regards to skeletal muscle atrophy conditions where inflammation is present. It is also reported that pathologic levels of the pro-inflammatory cytokine tumour necrosis factor (TNF-α are associated with muscle wasting, exerted through inhibition of myogenic differentiation and enhanced apoptosis. These findings led us to hypothesize that EPA may have a protective effect against skeletal muscle damage induced by the actions of TNF-α. Results The deleterious effects of TNF-α on C2C12 myogenesis were completely inhibited by co-treatment with EPA. Thus, EPA prevented the TNF-mediated loss of MyHC expression and significantly increased myogenic fusion (p p p p p p Conclusion In conclusion, EPA has a protective action against the damaging effects of TNF-α on C2C12 myogenesis. These findings support further investigations of EPA as a potential therapeutic agent during skeletal muscle regeneration following injury.

  18. A diacylglycerol kinase inhibitor, R59022, stimulates glucose transport through a MKK3/6-p38 signaling pathway in skeletal muscle cells.

    Science.gov (United States)

    Takahashi, Nobuhiko; Nagamine, Miho; Tanno, Satoshi; Motomura, Wataru; Kohgo, Yutaka; Okumura, Toshikatsu

    2007-08-17

    Diacylglycerol kinase (DGK) is one of lipid-regulating enzymes, catalyzes phosphorylation of diacylglycerol to phosphatidic acid. Because skeletal muscle, a major insulin-target organ for glucose disposal, expresses DGK, we investigated in the present study a role of DGK on glucose transport in skeletal muscle cells. PCR study showed that C2C12 myotubes expressed DGKalpha, delta, epsilon, zeta, or theta isoform mRNA. R59022, a specific inhibitor of DGK, significantly increased glucose transport, p38 and MKK3/6 activation in C2C12 myotubes. The R59022-induced glucose transport was blocked by SB203580, a specific p38 inhibitor. In contrast, R59022 failed to stimulate both possible known mechanisms to enhance glucose transport, an IRS1-PI3K-Akt pathway, muscle contraction signaling or GLUT1 and 4 expression. All these results suggest that DGK may play a role in glucose transport in the skeletal muscle cells through modulating a MKK3/6-p38 signaling pathway.

  19. Effects of a combined mechanical stimulation protocol: Value for skeletal muscle tissue engineering.

    Science.gov (United States)

    Boonen, Kristel J M; Langelaan, Marloes L P; Polak, Roderick B; van der Schaft, Daisy W J; Baaijens, Frank P T; Post, Mark J

    2010-05-28

    Skeletal muscle is an appealing topic for tissue engineering because of its variety in applications for regenerative medicine, in vitro physiological model systems, and in vitro meat production. Besides conventional biochemical cues to promote muscle tissue maturation in vitro, biophysical stimuli are necessary to reach the desired functionality and texture of the engineered tissue. Stretch, caused by active movements of the body, is an important factor present in the niche of muscle progenitor cells in vivo. We therefore investigated the effects of uniaxial ramp stretch (2%) followed by uniaxial intermittent dynamic stretch (4%) on C2C12 and murine muscle progenitor cells in a 2D and 3D environment and found that stretch negatively influenced maturation in all cases, demonstrated by decreased expression of MRFs and sarcomere proteins at the RNA level and a delay in the formation of cross striations. We therefore conclude that the current protocol is not recommended for skeletal muscle tissue engineering purposes. Copyright 2010 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2016-09-01

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

  1. Skeletal muscle ultrasonography: Visual versus quantitative evaluation.

    NARCIS (Netherlands)

    Pillen, S.; Keimpema, M. Van; Nievelstein, R.A.; Verrips, A.; Kruijsbergen-Raijmann, W. van; Zwarts, M.J.

    2006-01-01

    In this study, we compared the sensitivity and specificity of visual versus quantitative evaluation of skeletal muscle ultrasound in children suspected of having a neuromuscular disorder (NMD). Ultrasonography (US) scans of four muscles (biceps brachii, forearm flexors, quadriceps femoris, anterior

  2. Signaling pathways controlling skeletal muscle mass.

    Science.gov (United States)

    Egerman, Marc A; Glass, David J

    2014-01-01

    The molecular mechanisms underlying skeletal muscle maintenance involve interplay between multiple signaling pathways. Under normal physiological conditions, a network of interconnected signals serves to control and coordinate hypertrophic and atrophic messages, culminating in a delicate balance between muscle protein synthesis and proteolysis. Loss of skeletal muscle mass, termed "atrophy", is a diagnostic feature of cachexia seen in settings of cancer, heart disease, chronic obstructive pulmonary disease, kidney disease, and burns. Cachexia increases the likelihood of death from these already serious diseases. Recent studies have further defined the pathways leading to gain and loss of skeletal muscle as well as the signaling events that induce differentiation and post-injury regeneration, which are also essential for the maintenance of skeletal muscle mass. In this review, we summarize and discuss the relevant recent literature demonstrating these previously undiscovered mediators governing anabolism and catabolism of skeletal muscle.

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

    Science.gov (United States)

    Kim, SeungChan; Im, Wooseok

    2010-05-01

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

  4. Aging of Skeletal Muscle Fibers

    Science.gov (United States)

    Miljkovic, Natasa; Lim, Jae-Young; Miljkovic, Iva

    2015-01-01

    Aging has become an important topic for scientific research because life expectancy and the number of men and women in older age groups have increased dramatically in the last century. This is true in most countries of the world including the Republic of Korea and the United States. From a rehabilitation perspective, the most important associated issue is a progressive decline in functional capacity and independence. Sarcopenia is partly responsible for this decline. Many changes underlying the loss of muscle mass and force-generating capacity of skeletal muscle can be understood at the cellular and molecular levels. Muscle size and architecture are both altered with advanced adult age. Further, changes in myofibers include impairments in several physiological domains including muscle fiber activation, excitation-contraction coupling, actin-myosin cross-bridge interaction, energy production, and repair and regeneration. A thorough understanding of these alterations can lead to the design of improved preventative and rehabilitative interventions, such as personalized exercise training programs. PMID:25932410

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

    Directory of Open Access Journals (Sweden)

    Yue Wang

    2013-01-01

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

  6. Skeletal Muscle Microvasculature: A Highly Dynamic Lifeline.

    Science.gov (United States)

    Latroche, Claire; Gitiaux, Cyril; Chrétien, Fabrice; Desguerre, Isabelle; Mounier, Rémi; Chazaud, Bénédicte

    2015-11-01

    Skeletal muscle is highly irrigated by blood vessels. Beyond oxygen and nutrient supply, new vessel functions have been identified. This review presents vessel microanatomy and functions at tissue, cellular, and molecular levels. Mechanisms of vessel plasticity are described during skeletal muscle development and acute regeneration, and in physiological and pathological contexts. ©2015 Int. Union Physiol. Sci./Am. Physiol. Soc.

  7. Sympathetic actions on the skeletal muscle.

    Science.gov (United States)

    Roatta, Silvestro; Farina, Dario

    2010-01-01

    The sympathetic nervous system (SNS) modulates several functions in skeletal muscle fibers, including metabolism, ionic transport across the membrane, and contractility. These actions, together with the sympathetic control of other organ systems, support intense motor activity. However, some SNS actions on skeletal muscles may not always be functionally advantageous. Implications for motor control and sport performance are discussed.

  8. Dietary Flaxseed Mitigates Impaired Skeletal Muscle Regeneration: in Vivo, in Vitro and in Silico Studies

    Science.gov (United States)

    Carotenuto, Felicia; Costa, Alessandra; Albertini, Maria Cristina; Rocchi, Marco Bruno Luigi; Rudov, Alexander; Coletti, Dario; Minieri, Marilena; Di Nardo, Paolo; Teodori, Laura

    2016-01-01

    Background: Diets enriched with n-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to exert a positive impact on muscle diseases. Flaxseed is one of the richest sources of n-3 PUFA acid α-linolenic acid (ALA). The aim of this study was to assess the effects of flaxseed and ALA in models of skeletal muscle degeneration characterized by high levels of Tumor Necrosis Factor-α (TNF). Methods: The in vivo studies were carried out on dystrophic hamsters affected by muscle damage associated with high TNF plasma levels and fed with a long-term 30% flaxseed-supplemented diet. Differentiating C2C12 myoblasts treated with TNF and challenged with ALA represented the in vitro model. Skeletal muscle morphology was scrutinized by applying the Principal Component Analysis statistical method. Apoptosis, inflammation and myogenesis were analyzed by immunofluorescence. Finally, an in silico analysis was carried out to predict the possible pathways underlying the effects of n-3 PUFAs. Results: The flaxseed-enriched diet protected the dystrophic muscle from apoptosis and preserved muscle myogenesis by increasing the myogenin and alpha myosin heavy chain. Moreover, it restored the normal expression pattern of caveolin-3 thereby allowing protein retention at the sarcolemma. ALA reduced TNF-induced apoptosis in differentiating myoblasts and prevented the TNF-induced inhibition of myogenesis, as demonstrated by the increased expression of myogenin, myosin heavy chain and caveolin-3, while promoting myotube fusion. The in silico investigation revealed that FAK pathways may play a central role in the protective effects of ALA on myogenesis. Conclusions: These findings indicate that flaxseed may exert potent beneficial effects by preserving skeletal muscle regeneration and homeostasis partly through an ALA-mediated action. Thus, dietary flaxseed and ALA may serve as a useful strategy for treating patients with muscle dystrophies. PMID:26941581

  9. Human skeletal muscle releases leptin in vivo

    DEFF Research Database (Denmark)

    Wolsk, Emil; Grøndahl, Thomas Sahl; Pedersen, Bente Klarlund

    2012-01-01

    Leptin is considered an adipokine, however, cultured myocytes have also been found to release leptin. Therefore, as proof-of-concept we investigated if human skeletal muscle synthesized leptin by measuring leptin in skeletal muscle biopsies. Following this, we quantified human skeletal muscle...... and adipose tissue leptin release in vivo. We recruited 16 healthy male human participants. Catheters were inserted into the femoral artery and vein draining skeletal muscle, as well as an epigastric vein draining the abdominal subcutaneous adipose tissue. By combining the veno-arterial differences in plasma...... leptin with measurements of blood flow, leptin release from both tissues was quantified. To induce changes in leptin, the participants were infused with either saline or adrenaline in normo-physiological concentrations. The presence of leptin in skeletal muscle was confirmed by western blotting. Leptin...

  10. Channelopathies of skeletal muscle excitability

    Science.gov (United States)

    Cannon, Stephen C.

    2016-01-01

    Familial disorders of skeletal muscle excitability were initially described early in the last century and are now known to be caused by mutations of voltage-gated ion channels. The clinical manifestations are often striking, with an inability to relax after voluntary contraction (myotonia) or transient attacks of severe weakness (periodic paralysis). An essential feature of these disorders is fluctuation of symptoms that are strongly impacted by environmental triggers such as exercise, temperature, or serum K+ levels. These phenomena have intrigued physiologists for decades, and in the past 25 years the molecular lesions underlying these disorders have been identified and mechanistic studies are providing insights for therapeutic strategies of disease modification. These familial disorders of muscle fiber excitability are “channelopathies” caused by mutations of a chloride channel (ClC-1), sodium channel (NaV1.4), calcium channel (CaV1.1) and several potassium channels (Kir2.1, Kir2.6, Kir3.4). This review provides a synthesis of the mechanistic connections between functional defects of mutant ion channels, their impact on muscle excitability, how these changes cause clinical phenotypes, and approaches toward therapeutics. PMID:25880512

  11. Skeletal muscle is an endocrine organ.

    Science.gov (United States)

    Iizuka, Kenji; Machida, Takuji; Hirafuji, Masahiko

    2014-01-01

    Skeletal muscle plays a key role in postural retention as well as locomotion for maintaining the physical activities of human life. Skeletal muscle has a second role as an elaborate energy production and consumption system that influences the whole body's energy metabolism. Skeletal muscle is a specific organ that engenders a physical force, and exercise training has been known to bring about multiple benefits for human health maintenance and/or improvement. The mechanisms underlying the improvement of the human physical condition have been revealed: skeletal muscle synthesizes and secretes multiple factors, and these muscle-derived factors, so-called as myokines, exert beneficial effects on peripheral and remote organs. In this short review, we focus on the third aspect of skeletal muscle function - namely, the release of multiple types of myokines, which constitute a broad network for regulating the function of remote organs as well as skeletal muscle itself. We conclusively show that skeletal muscle is one of the endocrine organs and that understanding the mechanisms of production and secretion of myokines may lead to a new pharmacological approach for treatment of clinical disorders.

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

    Science.gov (United States)

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

    2001-05-01

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

  13. LECT2 functions as a hepatokine that links obesity to skeletal muscle insulin resistance.

    Science.gov (United States)

    Lan, Fei; Misu, Hirofumi; Chikamoto, Keita; Takayama, Hiroaki; Kikuchi, Akihiro; Mohri, Kensuke; Takata, Noboru; Hayashi, Hiroto; Matsuzawa-Nagata, Naoto; Takeshita, Yumie; Noda, Hiroyo; Matsumoto, Yukako; Ota, Tsuguhito; Nagano, Toru; Nakagen, Masatoshi; Miyamoto, Ken-ichi; Takatsuki, Kanako; Seo, Toru; Iwayama, Kaito; Tokuyama, Kunpei; Matsugo, Seiichi; Tang, Hong; Saito, Yoshiro; Yamagoe, Satoshi; Kaneko, Shuichi; Takamura, Toshinari

    2014-05-01

    Recent articles have reported an association between fatty liver disease and systemic insulin resistance in humans, but the causal relationship remains unclear. The liver may contribute to muscle insulin resistance by releasing secretory proteins called hepatokines. Here we demonstrate that leukocyte cell-derived chemotaxin 2 (LECT2), an energy-sensing hepatokine, is a link between obesity and skeletal muscle insulin resistance. Circulating LECT2 positively correlated with the severity of both obesity and insulin resistance in humans. LECT2 expression was negatively regulated by starvation-sensing kinase adenosine monophosphate-activated protein kinase in H4IIEC hepatocytes. Genetic deletion of LECT2 in mice increased insulin sensitivity in the skeletal muscle. Treatment with recombinant LECT2 protein impaired insulin signaling via phosphorylation of Jun NH2-terminal kinase in C2C12 myocytes. These results demonstrate the involvement of LECT2 in glucose metabolism and suggest that LECT2 may be a therapeutic target for obesity-associated insulin resistance.

  14. Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells

    Science.gov (United States)

    Koh, T. J.; Tidball, J. G.

    2000-01-01

    We tested the hypothesis that nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore on C(2)C(12) muscle cells, including preventing talin proteolysis and release into the cytosol and reducing loss of vinculin, cell detachment, and loss of cellular protein. These results indicate that nitric oxide inhibition of calpain protected the cells from ionophore-induced proteolysis. Calpain inhibitor I and a cell-permeable calpastatin peptide also protected the cells from proteolysis, confirming that ionophore-induced proteolysis was primarily calpain mediated. The activity of m-calpain in a casein zymogram was inhibited by sodium nitroprusside, and this inhibition was reversed by dithiothreitol. Previous incubation with the active site-targeted calpain inhibitor I prevented most of the sodium nitroprusside-induced inhibition of m-calpain activity. These data suggest that nitric oxide inhibited m-calpain activity via S-nitrosylation of the active site cysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.

  15. [In vitro construction of skeletal muscle tissues.

    Science.gov (United States)

    Morimoto, Yuya; Takeuchi, Shoji

    In conventional culture methods using culture dishes, myotubes formed by fusion of myoblasts adhere to the surface of the culture dishes. Because the adherence causes interruption of myotube contractions and immobilization of myotubes from the culture dishes, the conventional culture methods have limitations to applications of the myotubes into drug developments and medical treatments. In order to avoid their adherence, many researchers have proposed in vitro construction of skeletal muscle tissues which both ends are fixed to anchors. The skeletal muscle tissues achieve their contractions freely according to electrical stimulations or optical stimulations, and transfer of them to other experimental setup by releasing them form the anchors. By combining the skeletal muscle tissues with force sensors, the skeletal muscle tissues are available to drug screening tests based on contractile force as a functional index. Furthermore, survival of the skeletal muscle tissues are demonstrated by implantation of them to animals. Thus, in vitro constructed skeletal muscle tissues is now recognized as attractive tools in medical fields. This review will summarize fabrication methods, properties and medical applicability of the skeletal muscle tissues.

  16. Insulin resistance is associated with MCP1-mediated macrophage accumulation in skeletal muscle in mice and humans.

    Directory of Open Access Journals (Sweden)

    David Patsouris

    Full Text Available Inflammation is now recognized as a major factor contributing to type 2 diabetes (T2D. However, while the mechanisms and consequences associated with white adipose tissue inflammation are well described, very little is known concerning the situation in skeletal muscle. The aim of this study was to investigate, in vitro and in vivo, how skeletal muscle inflammation develops and how in turn it modulates local and systemic insulin sensitivity in different mice models of T2D and in humans, focusing on the role of the chemokine MCP1. Here, we found that skeletal muscle inflammation and macrophage markers are increased and associated with insulin resistance in mice models and humans. In addition, we demonstrated that intra-muscular TNFα expression is exclusively restricted to the population of intramuscular leukocytes and that the chemokine MCP1 was associated with skeletal muscle inflammatory markers in these models. Furthermore, we demonstrated that exposure of C2C12 myotubes to palmitate elevated the production of the chemokine MCP1 and that the muscle-specific overexpression of MCP1 in transgenic mice induced the local recruitment of macrophages and altered local insulin sensitivity. Overall our study demonstrates that skeletal muscle inflammation is clearly increased in the context of T2D in each one of the models we investigated, which is likely consecutive to the lipotoxic environment generated by peripheral insulin resistance, further increasing MCP1 expression in muscle. Consequently, our results suggest that MCP1-mediated skeletal muscle macrophages recruitment plays a role in the etiology of T2D.

  17. Satellite cells in human skeletal muscle plasticity.

    Science.gov (United States)

    Snijders, Tim; Nederveen, Joshua P; McKay, Bryon R; Joanisse, Sophie; Verdijk, Lex B; van Loon, Luc J C; Parise, Gianni

    2015-01-01

    Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models.

  18. Satellite cells: the architects of skeletal muscle.

    Science.gov (United States)

    Chang, Natasha C; Rudnicki, Michael A

    2014-01-01

    The outstanding regenerative capacity of skeletal muscle is attributed to the resident muscle stem cell termed satellite cell. Satellite cells are essential for skeletal muscle regeneration as they ultimately provide the myogenic precursors that rebuild damaged muscle tissue. Satellite cells characteristically are a heterogeneous population of stem cells and committed progenitor cells. Delineation of cellular hierarchy and understanding how lineage fate choices are determined within the satellite cell population will be invaluable for the advancement of muscle regenerative therapies. © 2014 Elsevier Inc. All rights reserved.

  19. Advances and challenges in skeletal muscle angiogenesis

    DEFF Research Database (Denmark)

    Olfert, I Mark; Baum, Oliver; Hellsten, Ylva

    2016-01-01

    during health, but poorly controlled in disease - resulting in either excessive capillary growth (pathological angiogenesis) or losses in capillarity (rarefaction). Given that skeletal muscle comprises nearly 40% of body mass in humans, skeletal muscle capillary density has a significant impact...... on metabolism, endocrine function, and locomotion, and is tightly regulated at many different levels. Skeletal muscle is also high adaptable, and thus one of the few organ systems which can be experimentally manipulated (e.g. by exercise) to study physiologic regulation of angiogenesis. This review will focus...

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

    Science.gov (United States)

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

    2000-01-01

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

  1. The Human Skeletal Muscle Proteome Project

    DEFF Research Database (Denmark)

    Gonzalez-Freire, Marta; Semba, Richard D.; Ubaida-Mohien, Ceereena

    2017-01-01

    Skeletal muscle is a large organ that accounts for up to half the total mass of the human body. A progressive decline in muscle mass and strength occurs with ageing and in some individuals configures the syndrome of ‘sarcopenia’, a condition that impairs mobility, challenges autonomy, and is a risk...... factor for mortality. The mechanisms leading to sarcopenia as well as myopathies are still little understood. The Human Skeletal Muscle Proteome Project was initiated with the aim to characterize muscle proteins and how they change with ageing and disease. We conducted an extensive review...... for the identification and quantification of proteins in skeletal muscle to discover new mechanisms for sarcopenia and specific muscle diseases that can be targeted for the prevention and treatment....

  2. Cardiac, Skeletal, and smooth muscle mitochondrial respiration

    DEFF Research Database (Denmark)

    Park, Song-Young; Gifford, Jayson R; Andtbacka, Robert H I

    2014-01-01

    , skeletal, and smooth muscle was harvested from a total of 22 subjects (53±6 yrs) and mitochondrial respiration assessed in permeabilized fibers. Complex I+II, state 3 respiration, an index of oxidative phosphorylation capacity, fell progressively from cardiac, skeletal, to smooth muscle (54±1; 39±4; 15......±1 pmol•s(-1)•mg (-1), pindex of mitochondrial density, also fell progressively from cardiac, skeletal, to smooth muscle (222±13; 115±2; 48±2 umol•g(-1)•min(-1), p... per mitochondrial content), oxidative phosphorylation capacity was no longer different between the three muscle types. Interestingly, Complex I state 2 normalized for CS activity, an index of non-phosphorylating respiration per mitochondrial content, increased progressively from cardiac, skeletal...

  3. Skeletal muscle glucose uptake during exercise

    DEFF Research Database (Denmark)

    Rose, Adam John; Richter, Erik

    2005-01-01

    The increase in skeletal muscle glucose uptake during exercise results from a coordinated increase in rates of glucose delivery (higher capillary perfusion), surface membrane glucose transport, and intracellular substrate flux through glycolysis. The mechanism behind the movement of GLUT4...

  4. Pathogenesis of Insulin Resistance in Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Muhammad A. Abdul-Ghani

    2010-01-01

    Full Text Available Insulin resistance in skeletal muscle is manifested by decreased insulin-stimulated glucose uptake and results from impaired insulin signaling and multiple post-receptor intracellular defects including impaired glucose transport, glucose phosphorylation, and reduced glucose oxidation and glycogen synthesis. Insulin resistance is a core defect in type 2 diabetes, it is also associated with obesity and the metabolic syndrome. Dysregulation of fatty acid metabolism plays a pivotal role in the pathogenesis of insulin resistance in skeletal muscle. Recent studies have reported a mitochondrial defect in oxidative phosphorylation in skeletal muscle in variety of insulin resistant states. In this review, we summarize the cellular and molecular defects that contribute to the development of insulin resistance in skeletal muscle.

  5. Fiber types in mammalian skeletal muscles

    National Research Council Canada - National Science Library

    Schiaffino, Stefano; Reggiani, Carlo

    2011-01-01

    Mammalian skeletal muscle comprises different fiber types, whose identity is first established during embryonic development by intrinsic myogenic control mechanisms and is later modulated by neural and hormonal factors...

  6. Space travel directly induces skeletal muscle atrophy

    Science.gov (United States)

    Vandenburgh, H.; Chromiak, J.; Shansky, J.; Del Tatto, M.; Lemaire, J.

    1999-01-01

    Space travel causes rapid and pronounced skeletal muscle wasting in humans that reduces their long-term flight capabilities. To develop effective countermeasures, the basis of this atrophy needs to be better understood. Space travel may cause muscle atrophy indirectly by altering circulating levels of factors such as growth hormone, glucocorticoids, and anabolic steroids and/or by a direct effect on the muscle fibers themselves. To determine whether skeletal muscle cells are directly affected by space travel, tissue-cultured avian skeletal muscle cells were tissue engineered into bioartificial muscles and flown in perfusion bioreactors for 9 to 10 days aboard the Space Transportation System (STS, i.e., Space Shuttle). Significant muscle fiber atrophy occurred due to a decrease in protein synthesis rates without alterations in protein degradation. Return of the muscle cells to Earth stimulated protein synthesis rates of both muscle-specific and extracellular matrix proteins relative to ground controls. These results show for the first time that skeletal muscle fibers are directly responsive to space travel and should be a target for countermeasure development.

  7. Mitochondria mediate tumor necrosis factor-alpha/NF-kappaB signaling in skeletal muscle myotubes

    Science.gov (United States)

    Li, Y. P.; Atkins, C. M.; Sweatt, J. D.; Reid, M. B.; Hamilton, S. L. (Principal Investigator)

    1999-01-01

    Tumor necrosis factor-alpha (TNF-alpha) is implicated in muscle atrophy and weakness associated with a variety of chronic diseases. Recently, we reported that TNF-alpha directly induces muscle protein degradation in differentiated skeletal muscle myotubes, where it rapidly activates nuclear factor kappaB (NF-kappaB). We also have found that protein loss induced by TNF-alpha is NF-kappaB dependent. In the present study, we analyzed the signaling pathway by which TNF-alpha activates NF-kappaB in myotubes differentiated from C2C12 and rat primary myoblasts. We found that activation of NF-kappaB by TNF-alpha was blocked by rotenone or amytal, inhibitors of complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, an inhibitor of complex III, enhanced TNF-alpha activation of NK-kappaB. These results suggest a key role of mitochondria-derived reactive oxygen species (ROS) in mediating NF-kappaB activation in muscle. In addition, we found that TNF-alpha stimulated protein kinase C (PKC) activity. However, other signal transduction mediators including ceramide, Ca2+, phospholipase A2 (PLA2), and nitric oxide (NO) do not appear to be involved in the activation of NF-kappaB.

  8. The skeletal muscle circadian clock: current insights

    Directory of Open Access Journals (Sweden)

    Nakao R

    2017-11-01

    Full Text Available Reiko Nakao,1 Takeshi Nikawa,2 Katsutaka Oishi1,3,4 1Biological Clock Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST, Tsukuba, 2Department of Nutritional Physiology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, 3Department of Applied Biological Science, Graduate School of Science and Technology, Tokyo University of Science, Noda, 4Department of Computational and Medical Sciences, Graduate School of Frontier Sciences, the University of Tokyo, Kashiwa, Japan Abstract: Skeletal muscle functions in locomotion, postural support, and energy metabolism. The loss of skeletal muscle mass and function leads to diseases such as sarcopenia and metabolic disorders. Inactivity (lack of exercise and an imbalanced diet (increased fat or decreased protein intake are thought to be involved in the prevalence of such pathologies. On the other hand, recent epidemiological studies of humans have suggested that circadian disruption caused by shift work, jet lag, and sleep disorders is associated with obesity and metabolic syndrome. Experimental studies of mice deficient in clock genes have also identified skeletal muscle defects, suggesting a molecular link between circadian clock machinery and skeletal muscle physiology. Furthermore, accumulating evidence about chronotherapy, including chronopharmacology, chrononutrition, and chronoexercise, has indicated that timing is important to optimize medical intervention for various diseases. The present review addresses current understanding of the functional roles of the molecular clock with respect to skeletal muscle and the potential of chronotherapy for diseases associated with skeletal muscle. Keywords: biological rhythm, metabolic syndrome, physical activity, neural signal, chronotherapy

  9. ISOLATION OF SKELETAL MUSCLE NUCLEI

    Science.gov (United States)

    Edelman, Jean C.; Edelman, P. Michael; Knigge, Karl M.; Schwartz, Irving L.

    1965-01-01

    A method employing aqueous media for isolation of nuclei from rat skeletal muscle is described. The technique involves (a) mincing and then homogenizing in a 0.32 M sucrose-salt solution with a Potter-Elvehjem type homogenizer using a Delrin (an acetal resin) pestle and a carefully controlled, relatively large pestle-to-glass clearance, (b) filtering through fiberglass and stainless steel screens of predetermined mesh size to remove myofibrils and connective tissue, and (c) centrifuging in a 2.15 M sucrose-salt solution containing 0.7 mM ATP. Electron and phase-contrast microscopic observations show that the nuclei are intact, unencumbered by cytoplasmic tags, and possess well preserved distinct nucleoli, nucleoplasm, and nuclear membranes. Cytoplasmic contamination is minimal and mainly mitochondrial. Chemical assays of the nuclear fraction show that the DNA/protein and RNA/DNA ratios are comparable to those obtained in other tissues. These ratios, as well as the low specific activity obtained for cytochrome c oxidase and the virtual absence of myofibrillar ATPase, indicate a high degree of purity with minimal mitochondrial and myofibrillar contamination. The steps comprising the technique and the reasons for their selection are discussed. PMID:4287141

  10. Intraurethral Injection of Autologous Minced Skeletal Muscle

    DEFF Research Database (Denmark)

    Gräs, Søren; Klarskov, Niels; Lose, Gunnar

    2014-01-01

    PURPOSE: Intraurethral injection of in vitro expanded autologous skeletal muscle derived cells is a new regenerative therapy for stress urinary incontinence. We examined the efficacy and safety of a simpler alternative strategy using freshly harvested, minced autologous skeletal muscle tissue...... with its inherent content of regenerative cells. MATERIALS AND METHODS: A total of 20 and 15 women with uncomplicated and complicated stress urinary incontinence, respectively, received intraurethral injections of minced autologous skeletal muscle tissue and were followed for 1 year. Efficacy was assessed...... noted. CONCLUSIONS: Intraurethral injection of minced autologous muscle tissue is a simple surgical procedure that appears safe and moderately effective in women with uncomplicated stress urinary incontinence. It compares well to a more complicated regenerative strategy using in vitro expanded muscle...

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

    Directory of Open Access Journals (Sweden)

    Lomax Michael A

    2007-03-01

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

  12. Skeletal muscle tensile strain dependence: hyperviscoelastic nonlinearity

    Science.gov (United States)

    Wheatley, Benjamin B; Morrow, Duane A; Odegard, Gregory M; Kaufman, Kenton R; Donahue, Tammy L Haut

    2015-01-01

    Introduction Computational modeling of skeletal muscle requires characterization at the tissue level. While most skeletal muscle studies focus on hyperelasticity, the goal of this study was to examine and model the nonlinear behavior of both time-independent and time-dependent properties of skeletal muscle as a function of strain. Materials and Methods Nine tibialis anterior muscles from New Zealand White rabbits were subject to five consecutive stress relaxation cycles of roughly 3% strain. Individual relaxation steps were fit with a three-term linear Prony series. Prony series coefficients and relaxation ratio were assessed for strain dependence using a general linear statistical model. A fully nonlinear constitutive model was employed to capture the strain dependence of both the viscoelastic and instantaneous components. Results Instantaneous modulus (p0.1). Additionally, the fully nonlinear hyperviscoelastic constitutive model provided an excellent fit to experimental data, while other models which included linear components failed to capture muscle function as accurately. Conclusions Material properties of skeletal muscle are strain-dependent at the tissue level. This strain dependence can be included in computational models of skeletal muscle performance with a fully nonlinear hyperviscoelastic model. PMID:26409235

  13. Ghrelin improves body weight loss and skeletal muscle catabolism associated with angiotensin II-induced cachexia in mice.

    Science.gov (United States)

    Sugiyama, Masako; Yamaki, Akira; Furuya, Mayumi; Inomata, Norio; Minamitake, Yoshiharu; Ohsuye, Kazuhiro; Kangawa, Kenji

    2012-10-10

    Ghrelin is a gastric peptide that regulates energy homeostasis. Angiotensin II (Ang II) is known to induce body weight loss and skeletal muscle catabolism through the ubiquitin-proteasome pathway. In this study, we investigated the effects of ghrelin on body weight and muscle catabolism in mice treated with Ang II. The continuous subcutaneous administration of Ang II to mice for 6 days resulted in cardiac hypertrophy and significant decreases in body weight gain, food intake, food efficiency, lean mass, and fat mass. In the gastrocnemius muscles of Ang II-treated mice, the levels of insulin-like growth factor 1 (IGF-1) were decreased, and the levels of mRNA expression of catabolic factors were increased. Although the repeated subcutaneous injections of ghrelin (1.0mg/kg, twice daily for 5 days) did not affect cardiac hypertrophy, they resulted in significant body weight gains and improved food efficiencies and tended to increase both lean and fat mass in Ang II-treated mice. Ghrelin also ameliorated the decreased IGF-1 levels and the increased mRNA expression levels of catabolic factors in the skeletal muscle. IGF-1 mRNA levels in the skeletal muscle significantly decreased 24h after Ang II infusion, and this was reversed by two subcutaneous injections of ghrelin. In C2C12-derived myocytes, the dexamethasone-induced mRNA expression of atrogin-1 was decreased by IGF-1 but not by ghrelin. In conclusion, we demonstrated that ghrelin improved body weight loss and skeletal muscle catabolism in mice treated with Ang II, possibly through the early restoration of IGF-1 mRNA in the skeletal muscle and the amelioration of nutritional status. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Skeletal muscle development and regeneration.

    NARCIS (Netherlands)

    Grefte, S.; Kuijpers-Jagtman, A.M.; Torensma, R.; Hoff, J.W. Von den

    2007-01-01

    In the late stages of muscle development, a unique cell population emerges that is a key player in postnatal muscle growth and muscle regeneration. The location of these cells next to the muscle fibers triggers their designation as satellite cells. During the healing of injured muscle tissue,

  15. Interleukin-6 myokine signaling in skeletal muscle

    DEFF Research Database (Denmark)

    Muñoz-Cánoves, Pura; Scheele, Camilla; Pedersen, Bente K

    2013-01-01

    Interleukin (IL)-6 is a cytokine with pleiotropic functions in different tissues and organs. Skeletal muscle produces and releases significant levels of IL-6 after prolonged exercise and is therefore considered as a myokine. Muscle is also an important target of the cytokine. IL-6 signaling has b...

  16. Connexins in skeletal muscle development and disease.

    Science.gov (United States)

    Merrifield, Peter A; Laird, Dale W

    2016-02-01

    Gap junctions consist of clusters of intercellular channels composed of connexins that connect adjacent cells and allow the exchange of small molecules. While the 21 member multi-gene family of connexins are ubiquitously found in humans, only Cx39, Cx40, Cx43 and Cx45 have been documented in developing myoblasts and injured adult skeletal muscle while healthy adult skeletal muscle is devoid of connexins. The use of gap junctional blockers and cultured myoblast cell lines have suggested that these connexins play a critical role in myotube formation and muscle regeneration. More recent genetically-modified mouse models where Cx43 function is greatly compromized or ablated have further supported a role for Cx43 in regulating skeletal muscle development. In the last decade, we have become aware of a cohort of patients that have a development disorder known as oculodentodigital dysplasia (ODDD). These patients harbor either gain or loss of Cx43 function gene mutations that result in many organ anomalies raising questions as to whether they suffer from defects in skeletal muscle formation or regeneration upon injury. Interesting, some ODDD patients report muscle weakness and loss of limb control but it is not clear if this is neurogenic or myogenic in origin. This review will focus on the role connexins play in muscle development and repair and discuss the impact of Cx43 mutants on muscle function. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  18. Vasodilatory mechanisms in contracting skeletal muscle

    DEFF Research Database (Denmark)

    Clifford, Philip S.; Hellsten, Ylva

    2004-01-01

    Skeletal muscle blood flow is closely coupled to metabolic demand, and its regulation is believed to be mainly the result of the interplay of neural vasoconstrictor activity and locally derived vasoactive substances. Muscle blood flow is increased within the first second after a single contraction...... and stabilizes within 30 s during dynamic exercise under normal conditions. Vasodilator substances may be released from contracting skeletal muscle, vascular endothelium, or red blood cells. The importance of specific vasodilators is likely to vary over the time course of flow, from the initial rapid rise...

  19. Effect of Exercise Intensity on Isoform-Specific Expressions of NT-PGC-1α mRNA in Mouse Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Xingyuan Wen

    2014-01-01

    Full Text Available PGC-1α is an inducible transcriptional coactivator that regulates mitochondrial biogenesis and cellular energy metabolism in skeletal muscle. Recent studies have identified two additional PGC-1α transcripts that are derived from an alternative exon 1 (exon 1b and induced by exercise. Given that the PGC-1α gene also produces NT-PGC-1α transcript by alternative 3′ splicing between exon 6 and exon 7, we have investigated isoform-specific expression of NT-PGC-1α mRNA in mouse skeletal muscle during physical exercise with different intensities. We report here that NT-PGC-1α-a mRNA expression derived from a canonical exon 1 (exon 1a is increased by high-intensity exercise and AMPK activator AICAR in mouse skeletal muscle but not altered by low- and medium-intensity exercise and β2-adrenergic receptor agonist clenbuterol. In contrast, the alternative exon 1b-driven NT-PGC-1α-b (PGC-1α4 and NT-PGC-1α-c are highly induced by low-, medium-, and high-intensity exercise, AICAR, and clenbuterol. Ectopic expression of NT-PGC-1α-a in C2C12 myotube cells upregulates myosin heavy chain (MHC I, MHC II a and Glut4, which represent oxidative fibers, and promotes the expression of mitochondrial genes (Cyc1, COX5B, and ATP5B. In line with gene expression data, citrate synthase activity was significantly increased by NT-PGC-1α-a in C2C12 myotube cells. Our results indicate the regulatory role for NT-PGC-1α-a in mitochondrial biogenesis and adaptation of skeletal muscle to endurance exercise.

  20. Skeletal muscle as an immunogenic organ

    DEFF Research Database (Denmark)

    Nielsen, Søren; Pedersen, Bente Klarlund

    2008-01-01

    During the past few years, a possible link between skeletal muscle contractile activity and immune changes has been established. This concept is based on the finding that exercise provokes an increase in a number of cytokines. We have suggested that cytokines and other peptides that are produced......; expressed and released by muscle fibers and exert either paracrine or endocrine effects should be classified as 'myokines'. Human skeletal muscle has the capacity to express several myokines belonging to distinct different cytokine classes and contractile activity plays a role in regulating the expression...... of cytokines in skeletal muscle. In the present review, we focus on the myokines interleukin (IL)-6, IL-8 and IL-15 and their possible anti-inflammatory, immunoregulatory and metabolic roles....

  1. Lactate oxidation in human skeletal muscle mitochondria

    DEFF Research Database (Denmark)

    Jacobs, Robert A; Meinild, Anne-Kristine; Nordsborg, Nikolai B

    2013-01-01

    Lactate is an important intermediate metabolite in human bioenergetics and is oxidized in many different tissues including the heart, brain, kidney, adipose tissue, liver, and skeletal muscle. The mechanism(s) explaining the metabolism of lactate in these tissues, however, remains unclear. Here, we...... analyze the ability of skeletal muscle to respire lactate by using an in situ mitochondrial preparation that leaves the native tubular reticulum and subcellular interactions of the organelle unaltered. Skeletal muscle biopsies were obtained from vastus lateralis muscle in 16 human subjects. Samples were...... of four separate and specific substrate titration protocols, the respirometric analysis revealed that mitochondria were capable of oxidizing lactate in the absence of exogenous LDH. The titration of lactate and NAD(+) into the respiration medium stimulated respiration (P = 0.003). The addition...

  2. The benefits of coffee on skeletal muscle.

    Science.gov (United States)

    Dirks-Naylor, Amie J

    2015-12-15

    Coffee is consumed worldwide with greater than a billion cups of coffee ingested every day. Epidemiological studies have revealed an association of coffee consumption with reduced incidence of a variety of chronic diseases as well as all-cause mortality. Current research has primarily focused on the effects of coffee or its components on various organ systems such as the cardiovascular system, with relatively little attention on skeletal muscle. Summary of current literature suggests that coffee has beneficial effects on skeletal muscle. Coffee has been shown to induce autophagy, improve insulin sensitivity, stimulate glucose uptake, slow the progression of sarcopenia, and promote the regeneration of injured muscle. Much more research is needed to reveal the full scope of benefits that coffee consumption may exert on skeletal muscle structure and function. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. STAT3 Activation in Skeletal Muscle Links Muscle Wasting and the Acute Phase Response in Cancer Cachexia

    Science.gov (United States)

    Kunzevitzky, Noelia; Guttridge, Denis C.; Khuri, Sawsan; Koniaris, Leonidas G.; Zimmers, Teresa A.

    2011-01-01

    Background Cachexia, or weight loss despite adequate nutrition, significantly impairs quality of life and response to therapy in cancer patients. In cancer patients, skeletal muscle wasting, weight loss and mortality are all positively associated with increased serum cytokines, particularly Interleukin-6 (IL-6), and the presence of the acute phase response. Acute phase proteins, including fibrinogen and serum amyloid A (SAA) are synthesized by hepatocytes in response to IL-6 as part of the innate immune response. To gain insight into the relationships among these observations, we studied mice with moderate and severe Colon-26 (C26)-carcinoma cachexia. Methodology/Principal Findings Moderate and severe C26 cachexia was associated with high serum IL-6 and IL-6 family cytokines and highly similar patterns of skeletal muscle gene expression. The top canonical pathways up-regulated in both were the complement/coagulation cascade, proteasome, MAPK signaling, and the IL-6 and STAT3 pathways. Cachexia was associated with increased muscle pY705-STAT3 and increased STAT3 localization in myonuclei. STAT3 target genes, including SOCS3 mRNA and acute phase response proteins, were highly induced in cachectic muscle. IL-6 treatment and STAT3 activation both also induced fibrinogen in cultured C2C12 myotubes. Quantitation of muscle versus liver fibrinogen and SAA protein levels indicates that muscle contributes a large fraction of serum acute phase proteins in cancer. Conclusions/Significance These results suggest that the STAT3 transcriptome is a major mechanism for wasting in cancer. Through IL-6/STAT3 activation, skeletal muscle is induced to synthesize acute phase proteins, thus establishing a molecular link between the observations of high IL-6, increased acute phase response proteins and muscle wasting in cancer. These results suggest a mechanism by which STAT3 might causally influence muscle wasting by altering the profile of genes expressed and translated in muscle such

  4. Tumor necrosis factor-α regulates distinct molecular pathways and gene networks in cultured skeletal muscle cells.

    Directory of Open Access Journals (Sweden)

    Shephali Bhatnagar

    2010-10-01

    Full Text Available Skeletal muscle wasting is a debilitating consequence of large number of disease states and conditions. Tumor necrosis factor-α (TNF-α is one of the most important muscle-wasting cytokine, elevated levels of which cause significant muscular abnormalities. However, the underpinning molecular mechanisms by which TNF-α causes skeletal muscle wasting are less well-understood.We have used microarray, quantitative real-time PCR (QRT-PCR, Western blot, and bioinformatics tools to study the effects of TNF-α on various molecular pathways and gene networks in C2C12 cells (a mouse myoblastic cell line. Microarray analyses of C2C12 myotubes treated with TNF-α (10 ng/ml for 18h showed differential expression of a number of genes involved in distinct molecular pathways. The genes involved in nuclear factor-kappa B (NF-kappaB signaling, 26s proteasome pathway, Notch1 signaling, and chemokine networks are the most important ones affected by TNF-α. The expression of some of the genes in microarray dataset showed good correlation in independent QRT-PCR and Western blot assays. Analysis of TNF-treated myotubes showed that TNF-α augments the activity of both canonical and alternative NF-κB signaling pathways in myotubes. Bioinformatics analyses of microarray dataset revealed that TNF-α affects the activity of several important pathways including those involved in oxidative stress, hepatic fibrosis, mitochondrial dysfunction, cholesterol biosynthesis, and TGF-β signaling. Furthermore, TNF-α was found to affect the gene networks related to drug metabolism, cell cycle, cancer, neurological disease, organismal injury, and abnormalities in myotubes.TNF-α regulates the expression of multiple genes involved in various toxic pathways which may be responsible for TNF-induced muscle loss in catabolic conditions. Our study suggests that TNF-α activates both canonical and alternative NF-κB signaling pathways in a time-dependent manner in skeletal muscle cells

  5. Skeletal muscle pathology in Huntington's disease.

    Science.gov (United States)

    Zielonka, Daniel; Piotrowska, Izabela; Marcinkowski, Jerzy T; Mielcarek, Michal

    2014-01-01

    Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by the expansion of a polyglutamine stretch within the huntingtin protein (HTT). The neurological symptoms, that involve motor, cognitive and psychiatric disturbances, are caused by neurodegeneration that is particularly widespread in the basal ganglia and cereberal cortex. HTT is ubiquitously expressed and in recent years it has become apparent that HD patients experience a wide array of peripheral organ dysfunction including severe metabolic phenotype, weight loss, HD-related cardiomyopathy and skeletal muscle wasting. Although skeletal muscles pathology became a hallmark of HD, the mechanisms underlying muscular atrophy in this disorder are unknown. Skeletal muscles account for approximately 40% of body mass and are highly adaptive to physiological and pathological conditions that may result in muscle hypertrophy (due to increased mechanical load) or atrophy (inactivity, chronic disease states). The atrophy is caused by degeneration of myofibers and their replacement by fibrotic tissue is the major pathological feature in many genetic muscle disorders. Under normal physiological conditions the muscle function is orchestrated by a network of intrinsic hypertrophic and atrophic signals linked to the functional properties of the motor units that are likely to be imbalanced in HD. In this article, we highlight the emerging field of research with particular focus on the recent studies of the skeletal muscle pathology and the identification of new disease-modifying treatments.

  6. β-agonists selectively modulate proinflammatory gene expression in skeletal muscle cells via non-canonical nuclear crosstalk mechanisms.

    Directory of Open Access Journals (Sweden)

    Krzysztof Kolmus

    Full Text Available The proinflammatory cytokine Tumour Necrosis Factor (TNF-α is implicated in a variety of skeletal muscle pathologies. Here, we have investigated how in vitro cotreatment of skeletal muscle C2C12 cells with β-agonists modulates the TNF-α-induced inflammatory program. We observed that C2C12 myotubes express functional TNF receptor 1 (TNF-R1 and β2-adrenoreceptors (β2-ARs. TNF-α activated the canonical Nuclear Factor-κB (NF-κB pathway and Mitogen-Activated Protein Kinases (MAPKs, culminating in potent induction of NF-κB-dependent proinflammatory genes. Cotreatment with the β-agonist isoproterenol potentiated the expression of inflammatory mediators, including Interleukin-6 (IL-6 and several chemokines. The enhanced production of chemotactic factors upon TNF-α/isoproterenol cotreatment was also suggested by the results from migrational analysis. Whereas we could not explain our observations by cytoplasmic crosstalk, we found that TNF-R1-and β2-AR-induced signalling cascades cooperate in the nucleus. Using the IL-6 promoter as a model, we demonstrated that TNF-α/isoproterenol cotreatment provoked phosphorylation of histone H3 at serine 10, concomitant with enhanced promoter accessibility and recruitment of the NF-κB p65 subunit, cAMP-response element-binding protein (CREB, CREB-binding protein (CBP and RNA polymerase II. In summary, we show that β-agonists potentiate TNF-α action, via nuclear crosstalk, that promotes chromatin relaxation at selected gene promoters. Our data warrant further study into the mode of action of β-agonists and urge for caution in their use as therapeutic agents for muscular disorders.

  7. High-density lipoprotein maintains skeletal muscle function by modulating cellular respiration in mice.

    Science.gov (United States)

    Lehti, Maarit; Donelan, Elizabeth; Abplanalp, William; Al-Massadi, Omar; Habegger, Kirk M; Weber, Jon; Ress, Chandler; Mansfeld, Johannes; Somvanshi, Sonal; Trivedi, Chitrang; Keuper, Michaela; Ograjsek, Teja; Striese, Cynthia; Cucuruz, Sebastian; Pfluger, Paul T; Krishna, Radhakrishna; Gordon, Scott M; Silva, R A Gangani D; Luquet, Serge; Castel, Julien; Martinez, Sarah; D'Alessio, David; Davidson, W Sean; Hofmann, Susanna M

    2013-11-26

    Abnormal glucose metabolism is a central feature of disorders with increased rates of cardiovascular disease. Low levels of high-density lipoprotein (HDL) are a key predictor for cardiovascular disease. We used genetic mouse models with increased HDL levels (apolipoprotein A-I transgenic [apoA-I tg]) and reduced HDL levels (apoA-I-deficient [apoA-I ko]) to investigate whether HDL modulates mitochondrial bioenergetics in skeletal muscle. ApoA-I ko mice exhibited fasting hyperglycemia and impaired glucose tolerance test compared with wild-type mice. Mitochondria isolated from gastrocnemius muscle of apoA-I ko mice displayed markedly blunted ATP synthesis. Endurance capacity during exercise exhaustion test was impaired in apoA-I ko mice. HDL directly enhanced glucose oxidation by increasing glycolysis and mitochondrial respiration rate in C2C12 muscle cells. ApoA-I tg mice exhibited lower fasting glucose levels, improved glucose tolerance test, increased lactate levels, reduced fat mass, associated with protection against age-induced decline of endurance capacity compared with wild-type mice. Circulating levels of fibroblast growth factor 21, a novel biomarker for mitochondrial respiratory chain deficiencies and inhibitor of white adipose lipolysis, were significantly reduced in apoA-I tg mice. Consistent with an increase in glucose utilization of skeletal muscle, genetically increased HDL and apoA-I levels in mice prevented high-fat diet-induced impairment of glucose homeostasis. In view of impaired mitochondrial function and decreased HDL levels in type 2 diabetes mellitus, our findings indicate that HDL-raising therapies may preserve muscle mitochondrial function and address key aspects of type 2 diabetes mellitus beyond cardiovascular disease.

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

    Directory of Open Access Journals (Sweden)

    Friedmann Theodore

    2009-08-01

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

  9. Role of skeletal muscle in lung development.

    Science.gov (United States)

    Baguma-Nibasheka, Mark; Gugic, Dijana; Saraga-Babic, Mirna; Kablar, Boris

    2012-07-01

    Skeletal (striated) muscle is one of the four basic tissue types, together with the epithelium, connective and nervous tissues. Lungs, on the other hand, develop from the foregut and among various cell types contain smooth, but not skeletal muscle. Therefore, during earlier stages of development, it is unlikely that skeletal muscle and lung depend on each other. However, during the later stages of development, respiratory muscle, primarily the diaphragm and the intercostal muscles, execute so called fetal breathing-like movements (FBMs), that are essential for lung growth and cell differentiation. In fact, the absence of FBMs results in pulmonary hypoplasia, the most common cause of death in the first week of human neonatal life. Most knowledge on this topic arises from in vivo experiments on larger animals and from various in vitro experiments. In the current era of mouse mutagenesis and functional genomics, it was our goal to develop a mouse model for pulmonary hypoplasia. We employed various genetically engineered mice lacking different groups of respiratory muscles or lacking all the skeletal muscle and established the criteria for pulmonary hypoplasia in mice, and therefore established a mouse model for this disease. We followed up this discovery with systematic subtractive microarray analysis approach and revealed novel functions in lung development and disease for several molecules. We believe that our approach combines elements of both in vivo and in vitro approaches and allows us to study the function of a series of molecules in the context of lung development and disease and, simultaneously, in the context of lung's dependence on skeletal muscle-executed FBMs.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-05-01

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

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

    DEFF Research Database (Denmark)

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

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

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  13. Omega-3 fatty acids differentially modulate enzymatic anti-oxidant systems in skeletal muscle cells.

    Science.gov (United States)

    da Silva, E P; Nachbar, R T; Levada-Pires, A C; Hirabara, S M; Lambertucci, R H

    2016-01-01

    During physical activity, increased reactive oxygen species production occurs, which can lead to cell damage and in a decline of individual's performance and health. The use of omega-3 polyunsaturated fatty acids as a supplement to protect the immune system has been increasing; however, their possible benefit to the anti-oxidant system is not well described. Thus, the aim of this study was to evaluate whether the omega-3 fatty acids (docosahexaenoic acid and eicosapentaenoic acid) can be beneficial to the anti-oxidant system in cultured skeletal muscle cells. C2C12 myocytes were differentiated and treated with either eicosapentaenoic acid or docosahexaenoic acid for 24 h. Superoxide content was quantified using the dihydroethidine oxidation method and superoxide dismutase, catalase, and glutathione peroxidase activity, and expression was quantified. We observed that the docosahexaenoic fatty acids caused an increase in superoxide production. Eicosapentaenoic acid induced catalase activity, while docosahexaenoic acid suppressed superoxide dismutase activity. In addition, we found an increased protein expression of the total manganese superoxide dismutase and catalase enzymes when cells were treated with eicosapentaenoic acid. Taken together, these data indicate that the use of eicosapentaenoic acid may present both acute and chronic benefits; however, the treatment with DHA may not be beneficial to muscle cells.

  14. Transplantation of Skeletal Muscle Stem Cells.

    Science.gov (United States)

    Hall, Monica N; Hall, John K; Cadwallader, Adam B; Pawlikowski, Bradley T; Doles, Jason D; Elston, Tiffany L; Olwin, Bradley B

    2017-01-01

    Transplanting adult stem cells provides a stringent test for self-renewal and the assessment of comparative engraftment in competitive transplant assays. Transplantation of satellite cells into mammalian skeletal muscle provided the first critical evidence that satellite cells function as adult muscle stem cells. Transplantation of a single satellite cell confirmed and extended this hypothesis, providing proof that the satellite cell is a bona fide adult skeletal muscle stem cell as reported by Sacco et al. (Nature 456(7221):502-506). Satellite cell transplantation has been further leveraged to identify culture conditions that maintain engraftment and to identify self-renewal deficits in satellite cells from aged mice. Conversion of iPSCs (induced pluripotent stem cells) to a satellite cell-like state, followed by transplantation, demonstrated that these cells possess adult muscle stem cell properties as reported by Darabi et al. (Stem Cell Rev Rep 7(4):948-957) and Mizuno et al. (FASEB J 24(7):2245-2253). Thus, transplantation strategies involving either satellite cells derived from adult muscles or derived from iPSCs may eventually be exploited as a therapy for treating patients with diseased or failing skeletal muscle. Here, we describe methods for isolating dispersed adult mouse satellite cells and satellite cells on intact myofibers for transplantation into recipient mice to study muscle stem cell function and behavior following engraftment .

  15. Satellite cells in human skeletal muscle plasticity

    Directory of Open Access Journals (Sweden)

    Tim eSnijders

    2015-10-01

    Full Text Available Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodelling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodelling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodelling in the wider context of satellite cell biology whose literature is largely based on animal and cell models.

  16. Mechanotransduction pathways in skeletal muscle hypertrophy.

    Science.gov (United States)

    Yamada, André Katayama; Verlengia, Rozangela; Bueno Junior, Carlos Roberto

    2012-02-01

    In the last decade, molecular biology has contributed to define some of the cellular events that trigger skeletal muscle hypertrophy. Recent evidence shows that insulin like growth factor 1/phosphatidyl inositol 3-kinase/protein kinase B (IGF-1/PI3K/Akt) signaling is not the main pathway towards load-induced skeletal muscle hypertrophy. During load-induced skeletal muscle hypertrophy process, activation of mTORC1 does not require classical growth factor signaling. One potential mechanism that would activate mTORC1 is increased synthesis of phosphatidic acid (PA). Despite the huge progress in this field, it is still early to affirm which molecular event induces hypertrophy in response to mechanical overload. Until now, it seems that mTORC1 is the key regulator of load-induced skeletal muscle hypertrophy. On the other hand, how mTORC1 is activated by PA is unclear, and therefore these mechanisms have to be determined in the following years. The understanding of these molecular events may result in promising therapies for the treatment of muscle-wasting diseases. For now, the best approach is a good regime of resistance exercise training. The objective of this point-of-view paper is to highlight mechanotransduction events, with focus on the mechanisms of mTORC1 and PA activation, and the role of IGF-1 on hypertrophy process.

  17. Disease-Induced Skeletal Muscle Atrophy and Fatigue

    NARCIS (Netherlands)

    Powers, Scott K.; Lynch, Gordon S.; Murphy, Kate T.; Reid, Michael B.; Zijdewind, Inge

    2016-01-01

    Numerous health problems including acute critical illness, cancer, diseases associated with chronic inflammation, and neurological disorders often result in skeletal muscle weakness and fatigue. Disease-related muscle atrophy and fatigue is an important clinical problem because acquired skeletal

  18. Resveratrol Ameliorates Palmitate-Induced Inflammation in Skeletal Muscle Cells by Attenuating Oxidative Stress and JNK/NF-κB Pathway in a SIRT1-Independent Mechanism.

    Science.gov (United States)

    Sadeghi, Asie; Seyyed Ebrahimi, Shadi Sadat; Golestani, Abolfazl; Meshkani, Reza

    2017-09-01

    Resveratrol has been shown to exert anti-inflammatory and anti-oxidant effects in a variety of cell types, however, its role in prevention of inflammatory responses mediated by palmitate in skeletal muscle cells remains unexplored. In the present study, we investigated the effects of resveratrol on palmitate-induced inflammation and elucidated the underlying mechanisms in skeletal muscle cells. The results showed that palmitate significantly enhanced TNF-α and IL-6 mRNA expression and protein secretion from C2C12 cells at 12, 24, and 36 h treatments. Increased expression of cytokines was accompanied by an enhanced phosphorylation of JNK, P38, ERK1/2, and IKKα/IKKβ. In addition, JNK and P38 inhibitors could significantly attenuate palmitate-induced mRNA expression of TNF-α and IL-6, respectively, whereas NF-κB inhibitor reduced the expression of both cytokines in palmitate-treated cells. Resveratrol pretreatment significantly prevented palmitate-induced TNF-α and IL-6 mRNA expression and protein secretion in C2C12 cells. Importantly, pre-treatment of the cells with resveratrol completely abrogated the phosphorylation of ERK1/2, JNK, and IKKα/IKKβ in palmitate treated cells. The protection from palmitate-induced inflammation by resveratrol was accompanied by a decrease in the generation of reactive oxygen species (ROS). N-acetyl cysteine (NAC), a known scavenger of ROS, could protect palmitate-induced expression of TNF-α and IL-6. Furthermore, inhibition of SIRT1 by shRNA or sirtinol demonstrated that the anti-inflammatory effect of resveratrol in muscle cells is mediated through a SIRT1-independent mechanism. Taken together, these findings suggest that resveratrol may represent a promising therapy for prevention of inflammation in skeletal muscle cells. J. Cell. Biochem. 118: 2654-2663, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  19. Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Sassoli, Chiara; Nosi, Daniele; Tani, Alessia; Chellini, Flaminia [Dept. of Experimental and Clinical Medicine—Section of Anatomy and Histology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy); Mazzanti, Benedetta [Dept. of Experimental and Clinical Medicine—Section of Haematology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy); Quercioli, Franco [CNR-National Institute of Optics (INO), Largo Enrico Fermi 6, 50125 Arcetri-Florence (Italy); Zecchi-Orlandini, Sandra [Dept. of Experimental and Clinical Medicine—Section of Anatomy and Histology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy); Formigli, Lucia, E-mail: formigli@unifi.it [Dept. of Experimental and Clinical Medicine—Section of Anatomy and Histology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy)

    2014-05-01

    Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7{sup +} satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration. - Highlights: • MSC-CM contains paracrine factors that up-regulate MMP expression and function in different skeletal muscle cells. • MSC-CM promotes myoblast and satellite cell migration, proliferation and differentiation. • MSC-CM negatively interferes with fibroblast-myoblast transition in primary skeletal fibroblasts. • Paracrine factors from MSCs modulate the fibrotic response and improve the endogenous mechanisms of muscle regeneration.

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

    Science.gov (United States)

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

    2016-01-04

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

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

    Science.gov (United States)

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

    2017-09-06

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

  2. Tissue engineering for skeletal muscle regeneration.

    Science.gov (United States)

    Rizzi, Roberto; Bearzi, Claudia; Mauretti, Arianna; Bernardini, Sergio; Cannata, Stefano; Gargioli, Cesare

    2012-07-01

    Stem cells and regenerative medicine have obtained a remarkable consent from the scientific community for their promising ability to recover aged, injured and diseased tissue. However, despite the noteworthy potential, hurdles currently hinder their use and clinical application: cell survival, immune response, tissue engraftment and efficient differentiation. Hence a new interdisciplinary scientific approach, such as tissue engineering, is going deep attempts to mimic neo-tissue-genesis as well as stem cell engraftment amelioration. Skeletal muscle tissue engineering represents a great potentiality in medicine for muscle regeneration exploiting new generation injectable hydrogel as scaffold supporting progenitor/stem cells for muscle differentiation reconstructing the natural skeletal muscle tissue architecture influenced by matrix mechanical and physical property and by a dynamic environment.

  3. YAP-Mediated Mechanotransduction in Skeletal Muscle.

    Science.gov (United States)

    Fischer, Martina; Rikeit, Paul; Knaus, Petra; Coirault, Catherine

    2016-01-01

    Skeletal muscle is not only translating chemical energy into mechanical work, it is also a highly adaptive and regenerative tissue whose architecture and functionality is determined by its mechanical and physical environment. Processing intra- and extracellular mechanical signaling cues contributes to the regulation of cell growth, survival, migration and differentiation. Yes-associated Protein (YAP), a transcriptional coactivator downstream of the Hippo pathway and its paralog, the transcriptional co-activator with PDZ-binding motif (TAZ), were recently found to play a key role in mechanotransduction in various tissues including skeletal muscle. Furthermore, YAP/TAZ modulate myogenesis and muscle regeneration and abnormal YAP activity has been reported in muscular dystrophy and rhabdomyosarcoma. Here, we summarize the current knowledge of mechanosensing and -signaling in striated muscle. We highlight the role of YAP signaling and discuss the different routes and hypotheses of its regulation in the context of mechanotransduction.

  4. Autophagy and skeletal muscles in sepsis.

    Directory of Open Access Journals (Sweden)

    Mahroo Mofarrahi

    Full Text Available Mitochondrial injury develops in skeletal muscles during the course of severe sepsis. Autophagy is a protein and organelle recycling pathway which functions to degrade or recycle unnecessary, redundant, or inefficient cellular components. No information is available regarding the degree of sepsis-induced mitochondrial injury and autophagy in the ventilatory and locomotor muscles. This study tests the hypotheses that the locomotor muscles are more prone to sepsis-induced mitochondrial injury, depressed biogenesis and autophagy induction compared with the ventilatory muscles.Adult male C57/Bl6 mice were injected with i.p. phosphate buffered saline (PBS or E. coli lipopolysaccharide (LPS, 20 mg/kg and sacrificed 24 h later. The tibialis anterior (TA, soleus (SOLD and diaphragm (DIA muscles were quickly excised and examined for mitochondrial morphological injury, Ca(++ retention capacity and biogenesis. Autophagy was detected with electron microscopy, lipidation of Lc3b proteins and by measuring gene expression of several autophagy-related genes. Electron microscopy revealed ultrastructural injuries in the mitochondria of each muscle, however, injuries were more severe in the TA and SOL muscles than they were in the DIA. Gene expressions of nuclear and mitochondrial DNA transcription factors and co-activators (indicators of biogenesis were significantly depressed in all treated muscles, although to a greater extent in the TA and SOL muscles. Significant autophagosome formation, Lc3b protein lipidation and upregulation of autophagy-related proteins were detected to a greater extent in the TA and SOL muscles and less so in the DIA. Lipidation of Lc3b and the degree of induction of autophagy-related proteins were significantly blunted in mice expressing a muscle-specific IκBα superrepresor.We conclude that locomotor muscles are more prone to sepsis-induced mitochondrial injury, decreased biogenesis and increased autophagy compared with the

  5. IL-6 regulation on skeletal muscle mitochondrial remodeling during cancer cachexia in the ApcMin/+ mouse

    Directory of Open Access Journals (Sweden)

    White James P

    2012-07-01

    Full Text Available Abstract Background Muscle protein turnover regulation during cancer cachexia is being rapidly defined, and skeletal muscle mitochondria function appears coupled to processes regulating muscle wasting. Skeletal muscle oxidative capacity and the expression of proteins regulating mitochondrial biogenesis and dynamics are disrupted in severely cachectic ApcMin/+ mice. It has not been determined if these changes occur at the onset of cachexia and are necessary for the progression of muscle wasting. Exercise and anti-cytokine therapies have proven effective in preventing cachexia development in tumor bearing mice, while their effect on mitochondrial content, biogenesis and dynamics is not well understood. The purposes of this study were to 1 determine IL-6 regulation on mitochondrial remodeling/dysfunction during the progression of cancer cachexia and 2 to determine if exercise training can attenuate mitochondrial dysfunction and the induction of proteolytic pathways during IL-6 induced cancer cachexia. Methods ApcMin/+ mice were examined during the progression of cachexia, after systemic interleukin (IL-6r antibody treatment, or after IL-6 over-expression with or without exercise. Direct effects of IL-6 on mitochondrial remodeling were examined in cultured C2C12 myoblasts. Results Mitochondrial content was not reduced during the initial development of cachexia, while muscle PGC-1α and fusion (Mfn1, Mfn2 protein expression was repressed. With progressive weight loss mitochondrial content decreased, PGC-1α and fusion proteins were further suppressed, and fission protein (FIS1 was induced. IL-6 receptor antibody administration after the onset of cachexia improved mitochondrial content, PGC-1α, Mfn1/Mfn2 and FIS1 protein expression. IL-6 over-expression in pre-cachectic mice accelerated body weight loss and muscle wasting, without reducing mitochondrial content, while PGC-1α and Mfn1/Mfn2 protein expression was suppressed and FIS1 protein expression

  6. Spot light on skeletal muscles: optogenetic stimulation to understand and restore skeletal muscle function.

    Science.gov (United States)

    van Bremen, Tobias; Send, Thorsten; Sasse, Philipp; Bruegmann, Tobias

    2017-09-16

    Damage of peripheral nerves results in paralysis of skeletal muscle. Currently, the only treatment option to restore proper function is electrical stimulation of the innervating nerve or of the skeletal muscles directly. However this approach has low spatial and temporal precision leading to co-activation of antagonistic muscles and lacks cell-type selectivity resulting in pain or discomfort by stimulation of sensible nerves. In contrast to electrical stimulation, optogenetic methods enable spatially confined and cell-type selective stimulation of cells expressing the light sensitive channel Channelrhodopsin-2 with precise temporal control over the membrane potential. Herein we summarize the current knowledge about the use of this technology to control skeletal muscle function with the focus on the direct, non-neuronal stimulation of muscle fibers. The high temporal flexibility of using light pulses allows new stimulation patterns to investigate skeletal muscle physiology. Furthermore, the high spatial precision of focused illumination was shown to be beneficial for selective stimulation of distinct nearby muscle groups. Finally, the cell-type specific expression of the light-sensitive effector proteins in muscle fibers will allow pain-free stimulation and open new options for clinical treatments. Therefore, we believe that direct optogenetic stimulation of skeletal muscles is a very potent method for basic scientists that also harbors several distinct advantages over electrical stimulation to be considered for clinical use in the future.

  7. The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease.

    Science.gov (United States)

    Davignon, Laurianne; Chauveau, Claire; Julien, Cédric; Dill, Corinne; Duband-Goulet, Isabelle; Cabet, Eva; Buendia, Brigitte; Lilienbaum, Alain; Rendu, John; Minot, Marie Christine; Guichet, Agnès; Allamand, Valérie; Vadrot, Nathalie; Fauré, Julien; Odent, Sylvie; Lazaro, Leïla; Leroy, Jean Paul; Marcorelles, Pascale; Dubourg, Odile; Ferreiro, Ana

    2016-04-15

    Despite recent progress in the genetic characterization of congenital muscle diseases, the genes responsible for a significant proportion of cases remain unknown. We analysed two branches of a large consanguineous family in which four patients presented with a severe new phenotype, clinically marked by neonatal-onset muscle weakness predominantly involving axial muscles, life-threatening respiratory failure, skin abnormalities and joint hyperlaxity without contractures. Muscle biopsies showed the unreported association of multi-minicores, caps and dystrophic lesions. Genome-wide linkage analysis followed by gene and exome sequencing in patients identified a homozygous nonsense mutation in TRIP4 encoding Activating Signal Cointegrator-1 (ASC-1), a poorly characterized transcription coactivator never associated with muscle or with human inherited disease. This mutation resulted in TRIP4 mRNA decay to around 10% of control levels and absence of detectable protein in patient cells. ASC-1 levels were higher in axial than in limb muscles in mouse, and increased during differentiation in C2C12 myogenic cells. Depletion of ASC-1 in cultured muscle cells from a patient and in Trip4 knocked-down C2C12 led to a significant reduction in myotube diameter ex vivo and in vitro, without changes in fusion index or markers of initial myogenic differentiation. This work reports the first TRIP4 mutation and defines a novel form of congenital muscle disease, expanding their histological, clinical and molecular spectrum. We establish the importance of ASC-1 in human skeletal muscle, identify transcriptional co-regulation as novel pathophysiological pathway, define ASC-1 as a regulator of late myogenic differentiation and suggest defects in myotube growth as a novel myopathic mechanism. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. Redox characterization of functioning skeletal muscle

    Directory of Open Access Journals (Sweden)

    Li eZuo

    2015-11-01

    Full Text Available Skeletal muscle physiology is influenced by the presence of chemically reactive molecules such as reactive oxygen species (ROS. These molecules regulate multiple redox-sensitive signaling pathways that play a critical role in cellular processes including gene expression and protein modification. While ROS have gained much attention for their harmful effects in muscle fatigue and dysfunction, research has also shown ROS to facilitate muscle adaptation after stressors such as physical exercise. This manuscript aims to provide a comprehensive review of the current understanding of redox signaling in skeletal muscle. ROS-induced oxidative stress and its role in the aging process are discussed. Mitochondria have been shown to generate large amounts of ROS during muscular contractions, and thus are susceptible to oxidative stress. ROS can modify proteins located in the mitochondrial membrane leading to cell death and osmotic swelling. ROS also contribute to the necrosis and inflammation of muscle fibers that is associated with muscular diseases including Duchenne muscular dystrophy (DMD. It is imperative that future research continues to investigate the exact role of ROS in normal skeletal muscle function as well as muscular dysfunction and disease.

  9. Gamma dosimetric parameters in some skeletal muscle relaxants

    Indian Academy of Sciences (India)

    H C MANJUNATHA

    2017-08-29

    Aug 29, 2017 ... But literature survey also reveals that there is no such studies on measurements in the skeletal muscle relaxants. Hence there is a need to measure the mass attenuation coefficient in skeletal muscle relaxants. To know the after-effects of radiation on skeletal muscle, it is important to consider the attenuation ...

  10. Training induced adaptation in horse skeletal muscle

    NARCIS (Netherlands)

    Dam, K.G. van

    2006-01-01

    It appears that the physiological and biochemical adaptation of skeletal muscle to training in equine species shows a lot of similarities with human and rodent physiological adaptation. On the other hand it is becoming increasingly clear that intra-cellular mechanisms of adaptation (substrate

  11. Signalling role of skeletal muscle during exercise

    NARCIS (Netherlands)

    Catoire, M.

    2014-01-01

    Abstract Upon  acute exercise skeletal muscle is immediately and heavily recruited, while other organs appear to play only a minor role during exercise. These other organs show significant changes and improvements in function, although they are not directly targeted by

  12. Valproic acid attenuates skeletal muscle wasting by inhibiting C/EBPβ-regulated atrogin1 expression in cancer cachexia.

    Science.gov (United States)

    Sun, Rulin; Zhang, Santao; Hu, Wenjun; Lu, Xing; Lou, Ning; Yang, Zhende; Chen, Shaoyong; Zhang, Xiaoping; Yang, Hongmei

    2016-07-01

    Muscle wasting is the hallmark of cancer cachexia and is associated with poor quality of life and increased mortality. Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, has important biological effects in the treatment of muscular dystrophy. To verify whether VPA could ameliorate muscle wasting induced by cancer cachexia, we explored the role of VPA in two cancer cachectic mouse models [induced by colon-26 (C26) adenocarcinoma or Lewis lung carcinoma (LLC)] and atrophied C2C12 myotubes [induced by C26 cell conditioned medium (CCM) or LLC cell conditioned medium (LCM)]. Our data demonstrated that treatment with VPA increased the mass and cross-sectional area of skeletal muscles in tumor-bearing mice. Furthermore, treatment with VPA also increased the diameter of myotubes cultured in conditioned medium. The skeletal muscles in cachectic mice or atrophied myotubes treated with VPA exhibited reduced levels of CCAAT/enhancer binding protein beta (C/EBPβ), resulting in atrogin1 downregulation and the eventual alleviation of muscle wasting and myotube atrophy. Moreover, atrogin1 promoter activity in myotubes was stimulated by CCM via activating the C/EBPβ-responsive cis-element and subsequently inhibited by VPA. In contrast to the effect of VPA on the levels of C/EBPβ, the levels of inactivating forkhead box O3 (FoxO3a) were unaffected. In summary, VPA attenuated muscle wasting and myotube atrophy and reduced C/EBPβ binding to atrogin1 promoter locus in the myotubes. Our discoveries indicate that HDAC inhibition by VPA might be a promising new approach for the preservation of skeletal muscle in cancer cachexia. Copyright © 2016 the American Physiological Society.

  13. Irbesartan enhances GLUT4 translocation and glucose transport in skeletal muscle cells.

    Science.gov (United States)

    Kobayashi, Tatsuo; Akiyama, Yuko; Akiyama, Nobuteru; Katoh, Hideaki; Yamamoto, Sachiko; Funatsuki, Kenzo; Yanagimoto, Toru; Notoya, Mitsuru; Asakura, Kenji; Shinosaki, Toshihiro; Hanasaki, Kohji

    2010-12-15

    Irbesartan, an angiotensin II type 1 receptor blocker has been reported to alleviate metabolic disorder in animal studies and human clinical trials. Although this effect may be related to the ability of irbesartan to serve as a partial agonist for the peroxisome proliferator-activated receptor (PPAR)-γ, the target tissues on which irbesartan acts remain poorly defined. As muscle glucose transport plays a major role in maintaining systemic glucose homeostasis, we investigated the effect of irbesartan on glucose uptake in skeletal muscle cells. In C2C12 myotubes, 24-h treatment with irbesartan significantly promoted both basal and insulin-stimulated glucose transport. In L6-GLUT4myc myoblasts, irbesartan caused a significant increase in glucose transport and GLUT4 translocation to the cell surface in a concentration-dependent manner. Valsartan, another angiotensin II type 1 receptor blocker had no effect on either glucose uptake or GLUT4 translocation, implying that these actions on glucose transport are independent of angiotensin II receptor blockade. Moreover, irbesartan exerted these effects in an additive manner with insulin, but not with acute treatment for 3 h, suggesting that they may require the synthesis of new proteins. Finally, in insulin-resistant Zucker fatty rat, irbesartan (50 mg/kg/day for 3 weeks) significantly ameliorated insulin resistance without increasing weight gain. We conclude that irbesartan has a direct action, which can be additive to insulin, of promoting glucose transport in skeletal muscle. This may be beneficial for ameliorating obesity-related glucose homeostasis derangement. Copyright © 2010 Elsevier B.V. All rights reserved.

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

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    Cheng-Hsiu Lin

    2017-11-01

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

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

    Science.gov (United States)

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

    2013-02-01

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

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

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    Sogayar Mari C

    2011-09-01

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

  17. Cellular Players in Skeletal Muscle Regeneration

    Directory of Open Access Journals (Sweden)

    Laura Cristina Ceafalan

    2014-01-01

    Full Text Available Skeletal muscle, a tissue endowed with remarkable endogenous regeneration potential, is still under focused experimental investigation mainly due to treatment potential for muscle trauma and muscular dystrophies. Resident satellite cells with stem cell features were enthusiastically described quite a long time ago, but activation of these cells is not yet controlled by any medical interventions. However, after thorough reports of their existence, survival, activation, and differentiation there are still many questions to be answered regarding the intimate mechanism of tissue regeneration. This review delivers an up-to-date inventory of the main known key players in skeletal muscle repair, revealed by various models of tissue injuries in mechanical trauma, toxic lesions, and muscular dystrophy. A better understanding of the spatial and temporal relationships between various cell populations, with different physical or paracrine interactions and phenotype changes induced by local or systemic signalling, might lead to a more efficient approach for future therapies.

  18. An extract of Urtica dioica L. mitigates obesity induced insulin resistance in mice skeletal muscle via protein phosphatase 2A (PP2A).

    Science.gov (United States)

    Obanda, Diana N; Ribnicky, David; Yu, Yongmei; Stephens, Jacqueline; Cefalu, William T

    2016-02-26

    The leaf extract of Urtica dioica L. (UT) has been reported to improve glucose homeostasis in vivo, but definitive studies on efficacy and mechanism of action are lacking. We investigated the effects of UT on obesity- induced insulin resistance in skeletal muscle. Male C57BL/6J mice were divided into three groups: low-fat diet (LFD), high-fat diet (HFD) and HFD supplemented with UT. Body weight, body composition, plasma glucose and plasma insulin were monitored. Skeletal muscle (gastrocnemius) was analyzed for insulin sensitivity, ceramide accumulation and the post translational modification and activity of protein phosphatase 2A (PP2A). PP2A is activated by ceramides and dephosphorylates Akt. C2C12 myotubes exposed to excess free fatty acids with or without UT were also evaluated for insulin signaling and modulation of PP2A. The HFD induced insulin resistance, increased fasting plasma glucose, enhanced ceramide accumulation and PP2A activity in skeletal muscle. Supplementation with UT improved plasma glucose homeostasis and enhanced skeletal muscle insulin sensitivity without affecting body weight and body composition. In myotubes, UT attenuated the ability of FFAs to induce insulin resistance and PP2A hyperactivity without affecting ceramide accumulation and PP2A expression. UT decreased PP2A activity through posttranslational modification that was accompanied by a reduction in Akt dephosphorylation.

  19. A Rhodiola rosea root extract protects skeletal muscle cells against chemically induced oxidative stress by modulating heat shock protein 70 (HSP70) expression.

    Science.gov (United States)

    Hernández-Santana, Aaron; Pérez-López, Verónica; Zubeldia, Jose María; Jiménez-del-Rio, Miguel

    2014-04-01

    Rhodiola rosea is a perennial plant in the Crassulaceae family, recently postulated to exert its adaptogenic functions partially by modulating the expression of molecular factors such as heat shock proteins (HSP). The aim of this study was to analyze the efficacy of a Rhodiola rosea extract (Rhodiolife) in protecting murine skeletal muscle cells (C2 C12 myotubes) from chemically induced oxidative stress and to establish whether modulation of HSP70 expression is observed. C2 C12 cells treated with Rhodiolife did not experience any loss of viability (p > 0.05) at concentrations of 1-100 µg/mL for up to 24 h. In control cultures, viability decreased 25% following exposure to 2 mM H2 O2 (1 h). However, no significant decrease in viability in cells pre-treated with extract at concentrations as low as 1 µg/mL was observed. HSP70 mRNA levels were up-regulated two-fold in cell cultures treated with Rhodiolife (10 µg/mL), and expression was further enhanced by exposure to H2 O2 (six-fold, p < 0.05). HSP70 protein levels were maintained in pre-treated cell cultures compared to controls but was significantly lower (-50%) in cells lacking treatment exposed to H2 O2 . The present results indicate that Rhodiolife protects C2 C12 myotubes against peroxide-induced oxidative stress through the modulation of the molecular chaperone HSP70. Copyright © 2013 John Wiley & Sons, Ltd.

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

    Science.gov (United States)

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

    2004-07-01

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

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

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    Calum J Redpath

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

  2. Alix Protein Is Substrate of Ozz-E3 Ligase and Modulates Actin Remodeling in Skeletal Muscle*

    Science.gov (United States)

    Bongiovanni, Antonella; Romancino, Daniele P.; Campos, Yvan; Paterniti, Gaetano; Qiu, Xiaohui; Moshiach, Simon; Di Felice, Valentina; Vergani, Naja; Ustek, Duran; d'Azzo, Alessandra

    2012-01-01

    Alix/AIP1 is a multifunctional adaptor protein that participates in basic cellular processes, including membrane trafficking and actin cytoskeleton assembly, by binding selectively to a variety of partner proteins. However, the mechanisms regulating Alix turnover, subcellular distribution, and function in muscle cells are unknown. We now report that Alix is expressed in skeletal muscle throughout myogenic differentiation. In myotubes, a specific pool of Alix colocalizes with Ozz, the substrate-binding component of the muscle-specific ubiquitin ligase complex Ozz-E3. We found that interaction of the two endogenous proteins in the differentiated muscle fibers changes Alix conformation and promotes its ubiquitination. This in turn regulates the levels of the protein in specific subcompartments, in particular the one containing the actin polymerization factor cortactin. In Ozz−/− myotubes, the levels of filamentous (F)-actin is perturbed, and Alix accumulates in large puncta positive for cortactin. In line with this observation, we show that the knockdown of Alix expression in C2C12 muscle cells affects the amount and distribution of F-actin, which consequently leads to changes in cell morphology, impaired formation of sarcolemmal protrusions, and defective cell motility. These findings suggest that the Ozz-E3 ligase regulates Alix at sites where the actin cytoskeleton undergoes remodeling. PMID:22334701

  3. Alix protein is substrate of Ozz-E3 ligase and modulates actin remodeling in skeletal muscle.

    Science.gov (United States)

    Bongiovanni, Antonella; Romancino, Daniele P; Campos, Yvan; Paterniti, Gaetano; Qiu, Xiaohui; Moshiach, Simon; Di Felice, Valentina; Vergani, Naja; Ustek, Duran; d'Azzo, Alessandra

    2012-04-06

    Alix/AIP1 is a multifunctional adaptor protein that participates in basic cellular processes, including membrane trafficking and actin cytoskeleton assembly, by binding selectively to a variety of partner proteins. However, the mechanisms regulating Alix turnover, subcellular distribution, and function in muscle cells are unknown. We now report that Alix is expressed in skeletal muscle throughout myogenic differentiation. In myotubes, a specific pool of Alix colocalizes with Ozz, the substrate-binding component of the muscle-specific ubiquitin ligase complex Ozz-E3. We found that interaction of the two endogenous proteins in the differentiated muscle fibers changes Alix conformation and promotes its ubiquitination. This in turn regulates the levels of the protein in specific subcompartments, in particular the one containing the actin polymerization factor cortactin. In Ozz(-/-) myotubes, the levels of filamentous (F)-actin is perturbed, and Alix accumulates in large puncta positive for cortactin. In line with this observation, we show that the knockdown of Alix expression in C2C12 muscle cells affects the amount and distribution of F-actin, which consequently leads to changes in cell morphology, impaired formation of sarcolemmal protrusions, and defective cell motility. These findings suggest that the Ozz-E3 ligase regulates Alix at sites where the actin cytoskeleton undergoes remodeling.

  4. Factors related to skeletal muscle mass in the frail elderly.

    Science.gov (United States)

    Sagawa, Keiichiro; Kikutani, Takeshi; Tamura, Fumiyo; Yoshida, Mitsuyoshi

    2017-01-01

    It is important for the elderly to maintain their skeletal muscle mass, which in turn helps to maintain physical functions. This study aimed to clarify factors related to skeletal muscle mass maintenance. Home-bound elderly (94 men and 216 women), at least 75 years of age, attending a day-care center in Tokyo, were enrolled in this study. Dentists specializing in dysphagia rehabilitation evaluated skeletal muscle mass, occlusal status and swallowing function. Physical function, cognitive function and nutritional status were also evaluated by interviewing caregivers. Correlations of skeletal muscle mass with various factors were determined in each gender group. Multiple regression analysis revealed that skeletal muscle mass was significantly related to nutritional status in both men and women. In men, there was a significant difference in skeletal muscle mass between those with and without occlusion of the natural teeth. Our results suggest that dental treatments and dentures would be useful for maintaining skeletal muscle mass, especially in men.

  5. Engineering vascularized skeletal muscle tissue

    NARCIS (Netherlands)

    Levenberg, Shulamit; Rouwkema, Jeroen; Macdonald, Mara; Garfein, Evan S.; Kohane, Daniel S.; Darland, Diane C.; Marini, Robert; van Blitterswijk, Clemens; Mulligan, Richard C.; D'Amore, Patricia A.; Langer, Robert

    2005-01-01

    One of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro could maintain cell viability during tissue growth, induce structural organization and promote vascularization upon implantation. Here we describe

  6. Skeletal Muscle Tissue Engineering: Methods to Form Skeletal Myotubes and Their Applications

    Science.gov (United States)

    Ostrovidov, Serge; Hosseini, Vahid; Ahadian, Samad; Fujie, Toshinori; Parthiban, Selvakumar Prakash; Ramalingam, Murugan; Bae, Hojae; Kaji, Hirokazu

    2014-01-01

    Skeletal muscle tissue engineering (SMTE) aims to repair or regenerate defective skeletal muscle tissue lost by traumatic injury, tumor ablation, or muscular disease. However, two decades after the introduction of SMTE, the engineering of functional skeletal muscle in the laboratory still remains a great challenge, and numerous techniques for growing functional muscle tissues are constantly being developed. This article reviews the recent findings regarding the methodology and various technical aspects of SMTE, including cell alignment and differentiation. We describe the structure and organization of muscle and discuss the methods for myoblast alignment cultured in vitro. To better understand muscle formation and to enhance the engineering of skeletal muscle, we also address the molecular basics of myogenesis and discuss different methods to induce myoblast differentiation into myotubes. We then provide an overview of different coculture systems involving skeletal muscle cells, and highlight major applications of engineered skeletal muscle tissues. Finally, potential challenges and future research directions for SMTE are outlined. PMID:24320971

  7. Reactive Oxygen Species in Skeletal Muscle Signaling

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    Elena Barbieri

    2012-01-01

    Full Text Available Generation of reactive oxygen species (ROS is a ubiquitous phenomenon in eukaryotic cells' life. Up to the 1990s of the past century, ROS have been solely considered as toxic species resulting in oxidative stress, pathogenesis and aging. However, there is now clear evidence that ROS are not merely toxic species but also—within certain concentrations—useful signaling molecules regulating physiological processes. During intense skeletal muscle contractile activity myotubes' mitochondria generate high ROS flows: this renders skeletal muscle a tissue where ROS hold a particular relevance. According to their hormetic nature, in muscles ROS may trigger different signaling pathways leading to diverging responses, from adaptation to cell death. Whether a “positive” or “negative” response will prevail depends on many variables such as, among others, the site of ROS production, the persistence of ROS flow or target cells' antioxidant status. In this light, a specific threshold of physiological ROS concentrations above which ROS exert negative, toxic effects is hard to determine, and the concept of “physiologically compatible” levels of ROS would better fit with such a dynamic scenario. In this review these concepts will be discussed along with the most relevant signaling pathways triggered and/or affected by ROS in skeletal muscle.

  8. Skeletal muscle proteomics in livestock production.

    Science.gov (United States)

    Picard, Brigitte; Berri, Cécile; Lefaucheur, Louis; Molette, Caroline; Sayd, Thierry; Terlouw, Claudia

    2010-05-01

    Proteomics allows studying large numbers of proteins, including their post-translational modifications. Proteomics has been, and still are, used in numerous studies on skeletal muscle. In this article, we focus on its use in the study of livestock muscle development and meat quality. Changes in protein profiles during myogenesis are described in cattle, pigs and fowl using comparative analyses across different ontogenetic stages. This approach allows a better understanding of the key stages of myogenesis and helps identifying processes that are similar or divergent between species. Genetic variability of muscle properties analysed by the study of hypertrophied cattle and sheep are discussed. Biological markers of meat quality, particularly tenderness in cattle, pigs and fowl are presented, including protein modifications during meat ageing in cattle, protein markers of PSE meat in turkeys and of post-mortem muscle metabolism in pigs. Finally, we discuss the interest of proteomics as a tool to understand better biochemical mechanisms underlying the effects of stress during the pre-slaughter period on meat quality traits. In conclusion, the study of proteomics in skeletal muscles allows generating large amounts of scientific knowledge that helps to improve our understanding of myogenesis and muscle growth and to control better meat quality.

  9. Quantitative skeletal muscle ultrasound: diagnostic value in childhood neuromuscular disease.

    NARCIS (Netherlands)

    Pillen, S.; Verrips, A.; Alfen, N. van; Arts, I.M.P.; Sie, L.T.L.; Zwarts, M.J.

    2007-01-01

    In this study we investigated the diagnostic value of quantitative skeletal muscle ultrasonography in 150 consecutively referred children with symptoms suspect for a neuromuscular disorder. Muscle thickness and quantitatively determined echo intensity of four muscles and the distribution of these

  10. Tissue Engineered Strategies for Skeletal Muscle Injury

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    Umile Giuseppe Longo

    2012-01-01

    Full Text Available Skeletal muscle injuries are common in athletes, occurring with direct and indirect mechanisms and marked residual effects, such as severe long-term pain and physical disability. Current therapy consists of conservative management including RICE protocol (rest, ice, compression, and elevation, nonsteroidal anti-inflammatory drugs, and intramuscular corticosteroids. However, current management of muscle injuries often does not provide optimal restoration to preinjury status. New biological therapies, such as injection of platelet-rich plasma and stem-cell-based therapy, are appealing. Although some studies support PRP application in muscle-injury management, reasons for concern persist, and further research is required for a standardized and safe use of PRP in clinical practice. The role of stem cells needs to be confirmed, as studies are still limited and inconsistent. Further research is needed to identify mechanisms involved in muscle regeneration and in survival, proliferation, and differentiation of stem cells.

  11. Training induced adaptation in horse skeletal muscle

    OpenAIRE

    Dam, K.G. van

    2006-01-01

    It appears that the physiological and biochemical adaptation of skeletal muscle to training in equine species shows a lot of similarities with human and rodent physiological adaptation. On the other hand it is becoming increasingly clear that intra-cellular mechanisms of adaptation (substrate transport, enzyme activity, etc) differ considerably between species. The major drawbacks in equine training physiological research are the lack of an appropriate training model and the lack of control o...

  12. MG53-IRS-1 (Mitsugumin 53-Insulin Receptor Substrate-1) Interaction Disruptor Sensitizes Insulin Signaling in Skeletal Muscle.

    Science.gov (United States)

    Lee, Hyun; Park, Jung-Jin; Nguyen, Nga; Park, Jun Sub; Hong, Jin; Kim, Seung-Hyeob; Song, Woon Young; Kim, Hak Joong; Choi, Kwangman; Cho, Sungchan; Lee, Jae-Seon; Kim, Bong-Woo; Ko, Young-Gyu

    2016-12-23

    Mitsugumin 53 (MG53) is an E3 ligase that interacts with and ubiquitinates insulin receptor substrate-1 (IRS-1) in skeletal muscle; thus, an MG53-IRS-1 interaction disruptor (MID), which potentially sensitizes insulin signaling with an elevated level of IRS-1 in skeletal muscle, is an excellent candidate for treating insulin resistance. To screen for an MID, we developed a bimolecular luminescence complementation system using an N-terminal luciferase fragment fused with IRS-1 and a C-terminal luciferase fragment fused with an MG53 C14A mutant that binds to IRS-1 but does not have E3 ligase activity. An MID, which was discovered using the bimolecular luminescence complementation system, disrupted the molecular association of MG53 with IRS-1, thus abolishing MG53-mediated IRS-1 ubiquitination and degradation. Thus, the MID sensitized insulin signaling and increased insulin-elicited glucose uptake with an elevated level of IRS-1 in C2C12 myotubes. These data indicate that this MID holds promise as a drug candidate for treating insulin resistance. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Skeletal Muscle Mitochondria and Aging: A Review

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    Courtney M. Peterson

    2012-01-01

    Full Text Available Aging is characterized by a progressive loss of muscle mass and muscle strength. Declines in skeletal muscle mitochondria are thought to play a primary role in this process. Mitochondria are the major producers of reactive oxygen species, which damage DNA, proteins, and lipids if not rapidly quenched. Animal and human studies typically show that skeletal muscle mitochondria are altered with aging, including increased mutations in mitochondrial DNA, decreased activity of some mitochondrial enzymes, altered respiration with reduced maximal capacity at least in sedentary individuals, and reduced total mitochondrial content with increased morphological changes. However, there has been much controversy over measurements of mitochondrial energy production, which may largely be explained by differences in approach and by whether physical activity is controlled for. These changes may in turn alter mitochondrial dynamics, such as fusion and fission rates, and mitochondrially induced apoptosis, which may also lead to net muscle fiber loss and age-related sarcopenia. Fortunately, strategies such as exercise and caloric restriction that reduce oxidative damage also improve mitochondrial function. While these strategies may not completely prevent the primary effects of aging, they may help to attenuate the rate of decline.

  14. Effect of limb immobilization on skeletal muscle

    Science.gov (United States)

    Booth, F. W.

    1982-01-01

    Current knowledge and questions remaining concerning the effects of limb immobilization on skeletal muscle is reviewed. The most dramatic of these effects is muscle atrophy, which has been noted in cases of muscles fixed at or below their resting length. Immobilization is also accompanied by a substantial decrease in motoneuronal discharges, which results in the conversion of slow-twitch muscle to muscle with fast-twitch characteristics. Sarcolemma effects include no change or a decrease in resting membrane potential, the appearance of extrajunctional acetylcholine receptors, and no change in acetylcholinesterase activity. Evidence of changes in motoneuron after hyperpolarization characteristics suggests that the muscle inactivity is responsible for neuronal changes, rather than vice versa. The rate of protein loss from atrophying muscles is determined solely by the first-order rate constant for degradation. Various other biochemical and functional changes have been noted, including decreased insulin responsiveness and protein synthesis. The model of limb immobilization may also be useful for related studies of muscle adaptation.

  15. Skeletal Muscle Tissue Engineering: Methods to Form Skeletal Myotubes and Their Applications

    OpenAIRE

    Ostrovidov, Serge; Hosseini, Vahid; Ahadian, Samad; Fujie, Toshinori; Parthiban, Selvakumar Prakash; Ramalingam, Murugan; Bae, Hojae; Kaji, Hirokazu; Khademhosseini, Ali

    2014-01-01

    Skeletal muscle tissue engineering (SMTE) aims to repair or regenerate defective skeletal muscle tissue lost by traumatic injury, tumor ablation, or muscular disease. However, two decades after the introduction of SMTE, the engineering of functional skeletal muscle in the laboratory still remains a great challenge, and numerous techniques for growing functional muscle tissues are constantly being developed. This article reviews the recent findings regarding the methodology and various technic...

  16. Disease-Induced Skeletal Muscle Atrophy and Fatigue.

    Science.gov (United States)

    Powers, Scott K; Lynch, Gordon S; Murphy, Kate T; Reid, Michael B; Zijdewind, Inge

    2016-11-01

    Numerous health problems, including acute critical illness, cancer, diseases associated with chronic inflammation, and neurological disorders, often result in skeletal muscle weakness and fatigue. Disease-related muscle atrophy and fatigue is an important clinical problem because acquired skeletal muscle weakness can increase the duration of hospitalization, result in exercise limitation, and contribute to a poor quality of life. Importantly, skeletal muscle atrophy is also associated with increased morbidity and mortality of patients. Therefore, improving our understanding of the mechanism(s) responsible for skeletal muscle weakness and fatigue in patients is a required first step to develop clinical protocols to prevent these skeletal muscle problems. This review will highlight the consequences and potential mechanisms responsible for skeletal muscle atrophy and fatigue in patients experiencing acute critical illness, cancer, chronic inflammatory diseases, and neurological disorders.

  17. Disease-Induced Skeletal Muscle Atrophy and Fatigue

    Science.gov (United States)

    Powers, Scott K.; Lynch, Gordon S.; Murphy, Kate T.; Reid, Michael B.; Zijdewind, Inge

    2016-01-01

    Numerous health problems including acute critical illness, cancer, diseases associated with chronic inflammation, and neurological disorders often result in skeletal muscle weakness and fatigue. Disease-related muscle atrophy and fatigue is an important clinical problem because acquired skeletal muscle weakness can increase the duration of hospitalization, result in exercise limitation, and contribute to a poor quality of life. Importantly, skeletal muscle atrophy is also associated with increased morbidity and mortality of patients. Therefore, improving our understanding of the mechanism(s) responsible for skeletal muscle weakness and fatigue in patients is a required first step to develop clinical protocols to prevent these skeletal muscle problems. This review will highlight the consequences and potential mechanisms responsible for skeletal muscle atrophy and fatigue in patients suffering from acute critical illness, cancer, chronic inflammatory diseases, and neurological disorders. PMID:27128663

  18. Hydrogen improves glycemic control in type1 diabetic animal model by promoting glucose uptake into skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Haruka Amitani

    Full Text Available Hydrogen (H(2 acts as a therapeutic antioxidant. However, there are few reports on H(2 function in other capacities in diabetes mellitus (DM. Therefore, in this study, we investigated the role of H(2 in glucose transport by studying cultured mouse C2C12 cells and human hepatoma Hep-G2 cells in vitro, in addition to three types of diabetic mice [Streptozotocin (STZ-induced type 1 diabetic mice, high-fat diet-induced type 2 diabetic mice, and genetically diabetic db/db mice] in vivo. The results show that H(2 promoted 2-[(14C]-deoxy-d-glucose (2-DG uptake into C2C12 cells via the translocation of glucose transporter Glut4 through activation of phosphatidylinositol-3-OH kinase (PI3K, protein kinase C (PKC, and AMP-activated protein kinase (AMPK, although it did not stimulate the translocation of Glut2 in Hep G2 cells. H(2 significantly increased skeletal muscle membrane Glut4 expression and markedly improved glycemic control in STZ-induced type 1 diabetic mice after chronic intraperitoneal (i.p. and oral (p.o. administration. However, long-term p.o. administration of H(2 had least effect on the obese and non-insulin-dependent type 2 diabetes mouse models. Our study demonstrates that H(2 exerts metabolic effects similar to those of insulin and may be a novel therapeutic alternative to insulin in type 1 diabetes mellitus that can be administered orally.

  19. IL-15 Activates the Jak3/STAT3 Signaling Pathway to Mediate Glucose Uptake in Skeletal Muscle Cells.

    Science.gov (United States)

    Krolopp, James E; Thornton, Shantaé M; Abbott, Marcia J

    2016-01-01

    Myokines are specialized cytokines that are secreted from skeletal muscle (SKM) in response to metabolic stimuli, such as exercise. Interleukin-15 (IL-15) is a myokine with potential to reduce obesity and increase lean mass through induction of metabolic processes. It has been previously shown that IL-15 acts to increase glucose uptake in SKM cells. However, the downstream signals orchestrating the link between IL-15 signaling and glucose uptake have not been fully explored. Here we employed the mouse SKM C2C12 cell line to examine potential downstream targets of IL-15-induced alterations in glucose uptake. Following differentiation, C2C12 cells were treated overnight with 100 ng/ml of IL-15. Activation of factors associated with glucose metabolism (Akt and AMPK) and known downstream targets of IL-15 (Jak1, Jak3, STAT3, and STAT5) were assessed with IL-15 stimulation. IL-15 stimulated glucose uptake and GLUT4 translocation to the plasma membrane. IL-15 treatment had no effect on phospho-Akt, phospho-Akt substrates, phospho-AMPK, phospho-Jak1, or phospho-STAT5. However, with IL-15, phospho-Jak3 and phospho-STAT3 levels were increased along with increased interaction of Jak3 and STAT3. Additionally, IL-15 induced a translocation of phospho-STAT3 from the cytoplasm to the nucleus. We have evidence that a mediator of glucose uptake, HIF1α, expression was dependent on IL-15 induced STAT3 activation. Finally, upon inhibition of STAT3 the positive effects of IL-15 on glucose uptake and GLUT4 translocation were abolished. Taken together, we provide evidence for a novel signaling pathway for IL-15 acting through Jak3/STAT3 to regulate glucose metabolism.

  20. IL-15 Activates the Jak3/STAT3 Signaling Pathway to Mediate Glucose Uptake in Skeletal Muscle Cells

    Directory of Open Access Journals (Sweden)

    James E Krolopp

    2016-12-01

    Full Text Available Myokines are specialized cytokines that are secreted from skeletal muscle (SKM in response to metabolic stimuli, such as exercise. Interleukin-15 (IL-15 is a myokine with potential to reduce obesity and increase lean mass through induction of metabolic processes. It has been previously shown that IL-15 acts to increase glucose uptake in SKM cells. However, the downstream signals orchestrating the link between IL-15 signaling and glucose uptake have not been fully explored. Here we employed the mouse SKM C2C12 cell line to examine potential downstream targets of IL-15-induced alterations in glucose uptake. Following differentiation, C2C12 cells were treated overnight with 100 ng/ml of IL-15. Activation of factors associated with glucose metabolism (Akt and AMPK and known downstream targets of IL-15 (Jak1, Jak3, STAT3, and STAT5 were assessed with IL-15 stimulation. IL-15 stimulated glucose uptake and GLUT4 translocation to the plasma membrane. IL-15 treatment had no effect on phospho-Akt, phospho-Akt substrates, phospho-AMPK, phospho-Jak1, or phospho-STAT5. However, with IL-15, phospho-Jak3 and phospho-STAT3 levels were increased along with increased interaction of Jak3 and STAT3. Additionally, IL-15 induced a translocation of phospho-STAT3 from the cytoplasm to the nucleus. We have evidence that a mediator of glucose uptake, HIF1α, expression was dependent on IL-15 induced STAT3 activation. Finally, upon inhibition of STAT3 the positive effects of IL-15 on glucose uptake and GLUT4 translocation were abolished. Taken together, we provide evidence for a novel signaling pathway for IL-15 acting through Jak3/STAT3 to regulate glucose metabolism.

  1. Measurement of skeletal muscle collagen breakdown by microdialysis

    DEFF Research Database (Denmark)

    Miller, B F; Ellis, D; Robinson, M M

    2011-01-01

    Exercise increases the synthesis of collagen in the extracellular matrix of skeletal muscle. Breakdown of skeletal muscle collagen has not yet been determined because of technical limitations. The purpose of the present study was to use local sampling to determine skeletal muscle collagen breakdown......, healthy male subjects performed a bout of resistance exercise with one leg, followed 17–21 h later by in vivo skeletal muscle sampling by microdialysis in exercised (EX) and control (CON) legs. Microdialysis reliably predicted [OHP] in vitro (R2=0.90). Analysis with GC–MS was strongly correlated...... to traditional analysis methods (CON: slope=1.03, R2=0.896, and Pskeletal muscle...

  2. GLUT-3 expression in human skeletal muscle

    Science.gov (United States)

    Stuart, C. A.; Wen, G.; Peng, B. H.; Popov, V. L.; Hudnall, S. D.; Campbell, G. A.

    2000-01-01

    Muscle biopsy homogenates contain GLUT-3 mRNA and protein. Before these studies, it was unclear where GLUT-3 was located in muscle tissue. In situ hybridization using a midmolecule probe demonstrated GLUT-3 within all muscle fibers. Fluorescent-tagged antibody reacting with affinity-purified antibody directed at the carboxy-terminus demonstrated GLUT-3 protein in all fibers. Slow-twitch muscle fibers, identified by NADH-tetrazolium reductase staining, possessed more GLUT-3 protein than fast-twitch fibers. Electron microscopy using affinity-purified primary antibody and gold particle-tagged second antibody showed that the majority of GLUT-3 was in association with triads and transverse tubules inside the fiber. Strong GLUT-3 signals were seen in association with the few nerves that traversed muscle sections. Electron microscopic evaluation of human peripheral nerve demonstrated GLUT-3 within the axon, with many of the particles related to mitochondria. GLUT-3 protein was found in myelin but not in Schwann cells. GLUT-1 protein was not present in nerve cells, axons, myelin, or Schwann cells but was seen at the surface of the peripheral nerve in the perineurium. These studies demonstrated that GLUT-3 mRNA and protein are expressed throughout normal human skeletal muscle, but the protein is predominantly found in the triads of slow-twitch muscle fibers.

  3. FGFR1 inhibits skeletal muscle atrophy associated with hindlimb suspension

    Directory of Open Access Journals (Sweden)

    Gerrard Dave

    2007-04-01

    Full Text Available Abstract Background Skeletal muscle atrophy can occur under many different conditions, including prolonged disuse or immobilization, cachexia, cushingoid conditions, secondary to surgery, or with advanced age. The mechanisms by which unloading of muscle is sensed and translated into signals controlling tissue reduction remains a major question in the field of musculoskeletal research. While the fibroblast growth factors (FGFs and their receptors are synthesized by, and intimately involved in, embryonic skeletal muscle growth and repair, their role maintaining adult muscle status has not been examined. Methods We examined the effects of ectopic expression of FGFR1 during disuse-mediated skeletal muscle atrophy, utilizing hindlimb suspension and DNA electroporation in mice. Results We found skeletal muscle FGF4 and FGFR1 mRNA expression to be modified by hind limb suspension,. In addition, we found FGFR1 protein localized in muscle fibers within atrophying mouse muscle which appeared to be resistant to atrophy. Electroporation and ectopic expression of FGFR1 significantly inhibited the decrease in muscle fiber area within skeletal muscles of mice undergoing suspension induced muscle atrophy. Ectopic FGFR1 expression in muscle also significantly stimulated protein synthesis in muscle fibers, and increased protein degradation in weight bearing muscle fibers. Conclusion These results support the theory that FGF signaling can play a role in regulation of postnatal skeletal muscle maintenance, and could offer potentially novel and efficient therapeutic options for attenuating muscle atrophy during aging, illness and spaceflight.

  4. L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an iNOS-Dependent Mechanism.

    Directory of Open Access Journals (Sweden)

    Daniel J Ham

    Full Text Available Dietary L-citrulline is thought to modulate muscle protein turnover by increasing L-arginine availability. To date, the direct effects of increased L-citrulline concentrations in muscle have been completely neglected. Therefore, we determined the role of L-citrulline in regulating cell size during catabolic conditions by depriving mature C2C12 myotubes of growth factors (serum free; SF or growth factors and nutrients (HEPES buffered saline; HBS. Cells were treated with L-citrulline or equimolar concentrations of L-arginine (positive control or L-alanine (negative control and changes in cell size and protein turnover were assessed. In myotubes incubated in HBS or SF media, L-citrulline improved rates of protein synthesis (HBS: +63%, SF: +37% and myotube diameter (HBS: +18%, SF: +29%. L-citrulline treatment substantially increased iNOS mRNA expression (SF: 350%, HBS: 750%. The general NOS inhibitor L-NAME and the iNOS specific inhibitor aminoguanidine prevented these effects in both models. Depriving myotubes in SF media of L-arginine or L-leucine, exacerbated wasting which was not attenuated by L-citrulline. The increased iNOS mRNA expression was temporally associated with increases in mRNA of the endogenous antioxidants SOD1, SOD3 and catalase. Furthermore, L-citrulline prevented inflammation (LPS and oxidative stress (H2O2 induced muscle cell wasting. In conclusion, we demonstrate a novel direct protective effect of L-citrulline on skeletal muscle cell size independent of L-arginine that is mediated through induction of the inducible NOS (iNOS isoform. This discovery of a nutritional modulator of iNOS mRNA expression in skeletal muscle cells could have substantial implications for the treatment of muscle wasting conditions.

  5. Sex hormones and skeletal muscle weakness

    DEFF Research Database (Denmark)

    Sipilä, Sarianna; Narici, Marco; Kjaer, Michael

    2013-01-01

    Human ageing is accompanied with deterioration in endocrine functions the most notable and well characterized of which being the decrease in the production of sex hormones. Current research literature suggests that low sex hormone concentration may be among the key mechanism for sarcopenia...... and muscle weakness. Within the European large scale MYOAGE project, the role of sex hormones, estrogens and testosterone, in causing the aging-related loss of muscle mass and function was further investigated. Hormone replacement therapy (HRT) in women is shown to diminish age-associated muscle loss, loss...... properties. HRT influences gene expression in e.g. cytoskeletal and cell-matrix proteins, has a stimulating effect upon IGF-I, and a role in IL-6 and adipokine regulation. Despite low circulating steroid-hormone level, postmenopausal women have a high local concentration of steroidogenic enzymes in skeletal...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-15

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

  7. PGC-1α-mediated adaptations in skeletal muscle

    DEFF Research Database (Denmark)

    Olesen, Jesper; Kiilerich, Kristian; Pilegaard, Henriette

    2010-01-01

    Lifestyle-related diseases are rapidly increasing at least in part due to less physical activity. The health beneficial effects of regular physical activity include metabolic adaptations in skeletal muscle, which are thought to be elicited by cumulative effects of transient gene responses to each...... to be an underlying mechanism for adaptations in skeletal muscle, when exercise is repeated. The current review presents some of the key findings in PGC-1alpha-mediated regulation of metabolically related, anti-oxidant and inflammatory proteins in skeletal muscle in the basal state and in response to exercise...... training, and describes functional significance of PGC-1alpha-mediated effects in skeletal muscle. In addition, regulation of PGC-1alpha expression and activity in skeletal muscle is described. The impact of changes in PGC-1alpha expression in mouse skeletal muscle and the ability of PGC-1alpha to regulate...

  8. Inferring crossbridge properties from skeletal muscle energetics.

    Science.gov (United States)

    Barclay, C J; Woledge, R C; Curtin, N A

    2010-01-01

    Work is generated in muscle by myosin crossbridges during their interaction with the actin filament. The energy from which the work is produced is the free energy change of ATP hydrolysis and efficiency quantifies the fraction of the energy supplied that is converted into work. The purpose of this review is to compare the efficiency of frog skeletal muscle determined from measurements of work output and either heat production or chemical breakdown with the work produced per crossbridge cycle predicted on the basis of the mechanical responses of contracting muscle to rapid length perturbations. We review the literature to establish the likely maximum crossbridge efficiency for frog skeletal muscle (0.4) and, using this value, calculate the maximum work a crossbridge can perform in a single attachment to actin (33 x 10(-21) J). To see whether this amount of work is consistent with our understanding of crossbridge mechanics, we examine measurements of the force responses of frog muscle to fast length perturbations and, taking account of filament compliance, determine the crossbridge force-extension relationship and the velocity dependences of the fraction of crossbridges attached and average crossbridge strain. These data are used in combination with a Huxley-Simmons-type model of the thermodynamics of the attached crossbridge to determine whether this type of model can adequately account for the observed muscle efficiency. Although it is apparent that there are still deficiencies in our understanding of how to accurately model some aspects of ensemble crossbridge behaviour, this comparison shows that crossbridge energetics are consistent with known crossbridge properties.

  9. Skeletal muscle disorders of glycogenolysis and glycolysis.

    Science.gov (United States)

    Godfrey, Richard; Quinlivan, Ros

    2016-07-01

    Skeletal muscle disorders of glycogenolysis and glycolysis account for most of the conditions collectively termed glycogen storage diseases (GSDs). These disorders are rare (incidence 1 in 20,000-43,000 live births), and are caused by autosomal or X-linked recessive mutations that result in a specific enzyme deficiency, leading to the inability to utilize muscle glycogen as an energy substrate. McArdle disease (GSD V) is the most common of these disorders, and is caused by mutations in the gene encoding muscle glycogen phosphorylase. Symptoms of McArdle disease and most other related GSDs include exercise intolerance, muscle contracture, acute rhabdomyolysis, and risk of acute renal failure. Older patients may exhibit muscle wasting and weakness involving the paraspinal muscles and shoulder girdle. For patients with these conditions, engaging with exercise is likely to be beneficial. Diagnosis is frequently delayed owing to the rarity of the conditions and lack of access to appropriate investigations. A few randomized clinical trials have been conducted, some focusing on dietary modification, although the quality of the evidence is low and no specific recommendations can yet be made. The development of EUROMAC, an international registry for these disorders, should improve our knowledge of their natural histories and provide a platform for future clinical trials.

  10. Resistance Exercise Training Alters Mitochondrial Function in Human Skeletal Muscle.

    Science.gov (United States)

    Porter, Craig; Reidy, Paul T; Bhattarai, Nisha; Sidossis, Labros S; Rasmussen, Blake B

    2015-09-01

    Loss of mitochondrial competency is associated with several chronic illnesses. Therefore, strategies that maintain or increase mitochondrial function will likely be of benefit in numerous clinical settings. Endurance exercise has long been known to increase mitochondrial function in the skeletal muscle. Comparatively little is known regarding the effect of resistance exercise training (RET) on skeletal muscle mitochondrial respiratory function. The purpose of the current study was to determine the effect of chronic resistance training on skeletal muscle mitochondrial respiratory capacity and function. Here, we studied the effect of a 12-wk RET program on skeletal muscle mitochondrial function in 11 young healthy men. Muscle biopsies were collected before and after the 12-wk training program, and mitochondrial respiratory capacity was determined in permeabilized myofibers by high-resolution respirometry. RET increased lean body mass and quadriceps muscle strength by 4% and 15%, respectively (P training (P function of skeletal muscle mitochondria.

  11. Cardiovascular regulation by skeletal muscle reflexes in health and disease

    National Research Council Canada - National Science Library

    Murphy, Megan N; Mizuno, Masaki; Mitchell, Jere H; Smith, Scott A

    2011-01-01

    .... These neurally mediated cardiovascular adjustments to physical activity are regulated, in part, by a peripheral reflex originating in contracting skeletal muscle termed the exercise pressor reflex...

  12. Regulation of PDH, GS and insulin signalling in skeletal muscle

    DEFF Research Database (Denmark)

    Biensø, Rasmus Sjørup

    The aims of the present thesis were to investigate 1) The impact of physical inactivity on insulinstimulated Akt, TBC1D4 and GS regulation in human skeletal muscle, 2) The impact of exercise training on glucose-mediated regulation of PDH and GS in skeletal muscle in elderly men, 3) The impact...... with physical inactivity in humans, and physical inactivity did not affect the ability of exercise to enhance insulinmediated skeletal muscle glucose extraction. 2) Exercise training-improved glucose handling in aged human skeletal muscle was associated with increased content of key proteins in glucose...

  13. Cryopreservation of human skeletal muscle impairs mitochondrial function

    DEFF Research Database (Denmark)

    Larsen, Steen; Wright-Paradis, C; Gnaiger, E

    2012-01-01

    Previous studies have investigated if cryopreservation is a viable approach for functional mitochondrial analysis. Different tissues have been studied, and conflicting results have been published. The aim of the present study was to investigate if mitochondria in human skeletal muscle maintain...... functionality after long term cryopreservation (1 year). Skeletal muscle samples were preserved in dimethyl sulfoxide (DMSO) for later analysis. Human skeletal muscle fibres were thawed and permeabilised with saponin, and mitochondrial respiration was measured by high-resolution respirometry. The capacity...... of oxidative phosphorylation was significantly (P cryopreserved human skeletal muscle samples. Cryopreservation impaired respiration with substrates linked to Complex I more than for Complex II (P

  14. Skeletal muscle increases FGF21 expression in mitochondrial disorders to compensate for energy metabolic insufficiency by activating the mTOR-YY1-PGC1α pathway.

    Science.gov (United States)

    Ji, Kunqian; Zheng, Jinfan; Lv, Jingwei; Xu, Jingwen; Ji, Xinbo; Luo, Yue-Bei; Li, Wei; Zhao, Yuying; Yan, Chuanzhu

    2015-07-01

    Fibroblast growth factor 21 (FGF21) is a growth factor with pleiotropic effects on regulating lipid and glucose metabolism. Its expression is increased in skeletal muscle of mice and humans with mitochondrial disorders. However, the effects of FGF21 on skeletal muscle in response to mitochondrial respiratory chain deficiency are largely unknown. Here we demonstrate that the increased expression of FGF21 is a compensatory response to respiratory chain deficiency. The mRNA and protein levels of FGF21 were robustly raised in skeletal muscle from patients with mitochondrial myopathy or MELAS. The mammalian target of rapamycin (mTOR) phosphorylation levels and its downstream targets, Yin Yang 1 (YY1) and peroxisome proliferator-activated receptor γ, coactivator 1α (PGC-1α), were increased by FGF21 treatment in C2C12 myoblasts. Activation of the mTOR-YY1-PGC1α pathway by FGF21 in myoblasts regulated energy homeostasis as demonstrated by significant increases in intracellular ATP synthesis, oxygen consumption rate, activity of citrate synthase, glycolysis, mitochondrial DNA copy number, and induction of the expression of key energy metabolic genes. The effects of FGF21 on mitochondrial function required phosphoinositide 3-kinase (PI3K), which activates mTOR. Inhibition of PI3K, mTOR, YY1, and PGC-1α activities attenuated the stimulating effects of FGF21 on intracellular ATP levels and mitochondrial gene expression. Our findings revealed that mitochondrial respiratory chain deficiency elicited a compensatory response in skeletal muscle by increasing the FGF21 expression levels in muscle, which resulted in enhanced mitochondrial function through an mTOR-YY1-PGC1α-dependent pathway in skeletal muscle. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Overexpression of SMPX in adult skeletal muscle does not change skeletal muscle fiber type or size.

    Directory of Open Access Journals (Sweden)

    Einar Eftestøl

    Full Text Available Mechanical factors such as stretch are thought to be important in the regulation of muscle phenotype. Small muscle protein X-linked (SMPX is upregulated by stretch in skeletal muscle and has been suggested to serve both as a transcription factor and a mechanosensor, possibly giving rise to changes in both fiber size and fiber type. We have used in vivo confocal imaging to study the subcellular localization of SMPX in skeletal muscle fibers of adult rats using a SMPX-EGFP fusion protein. The fusion protein was localized predominantly in repetitive double stripes flanking the Z-disc, and was excluded from all nuclei. This localization would be consistent with SMPX being a mechanoreceptor, but not with SMPX playing a role as a transcription factor. In vivo overexpression of ectopic SMPX in skeletal muscle of adult mice gave no significant changes in fiber type distribution or cross sectional area, thus a role of SMPX in regulating muscle phenotype remains unclear.

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

    Science.gov (United States)

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

    2016-12-01

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

  17. Effect of vitamin D on skeletal muscle.

    Science.gov (United States)

    Walrand, Stéphane

    2016-06-01

    Beyond its traditional biological roles on bone health, extra-skeletal effects of vitamin D are currently under extensive research. The expression of the vitamin D receptor in most tissues has also strengthened the argument for its multiple functions. Among these, the effect of vitamin D on the mass and muscle performance has long been discussed. In ancient Greece, Herodotus recommended the sun as a cure for the "weak and soft muscles" and former Olympians exposed to sunlight to improve their physical performance. In 1952, Dr Spellerberg, a sports physiologist, has conducted an extensive study on the effects of UV irradiation on the performance of elite athletes. Following the significant results of this investigation, the scientist has informed the Olympic Committee that UV irradiation had a "persuasive" effect on physical performance and motor skills. These data are consistent with many subsequent studies reporting an improvement in physical activity, speed and endurance in young subjects treated with UV or with supplements containing vitamin D. Additional observation indicates a significant effect on muscle strength, particularly in the lower limbs. Concerning the mechanisms involved, some recent fundamental studies have shown that vitamin D exerts some molecular effects within the muscle cell. Specifically, a regulatory effect of vitamin D on calcium flux, mineral homeostasis and signaling pathways controlling protein anabolism has been reported in muscle tissue. Several epidemiological studies show that low vitamin D status is always associated with a decrease in muscle mass, strength and contractile capacity in older people. Vitamin D deficiency accelerates muscle loss with age (sarcopenia), and therefore leads to a reduction in physical capacity and to an increased risk of falls and fractures. In contrast, an additional intake of vitamin D in older people significantly improves muscle function and physical performance.

  18. Osmoregulatory processes and skeletal muscle metabolism

    Science.gov (United States)

    Boschmann, Michael; Gottschalk, Simone; Adams, Frauke; Luft, Friedrich C.; Jordan, Jens

    Prolonged microgravity during space flight is associated with a decrease in blood and extracellular volume. These changes in water and electrolyte balance might activate catabolic processes which contribute finally to the loss of muscle and bone mass and strength. Recently, we found a prompt increase that energy expenditure by about 30% in both normal and overweight men and women after drinking 500 ml water. This effect is mediated by an increased sympathetic nervous system activity, obviously secondary to stimulation of osmosensitive afferent neurons in the liver, and skeletal muscle is possibly one effector organ. Therefore, we tested the hypothesis that this thermogenic response to water is accompanied by a stimulation of aerobic glucose metabolism in skeletal muscle. To this end, 16 young healthy volunteers (8 men) were studied. After an overnight fast (12h), a microdialysis probe was implanted into the right M. quadriceps femoris vastus lateralis and subsequently perfused with Ringer's solution (+50 mM ethanol). After 1h, volunteers were asked to drink 500 ml water (22° C) followed by continuing microdialysis for another 90 min. Dialysates (15 min fractions) were analyzed for [ethanol], [glucose], [lactate], [pyruvate], and [glycerol] in order to assess changes in muscle tissue perfusion (ethanol dilution technique), glycolysis and lipolysis. Blood samples were taken and heart rate (HR) and blood pressure (BP) were monitored. Neither HR and systolic and diastolic BP, nor plasma [glucose], [lactate], [insulin], and [C peptide] changed significantly after water drinking. Also, tissue perfusion and dialysate [glucose] did not change significantly. However, dialysate [lactate] increased by about 10 and 20% and dialysate [pyruvate] by about 100 and 200% in men and women, respectively. In contrast, dialysate [glycerol] decreased by about 30 and 20% in men and women, respectively. Therefore, drinking of 500 ml water stimulates aerobic glucose metabolism and inhibits

  19. Exercise-induced GLUT4 transcription via inactivation of HDAC4/5 in mouse skeletal muscle in an AMPKα2-dependent manner.

    Science.gov (United States)

    Niu, Yanmei; Wang, Tianyi; Liu, Sujuan; Yuan, Hairui; Li, Huige; Fu, Li

    2017-09-01

    Abnormal glucose metabolism induces various metabolic disorders such as insulin resistance and type 2 diabetes. Regular exercise improved glucose uptake and enhanced glucose oxidation by increasing GLUT4 transcription in skeletal muscle. However, the regulatory mechanisms of GLUT4 transcription in response to exercise are poorly understood. AMPK is a sensor of exercise and upstream kinase of class II HDACs that act as transcriptional repressors. We used 6-week treadmill exercise or one single-bout exercise wild type or AMPKα2 -/- C57BL/6J mice to explore how HDACs regulate GLUT4 transcription and the underlying molecular mechanisms mediated by AMPK in the physiologic process of exercise. We demonstrate that regular physical exercise significantly enhanced GLUT4 transcription by inactivating HDAC4/5 in skeletal muscle by ChIP experiment. HDAC4 coordinately regulated with HDAC5 represses transcriptional activity of GLUT4 promoter in C2C12 myotubes by Luciferase assay. If either HDAC4 or HDAC5 is silenced via RNAi technology, the functional compensation by the other will occur. In addition, a single-bout of exercise decreased HDAC4/5 activity in skeletal muscle of wild type but not in AMPKα2 -/- mice, suggesting an AMPKα2-dependent manner. Those findings provide new insight into the mechanisms responsible for AMPKα2-dependent regulation of GLUT4 transcription after exercise. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Increased skeletal muscle capillarization enhances insulin sensitivity

    DEFF Research Database (Denmark)

    Åkerström, Thorbjörn; Laub, Lasse; Vedel, Kenneth

    2014-01-01

    . Skeletal muscle specific angiogenesis was induced by adding the α1-adrenergic receptor antagonist Prazosin to the drinking water of Sprague Dawley rats (n=33) while 34 rats served as controls. Insulin sensitivity was measured ≥40 h after termination of the 3-week Prazosin treatment, which ensured...... that Prazosin was cleared from the blood stream. Whole-body insulin sensitivity was measured in conscious, unrestrained rats by hyperinsulinemic euglycemic clamp. Tissue specific insulin sensitivity was assessed by administration of 2-deoxy-[(3)H]-Glucose during the plateau phase of the clamp. Whole-body...

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

    Directory of Open Access Journals (Sweden)

    Lan eYe

    2012-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Manuel Antonio Riquelme

    2015-05-01

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

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

    Science.gov (United States)

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

    2011-11-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  5. Stretching skeletal muscle: chronic muscle lengthening through sarcomerogenesis.

    Directory of Open Access Journals (Sweden)

    Alexander M Zöllner

    Full Text Available Skeletal muscle responds to passive overstretch through sarcomerogenesis, the creation and serial deposition of new sarcomere units. Sarcomerogenesis is critical to muscle function: It gradually re-positions the muscle back into its optimal operating regime. Animal models of immobilization, limb lengthening, and tendon transfer have provided significant insight into muscle adaptation in vivo. Yet, to date, there is no mathematical model that allows us to predict how skeletal muscle adapts to mechanical stretch in silico. Here we propose a novel mechanistic model for chronic longitudinal muscle growth in response to passive mechanical stretch. We characterize growth through a single scalar-valued internal variable, the serial sarcomere number. Sarcomerogenesis, the evolution of this variable, is driven by the elastic mechanical stretch. To analyze realistic three-dimensional muscle geometries, we embed our model into a nonlinear finite element framework. In a chronic limb lengthening study with a muscle stretch of 1.14, the model predicts an acute sarcomere lengthening from 3.09[Formula: see text]m to 3.51[Formula: see text]m, and a chronic gradual return to the initial sarcomere length within two weeks. Compared to the experiment, the acute model error was 0.00% by design of the model; the chronic model error was 2.13%, which lies within the rage of the experimental standard deviation. Our model explains, from a mechanistic point of view, why gradual multi-step muscle lengthening is less invasive than single-step lengthening. It also explains regional variations in sarcomere length, shorter close to and longer away from the muscle-tendon interface. Once calibrated with a richer data set, our model may help surgeons to prevent muscle overstretch and make informed decisions about optimal stretch increments, stretch timing, and stretch amplitudes. We anticipate our study to open new avenues in orthopedic and reconstructive surgery and enhance

  6. Troponin T3 regulates nuclear localization of the calcium channel Ca{sub v}β{sub 1a} subunit in skeletal muscle

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Tan; Taylor, Jackson; Jiang, Yang [Department of Internal Medicine-Gerontology, Wake Forest School of Medicine, Winston-Salem, NC 27157 (United States); Pereyra, Andrea S. [Department of Histology, National University of La Plata, 1900 La Plata (Argentina); Messi, Maria Laura; Wang, Zhong-Min [Department of Internal Medicine-Gerontology, Wake Forest School of Medicine, Winston-Salem, NC 27157 (United States); Hereñú, Claudia [Department of Histology, National University of La Plata, 1900 La Plata (Argentina); Delbono, Osvaldo, E-mail: odelbono@wakehealth.edu [Department of Internal Medicine-Gerontology, Wake Forest School of Medicine, Winston-Salem, NC 27157 (United States); Neuroscience Program, Wake Forest School of Medicine, Winston-Salem, NC 27157 (United States)

    2015-08-15

    The voltage-gated calcium channel (Ca{sub v}) β{sub 1a} subunit (Ca{sub v}β{sub 1a}) plays an important role in excitation–contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Ca{sub v}β{sub 1a} subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160–244 aa) and Ca{sub v}β{sub 1a} NH{sub 2}-terminus (1–99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Ca{sub v}β{sub 1a}/YFP shows that TnT3 facilitates Ca{sub v}β{sub 1a} nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation. - Highlights: • Previously, we demonstrated that Ca{sub v}β{sub 1a} is a gene transcription regulator. • Here, we show that TnT3 interacts with Ca{sub v}β{sub 1a}. • We mapped TnT3 and Ca{sub v}β{sub 1a} interaction domain. • TnT3 facilitates Ca{sub v}β{sub 1a} nuclear enrichment. • The two proteins play a heretofore unknown role during early muscle differentiation.

  7. Masseter muscle thickness in different skeletal morphology: An ultrasonographic study

    Directory of Open Access Journals (Sweden)

    Rani Sushma

    2010-01-01

    Full Text Available Background: The thickness of the masseter muscle during relaxation and contraction states was measured by ultrasonography. Subjects were classified according to their sagittal skeletal relationships. The association between muscle thickness and facial morphology was studied. Context: Masseter muscle thickness influences the skeletal patterns. Aim: To measure and compare the thickness of the masseter muscle in individuals with skeletal class I occlusion and skeletal class II malocclusions and to correlate its relationship with craniofacial morphology. Settings and Design: The study was conducted in a hospital setup and was designed to study the thickness of the masseter muscle in different skeletal morphologies. Materials and Methods: Seventy two individuals between the ages of 18 and 25 years were divided into Group I, Group IIA and Group IIB according to their skeletal relationships. Masseter muscle thickness was measured by ultrasonography. Eight linear and six angular cephalometric measurements were assessed. Statistical Analysis Used: Analysis of variance and Pearson′s correlation analysis. Results: There was a statistically significant difference in muscle thickness between subjects of different skeletal patterns. Significant positive correlation between masseter muscle thickness and posterior total face height, jarabak ratio, ramus height, mandibular length and significant negative correlations with mandibular plane angle, gonial angle and PP-MP angle were observed. Conclusion: This study indicates the strong association between the masseter muscle and skeletal morphology.

  8. Endoplasmic Reticulum Stress in Skeletal Muscle Homeostasis and Disease

    OpenAIRE

    Rayavarapu, Sree; Coley, William; Nagaraju, Kanneboyina

    2012-01-01

    Our appreciation of the role of endoplasmic reticulum(ER) stress pathways in both skeletal muscle homeostasis and the progression of muscle diseases is gaining momentum. This review provides insight into ER stress mechanisms during physiologic and pathological disturbances in skeletal muscle. The role of ER stress in the response to dietary alterations and acute stressors, including its role in autoimmune and genetic muscle disorders, has been described. Recent studies identifying ER stress m...

  9. Effects of Ionizing Irradiation on Mouse Diaphragmatic Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Tingyang Zhou

    2017-07-01

    Full Text Available Undesirable exposure of diaphragm to radiation during thoracic radiation therapy has not been fully considered over the past decades. Our study aims to examine the potential biological effects on diaphragm induced by radiation. One-time ionizing irradiation of 10 Gy was applied either to the diaphragmatic region of mice or to the cultured C2C12 myocytes. Each sample was then assayed for muscle function, oxidative stress, or cell viability on days 1, 3, 5, and 7 after irradiation. Our mouse model shows that radiation significantly reduced muscle function on the 5th and 7th days and increased reactive oxygen species (ROS formation in the diaphragm tissue from days 3 to 7. Similarly, the myocytes exhibited markedly decreased viability and elevated oxidative stress from days 5 to 7 after radiation. These data together suggested that a single dose of 10-Gy radiation is sufficient to cause acute adverse effects on diaphragmatic muscle function, redox balance, and myocyte survival. Furthermore, using the collected data, we developed a physical model to formularize the correlation between diaphragmatic ROS release and time after irradiation, which can be used to predict the biological effects of radiation with a specific dosage. Our findings highlight the importance of developing protective strategies to attenuate oxidative stress and prevent diaphragm injury during radiotherapy.

  10. Satellite cell proliferation in adult skeletal muscle

    Science.gov (United States)

    Booth, Frank W. (Inventor); Thomason, Donald B. (Inventor); Morrison, Paul R. (Inventor); Stancel, George M. (Inventor)

    1995-01-01

    Novel methods of retroviral-mediated gene transfer for the in vivo corporation and stable expression of eukaryotic or prokaryotic foreign genes in tissues of living animals is described. More specifically, methods of incorporating foreign genes into mitotically active cells are disclosed. The constitutive and stable expression of E. coli .beta.-galactosidase gene under the promoter control of the Moloney murine leukemia virus long terminal repeat is employed as a particularly preferred embodiment, by way of example, establishes the model upon which the incorporation of a foreign gene into a mitotically-active living eukaryotic tissue is based. Use of the described methods in therapeutic treatments for genetic diseases, such as those muscular degenerative diseases, is also presented. In muscle tissue, the described processes result in genetically-altered satellite cells which proliferate daughter myoblasts which preferentially fuse to form a single undamaged muscle fiber replacing damaged muscle tissue in a treated animal. The retroviral vector, by way of example, includes a dystrophin gene construct for use in treating muscular dystrophy. The present invention also comprises an experimental model utilizable in the study of the physiological regulation of skeletal muscle gene expression in intact animals.

  11. Effects of dietary eicosapentaenoic acid (EPA supplementation in high-fat fed mice on lipid metabolism and apelin/APJ system in skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Chantal Bertrand

    Full Text Available Various studies have shown that eicosapentaenoic acid (EPA has beneficial effects on obesity and associated disorders. Apelin, the ligand of APJ receptor also exerts insulin-sensitizing effects especially by improving muscle metabolism. EPA has been shown to increase apelin production in adipose tissue but its effects in muscle have not been addressed. Thus, the effects of EPA supplementation (36 g/kg EPA in high-fat diet (HFD (45% fat, 20% protein, 35% carbohydrate were studied in mice with focus on muscle lipid metabolism and apelin/APJ expression. Compared with HFD mice, HFD+EPA mice had significantly less weight gain, fat mass, lower blood glucose, insulinemia and hepatic steatosis after 10 weeks of diet. In addition, EPA prevented muscle metabolism alterations since intramuscular triglycerides were decreased and β-oxidation increased. In soleus muscles of HFD+EPA mice, apelin and APJ expression were significantly increased compared to HFD mice. However, plasma apelin concentrations in HFD and HFD+EPA mice were similar. EPA-induced apelin expression was confirmed in differentiated C2C12 myocytes but in this model, apelin secretion was also increased in response to EPA treatment. In conclusion, EPA supplementation in HFD prevents obesity and metabolic alterations in mice, especially in skeletal muscle. Since EPA increases apelin/APJ expression in muscle, apelin may act in a paracrine/autocrine manner to contribute to these benefical effects.

  12. Current opportunities and challenges in skeletal muscle tissue engineering

    NARCIS (Netherlands)

    Koning, Merel; Harmsen, Martin C; van Luyn, Marja J A; Werker, Paul M N

    The purpose of this article is to give a concise review of the current state of the art in tissue engineering (TE) of skeletal muscle and the opportunities and challenges for future clinical applicability. The endogenous progenitor cells of skeletal muscle, i.e. satellite cells, show a high

  13. Regulatory factors and cell populations involved in skeletal muscle regeneration.

    NARCIS (Netherlands)

    Broek, R.W. Ten; Grefte, S.; Hoff, J.W. von den

    2010-01-01

    Skeletal muscle regeneration is a complex process, which is not yet completely understood. Satellite cells, the skeletal muscle stem cells, become activated after trauma, proliferate, and migrate to the site of injury. Depending on the severity of the myotrauma, activated satellite cells form new

  14. Mitochondrial biogenesis and angiogenesis in skeletal muscle of the elderly

    DEFF Research Database (Denmark)

    Iversen, Ninna; Krustrup, Peter; Rasmussen, Hans N

    2011-01-01

    The aim of this study was to test the hypotheses that 1) skeletal muscles of elderly subjects can adapt to a single endurance exercise bout and 2) endurance trained elderly subjects have higher expression/activity of oxidative and angiogenic proteins in skeletal muscle than untrained elderly people...

  15. Regulation of mechano growth factor in skeletal muscle and heart

    NARCIS (Netherlands)

    Ottens, M.

    2010-01-01

    The mechano growth factor (MGF) is expressed in mechanically overloaded skeletal muscle. MGF was discovered in 1996 as an alternative splice product of the IGF-1 gene. Since then, its significance has been investigated particularly in skeletal muscle, because the local expression of MGF could

  16. Skeletal muscle stem cells from animals I. Basic cell biology

    Science.gov (United States)

    Skeletal muscle stem cells from food-producing animals have been of interest to agricultural life scientists seeking to develop a better understanding of the molecular regulation of lean tissue (skeletal muscle protein hypertrophy) and intramuscular fat (marbling) development. Enhanced understanding...

  17. Insulin inhibits AMPK activity and phosphorylates AMPK Ser⁴⁸⁵/⁴⁹¹ through Akt in hepatocytes, myotubes and incubated rat skeletal muscle.

    Science.gov (United States)

    Valentine, Rudy J; Coughlan, Kimberly A; Ruderman, Neil B; Saha, Asish K

    2014-11-15

    Recent studies have highlighted the importance of an inhibitory phosphorylation site, Ser(485/491), on the α-subunit of AMP-activated protein kinase (AMPK); however, little is known about the regulation of this site in liver and skeletal muscle. We examined whether the inhibitory effects of insulin on AMPK activity may be mediated through the phosphorylation of this inhibitory Ser(485/491) site in hepatocytes, myotubes and incubated skeletal muscle. HepG2 and C2C12 cells were stimulated with or without insulin for 15-min. Similarly, rat extensor digitorum longus (EDL) muscles were treated +/- insulin for 10-min. Insulin significantly increased Ser(485/491) p-AMPK under all conditions, resulting in a subsequent reduction in AMPK activity, ranging from 40% to 70%, despite no change in p-AMPK Thr(172). Akt inhibition both attenuated the increase in Ser(485/491) p-AMPK caused by insulin, and prevented the decrease in AMPK activity. Similarly, the growth factor IGF-1 stimulated Ser(485/491) AMPK phosphorylation, and this too was blunted by inhibition of Akt. Inhibition of the mTOR pathway with rapamycin, however, had no effect on insulin-stimulated Ser(485/491) p-AMPK. These data suggest that insulin and IGF-1 diminish AMPK activity in hepatocytes and muscle, most likely through Akt activation and the inhibitory phosphorylation of Ser(485/491) on its α-subunit. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. TNF inhibits Notch-1 in skeletal muscle cells by Ezh2 and DNA methylation mediated repression: implications in duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Swarnali Acharyya

    2010-08-01

    Full Text Available Classical NF-kappaB signaling functions as a negative regulator of skeletal myogenesis through potentially multiple mechanisms. The inhibitory actions of TNFalpha on skeletal muscle differentiation are mediated in part through sustained NF-kappaB activity. In dystrophic muscles, NF-kappaB activity is compartmentalized to myofibers to inhibit regeneration by limiting the number of myogenic progenitor cells. This regulation coincides with elevated levels of muscle derived TNFalpha that is also under IKKbeta and NF-kappaB control.Based on these findings we speculated that in DMD, TNFalpha secreted from myotubes inhibits regeneration by directly acting on satellite cells. Analysis of several satellite cell regulators revealed that TNFalpha is capable of inhibiting Notch-1 in satellite cells and C2C12 myoblasts, which was also found to be dependent on NF-kappaB. Notch-1 inhibition occurred at the mRNA level suggesting a transcriptional repression mechanism. Unlike its classical mode of action, TNFalpha stimulated the recruitment of Ezh2 and Dnmt-3b to coordinate histone and DNA methylation, respectively. Dnmt-3b recruitment was dependent on Ezh2.We propose that in dystrophic muscles, elevated levels of TNFalpha and NF-kappaB inhibit the regenerative potential of satellite cells via epigenetic silencing of the Notch-1 gene.

  19. Angiotensin-(1-7 Prevents Skeletal Muscle Atrophy Induced by Transforming Growth Factor Type Beta (TGF-β via Mas Receptor Activation

    Directory of Open Access Journals (Sweden)

    Johanna Ábrigo

    2016-11-01

    Full Text Available Background: Transforming growth factor type beta 1 (TGF-β1 produces skeletal muscle atrophy. Angiotensin-(1-7 (Ang-(1-7, through the Mas receptor, prevents the skeletal muscle atrophy induced by sepsis, immobilization, or angiotensin II (Ang-II. However, the effect of Ang-(1-7 on muscle wasting induced by TGF-β1 is unknown. Aim: To evaluate whether Ang-(1-7/Mas receptor axis could prevent the skeletal muscle atrophy induced by TGF-β1. Methods: This study assessed the atrophic effect of TGF-β1 in C2C12 myotubes and mice in absence or presence of Ang-(1-7, and the receptor participation using A779, an antagonist of the Mas receptor. The levels of myosin heavy chain (MHC, polyubiquitination, and MuRF-1 were detected by western blot. Myotube diameter was also evaluated. In vivo analysis included the muscle strength, fibre diameter, MHC and MuRF-1 levels by western blot, and ROS levels by DCF probe detection. Results: The results showed that Ang-(1-7 prevented the increase in MuRF-1 and polyubiquitined protein levels, the decrease of MHC levels, the myotubes/fibre diameter diminution, and the increased production of reactive oxygen species (ROS induced by TGF-β1. Utilizing A779 inhibited the anti-atrophic effect of Ang-(1-7. Conclusion: The preventive effect of Ang-(1-7 on skeletal muscle atrophy induced by TGF-β1 is produced through inhibition of ROS production and proteasomal degradation of MHC.

  20. Regulation of Metabolic Signaling in Human Skeletal Muscle

    DEFF Research Database (Denmark)

    Albers, Peter Hjorth

    enzymes. Skeletal muscle consists of thousands of muscle fibers. These fibers can roughly be classified into type I and type II muscle fibers. The overall aim of this PhD thesis was to investigate the effect of insulin and exercise on human muscle fiber type specific metabolic signaling. The importance...

  1. Regulation of exercise-induced fiber type transformation, mitochondrial biogenesis, and angiogenesis in skeletal muscle

    National Research Council Canada - National Science Library

    Zhen Yan; Mitsuharu Okutsu; Yasir N. Akhtar; Vitor A. Lira

    2011-01-01

    .... Chronic increases of skeletal muscle contractile activity, such as endurance exercise, lead to a variety of physiological and biochemical adaptations in skeletal muscle, including mitochondrial...

  2. [Research progress of scaffold materials in skeletal muscle tissue engineering].

    Science.gov (United States)

    Huang, Weiyi; Liao, Hua

    2010-11-01

    To review the current researches of scaffold materials for skeletal muscle tissue engineering, to predict the development trend of scaffold materials in skeletal muscle tissue engineering in future. The related literature on skeletal muscle tissue engineering, involving categories and properties of scaffold materials, preparative technique and biocompatibility, was summarized and analyzed. Various scaffold materials were used in skeletal muscle tissue engineering, including inorganic biomaterials, biodegradable polymers, natural biomaterial, and biomedical composites. According to different needs of the research, various scaffolds were prepared due to different biomaterials, preparative techniques, and surface modifications. The development trend and perspective of skeletal muscle tissue engineering are the use of composite materials, and the preparation of composite scaffolds and surface modification according to the specific functions of scaffolds.

  3. Compatibility of hyaluronic acid hydrogel and skeletal muscle myoblasts

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-04-15

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

  4. Omega-3 Fatty Acids and Skeletal Muscle Health

    Science.gov (United States)

    Jeromson, Stewart; Gallagher, Iain J.; Galloway, Stuart D. R.; Hamilton, D. Lee

    2015-01-01

    Skeletal muscle is a plastic tissue capable of adapting and mal-adapting to physical activity and diet. The response of skeletal muscle to adaptive stimuli, such as exercise, can be modified by the prior nutritional status of the muscle. The influence of nutrition on skeletal muscle has the potential to substantially impact physical function and whole body metabolism. Animal and cell based models show that omega-3 fatty acids, in particular those of marine origin, can influence skeletal muscle metabolism. Furthermore, recent human studies demonstrate that omega-3 fatty acids of marine origin can influence the exercise and nutritional response of skeletal muscle. These studies show that the prior omega-3 status influences not only the metabolic response of muscle to nutrition, but also the functional response to a period of exercise training. Omega-3 fatty acids of marine origin therefore have the potential to alter the trajectory of a number of human diseases including the physical decline associated with aging. We explore the potential molecular mechanisms by which omega-3 fatty acids may act in skeletal muscle, considering the n-3/n-6 ratio, inflammation and lipidomic remodelling as possible mechanisms of action. Finally, we suggest some avenues for further research to clarify how omega-3 fatty acids may be exerting their biological action in skeletal muscle. PMID:26610527

  5. Omega-3 Fatty Acids and Skeletal Muscle Health.

    Science.gov (United States)

    Jeromson, Stewart; Gallagher, Iain J; Galloway, Stuart D R; Hamilton, D Lee

    2015-11-19

    Skeletal muscle is a plastic tissue capable of adapting and mal-adapting to physical activity and diet. The response of skeletal muscle to adaptive stimuli, such as exercise, can be modified by the prior nutritional status of the muscle. The influence of nutrition on skeletal muscle has the potential to substantially impact physical function and whole body metabolism. Animal and cell based models show that omega-3 fatty acids, in particular those of marine origin, can influence skeletal muscle metabolism. Furthermore, recent human studies demonstrate that omega-3 fatty acids of marine origin can influence the exercise and nutritional response of skeletal muscle. These studies show that the prior omega-3 status influences not only the metabolic response of muscle to nutrition, but also the functional response to a period of exercise training. Omega-3 fatty acids of marine origin therefore have the potential to alter the trajectory of a number of human diseases including the physical decline associated with aging. We explore the potential molecular mechanisms by which omega-3 fatty acids may act in skeletal muscle, considering the n-3/n-6 ratio, inflammation and lipidomic remodelling as possible mechanisms of action. Finally, we suggest some avenues for further research to clarify how omega-3 fatty acids may be exerting their biological action in skeletal muscle.

  6. Omega-3 Fatty Acids and Skeletal Muscle Health

    Directory of Open Access Journals (Sweden)

    Stewart Jeromson

    2015-11-01

    Full Text Available Skeletal muscle is a plastic tissue capable of adapting and mal-adapting to physical activity and diet. The response of skeletal muscle to adaptive stimuli, such as exercise, can be modified by the prior nutritional status of the muscle. The influence of nutrition on skeletal muscle has the potential to substantially impact physical function and whole body metabolism. Animal and cell based models show that omega-3 fatty acids, in particular those of marine origin, can influence skeletal muscle metabolism. Furthermore, recent human studies demonstrate that omega-3 fatty acids of marine origin can influence the exercise and nutritional response of skeletal muscle. These studies show that the prior omega-3 status influences not only the metabolic response of muscle to nutrition, but also the functional response to a period of exercise training. Omega-3 fatty acids of marine origin therefore have the potential to alter the trajectory of a number of human diseases including the physical decline associated with aging. We explore the potential molecular mechanisms by which omega-3 fatty acids may act in skeletal muscle, considering the n-3/n-6 ratio, inflammation and lipidomic remodelling as possible mechanisms of action. Finally, we suggest some avenues for further research to clarify how omega-3 fatty acids may be exerting their biological action in skeletal muscle.

  7. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed...... I NO synthase immunoreactivity and NADPH diaphorase activity. Type III NO synthase immunoreactivity was observed both in the endothelium of larger vessels and of microvessels. The results establish that human skeletal muscle expresses two different constitutive isoforms of NO synthase in different...... endothelium is consistent with a role for NO in the control of blood flow in human skeletal muscle....

  8. Skeletal Muscle Insulin Resistance in Endocrine Disease

    Directory of Open Access Journals (Sweden)

    Melpomeni Peppa

    2010-01-01

    Full Text Available We summarize the existing literature data concerning the involvement of skeletal muscle (SM in whole body glucose homeostasis and the contribution of SM insulin resistance (IR to the metabolic derangements observed in several endocrine disorders, including polycystic ovary syndrome (PCOS, adrenal disorders and thyroid function abnormalities. IR in PCOS is associated with a unique postbinding defect in insulin receptor signaling in general and in SM in particular, due to a complex interaction between genetic and environmental factors. Adrenal hormone excess is also associated with disrupted insulin action in peripheral tissues, such as SM. Furthermore, both hyper- and hypothyroidism are thought to be insulin resistant states, due to insulin receptor and postreceptor defects. Further studies are definitely needed in order to unravel the underlying pathogenetic mechanisms. In summary, the principal mechanisms involved in muscle IR in the endocrine diseases reviewed herein include abnormal phosphorylation of insulin signaling proteins, altered muscle fiber composition, reduced transcapillary insulin delivery, decreased glycogen synthesis, and impaired mitochondrial oxidative metabolism.

  9. Myofibre damage in human skeletal muscle

    DEFF Research Database (Denmark)

    Crameri, R M; Aagaard, P; Qvortrup, K

    2007-01-01

    Disruption to proteins within the myofibre after a single bout of unaccustomed eccentric exercise is hypothesized to induce delayed onset of muscle soreness and to be associated with an activation of satellite cells. This has been shown in animal models using electrical stimulation but not in hum......Disruption to proteins within the myofibre after a single bout of unaccustomed eccentric exercise is hypothesized to induce delayed onset of muscle soreness and to be associated with an activation of satellite cells. This has been shown in animal models using electrical stimulation...... but not in humans using voluntary exercise. Untrained males (n=8, range 22-27 years) performed 210 maximal eccentric contractions with each leg on an isokinetic dynamometer, voluntarily (VOL) with one leg and electrically induced (ES) with the other leg. Assessments from the skeletal muscle were obtained prior......, a significant disruption of cytoskeletal proteins (desmin) and a rise of myogenic growth factors (myogenin) occurred only in ES. Intracellular disruption and destroyed Z-lines were markedly more pronounced in ES (40%) compared with VOL (10%). Likewise, the increase in satellite cell markers [neural cell...

  10. The influence of skeletal muscle on systemic aging and lifespan

    OpenAIRE

    Demontis, Fabio; Piccirillo, Rosanna; Goldberg, Alfred L.; Perrimon, Norbert

    2013-01-01

    Epidemiological studies in humans suggest that skeletal muscle aging is a risk factor for the development of several age-related diseases such as metabolic syndrome, cancer, Alzheimer’s disease, and Parkinson’s disease. Here we review recent studies in mammals and Drosophila highlighting how nutrient- and stress-sensing in skeletal muscle can influence lifespan and overall aging of the organism. In addition to exercise and indirect effects of muscle metabolism, growing evidence suggests that ...

  11. Skeletal Muscle Hypertrophy and Cardiometabolic Benefits after Spinal Cord Injury

    Science.gov (United States)

    2016-10-01

    AWARD NUMBER: W81XWH-15-1-0671 TITLE: Skeletal Muscle Hypertrophy and Cardiometabolic Benefits after Spinal Cord Injury PRINCIPAL...29 Sep 2016 4. TITLE AND SUBTITLE Spinal Cord Injury 5a. CONTRACT NUMBER Skeletal Muscle Hypertrophy and Cardiometabolic Benefits after Spinal...metabolism, increasing leg muscle size and preventing an increase in leg fat mass. 15. SUBJECT TERMS RESISTANCE TRAINING, SPINAL CORD INJURY , BODY

  12. Ca(2+) calmodulin kinase and calcineurin mediate IGF-1-induced skeletal muscle dihydropyridine receptor alpha(1S) transcription.

    Science.gov (United States)

    Zheng, Z; Wang, Z M; Delbono, O

    2004-01-15

    The skeletal muscle L-type Ca(2+) channel or dihydropyridine(DHP)-sensitive receptor is a key molecule involved in membrane voltage-sensing, sarcoplasmic reticulum Ca(2+) release, and muscle contraction. Previous work from our laboratory has shown that the insulin-like growth factor-1 (IGF-1) increases skeletal muscle L-type Ca(2+) channel or dihydropyridine-sensitive receptor DHPRalpha(1S) transcriptional activity by acting on the cyclic AMP response element binding protein (CREB) element of the promoter region; however, the cellular signaling mediating this process is not known. In this study, we investigated the signaling pathway whereby IGF-1 enhances the expression of DHPRalpha(1S) in C2C12 myotubes, using a molecular, pharmacological and electrophysiological approach. We found that inhibition of the Ca(2+)/Calmodulin (CaM)-dependent protein kinase or calcineurin, influenced IGF-1-induced increase in DHPRalpha(1S) expression, as detected by recording the luminescence of the DHPRalpha(1S) promoter-luciferase fusion construct and by immunoblot analysis of the DHPR alpha1 subunit. IGF-1 significantly increased CaM kinase and calcineurin activity and the cellular levels of phosphorylated CREB in a time-dependent manner. The role of CaM kinase and calcineurin in DHPRalpha(1S) expression was confirmed by functional recording of the effects of the inhibition of the kinase and phosphatase on IGF-1-mediated enhancement of charge movement. These results support the conclusion that IGF-1 controls CREB phosphorylation by activating a phosphorylation and dephosphorylation cascade, which ultimately modulates the DHPRalpha(1S) gene transcription.

  13. Making muscle: skeletal myogenesis in vivo and in vitro.

    Science.gov (United States)

    Chal, Jérome; Pourquié, Olivier

    2017-06-15

    Skeletal muscle is the largest tissue in the body and loss of its function or its regenerative properties results in debilitating musculoskeletal disorders. Understanding the mechanisms that drive skeletal muscle formation will not only help to unravel the molecular basis of skeletal muscle diseases, but also provide a roadmap for recapitulating skeletal myogenesis in vitro from pluripotent stem cells (PSCs). PSCs have become an important tool for probing developmental questions, while differentiated cell types allow the development of novel therapeutic strategies. In this Review, we provide a comprehensive overview of skeletal myogenesis from the earliest premyogenic progenitor stage to terminally differentiated myofibers, and discuss how this knowledge has been applied to differentiate PSCs into muscle fibers and their progenitors in vitro. © 2017. Published by The Company of Biologists Ltd.

  14. Tissue Triage and Freezing for Models of Skeletal Muscle Disease

    Science.gov (United States)

    Meng, Hui; Janssen, Paul M.L.; Grange, Robert W.; Yang, Lin; Beggs, Alan H.; Swanson, Lindsay C.; Cossette, Stacy A.; Frase, Alison; Childers, Martin K.; Granzier, Henk; Gussoni, Emanuela; Lawlor, Michael W.

    2014-01-01

    Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease. PMID:25078247

  15. Tissue triage and freezing for models of skeletal muscle disease.

    Science.gov (United States)

    Meng, Hui; Janssen, Paul M L; Grange, Robert W; Yang, Lin; Beggs, Alan H; Swanson, Lindsay C; Cossette, Stacy A; Frase, Alison; Childers, Martin K; Granzier, Henk; Gussoni, Emanuela; Lawlor, Michael W

    2014-07-15

    Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

  16. Upregulation of skeletal muscle PGC-1α through the elevation of cyclic AMP levels by Cyanidin-3-glucoside enhances exercise performance.

    Science.gov (United States)

    Matsukawa, Toshiya; Motojima, Hideko; Sato, Yuki; Takahashi, Shinya; Villareal, Myra O; Isoda, Hiroko

    2017-03-20

    Regular exercise and physical training enhance physiological capacity and improve metabolic diseases. Skeletal muscles require peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) in the process of their adaptation to exercise owing to PGC-1α's ability to regulate mitochondrial biogenesis, angiogenesis, and oxidative metabolism. Cyanidin-3-glucoside (Cy3G) is a natural polyphenol and a nutraceutical factor, which has several beneficial effects on human health. Here, the effect of Cy3G on exercise performance and the underlying mechanisms involved were investigated. ICR mice were given Cy3G (1 mg/kg, orally) everyday and made to perform weight-loaded swimming exercise for 15 days. The endurance of mice orally administered with Cy3G was improved, enabling them to swim longer (time) and while the levels of exercise-induced lactate and fatigue markers (urea nitrogen, creatinine and total ketone bodies) were reduced. Additionally, the expression of lactate metabolism-related genes (lactate dehydrogenase B and monocarboxylate transporter 1) in gastrocnemius and biceps femoris muscles was increased in response to Cy3G-induced PGC-1α upregulation. In vitro, using C2C12 myotubes, Cy3G-induced elevation of intracellular cyclic AMP levels increased PGC-1α expression via the Ca(2+)/calmodulin-dependent protein kinase kinase pathway. This study demonstrates that Cy3G enhances exercise performance by activating lactate metabolism through skeletal muscle PGC-1α upregulation.

  17. Molecular Signals and Skeletal Muscle Adaptation to Exercise

    Directory of Open Access Journals (Sweden)

    Mark Wilson

    2013-08-01

    Full Text Available The phenotypic plasticity of skeletal muscle affords a considerable degree of adaptability not seen in other bodily tissues. The mechanical properties of skeletal muscle are highly dependent on loading conditions. The extent of skeletal muscle plasticity is distinctly highlighted by a loss of muscle mass, or atrophy, after a period of reduced weight-bearing activity, for example during periods of extended bed rest, space flight and in spinal cord injury. On the other hand, increased mechanical loading, or resistance training, induces muscle growth, or hypertrophy. Endurance exercise performance is also dependent on the adaptability of skeletal muscle, especially muscles that contribute to posture, locomotion and the mechanics of breathing. However, the molecular pathways governing skeletal muscle adaptations are yet to be satisfactorily delineated and require further investigation. Researchers in the areas of exercise physiology, physiotherapy and sports medicine are endeavoring to translate experimental knowledge into effective, innovative treatments and regimens in order to improve physical performance and health in both elite athletes and the general community. The efficacy of the translation of molecular biological paradigms in experimental exercise physiology has long been underappreciated. Indeed, molecular biology tools can now be used to answer questions regarding skeletal muscle adaptation in response to exercise and provide new frameworks to improve physical performance. Furthermore, transgenic animal models, knockout animal models and in vivo studies provide tools to test questions concerned with how exercise initiates adaptive changes in gene expression. In light of these perceived deficiencies, an attempt is made here to elucidate the molecular mechanisms of skeletal muscle adaptation to exercise. An examination will be made of the functional capacity of skeletal muscle to respond to a variety of exercise conditions, namely

  18. Molecular Signals and Skeletal Muscle Adaptation to Exercise

    Directory of Open Access Journals (Sweden)

    Mark Wilson

    2013-09-01

    Full Text Available The phenotypic plasticity of skeletal muscle affords a considerable degree of adaptability not seen in other bodily tissues. The mechanical properties of skeletal muscle are highly dependent on loading conditions. The extent of skeletal muscle plasticity is distinctly highlighted by a loss of muscle mass, or atrophy, after a period of reduced weight-bearing activity, for example during periods of extended bed rest, space flight and in spinal cord injury. On the other hand, increased mechanical loading, or resistance training, induces muscle growth, or hypertrophy. Endurance exercise performance is also dependent on the adaptability of skeletal muscle, especially muscles that contribute to posture, locomotion and the mechanics of breathing.  However, the molecular pathways governing skeletal muscle adaptations are yet to be satisfactorily delineated and require further investigation. Researchers in the areas of exercise physiology, physiotherapy and sports medicine are endeavoring to translate experimental knowledge into effective, innovative treatments and regimens in order to improve physical performance and health in both elite athletes and the general community. The efficacy of the translation of molecular biological paradigms in experimental exercise physiology has long been underappreciated. Indeed, molecular biology tools can now be used to answer questions regarding skeletal muscle adaptation in response to exercise and provide new frameworks to improve physical performance. Furthermore, transgenic animal models, knockout animal models and in vivo studies provide tools to test questions concerned with how exercise initiates adaptive changes in gene expression. In light of these perceived deficiencies, an attempt is made here to elucidate the molecular mechanisms of skeletal muscle adaptation to exercise. An examination will be made of the functional capacity of skeletal muscle to respond to a variety of exercise conditions, namely

  19. A metabolic link to skeletal muscle wasting and regeneration

    Directory of Open Access Journals (Sweden)

    René eKoopman

    2014-02-01

    Full Text Available Due to its essential role in movement, insulating the internal organs, generating heat to maintain core body temperature, and acting as a major energy storage depot, any impairment to skeletal muscle structure and function may lead to an increase in both morbidity and mortality. In the context of skeletal muscle, altered metabolism is directly associated with numerous pathologies and disorders, including diabetes, and obesity, while many skeletal muscle pathologies have secondary changes in metabolism, including cancer cachexia, sarcopenia and the muscular dystrophies. Furthermore, the importance of cellular metabolism in the regulation of skeletal muscle stem cells is beginning to receive significant attention. Thus, it is clear that skeletal muscle metabolism is intricately linked to the regulation of skeletal muscle mass and regeneration. The aim of this review is to discuss some of the recent findings linking a change in metabolism to changes in skeletal muscle mass, as well as describing some of the recent studies in developmental, cancer and stem-cell biology that have identified a role for cellular metabolism in the regulation of stem cell function, a process termed ‘metabolic reprogramming’.

  20. Arginylation of Myosin Heavy Chain Regulates Skeletal Muscle Strength

    Directory of Open Access Journals (Sweden)

    Anabelle S. Cornachione

    2014-07-01

    Full Text Available Protein arginylation is a posttranslational modification with an emerging global role in the regulation of actin cytoskeleton. To test the role of arginylation in the skeletal muscle, we generated a mouse model with Ate1 deletion driven by the skeletal muscle-specific creatine kinase (Ckmm promoter. Ckmm-Ate1 mice were viable and outwardly normal; however, their skeletal muscle strength was significantly reduced in comparison to controls. Mass spectrometry of isolated skeletal myofibrils showed a limited set of proteins, including myosin heavy chain, arginylated on specific sites. Atomic force microscopy measurements of contractile strength in individual myofibrils and isolated myosin filaments from these mice showed a significant reduction of contractile forces, which, in the case of myosin filaments, could be fully rescued by rearginylation with purified Ate1. Our results demonstrate that arginylation regulates force production in muscle and exerts a direct effect on muscle strength through arginylation of myosin.

  1. Regulatory mechanisms of skeletal muscle protein turnover during exercise

    DEFF Research Database (Denmark)

    Rose, Adam John; Richter, Erik

    2009-01-01

    Skeletal muscle protein turnover is a relatively slow metabolic process that is altered by various physiological stimuli such as feeding/fasting and exercise. During exercise, catabolism of amino acids contributes very little to ATP turnover in working muscle. With regards to protein turnover......, there is now consistent data from tracer studies in rodents and humans showing that global protein synthesis is blunted in working skeletal muscle. Whether there is altered skeletal muscle protein breakdown during exercise remains unclear. The blunting of protein synthesis is believed to be mediated...... downstream of changes in intracellular Ca(2+) and energy turnover. In particular, a signaling cascade involving Ca(2+)-calmodulin-eEF2 kinase-eEF2 is implicated. The possible functional significance of altered protein turnover in working skeletal muscle during exercise is discussed. Further work...

  2. Vasodilator interactions in skeletal muscle blood flow regulation

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Nyberg, Michael Permin; Jensen, Lasse Gliemann

    2012-01-01

    During exercise, oxygen delivery to skeletal muscle is elevated to meet the increased oxygen demand. The increase in blood flow to skeletal muscle is achieved by vasodilators formed locally in the muscle tissue, either on the intraluminal or the extraluminal side of the blood vessels. A number...... that this remaining hyperemia may be explained by cAMP and cGMP independent smooth muscle relaxation, such as effects of endothelial derived hyperpolarization factors (EDHFs) or through metabolic modulation of sympathetic effects. The nature and role of EDHF as well as potential novel mechanisms in muscle blood flow...

  3. Human skeletal muscle fibroblasts stimulate in vitro myogenesis and in vivo muscle regeneration

    DEFF Research Database (Denmark)

    Mackey, Abigail L.; Magnan, Mélanie; Chazaud, Bénédicte

    2017-01-01

    Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. In addition to the indispensable role satellite cells play in muscle regeneration, there is emerging evidence in rodents for a regulatory influence...... and strongly stimulate both MPC differentiation and MPC fusion. It thus appears, in humans, that fibroblasts exert a strong positive regulatory influence on MPC activity, in line with observations during in vivo skeletal muscle regeneration....

  4. Coordination of metabolic plasticity in skeletal muscle.

    Science.gov (United States)

    Hood, David A; Irrcher, Isabella; Ljubicic, Vladimir; Joseph, Anna-Maria

    2006-06-01

    Skeletal muscle is a highly malleable tissue, capable of pronounced metabolic and morphological adaptations in response to contractile activity (i.e. exercise). Each bout of contractile activity results in a coordinated alteration in the expression of a variety of nuclear DNA and mitochondrial DNA (mtDNA) gene products, leading to phenotypic adaptations. This results in an increase in muscle mitochondrial volume and changes in organelle composition, referred to as mitochondrial biogenesis. The functional consequence of this biogenesis is an improved resistance to fatigue. Signals initiated by the exercise bout involve changes in intracellular Ca2+ as well as alterations in energy status (i.e. ATP/ADP ratio) and the consequent activation of downstream kinases such as AMP kinase and Ca2+-calmodulin-activated kinases. These kinases activate transcription factors that bind DNA to affect the transcription of genes, the most evident manifestation of which occurs during the post-exercise recovery period when energy metabolism is directed toward anabolism, rather than contractile activity. An important protein that is affected by exercise is the transcriptional coactivator PGC-1alpha, which cooperates with multiple transcription factors to induce the expression of nuclear genes encoding mitochondrial proteins. Once translated in the cytosol, these mitochondrially destined proteins are imported into the mitochondrial outer membrane, inner membrane or matrix space via specific import machinery transport components. Contractile activity affects the expression of the import machinery, as well as the kinetics of import, thus facilitating the entry of newly synthesized proteins into the expanding organelle. An important set of proteins that are imported are the mtDNA transcription factors, which influence the expression and replication of mtDNA. While mtDNA contributes only 13 proteins to the synthesis of the organelle, these proteins are vital for the proper assembly of multi

  5. Fatty acid transport protein 1 (FATP1) localizes in mitochondria in mouse skeletal muscle and regulates lipid and ketone body disposal.

    Science.gov (United States)

    Guitart, Maria; Osorio-Conles, Oscar; Pentinat, Thais; Cebrià, Judith; García-Villoria, Judit; Sala, David; Sebastián, David; Zorzano, Antonio; Ribes, Antonia; Jiménez-Chillarón, Josep C; García-Martínez, Celia; Gómez-Foix, Anna M

    2014-01-01

    FATP1 mediates skeletal muscle cell fatty acid import, yet its intracellular localization and metabolic control role are not completely defined. Here, we examine FATP1 localization and metabolic effects of its overexpression in mouse skeletal muscle. The FATP1 protein was detected in mitochondrial and plasma membrane fractions, obtained by differential centrifugation, of mouse gastrocnemius muscle. FATP1 was most abundant in purified mitochondria, and in the outer membrane and soluble intermembrane, but not in the inner membrane plus matrix, enriched subfractions of purified mitochondria. Immunogold electron microscopy localized FATP1-GFP in mitochondria of transfected C2C12 myotubes. FATP1 was overexpressed in gastrocnemius mouse muscle, by adenovirus-mediated delivery of the gene into hindlimb muscles of newborn mice, fed after weaning a chow or high-fat diet. Compared to GFP delivery, FATP1 did not alter body weight, serum fed glucose, insulin and triglyceride levels, and whole-body glucose tolerance, in either diet. However, fatty acid levels were lower and β-hydroxybutyrate levels were higher in FATP1- than GFP-mice, irrespective of diet. Moreover, intramuscular triglyceride content was lower in FATP1- versus GFP-mice regardless of diet, and β-hydroxybutyrate content was unchanged in high-fat-fed mice. Electroporation-mediated FATP1 overexpression enhanced palmitate oxidation to CO2, but not to acid-soluble intermediate metabolites, while CO2 production from β-hydroxybutyrate was inhibited and that from glucose unchanged, in isolated mouse gastrocnemius strips. In summary, FATP1 was localized in mitochondria, in the outer membrane and intermembrane parts, of mouse skeletal muscle, what may be crucial for its metabolic effects. Overexpressed FATP1 enhanced disposal of both systemic fatty acids and intramuscular triglycerides. Consistently, it did not contribute to the high-fat diet-induced metabolic dysregulation. However, FATP1 lead to hyperketonemia

  6. Lifting the nebula: novel insights into skeletal muscle contractility.

    Science.gov (United States)

    Ottenheijm, Coen A C; Granzier, Henk

    2010-10-01

    Nebulin is a giant protein and a constituent of the skeletal muscle sarcomere. The name of this protein refers to its unknown (i.e., nebulous) function. However, recent rapid advances reveal that nebulin plays important roles in the regulation of muscle contraction. When these functions of nebulin are compromised, muscle weakness ensues, as is the case in patients with nemaline myopathy.

  7. Amla Enhances Mitochondrial Spare Respiratory Capacity by Increasing Mitochondrial Biogenesis and Antioxidant Systems in a Murine Skeletal Muscle Cell Line

    Directory of Open Access Journals (Sweden)

    Hirotaka Yamamoto

    2016-01-01

    Full Text Available Amla is one of the most important plants in Indian traditional medicine and has been shown to improve various age-related disorders while decreasing oxidative stress. Mitochondrial dysfunction is a proposed cause of aging through elevated oxidative stress. In this study, we investigated the effects of Amla on mitochondrial function in C2C12 myotubes, a murine skeletal muscle cell model with abundant mitochondria. Based on cell flux analysis, treatment with an extract of Amla fruit enhanced mitochondrial spare respiratory capacity, which enables cells to overcome various stresses. To further explore the mechanisms underlying these effects on mitochondrial function, we analyzed mitochondrial biogenesis and antioxidant systems, both proposed regulators of mitochondrial spare respiratory capacity. We found that Amla treatment stimulated both systems accompanied by AMPK and Nrf2 activation. Furthermore, we found that Amla treatment exhibited cytoprotective effects and lowered reactive oxygen species (ROS levels in cells subjected to t-BHP-induced oxidative stress. These effects were accompanied by increased oxygen consumption, suggesting that Amla protected cells against oxidative stress by using enhanced spare respiratory capacity to produce more energy. Thus we identified protective effects of Amla, involving activation of mitochondrial function, which potentially explain its various effects on age-related disorders.

  8. Effect of zinc-alpha2-glycoprotein (ZAG) on expression of uncoupling proteins in skeletal muscle and adipose tissue.

    Science.gov (United States)

    Sanders, Paul M; Tisdale, Michael J

    2004-08-20

    The plasma protein zinc-alpha2-glycoprotein (ZAG) has been shown to be identical with a lipid mobilizing factor capable of inducing loss of adipose tissue in cancer cachexia through an increased lipid mobilization and utilization. The ability of ZAG to induce uncoupling protein (UCP) expression has been determined using in vitro models of adipose tissue and skeletal muscle. ZAG induced a concentration-dependent increase in the expression of UCP-1 in primary cultures of brown, but not white, adipose tissue, and this effect was attenuated by the beta3-adrenergic receptor (beta3-AR) antagonist SR59230A. A 6.5-fold increase in UCP-1 expression was found in brown adipose tissue after incubation with 0.58 microM ZAG. ZAG also increased UCP-2 expression 3.5-fold in C2C12 murine myotubes, and this effect was also attenuated by SR59230A and potentiated by isobutylmethylxanthine, suggesting a cyclic AMP-mediated process through interaction with a beta3-AR. ZAG also produced a dose-dependent increase in UCP-3 in murine myotubes with a 2.5-fold increase at 0.58 microM ZAG. This effect was not mediated through the beta3-AR, but instead appeared to require mitogen activated protein kinase. These results confirm the ability of ZAG to directly influence UCP expression, which may play an important role in lipid utilization during cancer cachexia.

  9. Expression of androgen receptor target genes in skeletal muscle

    Directory of Open Access Journals (Sweden)

    Kesha Rana

    2014-10-01

    Full Text Available We aimed to determine the mechanisms of the anabolic actions of androgens in skeletal muscle by investigating potential androgen receptor (AR-regulated genes in in vitro and in vivo models. The expression of the myogenic regulatory factor myogenin was significantly decreased in skeletal muscle from testosterone-treated orchidectomized male mice compared to control orchidectomized males, and was increased in muscle from male AR knockout mice that lacked DNA binding activity (ARΔZF2 versus wildtype mice, demonstrating that myogenin is repressed by the androgen/AR pathway. The ubiquitin ligase Fbxo32 was repressed by 12 h dihydrotestosterone treatment in human skeletal muscle cell myoblasts, and c-Myc expression was decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle, and increased in AR∆ZF2 muscle. The expression of a group of genes that regulate the transition from myoblast proliferation to differentiation, Tceal7 , p57 Kip2, Igf2 and calcineurin Aa, was increased in AR∆ZF2 muscle, and the expression of all but p57 Kip2 was also decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle. We conclude that in males, androgens act via the AR in part to promote peak muscle mass by maintaining myoblasts in the proliferative state and delaying the transition to differentiation during muscle growth and development, and by suppressing ubiquitin ligase-mediated atrophy pathways to preserve muscle mass in adult muscle.

  10. Skeletal Muscle Cell Induction from Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Yusaku Kodaka

    2017-01-01

    Full Text Available Embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs have the potential to differentiate into various types of cells including skeletal muscle cells. The approach of converting ESCs/iPSCs into skeletal muscle cells offers hope for patients afflicted with the skeletal muscle diseases such as the Duchenne muscular dystrophy (DMD. Patient-derived iPSCs are an especially ideal cell source to obtain an unlimited number of myogenic cells that escape immune rejection after engraftment. Currently, there are several approaches to induce differentiation of ESCs and iPSCs to skeletal muscle. A key to the generation of skeletal muscle cells from ESCs/iPSCs is the mimicking of embryonic mesodermal induction followed by myogenic induction. Thus, current approaches of skeletal muscle cell induction of ESCs/iPSCs utilize techniques including overexpression of myogenic transcription factors such as MyoD or Pax3, using small molecules to induce mesodermal cells followed by myogenic progenitor cells, and utilizing epigenetic myogenic memory existing in muscle cell-derived iPSCs. This review summarizes the current methods used in myogenic differentiation and highlights areas of recent improvement.

  11. An extract of Artemisia dracunculus L. inhibits ubiquitin-proteasome activity and preserves skeletal muscle mass in a murine model of diabetes.

    Directory of Open Access Journals (Sweden)

    Heather Kirk-Ballard

    Full Text Available Impaired insulin signaling is a key feature of type 2 diabetes and is associated with increased ubiquitin-proteasome-dependent protein degradation in skeletal muscle. An extract of Artemisia dracunculus L. (termed PMI5011 improves insulin action by increasing insulin signaling in skeletal muscle. We sought to determine if the effect of PMI5011 on insulin signaling extends to regulation of the ubiquitin-proteasome system. C2C12 myotubes and the KK-A(y murine model of type 2 diabetes were used to evaluate the effect of PMI5011 on steady-state levels of ubiquitylation, proteasome activity and expression of Atrogin-1 and MuRF-1, muscle-specific ubiquitin ligases that are upregulated with impaired insulin signaling. Our results show that PMI5011 inhibits proteasome activity and steady-state ubiquitylation levels in vitro and in vivo. The effect of PMI5011 is mediated by PI3K/Akt signaling and correlates with decreased expression of Atrogin-1 and MuRF-1. Under in vitro conditions of hormonal or fatty acid-induced insulin resistance, PMI5011 improves insulin signaling and reduces Atrogin-1 and MuRF-1 protein levels. In the KK-A(y murine model of type 2 diabetes, skeletal muscle ubiquitylation and proteasome activity is inhibited and Atrogin-1 and MuRF-1 expression is decreased by PMI5011. PMI5011-mediated changes in the ubiquitin-proteasome system in vivo correlate with increased phosphorylation of Akt and FoxO3a and increased myofiber size. The changes in Atrogin-1 and MuRF-1 expression, ubiquitin-proteasome activity and myofiber size modulated by PMI5011 in the presence of insulin resistance indicate the botanical extract PMI5011 may have therapeutic potential in the preservation of muscle mass in type 2 diabetes.

  12. An In Vitro Model of Skeletal Muscle Volume Regulation

    OpenAIRE

    Anna Wibberley; Staunton, Caroline A; Feetham, Claire H.; Vereninov, Alexey A.; Richard Barrett-Jolley

    2015-01-01

    Introduction Hypertonic media causes cells to shrink due to water loss through aquaporin channels. After acute shrinkage, cells either regulate their volume or, alternatively, undergo a number of metabolic changes which ultimately lead to cell death. In many cell types, hypertonic shrinkage is followed by apoptosis. Due to the complex 3D morphology of skeletal muscle and the difficulty in obtaining isolated human tissue, we have begun skeletal muscle volume regulation studies using the human ...

  13. The effects of obesity on skeletal muscle regeneration

    Directory of Open Access Journals (Sweden)

    Dmitry eAkhmedov

    2013-12-01

    Full Text Available Obesity and metabolic disorders such as type 2 diabetes mellitus are accompanied by increased lipid deposition in adipose and non-adipose tissues including liver, pancreas, heart and skeletal muscle. Recent publications report impaired regenerative capacity of skeletal muscle following injury in obese mice. Although muscle regeneration has not been thoroughly studied in obese and type 2 diabetic humans and mechanisms leading to decreased muscle regeneration in obesity remain elusive, the initial findings point to the possibility that muscle satellite cell function is compromised under conditions of lipid overload. Elevated toxic lipid metabolites and increased proinflammatory cytokines as well as insulin and leptin resistance that occur in obese animals may contribute to decreased regenerative capacity of skeletal muscle. In addition, obesity-associated alterations in the metabolic state of skeletal muscle fibers and satellite cells may directly impair the potential for satellite cell-mediated repair. Here we discuss recent studies that expand our understanding of how obesity negatively impacts skeletal muscle maintenance and regeneration.

  14. Anisotropic Materials for Skeletal-Muscle-Tissue Engineering.

    Science.gov (United States)

    Jana, Soumen; Levengood, Sheeny K Lan; Zhang, Miqin

    2016-12-01

    Repair of damaged skeletal-muscle tissue is limited by the regenerative capacity of the native tissue. Current clinical approaches are not optimal for the treatment of large volumetric skeletal-muscle loss. As an alternative, tissue engineering represents a promising approach for the functional restoration of damaged muscle tissue. A typical tissue-engineering process involves the design and fabrication of a scaffold that closely mimics the native skeletal-muscle extracellular matrix (ECM), allowing organization of cells into a physiologically relevant 3D architecture. In particular, anisotropic materials that mimic the morphology of the native skeletal-muscle ECM, can be fabricated using various biocompatible materials to guide cell alignment, elongation, proliferation, and differentiation into myotubes. Here, an overview of fundamental concepts associated with muscle-tissue engineering and the current status of muscle-tissue-engineering approaches is provided. Recent advances in the development of anisotropic scaffolds with micro- or nanoscale features are reviewed, and how scaffold topographical, mechanical, and biochemical cues correlate to observed cellular function and phenotype development is examined. Finally, some recent developments in both the design and utility of anisotropic materials in skeletal-muscle-tissue engineering are highlighted, along with their potential impact on future research and clinical applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Glucose transporter expression in human skeletal muscle fibers

    DEFF Research Database (Denmark)

    Gaster, M; Handberg, A; Beck-Nielsen, H

    2000-01-01

    amplification (TSA) technique to detect the localization of glucose transporter expression in human skeletal muscle. We found expression of GLUT-1, GLUT-3, and GLUT-4 in developing human muscle fibers showing a distinct expression pattern. 1) GLUT-1 is expressed in human skeletal muscle cells during gestation......, but its expression is markedly reduced around birth and is further reduced to undetectable levels within the first year of life; 2) GLUT-3 protein expression appears at 18 wk of gestation and disappears after birth; and 3) GLUT-4 protein is diffusely expressed in muscle cells throughout gestation, whereas...

  16. Skeletal muscle proteomics: current approaches, technical challenges and emerging techniques

    Directory of Open Access Journals (Sweden)

    Ohlendieck Kay

    2011-02-01

    Full Text Available Abstract Background Skeletal muscle fibres represent one of the most abundant cell types in mammals. Their highly specialised contractile and metabolic functions depend on a large number of membrane-associated proteins with very high molecular masses, proteins with extensive posttranslational modifications and components that exist in highly complex supramolecular structures. This makes it extremely difficult to perform conventional biochemical studies of potential changes in protein clusters during physiological adaptations or pathological processes. Results Skeletal muscle proteomics attempts to establish the global identification and biochemical characterisation of all members of the muscle-associated protein complement. A considerable number of proteomic studies have employed large-scale separation techniques, such as high-resolution two-dimensional gel electrophoresis or liquid chromatography, and combined them with mass spectrometry as the method of choice for high-throughput protein identification. Muscle proteomics has been applied to the comprehensive biochemical profiling of developing, maturing and aging muscle, as well as the analysis of contractile tissues undergoing physiological adaptations seen in disuse atrophy, physical exercise and chronic muscle transformation. Biomedical investigations into proteome-wide alterations in skeletal muscle tissues were also used to establish novel biomarker signatures of neuromuscular disorders. Importantly, mass spectrometric studies have confirmed the enormous complexity of posttranslational modifications in skeletal muscle proteins. Conclusions This review critically examines the scientific impact of modern muscle proteomics and discusses its successful application for a better understanding of muscle biology, but also outlines its technical limitations and emerging techniques to establish new biomarker candidates.

  17. Frailty and skeletal muscle in older adults with cancer.

    Science.gov (United States)

    Williams, Grant R; Deal, Allison M; Muss, Hyman B; Weinberg, Marc S; Sanoff, Hanna K; Guerard, Emily J; Nyrop, Kirsten A; Pergolotti, Mackenzi; Shachar, Shlomit Strulov

    2017-08-24

    Computerized tomography (CT) imaging is routine in oncologic care and can be used to measure muscle quantity and composition that may improve prognostic assessment of older patients. This study examines the association of single-slice CT-assessed muscle measurements with a frailty index in older adults with cancer. Using the Carolina Senior Registry, we identified patients with CT imaging within 60days ± of geriatric assessment (GA). A 36-item Carolina Frailty Index was calculated. Cross-sectional skeletal muscle area (SMA) and Skeletal Muscle Density (SMD) were analyzed from CT scan L3 lumbar segments. SMA and patient height (m(2)) were used to calculate skeletal muscle index (SMI). Skeletal Muscle Gauge (SMG) was calculated by multiplying SMI×SMD. Of the 162 patients, mean age 73, 53% were robust, 27% pre-frail, and 21% frail. Significant differences were found between robust and frail patients for SMD (29.4 vs 24.1 HU, pfrailty increased by 20% (PR=1.20 [1.09, 1.32]) while the prevalence of frailty did not differ based on SMI. Muscle mass (measured as SMI) was poorly associated with a GA-based frailty index. Muscle density, which reflects muscle lipid content, was more associated with frailty. Although frailty and loss of muscle mass are both age-related conditions that are predictive of adverse outcomes, our results suggest they are separate entities. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Skeletal muscle proteomics: current approaches, technical challenges and emerging techniques

    LENUS (Irish Health Repository)

    Ohlendieck, Kay

    2011-02-01

    Abstract Background Skeletal muscle fibres represent one of the most abundant cell types in mammals. Their highly specialised contractile and metabolic functions depend on a large number of membrane-associated proteins with very high molecular masses, proteins with extensive posttranslational modifications and components that exist in highly complex supramolecular structures. This makes it extremely difficult to perform conventional biochemical studies of potential changes in protein clusters during physiological adaptations or pathological processes. Results Skeletal muscle proteomics attempts to establish the global identification and biochemical characterisation of all members of the muscle-associated protein complement. A considerable number of proteomic studies have employed large-scale separation techniques, such as high-resolution two-dimensional gel electrophoresis or liquid chromatography, and combined them with mass spectrometry as the method of choice for high-throughput protein identification. Muscle proteomics has been applied to the comprehensive biochemical profiling of developing, maturing and aging muscle, as well as the analysis of contractile tissues undergoing physiological adaptations seen in disuse atrophy, physical exercise and chronic muscle transformation. Biomedical investigations into proteome-wide alterations in skeletal muscle tissues were also used to establish novel biomarker signatures of neuromuscular disorders. Importantly, mass spectrometric studies have confirmed the enormous complexity of posttranslational modifications in skeletal muscle proteins. Conclusions This review critically examines the scientific impact of modern muscle proteomics and discusses its successful application for a better understanding of muscle biology, but also outlines its technical limitations and emerging techniques to establish new biomarker candidates.

  19. Exercise and obesity-induced insulin resistance in skeletal muscle

    Directory of Open Access Journals (Sweden)

    Hyo-Bum Kwak

    2013-12-01

    Full Text Available The skeletal muscle in our body is a major site for bioenergetics and metabolism during exercise. Carbohydrates and fats are the primary nutrients that provide the necessary energy required to maintain cellular activities during exercise. The metabolic responses to exercise in glucose and lipid regulation depend on the intensity and duration of exercise. Because of the increasing prevalence of obesity, recent studies have focused on the cellular and molecular mechanisms of obesity-induced insulin resistance in skeletal muscle. Accumulation of intramyocellular lipid may lead to insulin resistance in skeletal muscle. In addition, lipid intermediates (e.g., fatty acyl-coenzyme A, diacylglycerol, and ceramide impair insulin signaling in skeletal muscle. Recently, emerging evidence linking obesity-induced insulin resistance to excessive lipid oxidation, mitochondrial overload, and mitochondrial oxidative stress have been provided with mitochondrial function. This review will provide a brief comprehensive summary on exercise and skeletal muscle metabolism, and discuss the potential mechanisms of obesity-induced insulin resistance in skeletal muscle.

  20. The Impact of Shiftwork on Skeletal Muscle Health

    Directory of Open Access Journals (Sweden)

    Brad Aisbett

    2017-03-01

    Full Text Available (1 Background: About one in four workers undertake shift rosters that fall outside the traditional 7 a.m.–6 p.m. scheduling. Shiftwork alters workers’ exposure to natural and artificial light, sleep patterns, and feeding patterns. When compared to the rest of the working population, shiftworkers are at a greater risk of developing metabolic impairments over time. One fundamental component of metabolic health is skeletal muscle, the largest organ in the body. However, cause-and-effect relationships between shiftwork and skeletal muscle health have not been established; (2 Methods: A critical review of the literature was completed using online databases and reference lists; (3 Results: We propose a conceptual model drawing relationships between typical shiftwork consequences; altered light exposure, sleep patterns, and food and beverage consumption, and drivers of skeletal muscle health—protein intake, resistance training, and hormone release. At present, there is no study investigating the direct effect of shiftwork on skeletal muscle health. Instead, research findings showing that acute consequences of shiftwork negatively influence skeletal muscle homeostasis support the validity of our model; (4 Conclusion: Further research is required to test the potential relationships identified in our review, particularly in shiftwork populations. Part of this testing could include skeletal muscle specific interventions such as targeted protein intake and/or resistance-training.

  1. The Impact of Shiftwork on Skeletal Muscle Health.

    Science.gov (United States)

    Aisbett, Brad; Condo, Dominique; Zacharewicz, Evelyn; Lamon, Séverine

    2017-03-08

    (1) Background: About one in four workers undertake shift rosters that fall outside the traditional 7 a.m.-6 p.m. scheduling. Shiftwork alters workers' exposure to natural and artificial light, sleep patterns, and feeding patterns. When compared to the rest of the working population, shiftworkers are at a greater risk of developing metabolic impairments over time. One fundamental component of metabolic health is skeletal muscle, the largest organ in the body. However, cause-and-effect relationships between shiftwork and skeletal muscle health have not been established; (2) Methods: A critical review of the literature was completed using online databases and reference lists; (3) Results: We propose a conceptual model drawing relationships between typical shiftwork consequences; altered light exposure, sleep patterns, and food and beverage consumption, and drivers of skeletal muscle health-protein intake, resistance training, and hormone release. At present, there is no study investigating the direct effect of shiftwork on skeletal muscle health. Instead, research findings showing that acute consequences of shiftwork negatively influence skeletal muscle homeostasis support the validity of our model; (4) Conclusion: Further research is required to test the potential relationships identified in our review, particularly in shiftwork populations. Part of this testing could include skeletal muscle specific interventions such as targeted protein intake and/or resistance-training.

  2. Costameric proteins in human skeletal muscle during muscular inactivity.

    Science.gov (United States)

    Anastasi, Giuseppe; Cutroneo, Giuseppina; Santoro, Giuseppe; Arco, Alba; Rizzo, Giuseppina; Bramanti, Placido; Rinaldi, Carmen; Sidoti, Antonina; Amato, Aldo; Favaloro, Angelo

    2008-09-01

    Costameres are regions that are associated with the sarcolemma of skeletal muscle fibres and comprise proteins of the dystrophin-glycoprotein complex and vinculin-talin-integrin system. Costameres play both a mechanical and a signalling role, transmitting force from the contractile apparatus to the extracellular matrix in order to stabilize skeletal muscle fibres during contraction and relaxation. Recently, it was shown that bidirectional signalling occurs between sarcoglycans and integrins, with muscle agrin potentially interacting with both types of protein to enable signal transmission. Although numerous studies have been carried out on skeletal muscle diseases, such as Duchenne muscular dystrophy, recessive autosomal muscular dystrophies and other skeletal myopathies, insufficient data exist on the relationship between costameres and the pathology of the second motor nerve and between costameric proteins and muscle agrin in other conditions in which skeletal muscle atrophy occurs. Previously, we carried out a preliminary study on skeletal muscle from patients with sensitive-motor polyneuropathy, in which we analysed the distribution of sarcoglycans, integrins and agrin by immunostaining only. In the present study, we have examined the skeletal muscle fibres of ten patients with sensitive-motor polyneuropathy. We used immunofluorescence and reverse transcriptase PCR to examine the distribution of vinculin, talin and dystrophin, in addition to that of those proteins previously studied. Our aim was to characterize in greater detail the distribution and expression of costameric proteins and muscle agrin during this disease. In addition, we used transmission electron microscopy to evaluate the structural damage of the muscle fibres. The results showed that immunostaining of alpha 7B-integrin, beta 1D-integrin and muscle agrin appeared to be severely reduced, or almost absent, in the muscle fibres of the diseased patients, whereas staining of alpha 7A

  3. Growth factor-dependent and independent regulation of skeletal muscle mass - Is IGF-1 necessary for skeletal muscle hypertrophy?

    National Research Council Canada - National Science Library

    Miyazaki, Mitsunori

    2013-01-01

    .... IGF-1 has been indicated as a very effective anabolic agent, and thus considered a critical regulator of skeletal muscle hypertrophy in response to increased workload such as resistance exercise...

  4. Skeletal muscle degeneration and regeneration in mice and flies.

    Science.gov (United States)

    Rai, Mamta; Nongthomba, Upendra; Grounds, Miranda D

    2014-01-01

    Many aspects of skeletal muscle biology are remarkably similar between mammals and tiny insects, and experimental models of mice and flies (Drosophila) provide powerful tools to understand factors controlling the growth, maintenance, degeneration (atrophy and necrosis), and regeneration of normal and diseased muscles, with potential applications to the human condition. This review compares the limb muscles of mice and the indirect flight muscles of flies, with respect to the mechanisms of adult myofiber formation, homeostasis, atrophy, hypertrophy, and the response to muscle degeneration, with some comment on myogenic precursor cells and common gene regulatory pathways. There is a striking similarity between the species for events related to muscle atrophy and hypertrophy, without contribution of any myoblast fusion. Since the flight muscles of adult flies lack a population of reserve myogenic cells (equivalent to satellite cells), this indicates that such cells are not required for maintenance of normal muscle function. However, since satellite cells are essential in postnatal mammals for myogenesis and regeneration in response to myofiber necrosis, the extent to which such regeneration might be possible in flight muscles of adult flies remains unclear. Common cellular and molecular pathways for both species are outlined related to neuromuscular disorders and to age-related loss of skeletal muscle mass and function (sarcopenia). The commonality of events related to skeletal muscles in these disparate species (with vast differences in size, growth duration, longevity, and muscle activities) emphasizes the combined value and power of these experimental animal models. © 2014 Elsevier Inc. All rights reserved.

  5. Isolation and Culture of Satellite Cells from Mouse Skeletal Muscle.

    Science.gov (United States)

    Musarò, Antonio; Carosio, Silvia

    2017-01-01

    Skeletal muscle tissue is characterized by a population of quiescent mononucleated myoblasts, localized between the basal lamina and sarcolemma of myofibers, known as satellite cells. Satellite cells play a pivotal role in muscle homeostasis and are the major source of myogenic precursors in mammalian muscle regeneration.This chapter describes protocols for isolation and culturing satellite cells isolated from mouse skeletal muscles. The classical procedure, which will be discussed extensively in this chapter, involves the enzymatic dissociation of skeletal muscles, while the alternative method involves isolation of satellite cells from isolated myofibers in which the satellite cells remain in their in situ position underneath the myofiber basal lamina.In particular, we discuss the technical aspect of satellite cell isolation, the methods necessary to enrich the satellite cell fraction and the culture conditions that optimize proliferation and myotube formation of mouse satellite cells.

  6. The Mitochondrial Calcium Uniporter controls skeletal muscle trophism in vivo

    Science.gov (United States)

    Mammucari, Cristina; Gherardi, Gaia; Zamparo, Ilaria; Raffaello, Anna; Boncompagni, Simona; Chemello, Francesco; Cagnin, Stefano; Braga, Alessandra; Zanin, Sofia; Pallafacchina, Giorgia; Zentilin, Lorena; Sandri, Marco; De Stefani, Diego; Protasi, Feliciano; Lanfranchi, Gerolamo; Rizzuto, Rosario

    2015-01-01

    Summary Muscle atrophy contributes to the poor prognosis of many pathophysiological conditions, but pharmacological therapies are still limited. Muscle activity leads to major swings in mitochondrial [Ca2+] which control aerobic metabolism, cell death and survival pathways. We have investigated in vivo the effects of mitochondrial Ca2+ homeostasis in skeletal muscle function and trophism, by overexpressing or silencing the Mitochondrial Calcium Uniporter (MCU). The results demonstrate that both in developing and in adult muscles MCU-dependent mitochondrial Ca2+ uptake has a marked trophic effect that does not depend on aerobic control, but impinges on two major hypertrophic pathways of skeletal muscle, PGC-1α4 and IGF1-AKT/PKB. In addition, MCU overexpression protects from denervation-induced atrophy. These data reveal a novel Ca2+-dependent organelle-to-nucleus signaling route, which links mitochondrial function to the control of muscle mass and may represent a possible pharmacological target in conditions of muscle loss. PMID:25732818

  7. Expression of protocadherin gamma in skeletal muscle tissue is associated with age and muscle weakness

    NARCIS (Netherlands)

    Hangelbroek, R.W.J.; Fazelzadeh, P.; Tieland, C.A.B.; Boekschoten, M.V.; Hooiveld, G.J.E.J.; Duynhoven, van J.P.M.; Timmons, James; Verdijk, L.; Groot, de C.P.G.M.; Loon, van L.J.C.; Müller, M.R.

    2016-01-01

    Background
    The skeletal muscle system plays an important role in the independence of older adults. In this study we examine differences in the skeletal muscle transcriptome between healthy young and older subjects and (pre-)frail older adults. Additionally, we examine the effect of

  8. Reactive oxygen species are important mediators of taurine release from skeletal muscle cells

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Feveile Young, Jette; Oksbjerg, Niels

    2003-01-01

    C2C12, calcium, cell volume regulation, 5-lipoxygenase, melittin, anoxia, secretory phospholipase A2......C2C12, calcium, cell volume regulation, 5-lipoxygenase, melittin, anoxia, secretory phospholipase A2...

  9. Association between expression of FABPpm in skeletal muscle and insulin sensitivity in intramyocellular lipid-accumulated nonobese men.

    Science.gov (United States)

    Kawaguchi, Minako; Tamura, Yoshifumi; Kakehi, Saori; Takeno, Kageumi; Sakurai, Yuko; Watanabe, Takahiro; Funayama, Takashi; Sato, Fumihiko; Ikeda, Shinichi; Ogura, Yuji; Saga, Norio; Naito, Hisashi; Fujitani, Yoshio; Kanazawa, Akio; Kawamori, Ryuzo; Watada, Hirotaka

    2014-09-01

    Intramyocellular lipid (IMCL) accumulation is observed in both insulin-resistant subjects and insulin-sensitive endurance athletes (athlete's paradox). We hypothesized that the expression pattern of fatty acid transporters may influence oxidative capacity and determine the association between IMCL and insulin resistance. The objective of the study was to investigate the muscle expression of fatty acid transporters and their function related to insulin sensitivity in IMCL-accumulated subjects. The study subjects were 36 nonobese healthy men. Their IMCL levels were measured by (1)H-magnetic resonance spectroscopy, and their insulin sensitivity was evaluated by steady-state glucose infusion rate (GIR) during a euglycemic-hyperinsulinemic clamp. Gene expression levels in the vastus lateralis were evaluated by quantitative RT-PCR. We compared the clinical phenotypes and the expression levels of genes involved in lipid metabolism in skeletal muscle between IMCL-accumulated high-GIR (H-GIR) subjects (n = 8) and low-GIR subjects (n = 9). The functions of candidate fatty acid transporters were determined by in vitro analyses. Compared with the low-GIR group, body fat was lower and maximum oxygen uptake was higher in the H-GIR group. Several lipid oxidation genes in muscle were up-regulated in the H-GIR group, and this was associated with increased expression of higher plasma membrane-associated fatty acid-binding protein (FABPpm) and decreased expression of fatty acid transport protein (FATP)-1. Overexpression of FABPpm in C2C12 myotubes increased fatty acid oxidation coupled with the elevated expression of genes related to fatty acid oxidation. These changes were not observed in FATP1-overexpressed myotubes. Differences in the gene expression of fatty acid transporters may, at least in part, affect insulin sensitivity in IMCL-accumulated nonobese men.

  10. Action of Obestatin in Skeletal Muscle Repair: Stem Cell Expansion, Muscle Growth, and Microenvironment Remodeling

    Science.gov (United States)

    Gurriarán-Rodríguez, Uxía; Santos-Zas, Icía; González-Sánchez, Jessica; Beiroa, Daniel; Moresi, Viviana; Mosteiro, Carlos S; Lin, Wei; Viñuela, Juan E; Señarís, José; García-Caballero, Tomás; Casanueva, Felipe F; Nogueiras, Rubén; Gallego, Rosalía; Renaud, Jean-Marc; Adamo, Sergio; Pazos, Yolanda; Camiña, Jesús P

    2015-01-01

    The development of therapeutic strategies for skeletal muscle diseases, such as physical injuries and myopathies, depends on the knowledge of regulatory signals that control the myogenic process. The obestatin/GPR39 system operates as an autocrine signal in the regulation of skeletal myogenesis. Using a mouse model of skeletal muscle regeneration after injury and several cellular strategies, we explored the potential use of obestatin as a therapeutic agent for the treatment of trauma-induced muscle injuries. Our results evidenced that the overexpression of the preproghrelin, and thus obestatin, and GPR39 in skeletal muscle increased regeneration after muscle injury. More importantly, the intramuscular injection of obestatin significantly enhanced muscle regeneration by simulating satellite stem cell expansion as well as myofiber hypertrophy through a kinase hierarchy. Added to the myogenic action, the obestatin administration resulted in an increased expression of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) and the consequent microvascularization, with no effect on collagen deposition in skeletal muscle. Furthermore, the potential inhibition of myostatin during obestatin treatment might contribute to its myogenic action improving muscle growth and regeneration. Overall, our data demonstrate successful improvement of muscle regeneration, indicating obestatin is a potential therapeutic agent for skeletal muscle injury and would benefit other myopathies related to muscle regeneration. PMID:25762009

  11. Biomimetic Scaffolds for Regeneration of Volumetric Muscle Loss in Skeletal Muscle Injuries

    Science.gov (United States)

    Grasman, Jonathan M.; Zayas, Michelle J.; Page, Ray; Pins, George D.

    2015-01-01

    Skeletal muscle injuries typically result from traumatic incidents such as combat injuries where soft-tissue extremity injuries are present in one of four cases. Further, about 4.5 million reconstructive surgical procedures are performed annually as a result of car accidents, cancer ablation, or cosmetic procedures. These combat- and trauma-induced skeletal muscle injuries are characterized by volumetric muscle loss (VML), which significantly reduces the functionality of the injured muscle. While skeletal muscle has an innate repair mechanism, it is unable to compensate for VML injuries because large amounts of tissue including connective tissue and basement membrane are removed or destroyed. This results in in a significant need to develop off-the-shelf biomimetic scaffolds to direct skeletal muscle regeneration. Here, the structure and organization of native skeletal muscle tissue is described in order to reveal clear design parameters that are necessary for scaffolds to mimic in order to successfully regenerate muscular tissue. We review the literature with respect to the materials and methodologies used to develop scaffolds for skeletal muscle tissue regeneration as well as the limitations of these materials. We further discuss the variety of cell sources and different injury models to provide some context for the multiple approaches used to evaluate these scaffold materials. Recent findings are highlighted to address the state of the field and directions are outlined for future strategies, both in scaffold design and in the use of different injury models to evaluate these materials, for regenerating functional skeletal muscle. PMID:26219862

  12. Nutritional interventions to preserve skeletal muscle mass

    NARCIS (Netherlands)

    Backx, Evelien M.P.

    2016-01-01

    Muscle mass is the main predictor for muscle strength and physical function. The amount of muscle mass can decline rapidly during periods of reduced physical activity or during periods of energy intake restriction. For athletes, it is important to maintain muscle mass, since the loss of muscle is

  13. Protein and amino acid metabolism in skeletal muscle

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Guoyao.

    1989-01-01

    Isolated chick extensor digitorum communis (EDC) muscles and, in some experiments, rat skeletal muscles were used to study a number of aspects of protein and amino acid metabolism. (1) Chick EDC muscles synthesize and release large amounts of alanine and glutamine, which indirectly obtain their amino groups from branched-chain amino acids (BCAA). (2) Acetoacetate or DL-{beta}-hydroxybutyrate (4 mM) decrease (P < 0.01) alanine synthesis and BCAA transamination in EDC muscles from 24-h fasted chicks by decreasing (P < 0.01) intracellular concentrations of pyruvate due to inhibition of glycolysis. (3) Glutamine is extensively degraded in skeletal muscles from both chicks and rats, thus challenging the traditional view that glutamine oxidation is negligible in skeletal muscle. The cytosolic glutamine aminotransferases L and K in the rat and the mitochondrial phosphate-activated glutaminase in the chick play important roles in the conversion of glutamine to {alpha}-ketoglutarate for further oxidation. (4) Although methionine has been reported to be extensively transaminated in rat skeletal muscle preparations in the absence of other amino acids, transamination of methionine is absent or negligible in chick and rat skeletal muscles in the presence of physiological concentrations of amino acids. (5) Glutamine at 1.0-15 mM increases (P < 0.01) protein synthesis ({sup 3}H-phenylalanine incorporation), and at 10.0-15.0 mM decreases (P < 0.05) protein degradation ({sup 3}H-phenylalanine release from prelabelled protein in vivo) in EDC muscles from fed chicks as compared to muscles incubated in the absence of glutamine. (6) Acetoacetate or DL-{beta}-hydroxybutyrate (4 mM) has a small but significant inhibitory effect (P < 0.05) on the rate of protein synthesis, but has no effect (P > 0.05) on the rate of protein degradation in EDC muscles from fed chicks.

  14. Three-dimensional ultrasound strain imaging of skeletal muscles

    NARCIS (Netherlands)

    Gijsbertse, K.; Sprengers, A. M. J.; Nillesen, M. M.; Hansen, Hendrik H.G.; Lopata, R.G.P.; Verdonschot, N.; de Korte, C. L.

    2016-01-01

    In this study, a multi-dimensional strain estimation method is presented to assess local relative deformation in three orthogonal directions in 3D space of skeletal muscles during voluntary contractions. A rigid translation and compressive deformation of a block phantom, that mimics muscle

  15. Ultrastructure of skeletal muscle capillaries under conditions of space mission.

    Science.gov (United States)

    Volodina, A V; Pozdnyakov, O M

    2006-06-01

    Capillaries of the rat forepaw skeletal muscles were examined on day 14 of space mission and on days 1 and 14 after landing. Ultrastructural studies revealed apoptosis caused by muscle fiber atrophy and necrobiotic changes eventuating in coagulation or monocellular necrosis of endothelial cells. Formation of capillaries was detected, which can be regarded as an adaptive reaction to injuries caused by space mission factors.

  16. Skeletal muscle lipid metabolism in exercise and insulin resistance

    DEFF Research Database (Denmark)

    Kiens, Bente

    2006-01-01

    Lipids as fuel for energy provision originate from different sources: albumin-bound long-chain fatty acids (LCFA) in the blood plasma, circulating very-low-density lipoproteins-triacylglycerols (VLDL-TG), fatty acids from triacylglycerol located in the muscle cell (IMTG), and possibly fatty acids...... of insulin resistance in skeletal muscle, including possible molecular mechanisms involved, is discussed....

  17. Skeletal muscle deiodinase type 2 regulation during illness in mice

    NARCIS (Netherlands)

    Kwakkel, J.; van Beeren, H. C.; Ackermans, M. T.; Platvoet-ter Schiphorst, M. C.; Fliers, E.; Wiersinga, W. M.; Boelen, A.

    2009-01-01

    We have previously shown that skeletal muscle deiodinase type 2 (D2) mRNA (listed as Dio2 in MGI Database) is up-regulated in an animal model of acute illness. However, human Studies on the expression Of muscle D2 during illness report conflicting data. Therefore, we evaluated the expression of

  18. Purinergic receptors expressed in human skeletal muscle fibres

    DEFF Research Database (Denmark)

    Bornø, A; Ploug, Thorkil; Bune, L T

    2012-01-01

    Purinergic receptors are present in most tissues and thought to be involved in various signalling pathways, including neural signalling, cell metabolism and local regulation of the microcirculation in skeletal muscles. The present study aims to determine the distribution and intracellular content...... of purinergic receptors in skeletal muscle fibres in patients with type 2 diabetes and age-matched controls. Muscle biopsies from vastus lateralis were obtained from six type 2 diabetic patients and seven age-matched controls. Purinergic receptors were analysed using light and confocal microscopy...

  19. Assessment of the antidiabetic potential of selected medicinal plants using in vitro bioassays of muscle glucose transport and liver glucose production

    DEFF Research Database (Denmark)

    Beidokhti, M N; Sanchez Villavicencio, M L; Eid, H M

    2016-01-01

    by skeletal muscle cells are the principal contributors to the associated hyperglycemic state. The aim of this study was to assess the antidiabetic potential of five medicinal plant extracts using in vitro cell based assays targeting glucose uptake in C2C12 skeletal muscle cells [1] and glucose-6-phosphatase...... activity (G6Pase) [2] in rat hepatoma H4IIE. Cells were treated for 18h with maximal non-toxic concentrations (50 µg/mL) of the ethanolic extract of Psidium guajava (leaf and bark), Phyllanthus niruri (aerial parts), Eugenia jambolana (dried fruit) and Rhizophora mucronata (bark), which were determined...... by the lactate dehydrogenase (LDH) cytotoxicity assay. None of the extracts were able to reduce G6Pase activity. In contrast, one plant extract (P. guajava leaf extract) was found to significantly increase deoxyglucose uptake in C2C12 muscle cells (161%, p ≤0.001), to levels higher that of the positive control...

  20. Dystrophin muscle enhancer 1 is implicated in the activation of non-muscle isoforms in the skeletal muscle of patients with X-linked dilated cardiomyopathy

    NARCIS (Netherlands)

    Bastianutto, C.; Bestard, J. A.; Lahnakoski, K.; Broere, D.; de Visser, M.; Zaccolo, M.; Pozzan, T.; Ferlini, A.; Muntoni, F.; Patarnello, T.; Klamut, H. J.

    2001-01-01

    X-linked dilated cardiomyopathy (XLDC) is a dystrophinopathy characterized by severe cardiomyopathy with no skeletal muscle involvement. Several XLDC patients have been described with mutations that abolish dystrophin muscle (M) isoform expression. The absence of skeletal muscle degeneration

  1. Autophagy in Skeletal Muscle Homeostasis and in Muscular Dystrophies

    Directory of Open Access Journals (Sweden)

    Paolo Bonaldo

    2012-07-01

    Full Text Available Skeletal muscles are the agent of motion and one of the most important tissues responsible for the control of metabolism. The maintenance of muscle homeostasis is finely regulated by the balance between catabolic and anabolic process. Macroautophagy (or autophagy is a catabolic process that provides the degradation of protein aggregation and damaged organelles through the fusion between autophagosomes and lysosomes. Proper regulation of the autophagy flux is fundamental for the homeostasis of skeletal muscles during physiological situations and in response to stress. Defective as well as excessive autophagy is harmful for muscle health and has a pathogenic role in several forms of muscle diseases. This review will focus on the role of autophagy in muscle homeostasis and diseases.

  2. Mechanically induced alterations in cultured skeletal muscle growth

    Science.gov (United States)

    Vandenburgh, H. H.; Hatfaludy, S.; Karlisch, P.; Shansky, J.

    1991-01-01

    Model systems are available for mechanically stimulating cultured skeletal muscle cells by passive tensile forces which simulate those found in vivo. When applied to embryonic muscle cells in vitro these forces induce tissue organogenesis, metabolic adaptations, and muscle cell growth. The mechanical stimulation of muscle cell growth correlates with stretch-induced increases in the efflux of prostaglandins PGE2 and PGF2(alpha) in a time and frequency dependent manner. These prostaglandins act as mechanical 'second messengers' regulating skeletal muscle protein turnover rates. Since they also effect bone remodelling in response to tissue loading and unloading, secreted prostaglandins may serve as paracrine growth factors, coordinating the growth rates of muscle and bone in response to external mechanical forces. Cell culture model systems will supplement other models in understanding mechanical transduction processes at the molecular level.

  3. High skeletal muscle adenylate cyclase in malignant hyperthermia.

    OpenAIRE

    Willner, J H; Cerri, C G; Wood, D S

    1981-01-01

    Malignant hyperthermia occurs in humans with several congenital myopathies, usually in response to general anesthesia. Commonly, individuals who develop this syndrome lack symptoms of muscle disease, and their muscle lacks specific pathological changes. A biochemical marker for this myopathy has not previously been available; we found activity of adenylate cyclase and content of cyclic AMP to be abnormally high in skeletal muscle. Secondary modification of protein phosphorylation could explai...

  4. Decellularized Human Skeletal Muscle as Biologic Scaffold for Reconstructive Surgery

    Science.gov (United States)

    Porzionato, Andrea; Sfriso, Maria Martina; Pontini, Alex; Macchi, Veronica; Petrelli, Lucia; Pavan, Piero G.; Natali, Arturo N.; Bassetto, Franco; Vindigni, Vincenzo; De Caro, Raffaele

    2015-01-01

    Engineered skeletal muscle tissues have been proposed as potential solutions for volumetric muscle losses, and biologic scaffolds have been obtained by decellularization of animal skeletal muscles. The aim of the present work was to analyse the characteristics of a biologic scaffold obtained by decellularization of human skeletal muscles (also through comparison with rats and rabbits) and to evaluate its integration capability in a rabbit model with an abdominal wall defect. Rat, rabbit and human muscle samples were alternatively decellularized with two protocols: n.1, involving sodium deoxycholate and DNase I; n.2, trypsin-EDTA and Triton X-NH4OH. Protocol 2 proved more effective, removing all cellular material and maintaining the three-dimensional networks of collagen and elastic fibers. Ultrastructural analyses with transmission and scanning electron microscopy confirmed the preservation of collagen, elastic fibres, glycosaminoglycans and proteoglycans. Implantation of human scaffolds in rabbits gave good results in terms of integration, although recellularization by muscle cells was not completely achieved. In conclusion, human skeletal muscles may be effectively decellularized to obtain scaffolds preserving the architecture of the extracellular matrix and showing mechanical properties suitable for implantation/integration. Further analyses will be necessary to verify the suitability of these scaffolds for in vitro recolonization by autologous cells before in vivo implantation. PMID:26140375

  5. Functional classification of skeletal muscle networks. I. Normal physiology.

    Science.gov (United States)

    Wang, Yu; Winters, Jack; Subramaniam, Shankar

    2012-12-15

    Extensive measurements of the parts list of human skeletal muscle through transcriptomics and other phenotypic assays offer the opportunity to reconstruct detailed functional models. Through integration of vast amounts of data present in databases and extant knowledge of muscle function combined with robust analyses that include a clustering approach, we present both a protein parts list and network models for skeletal muscle function. The model comprises the four key functional family networks that coexist within a functional space; namely, excitation-activation family (forward pathways that transmit a motoneuronal command signal into the spatial volume of the cell and then use Ca(2+) fluxes to bind Ca(2+) to troponin C sites on F-actin filaments, plus transmembrane pumps that maintain transmission capacity); mechanical transmission family (a sophisticated three-dimensional mechanical apparatus that bidirectionally couples the millions of actin-myosin nanomotors with external axial tensile forces at insertion sites); metabolic and bioenergetics family (pathways that supply energy for the skeletal muscle function under widely varying demands and provide for other cellular processes); and signaling-production family (which represents various sensing, signal transduction, and nuclear infrastructure that controls the turn over and structural integrity and regulates the maintenance, regeneration, and remodeling of the muscle). Within each family, we identify subfamilies that function as a unit through analysis of large-scale transcription profiles of muscle and other tissues. This comprehensive network model provides a framework for exploring functional mechanisms of the skeletal muscle in normal and pathophysiology, as well as for quantitative modeling.

  6. Changes in skeletal muscle gene expression following clenbuterol administration

    Directory of Open Access Journals (Sweden)

    McIntyre Lauren M

    2006-12-01

    Full Text Available Abstract Background Beta-adrenergic receptor agonists (BA induce skeletal muscle hypertrophy, yet specific mechanisms that lead to this effect are not well understood. The objective of this research was to identify novel genes and physiological pathways that potentially facilitate BA induced skeletal muscle growth. The Affymetrix platform was utilized to identify gene expression changes in mouse skeletal muscle 24 hours and 10 days after administration of the BA clenbuterol. Results Administration of clenbuterol stimulated anabolic activity, as indicated by decreased blood urea nitrogen (BUN; P P Conclusion Global evaluation of gene expression after administration of clenbuterol identified changes in gene expression and overrepresented functional categories of genes that may regulate BA-induced muscle hypertrophy. Changes in mRNA abundance of multiple genes associated with myogenic differentiation may indicate an important effect of BA on proliferation, differentiation, and/or recruitment of satellite cells into muscle fibers to promote muscle hypertrophy. Increased mRNA abundance of genes involved in the initiation of translation suggests that increased levels of protein synthesis often associated with BA administration may result from a general up-regulation of translational initiators. Additionally, numerous other genes and physiological pathways were identified that will be important targets for further investigations of the hypertrophic effect of BA on skeletal muscle.

  7. Decellularized Human Skeletal Muscle as Biologic Scaffold for Reconstructive Surgery

    Directory of Open Access Journals (Sweden)

    Andrea Porzionato

    2015-07-01

    Full Text Available Engineered skeletal muscle tissues have been proposed as potential solutions for volumetric muscle losses, and biologic scaffolds have been obtained by decellularization of animal skeletal muscles. The aim of the present work was to analyse the characteristics of a biologic scaffold obtained by decellularization of human skeletal muscles (also through comparison with rats and rabbits and to evaluate its integration capability in a rabbit model with an abdominal wall defect. Rat, rabbit and human muscle samples were alternatively decellularized with two protocols: n.1, involving sodium deoxycholate and DNase I; n.2, trypsin-EDTA and Triton X-NH4OH. Protocol 2 proved more effective, removing all cellular material and maintaining the three-dimensional networks of collagen and elastic fibers. Ultrastructural analyses with transmission and scanning electron microscopy confirmed the preservation of collagen, elastic fibres, glycosaminoglycans and proteoglycans. Implantation of human scaffolds in rabbits gave good results in terms of integration, although recellularization by muscle cells was not completely achieved. In conclusion, human skeletal muscles may be effectively decellularized to obtain scaffolds preserving the architecture of the extracellular matrix and showing mechanical properties suitable for implantation/integration. Further analyses will be necessary to verify the suitability of these scaffolds for in vitro recolonization by autologous cells before in vivo implantation.

  8. Muscle size explains low passive skeletal muscle force in heart failure patients

    OpenAIRE

    Panizzolo, FA; Maiorana, AJ; Naylor, LH; Dembo, LG; Lloyd, DG; Green, DJ; Rubenson, J

    2016-01-01

    BACKGROUND: Alterations in skeletal muscle function and architecture have been linked to the compromised exercise capacity characterizing chronic heart failure (CHF). However, how passive skeletal muscle force is affected in CHF is not clear. Understanding passive force characteristics in CHF can help further elucidate the extent to which altered contractile properties and/or architecture might affect muscle and locomotor function. Therefore, the aim of this study was to investigate passive f...

  9. Force Transmission between Synergistic Skeletal Muscles through Connective Tissue Linkages

    Directory of Open Access Journals (Sweden)

    Huub Maas

    2010-01-01

    Full Text Available The classic view of skeletal muscle is that force is generated within its muscle fibers and then directly transmitted in-series, usually via tendon, onto the skeleton. In contrast, recent results suggest that muscles are mechanically connected to surrounding structures and cannot be considered as independent actuators. This article will review experiments on mechanical interactions between muscles mediated by such epimuscular myofascial force transmission in physiological and pathological muscle conditions. In a reduced preparation, involving supraphysiological muscle conditions, it is shown that connective tissues surrounding muscles are capable of transmitting substantial force. In more physiologically relevant conditions of intact muscles, however, it appears that the role of this myofascial pathway is small. In addition, it is hypothesized that connective tissues can serve as a safety net for traumatic events in muscle or tendon. Future studies are needed to investigate the importance of intermuscular force transmission during movement in health and disease.

  10. Circadian clock regulation of skeletal muscle growth and repair.

    Science.gov (United States)

    Chatterjee, Somik; Ma, Ke

    2016-01-01

    Accumulating evidence indicates that the circadian clock, a transcriptional/translational feedback circuit that generates ~24-hour oscillations in behavior and physiology, is a key temporal regulatory mechanism involved in many important aspects of muscle physiology. Given the clock as an evolutionarily-conserved time-keeping mechanism that synchronizes internal physiology to environmental cues, locomotor activities initiated by skeletal muscle enable entrainment to the light-dark cycles on earth, thus ensuring organismal survival and fitness. Despite the current understanding of the role of molecular clock in preventing age-related sarcopenia, investigations into the underlying molecular pathways that transmit clock signals to the maintenance of skeletal muscle growth and function are only emerging. In the current review, the importance of the muscle clock in maintaining muscle mass during development, repair and aging, together with its contribution to muscle metabolism, will be discussed. Based on our current understandings of how tissue-intrinsic muscle clock functions in the key aspects muscle physiology, interventions targeting the myogenic-modulatory activities of the clock circuit may offer new avenues for prevention and treatment of muscular diseases. Studies of mechanisms underlying circadian clock function and regulation in skeletal muscle warrant continued efforts.

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  10. Myostatin in the Pathophysiology of Skeletal Muscle

    OpenAIRE

    Carnac, Gilles; Vernus, Barbara; Bonnieu, Anne

    2007-01-01

    Myostatin is an endogenous, negative regulator of muscle growth determining both muscle fiber number and size. The myostatin pathway is conserved across diverse species ranging from zebrafish to humans. Experimental models of muscle growth and regeneration have implicated myostatin as an important mediator of catabolic pathways in muscle cells. Inhibition of this pathway has emerged as a promising therapy for muscle wasting. Here we discuss the recent developments and the controversies in myo...

  11. Growth Factors and Tension-Induced Skeletal Muscle Growth

    Science.gov (United States)

    Vandenburgh, Herman H.

    1994-01-01

    The project investigated biochemical mechanisms to enhance skeletal muscle growth, and developed a computer based mechanical cell stimulator system. The biochemicals investigated in this study were insulin/(Insulin like Growth Factor) IGF-1 and Steroids. In order to analyze which growth factors are essential for stretch-induced muscle growth in vitro, we developed a defined, serum-free medium in which the differentiated, cultured avian muscle fibers could be maintained for extended periods of time. The defined medium (muscle maintenance medium, MM medium) maintains the nitrogen balance of the myofibers for 3 to 7 days, based on myofiber diameter measurements and myosin heavy chain content. Insulin and IGF-1, but not IGF-2, induced pronounced myofiber hypertrophy when added to this medium. In 5 to 7 days, muscle fiber diameters increase by 71 % to 98% compared to untreated controls. Mechanical stimulation of the avian muscle fibers in MM medium increased the sensitivity of the cells to insulin and IGF-1, based on a leftward shift of the insulin dose/response curve for protein synthesis rates. (54). We developed a ligand binding assay for IGF-1 binding proteins and found that the avian skeletal muscle cultures produced three major species of 31, 36 and 43 kD molecular weight (54) Stretch of the myofibers was found to have no significant effect on the efflux of IGF-1 binding proteins, but addition of exogenous collagen stimulated IGF-1 binding protein production 1.5 to 5 fold. Steroid hormones have a profound effect on muscle protein turnover rates in vivo, with the stress-related glucocorticoids inducing rapid skeletal muscle atrophy while androgenic steroids induce skeletal muscle growth. Exercise in humans and animals reduces the catabolic effects of glucocorticoids and may enhance the anabolic effects of androgenic steroids on skeletal muscle. In our continuing work on the involvement of exogenrus growth factors in stretch-induced avian skeletal muscle growth, we

  12. The adipokine leptin increases skeletal muscle mass and significantly alters skeletal muscle miRNA expression profile in aged mice

    Energy Technology Data Exchange (ETDEWEB)

    Hamrick, Mark W., E-mail: mhamrick@mail.mcg.edu [Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Department of Orthopaedic Surgery, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Herberg, Samuel; Arounleut, Phonepasong [Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Department of Orthopaedic Surgery, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); He, Hong-Zhi [Henry Ford Immunology Program, Henry Ford Health System, Detroit, MI (United States); Department of Dermatology, Henry Ford Health System, Detroit, MI (United States); Shiver, Austin [Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Department of Orthopaedic Surgery, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Qi, Rui-Qun [Henry Ford Immunology Program, Henry Ford Health System, Detroit, MI (United States); Department of Dermatology, Henry Ford Health System, Detroit, MI (United States); Zhou, Li [Henry Ford Immunology Program, Henry Ford Health System, Detroit, MI (United States); Department of Dermatology, Henry Ford Health System, Detroit, MI (United States); Department of Internal Medicine, Henry Ford Health System, Detroit, MI (United States); Isales, Carlos M. [Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Department of Orthopaedic Surgery, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); and others

    2010-09-24

    Research highlights: {yields} Aging is associated with muscle atrophy and loss of muscle mass, known as the sarcopenia of aging. {yields} We demonstrate that age-related muscle atrophy is associated with marked changes in miRNA expression in muscle. {yields} Treating aged mice with the adipokine leptin significantly increased muscle mass and the expression of miRNAs involved in muscle repair. {yields} Recombinant leptin therapy may therefore be a novel approach for treating age-related muscle atrophy. -- Abstract: Age-associated loss of muscle mass, or sarcopenia, contributes directly to frailty and an increased risk of falls and fractures among the elderly. Aged mice and elderly adults both show decreased muscle mass as well as relatively low levels of the fat-derived hormone leptin. Here we demonstrate that loss of muscle mass and myofiber size with aging in mice is associated with significant changes in the expression of specific miRNAs. Aging altered the expression of 57 miRNAs in mouse skeletal muscle, and many of these miRNAs are now reported to be associated specifically with age-related muscle atrophy. These include miR-221, previously identified in studies of myogenesis and muscle development as playing a role in the proliferation and terminal differentiation of myogenic precursors. We also treated aged mice with recombinant leptin, to determine whether leptin therapy could improve muscle mass and alter the miRNA expression profile of aging skeletal muscle. Leptin treatment significantly increased hindlimb muscle mass and extensor digitorum longus fiber size in aged mice. Furthermore, the expression of 37 miRNAs was altered in muscles of leptin-treated mice. In particular, leptin treatment increased the expression of miR-31 and miR-223, miRNAs known to be elevated during muscle regeneration and repair. These findings suggest that aging in skeletal muscle is associated with marked changes in the expression of specific miRNAs, and that nutrient

  13. Regulation of insulin-like growth factor-I in skeletal muscle and muscle cells.

    Science.gov (United States)

    Frost, R A; Lang, C H

    2003-03-01

    Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are potent regulators of muscle mass. Transgenic mice that over-express these proteins exhibit dramatically enlarged skeletal muscles. In contrast, malnutrition, critical illness, sepsis, and aging are all associated with a dramatic reduction in muscle mass and function. The circulating concentration of IGF-I and the expression of IGF-I in skeletal muscle are also reduced during catabolic states. Consequently, GH has been used clinically to increase lean body mass in patients with muscle wasting. Likewise, delivery of IGF-I specifically into muscle has been proposed as a genetic therapy for muscle disorders. A better understanding of the regulation of IGF-I expression in skeletal muscle and muscle cells is therefore of importance. Yet, our knowledge in this area has been limited by a lack of GH responsive muscle cells. In addition the IGF-I gene spans over 90 kb of genomic DNA and it exhibits a very complex regulatory pattern. This review will summarize our knowledge of the control of muscle mass by GH, IGF-I, anabolic steroids, exercise and other growth enhancing hormones. We will also highlight recent advances in the regulation of IGF-I and signal transducers and activators of transcription (Stats) by GH. A special emphasis will be placed on the interaction of IGF-I and proinflammatory cytokines in skeletal muscle and muscle cells.

  14. Effect of repeated forearm muscle cooling on the adaptation of skeletal muscle metabolism in humans

    Science.gov (United States)

    Wakabayashi, Hitoshi; Nishimura, Takayuki; Wijayanto, Titis; Watanuki, Shigeki; Tochihara, Yutaka

    2017-07-01

    This study aimed to investigate the effect of repeated cooling of forearm muscle on adaptation in skeletal muscle metabolism. It is hypothesized that repeated decreases of muscle temperature would increase the oxygen consumption in hypothermic skeletal muscle. Sixteen healthy males participated in this study. Their right forearm muscles were locally cooled to 25 °C by cooling pads attached to the skin. This local cooling was repeated eight times on separate days for eight participants (experimental group), whereas eight controls received no cold exposure. To evaluate adaptation in skeletal muscle metabolism, a local cooling test was conducted before and after the repeated cooling period. Change in oxy-hemoglobin content in the flexor digitorum at rest and during a 25-s isometric handgrip (10% maximal voluntary construction) was measured using near-infrared spectroscopy at every 2 °C reduction in forearm muscle temperature. The arterial blood flow was occluded for 15 s by upper arm cuff inflation at rest and during the isometric handgrip. The oxygen consumption in the flexor digitorum muscle was evaluated by a slope of the oxy-hemoglobin change during the arterial occlusion. In the experimental group, resting oxygen consumption in skeletal muscle did not show any difference between pre- and post-intervention, whereas muscle oxygen consumption during the isometric handgrip was significantly higher in post-intervention than in pre-test from thermoneutral baseline to 31 °C muscle temperature ( P < 0.05). This result indicated that repeated local muscle cooling might facilitate oxidative metabolism in the skeletal muscle. In summary, skeletal muscle metabolism during submaximal isometric handgrip was facilitated after repeated local muscle cooling.

  15. Relative appendicular skeletal muscle mass is associated with isokinetic muscle strength and balance in healthy collegiate men.

    Science.gov (United States)

    Kim, Sung-Eun; Hong, Ju; Cha, Jun-Youl; Park, Jung-Min; Eun, Denny; Yoo, Jaehyun; Jee, Yong-Seok

    2016-11-01

    There are few studies on the relationship between skeletal muscle mass and balance in the young ages. We investigated the relationship between appendicular skeletal muscle mass, isokinetic muscle strength of lower extremity, and balance among healthy young men using relative skeletal muscle index. Thirty men were grouped according to relative appendicular skeletal muscle mass index: higher skeletal muscle group (n = 15) and lower skeletal muscle group (n = 15). Static and dynamic balance abilities were measured using the following: a test where participants stood on one leg with eyes closed, a modified Clinical Test of Sensory Interaction on Balance (mCTSIB) with eyes open and eyes closed, a stability test, and limits of stability test. The muscle strength of lower extremities was measured with an isokinetic analyser in hip, knee, and ankle joints. Participants with higher appendicular skeletal muscle mass were significantly more stable in maintaining dynamic balance than those with lower appendicular skeletal muscle mass. Moreover, appendicular skeletal muscle mass index was positively correlated with dynamic balance ability. Participants with higher appendicular skeletal muscle mass had stronger strength in the lower extremity, and there were significant differences in the isokinetic torque ratios between groups. From these results, it can be inferred that higher appendicular skeletal muscle mass relates to muscle strength and the alteration in the peak torque ratio of the lower extremity, contributing to the maintenance of balance.

  16. Response and function of skeletal muscle heat shock protein 70.

    Science.gov (United States)

    Liu, Yuefei; Gampert, Larissa; Nething, Katja; Steinacker, Jürgen M

    2006-09-01

    In response to stress, cells produce a series of heat shock proteins (Hsps). One of the most prominent Hsps, is the 70 kDa Hsp (Hsp70). Hsp70 is a highly conserved and essential protein against stress. The skeletal muscle responds to a diverse group of stress signals namely, muscle contraction linked energy and milieu challenges, ischemia and exercise by producing Hsp70. The extent of this Hsp70 response in skeletal muscle depends on the type and intensity of the signal, and is characterized in a muscle fiber specific manner by a special time course. Hsp70 in the skeletal muscle is regulated at transcriptional, translational and posttranslational levels. Hsp70 serves as an indicator for cellular stress as a molecular chaperone, plays pivotal role in maintaining cellular homeostasis by preventing apoptosis, influences energy metabolism, facilitates cellular processes in terms of muscular adaptation and interacts with other signalling pathways. This review summarizes our current knowledge on the skeletal muscle Hsp70 response.

  17. Altered cross-bridge properties in skeletal muscle dystrophies

    Directory of Open Access Journals (Sweden)

    Aziz eGuellich

    2014-10-01

    Full Text Available Force and motion generated by skeletal muscle ultimately depends on the cyclical interaction of actin with myosin. This mechanical process is regulated by intracellular Ca2+ through the thin filament-associated regulatory proteins i.e.; troponins and tropomyosin. Muscular dystrophies are a group of heterogeneous genetic affections characterized by progressive degeneration and weakness of the skeletal muscle as a consequence of loss of muscle tissue which directly reduces the number of potential myosin cross-bridges involved in force production. Mutations in genes responsible for skeletal muscle dystrophies have been shown to modify the function of contractile proteins and cross-bridge interactions. Altered gene expression or RNA splicing or post-translational modifications of contractile proteins such as those related to oxidative stress, may affect cross-bridge function by modifying key proteins of the excitation-contraction coupling. Micro-architectural change in myofilament is another mechanism of altered cross-bridge performance. In this review, we provide an overview about changes in cross-bridge performance in skeletal muscle dystrophies and discuss their ultimate impacts on striated muscle function.

  18. Skeletal muscle as a regulator of the longevity protein, Klotho

    Directory of Open Access Journals (Sweden)

    Keith G Avin

    2014-06-01

    Full Text Available Klotho is a powerful longevity protein that has been linked to the prevention of muscle atrophy, osteopenia, and cardiovascular disease. Similar anti-aging effects have also been ascribed to exercise and physical activity. While an association between muscle function and klotho expression has been previously suggested from longitudinal cohort studies, a direct relationship between circulating klotho and skeletal muscle has not been investigated. In this paper, we present a review of the literature and preliminary evidence that, together, suggests klotho expression may be modulated by skeletal muscle activity. Our pilot clinical findings performed in young and aged individuals suggest that circulating klotho levels are upregulated in response to an acute exercise bout, but that the response may be dependent on fitness level. A similar upregulation of circulating klotho is also observed in response to an acute exercise in young and old mice, suggesting this may be a good model for mechanistically probing the role of physical activity on klotho expression. Finally, we highlight overlapping signaling pathways that are modulated by both klotho and skeletal muscle and propose potential mechanisms for cross-talk between the two. It is hoped that this review will stimulate further consideration of the relationship between skeletal muscle activity and klotho expression, potentially leading to important insights into the well-documented systemic anti-aging effects of exercise.

  19. Regulation of the skeletal muscle blood flow in humans

    DEFF Research Database (Denmark)

    Mortensen, Stefan; Saltin, Bengt

    2014-01-01

    hyperaemia whereas the role of ATP remains uncertain due to lack of specific purinergic receptor blockers for human use. The purpose of this review is to address the interaction between vasodilator systems and to discuss the multiple proposed roles of ATP in human skeletal muscle blood flow regulation......In humans, skeletal muscle blood flow is regulated by an interaction between several locally formed vasodilators including nitric oxide (NO) and prostaglandins. In plasma, ATP is a potent vasodilator that stimulates the formation of NO and prostaglandins and very importantly can offset local...... sympathetic vasoconstriction. ATP is released into plasma from erythrocytes and endothelial cells and the plasma concentration increases in both the feeding artery and the vein draining the contracting skeletal muscle. Adenosine also stimulates the formation of NO and prostaglandins, but the plasma adenosine...

  20. Intracellular compartmentalization of skeletal muscle glycogen metabolism and insulin signalling

    DEFF Research Database (Denmark)

    Prats Gavalda, Clara; Gomez-Cabello, Alba; Vigelsø Hansen, Andreas

    2011-01-01

    The interest in skeletal muscle metabolism and insulin signalling has increased exponentially in recent years as a consequence of their role in the development of type 2 diabetes mellitus. Despite this, the exact mechanisms involved in the regulation of skeletal muscle glycogen metabolism...... and insulin signalling transduction remain elusive. We believe that one of the reasons is that the role of intracellular compartmentalization as a regulator of metabolic pathways and signalling transduction has been rather ignored. This paper briefly reviews the literature to discuss the role of intracellular...... compartmentalization in the regulation of skeletal muscle glycogen metabolism and insulin signalling. As a result, a hypothetical regulatory mechanism is proposed by which cells could direct glycogen resynthesis towards different pools of glycogen particles depending on the metabolic needs. Furthermore, we discuss...

  1. Changes in skeletal muscle gene expression following clenbuterol administration.

    Science.gov (United States)

    Spurlock, Diane M; McDaneld, Tara G; McIntyre, Lauren M

    2006-12-20

    Beta-adrenergic receptor agonists (BA) induce skeletal muscle hypertrophy, yet specific mechanisms that lead to this effect are not well understood. The objective of this research was to identify novel genes and physiological pathways that potentially facilitate BA induced skeletal muscle growth. The Affymetrix platform was utilized to identify gene expression changes in mouse skeletal muscle 24 hours and 10 days after administration of the BA clenbuterol. Administration of clenbuterol stimulated anabolic activity, as indicated by decreased blood urea nitrogen (BUN; P clenbuterol treatment. A total of 22,605 probesets were evaluated with 52 probesets defined as differentially expressed based on a false discovery rate of 10%. Differential mRNA abundance of four of these genes was validated in an independent experiment by quantitative PCR. Functional characterization of differentially expressed genes revealed several categories that participate in biological processes important to skeletal muscle growth, including regulators of transcription and translation, mediators of cell-signalling pathways, and genes involved in polyamine metabolism. Global evaluation of gene expression after administration of clenbuterol identified changes in gene expression and overrepresented functional categories of genes that may regulate BA-induced muscle hypertrophy. Changes in mRNA abundance of multiple genes associated with myogenic differentiation may indicate an important effect of BA on proliferation, differentiation, and/or recruitment of satellite cells into muscle fibers to promote muscle hypertrophy. Increased mRNA abundance of genes involved in the initiation of translation suggests that increased levels of protein synthesis often associated with BA administration may result from a general up-regulation of translational initiators. Additionally, numerous other genes and physiological pathways were identified that will be important targets for further investigations of the

  2. Localisation of AMPK γ subunits in cardiac and skeletal muscles.

    Science.gov (United States)

    Pinter, Katalin; Grignani, Robert T; Watkins, Hugh; Redwood, Charles

    2013-12-01

    The trimeric protein AMP-activated protein kinase (AMPK) is an important sensor of energetic status and cellular stress, and mutations in genes encoding two of the regulatory γ subunits cause inherited disorders of either cardiac or skeletal muscle. AMPKγ2 mutations cause hypertrophic cardiomyopathy with glycogen deposition and conduction abnormalities; mutations in AMPKγ3 result in increased skeletal muscle glycogen. In order to gain further insight into the roles of the different γ subunits in muscle and into possible disease mechanisms, we localised the γ2 and γ3 subunits, along with the more abundant γ1 subunit, by immunofluorescence in cardiomyocytes and skeletal muscle fibres. The predominant cardiac γ2 variant, γ2-3B, gave a striated pattern in cardiomyocytes, aligning with the Z-disk but with punctate staining similar to T-tubule (L-type Ca(2+) channel) and sarcoplasmic reticulum (SERCA2) markers. In skeletal muscle fibres AMPKγ3 localises to the I band, presenting a uniform staining that flanks the Z-disk, also coinciding with the position of Ca(2+) influx in these muscles. The localisation of γ2-3B- and γ3-containing AMPK suggests that these trimers may have similar functions in the different muscles. AMPK containing γ2-3B was detected in oxidative skeletal muscles which had low expression of γ3, confirming that these two regulatory subunits may be co-ordinately regulated in response to metabolic requirements. Compartmentalisation of AMPK complexes is most likely dependent on the regulatory γ subunit and this differential localisation may direct substrate selection and specify particular functional roles.

  3. Muscle interleukin-6 and fasting-induced PDH regulation in mouse skeletal muscle.

    Science.gov (United States)

    Gudiksen, Anders; Bertholdt, Laerke; Vingborg, Mikkel Birkkjaer; Hansen, Henriette Watson; Ringholm, Stine; Pilegaard, Henriette

    2017-03-01

    Fasting prompts a metabolic shift in substrate utilization from carbohydrate to predominant fat oxidation in skeletal muscle, and pyruvate dehydrogenase (PDH) is seen as a controlling link between the competitive oxidation of carbohydrate and fat during metabolic challenges like fasting. Interleukin (IL)-6 has been proposed to be released from muscle with concomitant effects on both glucose and fat utilization. The aim was to test the hypothesis that muscle IL-6 has a regulatory impact on fasting-induced suppression of skeletal muscle PDH. Skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice and floxed littermate controls (control) were either fed or fasted for 6 or 18 h. Lack of muscle IL-6 elevated the respiratory exchange ratio in the fed and early fasting state, but not with prolonged fasting. Activity of PDH in the active form (PDHa) was higher in fed and fasted IL-6 MKO than in control mice at 18 h, but not at 6 h, whereas lack of muscle IL-6 did not prevent downregulation of PDHa activity in skeletal muscle or changes in plasma and muscle substrate levels in response to 18 h of fasting. Phosphorylation of three of four sites on PDH-E1α increased with 18 h of fasting, but was lower in IL-6 MKO mice than in control. In addition, both PDK4 mRNA and protein increased with 6 and 18 h of fasting in both genotypes, but PDK4 protein was lower in IL-6 MKO than in control. In conclusion, skeletal muscle IL-6 seems to regulate whole body substrate utilization in the fed, but not fasted, state and influence skeletal muscle PDHa activity in a circadian manner. However, skeletal muscle IL-6 is not required for maintaining metabolic flexibility in response to fasting. Copyright © 2017 the American Physiological Society.

  4. STRUCTURAL ALTERATIONS OF SKELETAL MUSCLE IN COPD

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    Sunita eMathur

    2014-03-01

    Full Text Available Background: Chronic obstructive pulmonary disease (COPD is a respiratory disease associated with a systemic inflammatory response. Peripheral muscle dysfunction has been well characterized in individuals with COPD and results from a complex interaction between systemic and local factors. Objective: In this narrative review, we will describe muscle wasting in people with COPD, the associated structural changes, muscle regenerative capacity and possible mechanisms for muscle wasting. We will also discuss how structural changes relate to impaired muscle function and mobility in people with COPD. Key Observations: Approximately 30-40% of individuals with COPD experience muscle mass depletion. Furthermore, muscle atrophy is a predictor of physical function and mortality in this population. Associated structural changes include a decreased proportion and size of type-I fibers, reduced oxidative capacity and mitochondrial density mainly in the quadriceps. Observations related to impaired muscle regenerative capacity in individuals with COPD include a lower proportion of central nuclei in the presence or absence of muscle atrophy and decreased maximal telomere length, which has been correlated with reduced muscle cross-sectional area. Potential mechanisms for muscle wasting in COPD may include excessive production of reactive oxygen species, altered amino acid metabolism and lower expression of peroxisome proliferator-activated receptors-gamma-coactivator 1-alpha mRNA. Despite a moderate relationship between muscle atrophy and function, impairments in oxidative metabolism only seems weakly related to muscle function. Conclusion: This review article demonstrates the cellular modifications in the peripheral muscle of people with COPD and describes the evidence of its relationship to muscle function. Future research will focus on rehabilitation strategies to improve muscle wasting and maximize function.

  5. Effects of oral phosphatidic acid feeding with or without whey protein on muscle protein synthesis and anabolic signaling in rodent skeletal muscle.

    Science.gov (United States)

    Mobley, C Brooks; Hornberger, Troy A; Fox, Carlton D; Healy, James C; Ferguson, Brian S; Lowery, Ryan P; McNally, Rachel M; Lockwood, Christopher M; Stout, Jeffrey R; Kavazis, Andreas N; Wilson, Jacob M; Roberts, Michael D

    2015-01-01

    Phosphatidic acid (PA) is a diacyl-glycerophospholipid that acts as a signaling molecule in numerous cellular processes. Recently, PA has been proposed to stimulate skeletal muscle protein accretion, but mechanistic studies are lacking. Furthermore, it is unknown whether co-ingesting PA with other leucine-containing ingredients can enhance intramuscular anabolic signaling mechanisms. Thus, the purpose of this study was to examine if oral PA feeding acutely increases anabolic signaling markers and muscle protein synthesis (MPS) in gastrocnemius with and without whey protein concentrate (WPC). Overnight fasted male Wistar rats (~250 g) were randomly assigned to four groups: control (CON, n = 6-13), PA (29 mg; n = 8), WPC (197 mg; n = 8), or PA + WPC (n = 8). Three hours post-feeding, gastrocnemius muscle was removed for markers of Akt-mTOR signaling, gene expression patterns related to skeletal muscle mass regulation and metabolism, and MPS analysis via the SUnSET method. Compared to CON rats, PA, WPC and PA + WPC resulted in a significant elevation in the phosphorylation of mTOR (Ser2481) and rps6 (Ser235/236) (p < 0.05) in the gastrocnemius though there were no differences between the supplemented groups. MPS levels in the gastrocnemius were significantly (p < 0.05) elevated in WPC versus CON rats, and tended to be elevated in PA versus CON rats (p = 0.08), though MPS was less in PA + WPC versus WPC rats (p < 0.05) in spite of robust increases in mTOR pathway activity markers in the former group. C2C12 myoblast data agreed with the in vivo data herein showing that PA increased MPS levels 51% (p < 0.001) phosphorylated p70s6k (Thr389) levels 67% (p < 0.001). Our results are the first in vivo evidence to demonstrate that PA tends to increases MPS 3 h post-feeding, though PA may delay WPC-mediated MPS kinetics within a 3 h post-feeding window.

  6. Growth factor involvement in tension-induced skeletal muscle growth

    Science.gov (United States)

    Vandenburgh, Herman H.

    1993-01-01

    Long-term manned space travel will require a better understanding of skeletal muscle atrophy which results from microgravity. Astronaut strength and dexterity must be maintained for normal mission operations and for emergency situations. Although exercise in space slows the rate of muscle loss, it does not prevent it. A biochemical understanding of how gravity/tension/exercise help to maintain muscle size by altering protein synthesis and/or degradation rate should ultimately allow pharmacological intervention to prevent muscle atrophy in microgravity. The overall objective is to examine some of the basic biochemical processes involved in tension-induced muscle growth. With an experimental in vitro system, the role of exogenous and endogenous muscle growth factors in mechanically stimulated muscle growth are examined. Differentiated avian skeletal myofibers can be 'exercised' in tissue culture using a newly developed dynamic mechanical cell stimulator device which simulates different muscle activity patterns. Patterns of mechanical activity which significantly affect muscle growth and metabolic characteristics were found. Both exogenous and endogenous growth factors are essential for tension-induced muscle growth. Exogenous growth factors found in serum, such as insulin, insulin-like growth factors, and steroids, are important regulators of muscle protein turnover rates and mechanically-induced muscle growth. Endogenous growth factors are synthesized and released into the culture medium when muscle cells are mechanically stimulated. At least one family of mechanically induced endogenous factors, the prostaglandins, help to regulate the rates of protein turnover in muscle cells. Endogenously synthesized IGF-1 is another. The interaction of muscle mechanical activity and these growth factors in the regulation of muscle protein turnover rates with our in vitro model system is studied.

  7. Substrate kinetics in patients with disorders of skeletal muscle metabolism.

    Science.gov (United States)

    Ørngreen, Mette Cathrine

    2016-07-01

    The main purpose of the following studies was to investigate pathophysiological mechanisms in fat and carbohydrate metabolism and effect of nutritional interventions in patients with metabolic myopathies and in patients with severe muscle wasting. Yet there is no cure for patients with skeletal muscle disorders. The group of patients is heterozygous and this thesis is focused on patients with metabolic myopathies and low muscle mass due to severe muscle wasting. Disorders of fatty acid oxidation (FAO) are, along with myophosphorylase deficiency (McArdle disease), the most common inborn errors of metabolism leading to recurrent episodes of rhabdomyolysis in adults. Prolonged exercise, fasting, and fever are the main triggering factors for rhabdomyolysis in these conditions, and can be complicated by acute renal failure. Patients with low muscle mass are in risk of loosing their functional skills and depend on a wheel chair and respiratory support. We used nutritional interventions and metabolic studies with stable isotope technique and indirect calorimetry in patients with metabolic myopathies and patients with low muscle mass to get information of the metabolism of the investigated diseases, and to gain knowledge of the biochemical pathways of intermediary metabolism in human skeletal muscle. We have shown that patients with fat metabolism disorders in skeletal muscle affecting the transporting enzyme of fat into the mitochondria (carnitine palmitoyltransferase II deficiency) and affecting the enzyme responsible for breakdown of the long-chain fatty acids (very long chain acyl-CoA dehydrogenase deficiency) have a normal fatty acid oxidation at rest, but enzyme activity is too low to increase fatty acid oxidation during exercise. Furthermore, these patients benefit from a carbohydrate rich diet. Oppositely is exercise capacity worsened by a fat-rich diet in these patients. The patients also benefit from IV glucose, however, when glucose is given orally just before

  8. Skeletal Muscle Satellite Cell Activation Following Cutaneous Burn in Rats

    Science.gov (United States)

    2013-12-01

    cultures of SJL/J and BALB/C skeletal muscle. Exp Cell Res 1994;211(1):99–107. [37] Yablonka-Reuveni Z, Rivera AJ. Temporal expression of regulatory...precursor cells. Am J Physiol Cell Physiol 2004;287(6):C1753–62. [41] Yasuhara S, Perez ME, Kanakubo E, Yasuhara Y, Shin YS, Kaneki M, Fujita T, Martyn JA...Yasuhara S, Kanakubo E, Perez ME, Kaneki M, Fujita T, Okamoto T, Martyn JA. The 1999 Moyer award, Burn injury induces skeletal muscle apoptosis and

  9. Skeletal Muscle Laminopathies: A Review of Clinical and Molecular Features

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    Lorenzo Maggi

    2016-08-01

    Full Text Available LMNA-related disorders are caused by mutations in the LMNA gene, which encodes for the nuclear envelope proteins, lamin A and C, via alternative splicing. Laminopathies are associated with a wide range of disease phenotypes, including neuromuscular, cardiac, metabolic disorders and premature aging syndromes. The most frequent diseases associated with mutations in the LMNA gene are characterized by skeletal and cardiac muscle involvement. This review will focus on genetics and clinical features of laminopathies affecting primarily skeletal muscle. Although only symptomatic treatment is available for these patients, many achievements have been made in clarifying the pathogenesis and improving the management of these diseases.

  10. Length dependence of active force production in skeletal muscle.

    Science.gov (United States)

    Rassier, D E; MacIntosh, B R; Herzog, W

    1999-05-01

    The sliding filament and cross-bridge theories of muscle contraction provide discrete predictions of the tetanic force-length relationship of skeletal muscle that have been tested experimentally. The active force generated by a maximally activated single fiber (with sarcomere length control) is maximal when the filament overlap is optimized and is proportionally decreased when overlap is diminished. The force-length relationship is a static property of skeletal muscle and, therefore, it does not predict the consequences of dynamic contractions. Changes in sarcomere length during muscle contraction result in modulation of the active force that is not necessarily predicted by the cross-bridge theory. The results of in vivo studies of the force-length relationship suggest that muscles that operate on the ascending limb of the force-length relationship typically function in stretch-shortening cycle contractions, and muscles that operate on the descending limb typically function in shorten-stretch cycle contractions. The joint moments produced by a muscle depend on the moment arm and the sarcomere length of the muscle. Moment arm magnitude also affects the excursion (length change) of a muscle for a given change in joint angle, and the number of sarcomeres arranged in series within a muscle fiber determines the sarcomere length change associated with a given excursion.

  11. Lactate and force production in skeletal muscle

    DEFF Research Database (Denmark)

    Kristensen, Michael; Albertsen, Janni; Rentsch, Maria

    2005-01-01

    Lactic acid accumulation is generally believed to be involved in muscle fatigue. However, one study reported that in rat soleus muscle (in vitro), with force depressed by high external K+ concentrations a subsequent incubation with lactic acid restores force and thereby protects against fatigue...... muscle. Three incubation solutions were used: 20 mm Na-lactate (which acidifies internal pH), 12 mm Na-lactate +8 mm lactic acid (which mimics the pH changes during muscle activity), and 20 mm lactic acid (which acidifies external pH more than internal pH). All three solutions improved force in K+-depressed...... development in repetitively stimulated muscle (Na-lactate had a negative effect). It is concluded that although lactate/lactic acid incubation regains force in K+-depressed resting muscle, a similar incubation has no or a negative effect on force development in active muscle. It is suggested...

  12. Supplemental epilactose prevents metabolic disorders through uncoupling protein-1 induction in the skeletal muscle of mice fed high-fat diets.

    Science.gov (United States)

    Murakami, Yuki; Ojima-Kato, Teruyo; Saburi, Wataru; Mori, Haruhide; Matsui, Hirokazu; Tanabe, Soichi; Suzuki, Takuya

    2015-12-14

    Obesity is one of the major health problems throughout the world. The present study investigated the preventive effect of epilactose--a rare non-digestible disaccharide--on obesity and metabolic disorders in mice fed high-fat (HF) diets. Feeding with HF diets increased body weight gain, fat pad weight and adipocyte size in mice (Pmuscle (P=0·04) and to 1·3-fold in the brown adipose tissue (P=0·02) in mice fed HF diets. Feeding HF diets induced pro-inflammatory macrophage infiltration into white adipose tissue, as indicated by the increased expression of monocyte chemotactic protein-1, TNF-α and F4/80, and these increases were attenuated by supplemental epilactose. In differentiated myogenic-like C2C12 cells, propionic acid, but not acetic or n-butyric acids, directly enhanced UCP-1 expression by approximately 2-fold (Pskeletal muscle and brown adipose tissue can enhance whole-body energy expenditure, leading to effective prevention of obesity and metabolic disorders in mice fed HF diets. It is suggested that propionic acid--a bacterial metabolite--acts as a mediator to induce UCP-1 expression in skeletal muscles.

  13. Functional classification of skeletal muscle networks. II. Applications to pathophysiology.

    Science.gov (United States)

    Wang, Yu; Winters, Jack; Subramaniam, Shankar

    2012-12-15

    In our preceding companion paper (Wang Y, Winters J, Subramaniam S. J Appl Physiol. doi: 10.1152/japplphysiol.01514.2011), we used extensive expression profile data on normal human subjects, in combination with legacy knowledge to classify skeletal muscle function into four models, namely excitation-activation, mechanical, metabolic, and signaling-production model families. In this paper, we demonstrate how this classification can be applied to study two well-characterized myopathies: amyotrophic lateral sclerosis (ALS) and Duchenne muscular dystrophy (DMD). Using skeletal muscle profile data from ALS and DMD patients compared with that from normal subjects, normal young in the case of DMD, we delineate molecular mechanisms that are causative and consequential to skeletal muscle dysfunction. In ALS, our analysis establishes the metabolic role and specifically identifies the mechanisms of calcium dysregulation and defects in mitochondrial transport of materials as important for muscle dysfunction. In DMD, we illustrate how impaired mechanical function is strongly coordinated with other three functional networks, resulting in transformation of the skeletal muscle into hybrid forms as a compensatory mechanism. Our functional models also provide, in exquisite detail, the mechanistic role of myriad proteins in these four families in normal and disease function.

  14. Needle muscle biopsy: technique validation and histological and histochemical methods for evaluating canine skeletal muscles

    Directory of Open Access Journals (Sweden)

    Sérgio de Almeida Braga

    2017-05-01

    Full Text Available This study evaluated the needle muscle biopsy technique using a 6G Bergström percutaneous needle combined with histological and histochemical methods to analyze the skeletal muscle of dogs. There are few studies about canine skeletal muscles and a lack of reports in the literature about tissue collection and analysis for canine species. Evaluation of 32 German Shepherd samples collected from the gluteus medius, at a depth of 3 cm, was performed. The choice of gluteus medius and the 3-cm depth provided good quantity fragments with sufficient sizes (3–5 mm, which permitted optimal visualization of muscle fibers. Myosin ATPase, at pH 9.4, 4.6, and 4.3, and SDH reactions revealed that all muscle samples analyzed had fibers in the classic mosaic arrangement, enabling counting and typification. The mean percentages of fibers were 29.95% for type I and 70.05% for type II. On the basis of these results, we concluded that the percutaneous needle biopsy technique for canine skeletal muscles is a safe and easy procedure that obtains fragments of proper sizes, thereby enabling the study of muscle fibers. Standardization of the muscle of choice and the depth of muscle sample collection significantly contributed to this success. This is an important method to evaluate muscle fiber types of dogs and diagnose important diseases affecting the skeletal muscles.

  15. Erythropoietin receptor in human skeletal muscle and the effects of acute and long-term injections with recombinant human erythropoietin on the skeletal muscle

    DEFF Research Database (Denmark)

    Lundby, Carsten; Hellsten, Ylva; Jensen, Mie B. F.

    2008-01-01

    The presence and potential physiological role of the erythropoietin receptor (Epo-R) were examined in human skeletal muscle. In this study we demonstrate that Epo-R is present in the endothelium, smooth muscle cells, and in fractions of the sarcolemma of skeletal muscle fibers. To study...... the potential effects of Epo in human skeletal muscle, two separate studies were conducted: one to study the acute effects of a single Epo injection on skeletal muscle gene expression and plasma hormones and another to study the effects of long-term (14 wk) Epo treatment on skeletal muscle structure. Subjects....... In conclusion, the Epo-R is present in the vasculature and myocytes in human skeletal muscle, suggesting a role in both cell types. In accordance, a single injection of Epo regulates myoglobin, MRF-4, and transferrin receptor mRNA levels. However, in contrast to our hypothesis, prolonged Epo administration had...

  16. Noncoding RNAs in the regulation of skeletal muscle biology in health and disease

    OpenAIRE

    Simionescu-Bankston, Adriana; Kumar, Ashok

    2016-01-01

    Skeletal muscle is composed of multinucleated myofibers that arise from the fusion of myoblasts during development. Skeletal muscle is essential for various body functions such as maintaining posture, locomotion, breathing, and metabolism. Skeletal muscle undergoes remarkable adaptations in response to environmental stimuli leading to atrophy or hypertrophy. Moreover, degeneration of skeletal muscle is a common feature in a number of muscular disorders including muscular dystrophy. Emerging e...

  17. Pixel-based meshfree modelling of skeletal muscles

    OpenAIRE

    Chen, Jiun-Shyan; Basava, Ramya Rao; Zhang, Yantao; Csapo, Robert; Malis, Vadim; Sinha, Usha; Hodgson, John; Sinha, Shantanu

    2015-01-01

    This paper introduces the meshfree Reproducing Kernel Particle Method (RKPM) for 3D image-based modeling of skeletal muscles. This approach allows for construction of simulation model based on pixel data obtained from medical images. The material properties and muscle fiber direction obtained from Diffusion Tensor Imaging (DTI) are input at each pixel point. The reproducing kernel (RK) approximation allows a representation of material heterogeneity with smooth transition. A ...

  18. Statin Therapy Alters Lipid Storage in Diabetic Skeletal Muscle.

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    Irena A Rebalka

    2016-07-01

    Full Text Available While statins significantly reduce cholesterol levels and thereby reduce the risk of cardiovascular disease, the development of myopathy with statin use is a significant clinical side-effect. Recent guidelines recommend increasing inclusion criteria for statin treatment in diabetic individuals; however, the impact of statins on skeletal muscle health in those with diabetes (who already suffer from impairments in muscle health is ill-defined. Here we investigate the effects of Fluvastatin treatment on muscle health in wild-type and streptozotocin (STZ-induced diabetic mice. Wild-type and STZ-diabetic mice received diet enriched with 600 mg/kg Fluvastatin or control chow for 24 days. Muscle morphology, intra and extracellular lipid levels, and lipid transporter content was investigated. Our findings indicate that short-term Fluvastatin administration induced a myopathy that was not exacerbated by the presence of STZ-induced diabetes. Fluvastatin significantly increased ectopic lipid deposition within the muscle of STZ-diabetic animals, findings that were not seen with diabetes or statin treatment alone. Consistent with this observation, only Fluvastatin-treated diabetic mice downregulated protein expression of lipid transporters FAT/CD36 and FABPpm in their skeletal muscle. No differences in FAT/CD36 or FABPpm mRNA content were observed. Altered lipid compartmentalization resultant of a downregulation in lipid transporter content in STZ-induced diabetic skeletal muscle was apparent in the current investigation. Given the association between ectopic lipid deposition in skeletal muscle and the development of insulin-resistance, our findings highlight the necessity for more thorough investigations into the impact of statins in humans with diabetes.

  19. Skeletal and cardiac muscle pericytes: Functions and therapeutic potential

    OpenAIRE

    Murray, IR; Baily, JE; Chen, WCW; Dar, A; Gonzalez, ZN; Jensen, AR; Petrigliano, FA; Deb, A; Henderson, NC

    2017-01-01

    Pericytes are periendothelial mesenchymal cells residing within the microvasculature. Skeletal muscle and cardiac pericytes are now recognized to fulfill an increasing number of functions in normal tissue homeostasis, including contributing to microvascular function by maintaining vessel stability and regulating capillary flow. In the setting of muscle injury, pericytes contribute to a regenerative microenvironment through release of trophic factors and by modulating local immune responses. I...

  20. A simplified immunohistochemical classification of skeletal muscle fibres in mouse

    OpenAIRE

    Kammoun, M.; Cassar-malek, I.; Meunier, B; Picard, B.

    2014-01-01

    The classification of muscle fibres is of particular interest for the study of the skeletal muscle properties in a wide range of scientific fields, especially animal phenotyping. It is therefore important to define a reliable method for classifying fibre types. The aim of this study was to establish a simplified method for the immunohistochemical classification of fibres in mouse. To carry it out, we first tested a combination of several anti myosin heavy chain (MyHC) antibodies in order to c...

  1. Structural, biochemical, cellular, and functional changes in skeletal muscle extracellular matrix with aging

    DEFF Research Database (Denmark)

    Kragstrup, Tue Wenzel; Kjaer, M; Mackey, A L

    2011-01-01

    The extracellular matrix (ECM) of skeletal muscle is critical for force transmission and for the passive elastic response of skeletal muscle. Structural, biochemical, cellular, and functional changes in skeletal muscle ECM contribute to the deterioration in muscle mechanical properties with aging...... of the changes in skeletal muscle ECM with aging may be preventable with resistance or weight training, but it is clear that more human studies are needed on the topic........ Structural changes include an increase in the collagen concentration, a change in the elastic fiber system, and an increase in fat infiltration of skeletal muscle. Biochemical changes include a decreased turnover of collagen with potential accumulation of enzymatically mediated collagen cross...

  2. Regenerated rat skeletal muscle after periodic contusions

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    V.B. Minamoto

    2001-11-01

    Full Text Available In the present study we evaluated the morphological aspect and changes in the area and incidence of muscle fiber types of long-term regenerated rat tibialis anterior (TA muscle previously submitted to periodic contusions. Animals received eight consecutive traumas: one trauma per week, for eight weeks, and were evaluated one (N = 8 and four (N = 9 months after the last contusion. Serial cross-sections were evaluated by toluidine blue staining, acid phosphatase and myosin ATPase reactions. The weight of injured muscles was decreased compared to the contralateral intact one (one month: 0.77 ± 0.15 vs 0.91 ± 0.09 g, P = 0.03; four months: 0.79 ± 0.14 vs 1.02 ± 0.07 g, P = 0.0007, respectively and showed abundant presence of split fibers and fibers with centralized nuclei, mainly in the deep portion. Damaged muscles presented a higher incidence of undifferentiated fibers when compared to the intact one (one month: 3.4 ± 2.1 vs 0.5 ± 0.3%, P = 0.006; four months: 2.3 ± 1.6 vs 0.3 ± 0.3%, P = 0.007, respectively. Injured TA evaluated one month later showed a decreased area of muscle fibers when compared to the intact one (P = 0.003. Thus, we conclude that: a muscle fibers were damaged mainly in the deep portion, probably because they were compressed against the tibia; b periodic contusions in the TA muscle did not change the percentage of type I and II muscle fibers; c periodically injured TA muscles took four months to reach a muscle fiber area similar to that of the intact muscle.

  3. Impact of placental insufficiency on fetal skeletal muscle growth.

    Science.gov (United States)

    Brown, Laura D; Hay, William W

    2016-11-05

    Intrauterine growth restriction (IUGR) caused by placental insufficiency is one of the most common and complex problems in perinatology, with no known cure. In pregnancies affected by placental insufficiency, a poorly functioning placenta restricts nutrient supply to the fetus and prevents normal fetal growth. Among other significant deficits in organ development, the IUGR fetus characteristically has less lean body and skeletal muscle mass than their appropriately-grown counterparts. Reduced skeletal muscle growth is not fully compensated after birth, as individuals who were born small for gestational age (SGA) from IUGR have persistent reductions in muscle mass and strength into adulthood. The consequences of restricted muscle growth and accelerated postnatal "catch-up" growth in the form of adiposity may contribute to the increased later life risk for visceral adiposity, peripheral insulin resistance, diabetes, and cardiovascular disease in individuals who were formerly IUGR. This review will discuss how an insufficient placenta results in impaired fetal skeletal muscle growth and how lifelong reductions in muscle mass might contribute to increased metabolic disease risk in this vulnerable population. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. The effects of ectopic UCP1 expression on gene expression in skeletal muscle [Mus Musculus

    NARCIS (Netherlands)

    Schothorst, van E.M.

    2015-01-01

    This SuperSeries is composed of the following subset Series: GSE45991: Amino acid deprivation due to overexpression of UCP1 in skeletal muscle: signalling via FGF-21 GSE45992: Transgenic overexpression of UCP1 in skeletal muscle in mice fed a HFD: signalling via FGF-21 Skeletal muscle FGF21

  5. Regulation of skeletal muscle glycogenolysis during exercise

    DEFF Research Database (Denmark)

    Hargreaves, M; Richter, Erik

    1988-01-01

    Muscle-glycogen breakdown during exercise is influenced by both local and systemic factors. Contractions per se increase glycogenolysis via a calci