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Sample records for dumbfounded affects myoblast

  1. The Intracellular Domain of Dumbfounded Affects Myoblast Fusion Efficiency and Interacts with Rolling Pebbles and Loner

    PubMed Central

    Bulchand, Sarada; Menon, Sree Devi; George, Simi Elizabeth; Chia, William

    2010-01-01

    Drosophila body wall muscles are multinucleated syncytia formed by successive fusions between a founder myoblast and several fusion competent myoblasts. Initial fusion gives rise to a bi/trinucleate precursor followed by more fusion cycles forming a mature muscle. This process requires the functions of various molecules including the transmembrane myoblast attractants Dumbfounded (Duf) and its paralogue Roughest (Rst), a scaffold protein Rolling pebbles (Rols) and a guanine nucleotide exchange factor Loner. Fusion completely fails in a duf, rst mutant, and is blocked at the bi/trinucleate stage in rols and loner single mutants. We analysed the transmembrane and intracellular domains of Duf, by mutating conserved putative signaling sites and serially deleting the intracellular domain. These were tested for their ability to translocate and interact with Rols and Loner and to rescue the fusion defect in duf, rst mutant embryos. Studying combinations of double mutants, further tested the function of Rols, Loner and other fusion molecules. Here we show that serial truncations of the Duf intracellular domain successively compromise its function to translocate and interact with Rols and Loner in addition to affecting myoblast fusion efficiency in embryos. Putative phosphorylation sites function additively while the extreme C terminus including a PDZ binding domain is dispensable for its function. We also show that fusion is completely blocked in a rols, loner double mutant and is compromised in other double mutants. These results suggest an additive function of the intracellular domain of Duf and an early function of Rols and Loner which is independent of Duf. PMID:20186342

  2. Myoblasts from affected and non-affected FSHD muscles exhibit morphological differentiation defects

    PubMed Central

    Barro, Marietta; Carnac, Gilles; Flavier, Sébastien; Mercier, Jacques; Vassetzky, Yegor; Laoudj-Chenivesse, Dalila

    2010-01-01

    Abstract Facioscapulohumeral dystrophy (FSHD) is a muscular hereditary disease with a prevalence of 1 in 20,000 caused by a partial deletion of a subtelomeric repeat array on chromosome 4q. However, very little is known about the pathogenesis as well as the molecular and biochemical changes linked to the progressive muscle degeneration observed in these patients. Several studies have investigated possible pathophysiological pathways in FSHD myoblasts and mature muscle cells but some of these reports were apparently in contradiction. The discrepancy between these studies may be explained by differences between the sources of myoblasts. Therefore, we decided to thoroughly analyze affected and unaffected muscles from patients with FSHD in terms of vulnerability to oxidative stress, differentiation capacity and morphological abnormalities. We have established a panel of primary myoblast cell cultures from patients affected with FSHD and matched healthy individuals. Our results show that primary myoblasts are more susceptible to an induced oxidative stress than control myoblasts. Moreover, we demonstrate that both types of FSHD primary myoblasts differentiate into multi-nucleated myotubes, which present morphological abnormalities. Whereas control myoblasts fuse to form branched myotubes with aligned nuclei, FSHD myoblasts fuse to form either thin and branched myotubes with aligned nuclei or large myotubes with random nuclei distribution. In conclusion, we postulate that these abnormalities could be responsible for muscle weakness in patients with FSHD and provide an important marker for FSHD myoblasts. PMID:18505476

  3. Substrate stiffness affects skeletal myoblast differentiation in vitro

    NASA Astrophysics Data System (ADS)

    Romanazzo, Sara; Forte, Giancarlo; Ebara, Mitsuhiro; Uto, Koichiro; Pagliari, Stefania; Aoyagi, Takao; Traversa, Enrico; Taniguchi, Akiyoshi

    2012-12-01

    To maximize the therapeutic efficacy of cardiac muscle constructs produced by stem cells and tissue engineering protocols, suitable scaffolds should be designed to recapitulate all the characteristics of native muscle and mimic the microenvironment encountered by cells in vivo. Moreover, so not to interfere with cardiac contractility, the scaffold should be deformable enough to withstand muscle contraction. Recently, it was suggested that the mechanical properties of scaffolds can interfere with stem/progenitor cell functions, and thus careful consideration is required when choosing polymers for targeted applications. In this study, cross-linked poly-ɛ-caprolactone membranes having similar chemical composition and controlled stiffness in a supra-physiological range were challenged with two sources of myoblasts to evaluate the suitability of substrates with different stiffness for cell adhesion, proliferation and differentiation. Furthermore, muscle-specific and non-related feeder layers were prepared on stiff surfaces to reveal the contribution of biological and mechanical cues to skeletal muscle progenitor differentiation. We demonstrated that substrate stiffness does affect myogenic differentiation, meaning that softer substrates can promote differentiation and that a muscle-specific feeder layer can improve the degree of maturation in skeletal muscle stem cells.

  4. Culture Conditions Affect Expression of DUX4 in FSHD Myoblasts.

    PubMed

    Pandey, Sachchida Nand; Khawaja, Hunain; Chen, Yi-Wen

    2015-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is believed to be caused by aberrant expression of double homeobox 4 (DUX4) due to epigenetic changes of the D4Z4 region at chromosome 4q35. Detecting DUX4 is challenging due to its stochastic expression pattern and low transcription level. In this study, we examined different cDNA synthesis strategies and the sensitivity for DUX4 detection. In addition, we investigated the effects of dexamethasone and knockout serum replacement (KOSR) on DUX4 expression in culture. Our data showed that DUX4 was consistently detected in cDNA samples synthesized using Superscript III. The sensitivity of DUX4 detection was higher in the samples synthesized using oligo(dT) primers compared to random hexamers. Adding dexamethasone to the culture media significantly suppressed DUX4 expression in immortalized (1.3 fold, p < 0.01) and primary (4.7 fold, p < 0.01) FSHD myoblasts, respectively. Culture medium with KOSR increased DUX4 expression and the response is concentration dependent. The findings suggest that detection strategies and culture conditions should be carefully considered when studying DUX4 in cultured cells. PMID:26007167

  5. In ovo L-arginine supplementation stimulates myoblast differentiation but negatively affects muscle development of broiler chicken after hatching.

    PubMed

    Li, Y; Wang, Y; Willems, E; Willemsen, H; Franssens, L; Buyse, J; Decuypere, E; Everaert, N

    2016-02-01

    In this study, we tested the hypothesis that in ovo feeding (IOF) of L-arginine (L-Arg) enhances nitric oxide (NO) production, stimulates the process of myogenesis, and regulates post-hatching muscle growth. Different doses of L-Arg were injected into the amnion of chicken embryos at embryonic day (ED) 16. After hatching, the body weight of individual male chickens was recorded weekly for 3 weeks. During in vitro experiments, myoblasts of the pectoralis major (PM) were extracted at ED16 and were incubated in medium containing 0.01 mm L-Arg, 0.05 mm L-Arg, and (or) 0.05 mm L-nitro-arginine-methyl-ester (L-NAME), an inhibitor of nitric oxide synthase (NOS). When 25 mg/kg L-Arg/initial egg weight was injected, no difference was observed in body weight at hatch, but a significant decrease was found during the following 3 weeks compared to that of the non-injected and saline-injected control, and this also affected the growth of muscle mass. L-NAME inhibited gene expression of myogenic differentiation antigen (MyoD), myogenin, NOS, and follistatin, decreased the cell viability, and increased myostatin (MSTN) gene expression. 0.05 mm L-Arg stimulated myogenin gene expression but also depressed muscle cell viability. L-NAME blocked the effect of 0.05 mm L-Arg on myogenin mRNA levels when co-incubated with 0.05 mm L-Arg. L-Arg treatments had no significant influence on NOS mRNA gene expression, but had inhibiting effect on follistatin gene expression, while L-NAME treatments had effects on both. These results suggested that L-Arg stimulated myoblast differentiation, but the limited number of myoblasts would form less myotubes and then less myofibers, while the latter limited the growth of muscle mass. PMID:25846259

  6. Transfection of L6 myoblasts with adipocyte fatty acid-binding protein cDNA does not affect fatty acid uptake but disturbs lipid metabolism and fusion.

    PubMed Central

    Prinsen, C F; Veerkamp, J H

    1998-01-01

    We studied the involvement of fatty acid-binding protein (FABP) in growth, differentiation and fatty acid metabolism of muscle cells by lipofection of rat L6 myoblasts with rat heart (H) FABP cDNA or with rat adipocyte (A) FABP cDNA in a eukaryotic expression vector which contained a puromycin acetyltransferase cassette. Stable transfectants showed integration into the genome for all constructs and type-specific overexpression at the mRNA and protein level for the clones with H-FABP and A-FABP cDNA constructs. The rate of proliferation of myoblasts transfected with rat A-FABP cDNA was 2-fold higher compared with all other transfected cells. In addition, these myoblasts showed disturbed fusion and differentiation, as assessed by morphological examination and creatine kinase activity. Uptake rates of palmitate were equal for all clone types, in spite of different FABP content and composition. Palmitate oxidation over a 3 h period was similar in all clones from growth medium. After being cultured in differentiation medium, mock- and H-FABP-cDNA-transfected cells showed a lower fatty acid-oxidation rate, in contrast with A-FABP-cDNA-transfected clones. The ratio of [14C]palmitic acid incorporation into phosphatidylcholine and phosphatidylethanolamine of A-FABP-cDNA-transfected clones changed in the opposite direction in differentiation medium from that of mock- and H-FABP-cDNA-transfected clones. In conclusion, transfection of L6 myoblasts with A-FABP cDNA does not affect H-FABP content and fatty acid uptake, but changes fatty acid metabolism. The latter changes may be related to the observed fusion defect. PMID:9425108

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

    PubMed Central

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

    2016-01-01

    The testis of Drosophila resembles an individual testis tubule of mammals. Both are surrounded by a sheath of smooth muscles, which in Drosophila are multinuclear and originate from a pool of myoblasts that are set aside in the embryo and accumulate on the genital disc later in development. These muscle stem cells start to differentiate early during metamorphosis and give rise to all muscles of the inner male reproductive system. Shortly before the genital disc and the developing testes connect, multinuclear nascent myotubes appear on the anterior tips of the seminal vesicles. Here, we show that adhesion molecules are distinctly localized on the seminal vesicles; founder cell (FC)-like myoblasts express Dumbfounded (Duf) and Roughest (Rst), and fusion-competent myoblast (FCM)-like cells mainly express Sticks and stones (Sns). The smooth but multinuclear myotubes of the testes arose by myoblast fusion. RNAi-mediated attenuation of Sns or both Duf and Rst severely reduced the number of nuclei in the testes muscles. Duf and Rst probably act independently in this context. Despite reduced fusion in all of these RNAi-treated animals, myotubes migrated onto the testes, testes were shaped and coiled, muscle filaments were arranged as in the wild type and spermatogenesis proceeded normally. Hence, the testes muscles compensate for fusion defects so that the myofibres encircling the adult testes are indistinguishable from those of the wild type and male fertility is guaranteed. PMID:26657767

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

    PubMed

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

    2016-01-15

    The testis of Drosophila resembles an individual testis tubule of mammals. Both are surrounded by a sheath of smooth muscles, which in Drosophila are multinuclear and originate from a pool of myoblasts that are set aside in the embryo and accumulate on the genital disc later in development. These muscle stem cells start to differentiate early during metamorphosis and give rise to all muscles of the inner male reproductive system. Shortly before the genital disc and the developing testes connect, multinuclear nascent myotubes appear on the anterior tips of the seminal vesicles. Here, we show that adhesion molecules are distinctly localized on the seminal vesicles; founder cell (FC)-like myoblasts express Dumbfounded (Duf) and Roughest (Rst), and fusion-competent myoblast (FCM)-like cells mainly express Sticks and stones (Sns). The smooth but multinuclear myotubes of the testes arose by myoblast fusion. RNAi-mediated attenuation of Sns or both Duf and Rst severely reduced the number of nuclei in the testes muscles. Duf and Rst probably act independently in this context. Despite reduced fusion in all of these RNAi-treated animals, myotubes migrated onto the testes, testes were shaped and coiled, muscle filaments were arranged as in the wild type and spermatogenesis proceeded normally. Hence, the testes muscles compensate for fusion defects so that the myofibres encircling the adult testes are indistinguishable from those of the wild type and male fertility is guaranteed. PMID:26657767

  9. Hyperthermia Differently Affects Connexin43 Expression and Gap Junction Permeability in Skeletal Myoblasts and HeLa Cells

    PubMed Central

    Antanavičiūtė, Ieva; Mildažienė, Vida; Stankevičius, Edgaras; Herdegen, Thomas; Skeberdis, Vytenis Arvydas

    2014-01-01

    Stress kinases can be activated by hyperthermia and modify the expression level and properties of membranous and intercellular channels. We examined the role of c-Jun NH2-terminal kinase (JNK) in hyperthermia-induced changes of connexin43 (Cx43) expression and permeability of Cx43 gap junctions (GJs) in the rabbit skeletal myoblasts (SkMs) and Cx43-EGFP transfected HeLa cells. Hyperthermia (42°C for 6 h) enhanced the activity of JNK and its target, the transcription factor c-Jun, in both SkMs and HeLa cells. In SkMs, hyperthermia caused a 3.2-fold increase in the total Cx43 protein level and enhanced the efficacy of GJ intercellular communication (GJIC). In striking contrast, hyperthermia reduced the total amount of Cx43 protein, the number of Cx43 channels in GJ plaques, the density of hemichannels in the cell membranes, and the efficiency of GJIC in HeLa cells. Both in SkMs and HeLa cells, these changes could be prevented by XG-102, a JNK inhibitor. In HeLa cells, the changes in Cx43 expression and GJIC under hyperthermic conditions were accompanied by JNK-dependent disorganization of actin cytoskeleton stress fibers while in SkMs, the actin cytoskeleton remained intact. These findings provide an attractive model to identify the regulatory players within signalosomes, which determine the cell-dependent outcomes of hyperthermia. PMID:25143668

  10. Myoblast fusion in Drosophila

    SciTech Connect

    Haralalka, Shruti; Abmayr, Susan M.

    2010-11-01

    The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.

  11. G-protein coupled receptor BAI3 promotes myoblast fusion in vertebrates.

    PubMed

    Hamoud, Noumeira; Tran, Viviane; Croteau, Louis-Philippe; Kania, Artur; Côté, Jean-François

    2014-03-11

    Muscle fibers form as a result of myoblast fusion, yet the cell surface receptors regulating this process are unknown in vertebrates. In Drosophila, myoblast fusion involves the activation of the Rac pathway by the guanine nucleotide exchange factor Myoblast City and its scaffolding protein ELMO, downstream of cell-surface cell-adhesion receptors. We previously showed that the mammalian ortholog of Myoblast City, DOCK1, functions in an evolutionarily conserved manner to promote myoblast fusion in mice. In search for regulators of myoblast fusion, we identified the G-protein coupled receptor brain-specific angiogenesis inhibitor (BAI3) as a cell surface protein that interacts with ELMO. In cultured cells, BAI3 or ELMO1/2 loss of function severely impaired myoblast fusion without affecting differentiation and cannot be rescued by reexpression of BAI3 mutants deficient in ELMO binding. The related BAI protein family member, BAI1, is functionally distinct from BAI3, because it cannot rescue the myoblast fusion defects caused by the loss of BAI3 function. Finally, embryonic muscle precursor expression of a BAI3 mutant unable to bind ELMO was sufficient to block myoblast fusion in vivo. Collectively, our findings provide a role for BAI3 in the relay of extracellular fusion signals to their intracellular effectors, identifying it as an essential transmembrane protein for embryonic vertebrate myoblast fusion. PMID:24567399

  12. Skeletal myoblasts for cardiac repair

    PubMed Central

    Durrani, Shazia; Konoplyannikov, Mikhail; Ashraf, Muhammad; Haider, Khawaja Husnain

    2011-01-01

    Stem cells provide an alternative curative intervention for the infarcted heart by compensating for the cardiomyocyte loss subsequent to myocardial injury. The presence of resident stem and progenitor cell populations in the heart, and nuclear reprogramming of somatic cells with genetic induction of pluripotency markers are the emerging new developments in stem cell-based regenerative medicine. However, until safety and feasibility of these cells are established by extensive experimentation in in vitro and in vivo experimental models, skeletal muscle-derived myoblasts, and bone marrow cells remain the most well-studied donor cell types for myocardial regeneration and repair. This article provides a critical review of skeletal myoblasts as donor cells for transplantation in the light of published experimental and clinical data, and indepth discussion of the advantages and disadvantages of skeletal myoblast-based therapeutic intervention for augmentation of myocardial function in the infarcted heart. Furthermore, strategies to overcome the problems of arrhythmogenicity and failure of the transplanted skeletal myoblasts to integrate with the host cardiomyocytes are discussed. PMID:21082891

  13. Determining the mechanical properties of plectin in mouse myoblasts and keratinocytes

    PubMed Central

    Bonakdar, Navid; Schilling, Achim; Spörrer, Marina; Lennert, Pablo; Mainka, Astrid; Winter, Lilli; Walko, Gernot; Wiche, Gerhard; Fabry, Ben; Goldmann, Wolfgang H.

    2015-01-01

    Plectin is the prototype of an intermediate filament (IF)-based cytolinker protein. It affects cells mechanically by interlinking and anchoring cytoskeletal filaments and acts as scaffolding and docking platform for signaling proteins to control cytoskeleton dynamics. The most common disease caused by mutations in the human plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Therefore, we compared the biomechanical properties and the response to mechanical stress of murine plectin-deficient myoblasts and keratinocytes with wild-type cells. Using a cell stretching device, plectin-deficient myoblasts exhibited lower mechanical vulnerability upon external stress compared to wild-type cells, which we attributed to lower cellular pre-stress. Contrary to myoblasts, wild-type and plectin-deficient keratinocytes showed no significant differences. In magnetic tweezer measurements using fibronectin-coated paramagnetic beads, the stiffness of keratinocytes was higher than of myoblasts. Interestingly, cell stiffness, adhesion strength, and cytoskeletal dynamics were strikingly altered in plectin-deficient compared to wild-type myoblasts, whereas smaller differences were observed between plectin-deficient and wild-type keratinocytes, indicating that plectin might be more important for stabilizing cytoskeletal structures in myoblasts than in keratinocytes. Traction forces strongly correlated with the stiffness of plectin-deficient and wild-type myoblasts and keratinocytes. Contrary to that cell motility was comparable in plectin-deficient and wild-type myoblasts, but was significantly increased in plectin-deficient compared to wild-type keratinocytes. Thus, we postulate that the lack of plectin has divergent implications on biomechanical properties depending on the respective cell type. PMID:25447312

  14. Myoblasts and myoblast-conditioned medium attract the earliest spinal neurites from frog embryos.

    PubMed Central

    McCaig, C D

    1986-01-01

    A study was made of the capacity of newly segmented somites, unsegmented mesoderm and medium conditioned by each of these tissues to attract the growth of the earliest spinal neurites from the neural tube of Xenopus laevis in tissue culture. When presented with segmented somitic myoblasts or sheets of skin, spinal neurites grew selectively towards the somitic myoblasts. Neurites were not attracted specifically to somitic myoblasts from their own rostrocaudal level. A variable proportion of myoblasts from unsegmented caudal mesoderm differentiated and elongated in co-culture with neural tube and skin. These myoblasts also attracted neural outgrowths, but only if present in sufficient numbers. An agar slab containing medium conditioned by the presence of segmented myoblasts for 1 day attracted neurite outgrowths. A source of medium conditioned by the presence of undifferentiated, unsegmented myotomal mesoderm alone did not attract neurite outgrowths. Nerve growth factor (NGF) at a range of concentrations in the agar source (500-10,000 ng/ml) did not attract the earliest neurite outgrowths. It is concluded that the earliest skeletal myoblasts from Xenopus laevis embryos may attract neural outgrowths by releasing a soluble factor. Myoblasts may have to develop to the stage of somite segmentation before secretion of such an agent begins. The release of a myoblast-derived factor so early in development may assist directed nerve growth in vivo. Images Plate 1 Plate 2 PMID:3795063

  15. Myoblast fusion: lessons from flies and mice

    PubMed Central

    Abmayr, Susan M.; Pavlath, Grace K.

    2012-01-01

    The fusion of myoblasts into multinucleate syncytia plays a fundamental role in muscle function, as it supports the formation of extended sarcomeric arrays, or myofibrils, within a large volume of cytoplasm. Principles learned from the study of myoblast fusion not only enhance our understanding of myogenesis, but also contribute to our perspectives on membrane fusion and cell-cell fusion in a wide array of model organisms and experimental systems. Recent studies have advanced our views of the cell biological processes and crucial proteins that drive myoblast fusion. Here, we provide an overview of myoblast fusion in three model systems that have contributed much to our understanding of these events: the Drosophila embryo; developing and regenerating mouse muscle; and cultured rodent muscle cells. PMID:22274696

  16. Losartan enhances the success of myoblast transplantation.

    PubMed

    Fakhfakh, Raouia; Lamarre, Yann; Skuk, Daniel; Tremblay, Jacques P

    2012-01-01

    Duchenne muscular dystrophy is a recessive X-linked genetic disease caused by dystrophin gene mutations. Cell therapy can be a potential approach aiming to introduce a functional dystrophin in the dystrophic patient myofibers. However, this strategy produced so far limited results. Transforming growth factor-β (TGF-β) is a negative regulator of skeletal muscle development and is responsible for limiting myogenic regeneration. The combination of TGF-β signaling inhibition with myoblast transplantation can be an effective therapeutic approach in dystrophin-deficient patients. Our aim was to verify whether the success of human myoblast transplantation in immunodeficient dystrophic mice is enhanced with losartan, a molecule that downregulates TGF-β expression. In vitro, blocking TGF-β activity with losartan increased proliferation and fusion and decreased apoptosis in human myoblasts. In vivo, human myoblasts were transplanted in mice treated with oral losartan. Immunodetection of human dystrophin in tibialis anterior cross sections 1 month posttransplantation revealed more human dystrophin-positive myofibers in these mice than in nontreated dystrophic mice. Thus, blocking the TGF-β signal with losartan treatment improved the success of myoblast transplantation probably by increasing myoblast proliferation and fusion, decreasing macrophage activation, and changing the expression of myogenic regulator factors. PMID:21535912

  17. Monoamine oxidase inhibition prevents mitochondrial dysfunction and apoptosis in myoblasts from patients with collagen VI myopathies

    PubMed Central

    Sorato, E.; Menazza, S.; Zulian, A.; Sabatelli, P.; Gualandi, F.; Merlini, L.; Bonaldo, P.; Canton, M.; Bernardi, P.; Di Lisa, F.

    2014-01-01

    Although mitochondrial dysfunction and oxidative stress have been proposed to play a crucial role in several types of muscular dystrophy (MD), whether a causal link between these two alterations exists remains an open question. We have documented that mitochondrial dysfunction through opening of the permeability transition pore plays a key role in myoblasts from patients as well as in mouse models of MD, and that oxidative stress caused by monoamine oxidases (MAO) is involved in myofiber damage. In the present study we have tested whether MAO-dependent oxidative stress is a causal determinant of mitochondrial dysfunction and apoptosis in myoblasts from patients affected by collagen VI myopathies. We find that upon incubation with hydrogen peroxide or the MAO substrate tyramine myoblasts from patients upregulate MAO-B expression and display a significant rise in reactive oxygen species (ROS) levels, with concomitant mitochondrial depolarization. MAO inhibition by pargyline significantly reduced both ROS accumulation and mitochondrial dysfunction, and normalized the increased incidence of apoptosis in myoblasts from patients. Thus, MAO-dependent oxidative stress is causally related to mitochondrial dysfunction and cell death in myoblasts from patients affected by collagen VI myopathies, and inhibition of MAO should be explored as a potential treatment for these diseases. PMID:25017965

  18. Overexpression of calpastatin inhibits L8 myoblast fusion

    SciTech Connect

    Barnoy, Sivia; E-mail: sivia@post.tau.ac.il; Maki, Masatoshi; Kosower, Nechama S.

    2005-07-08

    The formation of skeletal muscle fibers involves cessation of myoblast division, myoblast alignment, and fusion to multinucleated myofibers. Calpain is one of the factors shown to be involved in myoblast fusion. Using L8 rat myoblasts, we found that calpain levels did not change significantly during myoblast differentiation, whereas calpastatin diminished prior to myoblast fusion and reappeared after fusion. The transient diminution in calpastatin allows the Ca{sup 2+}-promoted activation of calpain and calpain-induced membrane proteolysis, which is required for myoblast fusion. Here we show that calpastatin overexpression in L8 myoblasts does not inhibit cell proliferation and alignment, but prevents myoblast fusion and fusion-associated protein degradation. In addition, calpastatin appears to modulate myogenic gene expression, as indicated by the lack of myogenin (a transcription factor expressed in differentiating myoblasts) in myoblasts overexpressing calpastatin. These results suggest that, in addition to the role in membrane disorganization in the fusing myoblasts, the calpain-calpastatin system may also modulate the levels of factors required for myoblast differentiation.

  19. Isolation and Characterization of Human Fetal Myoblasts

    PubMed Central

    Lapan, Ariya D.; Gussoni, Emanuela

    2011-01-01

    Dissociated human fetal skeletal muscle contains myogenic cells, as well as non-myogenic cells such as adipocytes, fibroblasts, and lymphocytes. It is therefore important to determine an efficient and reliable isolation method to obtain a purer population of myoblasts. Toward this end, fluorescence-activated cell sorting in conjunction with robust myogenic cell surface markers can be utilized to enrich for myoblasts in dissociated muscle. In this chapter, we describe a method to significantly enrich for myoblasts using melanoma cell adhesion molecule (MCAM), which we have determined to be an excellent marker of human fetal myoblasts. The myoblasts resulting from this isolation method can then be expanded in vitro and still retain significant myogenic activity as shown by an in vitro fusion assay. The ability to isolate a highly myogenic population from dissociated muscle facilitates the in vitro study of skeletal muscle development and muscle diseases. Furthermore, robust expansion of these cells will lead to new insights in the development of cell-based therapies for human muscle disorders. PMID:22130828

  20. Utilization of myoblasts from transgenic mice to evaluate the efficacy of myoblast transplantation.

    PubMed

    Kinoshita, I; Huard, J; Tremblay, J P

    1994-09-01

    A possible treatment for Duchenne muscular dystrophy is the injection of normal myoblasts into dystrophic muscles to induce the formation of new, healthy, and dystrophin-positive muscle fibers. To develop this therapy, it is important to identify the muscle fibers formed by the injected myoblasts in the host muscles. In this study, we used myoblasts from transgenic mice which have a gene expressing beta-galactosidase under the control of the promoter of quail fast skeletal muscle troponin I. This transgene is expressed in myotubes and muscle fibers, but not in myoblasts. Twenty-eight days after myoblast transplantation in nude and in mdx mice, muscle fibers containing of beta-galactosidase were identified by x-gal staining. In mdx mice, most of the beta-galactosidase-positive muscle fibers resulting from the myoblast transplantation were also dystrophin positive. This technique could make it possible to follow the success of myoblast transplantation even in mice that are not depleted of dystrophin. PMID:8065399

  1. Correction of the FSHD myoblast differentiation defect by fusion with healthy myoblasts.

    PubMed

    Dib, Carla; Bou Saada, Yara; Dmitriev, Petr; Richon, Catherine; Dessen, Philippe; Laoudj-Chenivesse, Dalila; Carnac, Gilles; Lipinski, Marc; Vassetzky, Yegor S

    2016-01-01

    Facioscapulohumeral dystrophy (FSHD) is a neuromuscular disease with a prevalence that could reach 1 in 8,000 characterized by progressive asymmetric muscle weakness. Myoblasts isolated from FSHD muscles exhibit morphological differentiation defects and show a distinct transcription profile. These abnormalities may be linked to the muscle weakness in FSHD patients. We have tested whether fusion of FSHD myoblasts with primary myoblasts isolated from healthy individuals could correct the differentiation defects. Our results show that the number of hybrid myotubes with normal phenotype increased with the percentage of normal myoblasts initially cultured. We demonstrated that a minimum of 50% of normal nuclei is required for a phenotypic correction of the FSHD phenotype. Moreover, transcriptomic profiles of phenotypically corrected hybrid myotubes showed that the expression of deregulated genes in FSHD myotubes became almost normal. The number of deregulated pathways also decreased from 39 in FSHD myotubes to one in hybrid myotubes formed with 40% FSHD and 60% normal myoblasts. We thus propose that while phenotypical and functional correction of FSHD is feasible, it requires more than 50% of normal myoblasts, it creates limitations for cell therapy in the FSHD context. PMID:26218298

  2. miR-411 is up-regulated in FSHD myoblasts and suppresses myogenic factors

    PubMed Central

    2013-01-01

    Background Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscle disorder, which is linked to the contraction of the D4Z4 array at chromosome 4q35. Recent studies suggest that this shortening of the D4Z4 array leads to aberrant expression of double homeobox protein 4 (DUX4) and causes FSHD. In addition, misregulation of microRNAs (miRNAs) has been reported in muscular dystrophies including FSHD. In this study, we identified a miRNA that is differentially expressed in FSHD myoblasts and investigated its function. Methods To identify misregulated miRNAs and their potential targets in FSHD myoblasts, we performed expression profiling of both miRNA and mRNA using TaqMan Human MicroRNA Arrays and Affymetrix Human Genome U133A plus 2.0 microarrays, respectively. In addition, we over-expressed miR-411 in C2C12 cells to determine the effect of miR-411 on myogenic markers. Results Using miRNA and mRNA expression profiling, we identified 8 miRNAs and 1,502 transcripts that were differentially expressed in FSHD myoblasts during cell proliferation. One of the 8 differentially expressed miRNAs, miR-411, was validated by quantitative RT-PCR in both primary (2.1 fold, p<0.01) and immortalized (2.7 fold, p<0.01) myoblasts. In situ hybridization showed cytoplasmic localization of miR-411 in FSHD myoblasts. By analyzing both miRNA and mRNA data using Partek Genomics Suite, we identified 4 mRNAs potentially regulated by miR-411 including YY1 associated factor 2 (YAF2). The down-regulation of YAF2 in immortalized myoblasts was validated by immunoblotting (−3.7 fold, p<0.01). C2C12 cells were transfected with miR-411 to determine whether miR-411 affects YAF2 expression in myoblasts. The results showed that over-expression of miR-411 reduced YAF2 mRNA expression. In addition, expression of myogenic markers including Myod, myogenin, and myosin heavy chain 1 (Myh1) were suppressed by miR-411. Conclusions The study demonstrated that miR-411 was differentially

  3. The effect of palmitate supplementation on gene expression profile in proliferating myoblasts.

    PubMed

    Grabiec, K; Majewska, A; Wicik, Z; Milewska, M; Błaszczyk, M; Grzelkowska-Kowalczyk, K

    2016-06-01

    High-fat diet, exposure to saturated fatty acids, or the presence of adipocytes in myoblast microenvironment affects skeletal muscle growth and function. The aim of the present study was to investigate the effect of palmitate supplementation on transcriptomic profile of mouse C2C12 myoblasts. Global gene expression was evaluated using whole mouse genome oligonucleotide microarrays, and the results were validated through qPCR. A total of 4047 genes were identified as differentially expressed, including 3492 downregulated and 555 upregulated genes, during a 48-h exposure to palmitate (0.1 mmol/l). Functional classification showed the involvement of these genes in several processes which regulate cell growth. In conclusion, the addition of palmitate modifies the expression of genes associated with (1) myoblast responsiveness to hormones and growth factors, (2) cytokine and growth factor expression, and (3) regulation of cell-cell and cell-matrix communication. Such alterations can affect myoblast growth and differentiation; however, further studies in this field are required. PMID:27114085

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

    PubMed Central

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

    2016-01-01

    Objectives Most cell culture studies have been performed at atmospheric oxygen tension of 21%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G0 arrested myoblasts in 21% O2 and in 1% O2 in order to see how oxygen tension affects activation and proliferation of human myoblasts. Materials and Methods Human myoblasts were isolated from skeletal muscle tissue and G0 arrested in vitro followed by reactivation at 21% O2 and 1% O2. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot. Results and Conclusions We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β-catenin indicated that notch signaling may be induced in 21% O2, while the canonical Wnt signaling may be induced in 1% O2 during activation and proliferation of myoblasts. PMID:27442119

  5. Network formation through active migration of human vascular endothelial cells in a multilayered skeletal myoblast sheet.

    PubMed

    Nagamori, Eiji; Ngo, Trung Xuan; Takezawa, Yasunori; Saito, Atsuhiro; Sawa, Yoshiki; Shimizu, Tatsuya; Okano, Teruo; Taya, Masahito; Kino-oka, Masahiro

    2013-01-01

    Autologous transplantation of myoblast sheet has attracted attention as a new technique for curing myocardial infarction. Myoblast sheet has the ability to secret cytokines that improve heart function via the facilitation of angiogenesis on affected part. To mimic the in vivo angiogenesis in the myoblast sheet after transplantation, a five-layered cell sheet of human skeletal muscle myoblasts (HSMMs) was overlaid on human umbilical vein endothelial cells (HUVECs) which enables evaluation of dynamic HUVEC behavior. HUVECs existing initially at the bottom of the sheet changed to be a stretched shape and migrated upward compared with the surrounding HSMMs in the sheet. Prolonged incubation resulted in network formation of HUVECs in the middle of the sheet, although non-networked HUVECs continued to migrate to the top of the sheet, which meant the spatial habitation of HUVECs in the cell sheet. Image processing was performed to determine the variation in the extent of network formation at different HUVEC densities. It was found that the extent of formed network depended on the frequency of encounters among HUVECs in the middle of the sheet. The present system, which can evaluate network formation, is considered to be a promising in vitro angiogenesis model. PMID:23117213

  6. Creatine kinase B is necessary to limit myoblast fusion during myogenesis

    PubMed Central

    Simionescu-Bankston, Adriana; Pichavant, Christophe; Canner, James P.; Apponi, Luciano H.; Wang, Yanru; Steeds, Craig; Olthoff, John T.; Belanto, Joseph J.; Ervasti, James M.

    2015-01-01

    Myoblast fusion is critical for proper muscle growth and regeneration. During myoblast fusion, the localization of some molecules is spatially restricted; however, the exact reason for such localization is unknown. Creatine kinase B (CKB), which replenishes local ATP pools, localizes near the ends of cultured primary mouse myotubes. To gain insights into the function of CKB, we performed a yeast two-hybrid screen to identify CKB-interacting proteins. We identified molecules with a broad diversity of roles, including actin polymerization, intracellular protein trafficking, and alternative splicing, as well as sarcomeric components. In-depth studies of α-skeletal actin and α-cardiac actin, two predominant muscle actin isoforms, demonstrated their biochemical interaction and partial colocalization with CKB near the ends of myotubes in vitro. In contrast to other cell types, specific knockdown of CKB did not grossly affect actin polymerization in myotubes, suggesting other muscle-specific roles for CKB. Interestingly, knockdown of CKB resulted in significantly increased myoblast fusion and myotube size in vitro, whereas knockdown of creatine kinase M had no effect on these myogenic parameters. Our results suggest that localized CKB plays a key role in myotube formation by limiting myoblast fusion during myogenesis. PMID:25810257

  7. Cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser.

    PubMed

    Alexsandra da Silva Neto Trajano, Larissa; da Silva, Camila Luna; de Carvalho, Simone Nunes; Cortez, Erika; Mencalha, André Luiz; de Souza da Fonseca, Adenilson; Stumbo, Ana Carolina

    2016-07-01

    Low-level infrared laser is considered safe and effective for treatment of muscle injuries. However, the mechanism involved on beneficial effects of laser therapy are not understood. The aim was to evaluate cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser at therapeutic fluences. C2C12 myoblast cultures at different (2 and 10 %) fetal bovine serum (FBS) concentrations were exposed to low-level infrared laser (808 nm, 100 mW) at different fluences (10, 35, and 70 J/cm(2)) and evaluated after 24, 48, and 72 h. Cell viability was evaluated by WST-1 assay; reactive oxygen species (ROS), apoptosis, and necrosis were evaluated by flow cytometry. Cell viability was decreased atthe lowest FBS concentration. Laser exposure increased the cell viability in myoblast cultures at 2 % FBS after 48 and 72 h, but no significant increase in ROS was observed. Apoptosis was decreased at the higher fluence and necrosis was increased at lower fluence in myoblast cultures after 24 h of laser exposure at 2 % FBS. No laser-induced alterations were obtained at 10 % FBS. Results show that level of reactive oxygen species is not altered, at least to those evaluated in this study, but low-level infrared laser exposure affects cell viability, apoptosis, and necrosis in myoblast cultures depending on laser fluence and physiologic conditions of cells. PMID:26886589

  8. Systemic delivery of recombinant proteins by genetically modified myoblasts

    SciTech Connect

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

    1991-12-06

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

  9. Heat shock attenuates VEGF expression in three-dimensional myoblast sheets deteriorating therapeutic efficacy in heart failure

    PubMed Central

    Augustin, Mona; Mahar, Muhammad Ali Asim; Lakkisto, Päivi; Tikkanen, Ilkka; Vento, Antti; Pätilä, Tommi; Harjula, Ari

    2011-01-01

    Summary Background Myoblast sheet transplantation is a promising novel treatment for ischemic heart failure. The aim of this study was to test the hypothesis that heat shock (HS) pre-treatment affects the angiogenic properties of myoblast sheets in vivo and in vitro. Material/Methods We studied HS preconditioning of L6 myoblast sheets in relation to their apoptosis, proliferation, and vascular endothelial growth factor (VEGF)-associated responses under normoxia and under hypoxia in vitro. In vivo evaluation of their therapeutic effect was performed with 60 male Wistar rats divided into 3 groups (20 each): sole left anterior descending (LAD) ligation (control); LAD ligation and non-conditioned sheet transplantation (L6 No-Shock); and LAD ligation and L6-heat shock conditioned sheet transplantation (L6 Heat-Shock). Left ventricular function was evaluated by echocardiography after 3, 10, and 28 days. Results Expression of HSP70/72 was strongly induced 24 hours after HS, and thereafter it decreased notably during 72 hours in hypoxia. Under normal growth conditions, HSP70/72 expression remained stable. HS delayed apoptosis-associated caspase-3 expression during 24-hour hypoxia compared to non-treated controls. However, VEGF expression reduced significantly in the heat shock pretreated sheets. Ejection fraction of the L6-myoblast HS pre-treatment group (L6 Heat-Shock) decreased gradually during follow-up, in the same pattern as the controls. However, these functional parameters improved in the L6-myoblast normal sheet group (L6 No-Shock) at the tenth day and remained significantly better. Conclusions HS protects myoblast sheets from hypoxia-associated apoptosis in vitro, but reduces VEGF expression of the sheet, leading to lower therapeutic effect in heart failure. PMID:22129892

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

    PubMed

    Jeong, Jaemin; Conboy, Irina M

    2011-10-14

    Cell membrane consists of various lipids such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Among them, PS is a molecular marker of apoptosis, because it is located to the inner leaflet of plasma membrane generally but it is moved to the outer leaflet during programmed cell death. The process of apoptosis has been implicated in the fusion of muscle progenitor cells, myoblasts, into myotubes. However, it remained unclear whether PS regulates muscle cell differentiation directly. In this paper, localization of PS to the outer leaflet of plasma membrane in proliferating primary myoblasts and during fusion of these myoblasts into myotubes is validated using Annexin V. Moreover, we show the presence of PS clusters at the cell-cell contact points, suggesting the importance of membrane ruffling and PS exposure for the myogenic cell fusion. Confirming this conclusion, experimentally constructed PS, but not PC liposomes dramatically enhance the formation of myotubes from myoblasts, thus demonstrating a direct positive effect of PS on the muscle cell fusion. In contrast, myoblasts exposed to PC liposomes produce long myotubes with low numbers of myonuclei. Moreover, pharmacological masking of PS on the myoblast surface inhibits fusion of these cells into myotubes in a dose-dependent manner. PMID:21910971

  11. Identification of singles bar as a direct transcriptional target of Drosophila Myocyte enhancer factor-2 and a regulator of adult myoblast fusion

    PubMed Central

    Brunetti, Tonya M.; Fremin, Brayon J.; Cripps, Richard M.

    2015-01-01

    In Drosophila, myoblast fusion is a conserved process in which founder cells (FCs) and fusion competent myoblasts (FCMs) fuse to form a syncytial muscle fiber. Mutants for the myogenic regulator Myocyte enhancer factor-2 (MEF2) show a failure of myoblast fusion, indicating that MEF2 regulates the fusion process. Indeed, chromatin immunoprecipitation studies show that several genes involved in myoblast fusion are bound by MEF2 during embryogenesis. Of these, the MARVEL domain gene singles bar (sing), is down-regulated in MEF2 knockdown pupae, and has five consensus MEF2 binding sites within a 9000-bp region. To determine if MEF2 is an essential and direct regulator of sing during pupal muscle development, we identified a 315-bp myoblast enhancer of sing. This enhancer was active during myoblast fusion, and mutation of two MEF2 sites significantly decreased enhancer activity. We show that lack of sing expression resulted in adult lethality and muscle loss, due to a failure of fusion during the pupal stage. Additionally, we sought to determine if sing was required in either FCs or FCMs to support fusion. Interestingly, knockdown of sing in either population did not significantly affect fusion, however, knockdown in both FCs and FCMs resulted in muscles with significantly reduced nuclei numbers, provisionally indicating that sing function is required in either cell type, but not both. Finally, we found that MEF2 regulated sing expression at the embryonic stage through the same 315-bp enhancer, indicating that sing is a MEF2 target at both critical stages of myoblast fusion. Our studies define for the first time how MEF2 directly controls fusion at multiple stages of the life cycle, and provide further evidence that the mechanisms of fusion characterized in Drosophila embryos is also used in the formation of the more complex adult muscles. PMID:25797154

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

    SciTech Connect

    Jeong, Jaemin; Conboy, Irina M.

    2011-10-14

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

  13. Change in viability of C2C12 myoblasts under compression, shear and oxidative challenges.

    PubMed

    Hong, Ye; Yao, Yifei; Wong, Singwan; Bian, Liming; Mak, Arthur F T

    2016-05-24

    Skeletal and epidermal loadings can damage muscle cells and contribute to the development of deep tissue injury (DTI) - a severe kind of pressure ulcers affecting many people with disability. Important predisposing factors include the multiaxial stress and strain fields in the internal tissues, particularly the vulnerable muscles around bony prominences. A careful study of the mechanical damage thresholds for muscle cell death is critical not only to the understanding of the formation of DTI, but also to the design of various body support surfaces for prevention. In this paper, we measured the mechanical damage thresholds of C2C12 myoblasts under prescribed compressive strains (15% and 30%) and shear strains (from 0% to 100%), and studied how oxidative stress, as caused potentially by reperfusion or inflammation, may affect such damage thresholds. A flat plate was used to apply a uniform compressive strain and a radially increasing shear strain on disks of Gelatin-methacrylate (GelMA) hydrogel with myoblasts encapsulated within. The percentages of cell death were estimated with propidium iodide (PI) and calcein AM staining. Results suggested that cell death depended on both the level and duration of the applied strain. There seemed to be a non-linear coupling between compression and shear. Muscle cells often need to function biomechanically in challenging oxidative environments. To study how oxidative stress may affect the mechanical damage thresholds of myoblasts, cell viability under compressive and shear strains was also studied after the cells were pre-treated for different durations (1h and 20h) with different concentrations (0.1mM and 0.5mM) of hydrogen peroxide (H2O2). Oxidative stress can either compromise or enhance the cellular resistance to shear damage, depending on the level and duration of the oxidative exposure. PMID:27017299

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

    PubMed Central

    Du, Jingjing; Li, Qiang; Shen, Linyuan; Lei, Huaigang; Luo, Jia; Liu, Yihui; Zhang, Peiwen; Pu, Qiang; Zhang, Yi; Shuai, Surong; Li, Xuewei; Zhang, Shunhua; Zhu, Li

    2016-01-01

    MicroRNAs are a class of 18–22-nucleotide noncoding RNAs that posttranscriptionally regulate gene expression and have been shown to play an important role during myoblast differentiation. In this study, we found that the expression of miR-145a-5p was gradually increased during C2C12 myoblast differentiation, and miR-145a-5p inhibitors or mimics significantly suppressed or promoted the relative expression of specific myogenesis related marker genes. Moreover, overexpression or inhibition of miR-145a-5p enhanced or repressed the expression of some special genes involved in the endogenous Wnt signaling pathway during C2C12 myoblast differentiation, including Wnt5a, LRP5, Axin2, and β-catenin. These results indicated that miR-145a-5p might be considered as a new myogenic differentiation-associated microRNA that can promote C2C12 myoblast differentiation by enhancing genes related to myoblasts differentiation. PMID:27239472

  15. Stabilin-2 modulates the efficiency of myoblast fusion during myogenic differentiation and muscle regeneration

    PubMed Central

    Park, Seung-Yoon; Yun, Youngeun; Lim, Jung-Suk; Kim, Mi-Jin; Kim, Sang-Yeob; Kim, Jung-Eun; Kim, In-San

    2016-01-01

    Myoblast fusion is essential for the formation of skeletal muscle myofibres. Studies have shown that phosphatidylserine is necessary for myoblast fusion, but the underlying mechanism is not known. Here we show that the phosphatidylserine receptor stabilin-2 acts as a membrane protein for myoblast fusion during myogenic differentiation and muscle regeneration. Stabilin-2 expression is induced during myogenic differentiation, and is regulated by calcineurin/NFAT signalling in myoblasts. Forced expression of stabilin-2 in myoblasts is associated with increased myotube formation, whereas deficiency of stabilin-2 results in the formation of small, thin myotubes. Stab2-deficient mice have myofibres with small cross-sectional area and few myonuclei and impaired muscle regeneration after injury. Importantly, myoblasts lacking stabilin-2 have reduced phosphatidylserine-dependent fusion. Collectively, our results show that stabilin-2 contributes to phosphatidylserine-dependent myoblast fusion and provide new insights into the molecular mechanism by which phosphatidylserine mediates myoblast fusion during muscle growth and regeneration. PMID:26972991

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

    PubMed

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

    2016-08-01

    Unloading stress, such as bed rest, inhibits the regenerative potential of skeletal muscles; however, the underlying mechanisms remain largely unknown. FOXO1 expression, which induces the upregulated expression of the cell cycle inhibitors p57 and Gadd45α, is known to be increased in the skeletal muscle under unloading conditions. However, there is no report addressing FOXO1-induced inhibition of myoblast proliferation. Therefore, we induced muscle injury by cardiotoxin in transgenic mice overexpressing FOXO1 in the skeletal muscle (FOXO1-Tg mice) and observed regeneration delay in skeletal muscle mass and cross-sectional area in FOXO1-Tg mice. Increased p57 and Gadd45α mRNA levels, and decreased proliferation capacity were observed in C2C12 myoblasts expressing a tamoxifen-inducible active form of FOXO1. These results suggest that decreased proliferation capacity of myoblasts by FOXO1 disrupts skeletal muscle regeneration under FOXO1-increased conditions, such as unloading. PMID:27010781

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

    SciTech Connect

    Liu, Chenxi; Li, Wenrong; Zhang, Xuemei; Zhang, Ning; He, Sangang; Huang, Juncheng; Ge, Yubin; Liu, Mingjun

    2012-06-08

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

  18. Macrophage migration inhibitory factor in the regulation of myoblast proliferation and differentiation.

    PubMed

    Wen, Fengyun; Zheng, Jin; Yu, Jing; Gao, Mingju; Gao, Sumin; Zhou, Yingying; Liu, Jianyu; Yang, Zaiqing

    2016-07-01

    Obesity is documented to be a state of chronic mild inflammation associated with increased macrophage infiltration into adipose tissue and liver and skeletal muscle. As a pleiotropic inflammatory mediator, macrophage migration inhibitory factor (MIF) is associated with metabolic disease, so MIF may signal molecular links between adipocytes and myocytes. MIF expression was modified during myoblast differentiation, but the role of MIF during this process is unclear. C2C12 cells were transfected with MIF to investigate their role during differentiation. MIF expression attenuated C2C12 differentiation. It did not change proliferation, but downregulated cyclin D1 and CDK4, causing cell accumulation in the G1 phase. p21 protein was increased significantly and MyoD, MyoG, and p21 mRNA also increased significantly in the C2C12 cells treated with ISO-1, suggesting that inhibition of MIF promotes differentiation. MIF inhibits the myoblast differentiation by affecting the cell cycle progression, but does not affect proliferation. PMID:26927414

  19. Maduramicin Inhibits Proliferation and Induces Apoptosis in Myoblast Cells

    PubMed Central

    Chen, Xin; Gu, Ying; Singh, Karnika; Shang, Chaowei; Barzegar, Mansoureh; Jiang, Shanxiang; Huang, Shile

    2014-01-01

    Maduramicin, a polyether ionophore antibiotic derived from the bacterium Actinomadura yumaensis, is currently used as a feed additive against coccidiosis in poultry worldwide. It has been clinically observed that maduramicin can cause skeletal muscle and heart cell damage, resulting in skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. However, the mechanism of its toxic action in myoblasts is not well understood. Using mouse myoblasts (C2C12) and human rhabdomyosarcoma (RD and Rh30) cells as an experimental model for myoblasts, here we found that maduramicin inhibited cell proliferation and induced cell death in a concentration-dependent manner. Further studies revealed that maduramicin induced accumulation of the cells at G0/G1 phase of the cell cycle, and induced apoptosis in the cells. Concurrently, maduramicin downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and CDC25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in decreased phosphorylation of Rb. Maduramicin also induced expression of BAK, BAD, DR4, TRADD and TRAIL, leading to activation of caspases 8, 9 and 3 as well as cleavage of poly ADP ribose polymerase (PARP). Taken together, our results suggest that maduramicin executes its toxicity in myoblasts at least by inhibiting cell proliferation and inducing apoptotic cell death. PMID:25531367

  20. Maduramicin inhibits proliferation and induces apoptosis in myoblast cells.

    PubMed

    Chen, Xin; Gu, Ying; Singh, Karnika; Shang, Chaowei; Barzegar, Mansoureh; Jiang, Shanxiang; Huang, Shile

    2014-01-01

    Maduramicin, a polyether ionophore antibiotic derived from the bacterium Actinomadura yumaensis, is currently used as a feed additive against coccidiosis in poultry worldwide. It has been clinically observed that maduramicin can cause skeletal muscle and heart cell damage, resulting in skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. However, the mechanism of its toxic action in myoblasts is not well understood. Using mouse myoblasts (C2C12) and human rhabdomyosarcoma (RD and Rh30) cells as an experimental model for myoblasts, here we found that maduramicin inhibited cell proliferation and induced cell death in a concentration-dependent manner. Further studies revealed that maduramicin induced accumulation of the cells at G0/G1 phase of the cell cycle, and induced apoptosis in the cells. Concurrently, maduramicin downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and CDC25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in decreased phosphorylation of Rb. Maduramicin also induced expression of BAK, BAD, DR4, TRADD and TRAIL, leading to activation of caspases 8, 9 and 3 as well as cleavage of poly ADP ribose polymerase (PARP). Taken together, our results suggest that maduramicin executes its toxicity in myoblasts at least by inhibiting cell proliferation and inducing apoptotic cell death. PMID:25531367

  1. Combinations of Kinase Inhibitors Protecting Myoblasts against Hypoxia

    PubMed Central

    Kang, Yunyi; Tierney, Matthew; Ong, Edison; Zhang, Linda; Piermarocchi, Carlo; Sacco, Alessandra; Paternostro, Giovanni

    2015-01-01

    Cell-based therapies to treat skeletal muscle disease are limited by the poor survival of donor myoblasts, due in part to acute hypoxic stress. After confirming that the microenvironment of transplanted myoblasts is hypoxic, we screened a kinase inhibitor library in vitro and identified five kinase inhibitors that protected myoblasts from cell death or growth arrest in hypoxic conditions. A systematic, combinatorial study of these compounds further improved myoblast viability, showing both synergistic and additive effects. Pathway and target analysis revealed CDK5, CDK2, CDC2, WEE1, and GSK3β as the main target kinases. In particular, CDK5 was the center of the target kinase network. Using our recently developed statistical method based on elastic net regression we computationally validated the key role of CDK5 in cell protection against hypoxia. This method provided a list of potential kinase targets with a quantitative measure of their optimal amount of relative inhibition. A modified version of the method was also able to predict the effect of combinations using single-drug response data. This work is the first step towards a broadly applicable system-level strategy for the pharmacology of hypoxic damage. PMID:26042811

  2. Identification and functional characterization of TRPA1 in human myoblasts.

    PubMed

    Osterloh, Markus; Böhm, Mario; Kalbe, Benjamin; Osterloh, Sabrina; Hatt, Hanns

    2016-02-01

    The proper function of the skeletal muscle is essential for the survival of most animals. Thus, efficient and rapid repair of muscular damage following injury is crucial. In recent years, satellite cells have emerged as key players of muscle repair, capable of undergoing extensive proliferation after injury, fusing into myotubes and restoring muscle function. Furthermore, it has been shown that Ca(2+)/calmodulin-dependent generation of nitric oxide (NO) is an important regulator of muscle repair. Here, we demonstrate the functional expression of transient receptor potential, subfamily A1 (TRPA1) channel in human primary myoblasts. Stimulation of these cells with well-known TRPA1 ligands led to robust intracellular Ca(2+) rises which could be inhibited by specific TRPA1 antagonists. Moreover, we show that TRPA1 activation enhances important aspects of skeletal muscle repair such as cell migration and myoblast fusion in vitro. Interestingly, TRPA1 levels and inducible Ca(2+) transients decline with ongoing myoblast differentiation. We suggest that TRPA1 might serve as a physiological mediator for inflammatory signals and appears to have a functional role in promoting myoblast migration, fusion, and potentially also in activating satellite cells in humans. PMID:26328519

  3. Retardation of C2C12 myoblast cell proliferation by exposure to low-temperature atmospheric plasma.

    PubMed

    Nakai, Naoya; Fujita, Ryo; Kawano, Fuminori; Takahashi, Kazuo; Ohira, Takashi; Shibaguchi, Tsubasa; Nakata, Ken; Ohira, Yoshinobu

    2014-09-01

    As the first step in evaluating the possibility of low-temperature atmospheric plasma for clinical applications in the treatment of rhabdomyosarcoma (RMS), we determined the effects of plasma exposure on C2C12 myoblasts. The low-temperature atmospheric plasma was generated through an electrical discharge in argon gas. One minute of plasma exposure every 24 h inhibited the cell proliferation, whereas myoblast differentiation was not affected. Plasma exposure increased the phosphorylation of ERK and JNK at 30 min after the exposure, but the phosphorylation of both was decreased to less than control levels at 1 and 4 h after the exposure. Plasma exposure increased the percentage of cells in the G2/M phase at 8 h after the exposure. In conclusion, plasma exposure retarded the proliferation of C2C12 myoblasts by G2/M arrest. Therefore, plasma exposure can be a possible treatment for the anti-proliferative effects of malignant tumors, such as RMS, without affecting differentiated skeletal muscle cells. PMID:25034108

  4. Microfluidic analysis of extracellular matrix-bFGF crosstalk on primary human myoblast chemoproliferation, chemokinesis, and chemotaxis

    PubMed Central

    Ferreira, Meghaan M.; Dewi, Ruby E.; Heilshorn, Sarah C.

    2015-01-01

    Exposing myoblasts to basic fibroblast growth factor (bFGF), which is released after muscle injury, results in receptor phosphorylation, faster migration, and increased proliferation. These effects occur on time scales that extend across three orders of magnitude (100 – 103 minutes). Finite element modeling of Transwell assays, which are traditionally used to assess chemotaxis, revealed that the bFGF gradient formed across the membrane pore is short-lived and diminishes 45% within the first minute. Thus, to evaluate bFGF-induced migration over 102 minutes, we employed a microfluidic assay capable of producing a stable, linear concentration gradient to perform single-cell analyses of chemokinesis and chemotaxis. We hypothesized that the composition of the underlying extracellular matrix (ECM) may affect the behavioral response of myoblasts to soluble bFGF, as previous work with other cell types has suggested crosstalk between integrin and fibroblast growth factor (FGF) receptors. Consistent with this notion, we found that bFGF significantly reduced the doubling time of myoblasts cultured on laminin but not fibronectin or collagen. Laminin also promoted significantly faster migration speeds (13.4 μm/h) than either fibronectin (10.6 μm/h) or collagen (7.6 μm/h) without bFGF stimulation. Chemokinesis driven by bFGF further increased migration speed in a strictly additive manner, resulting in an average increase of 2.3 μm/h across all ECMs tested. We observed relatively mild chemoattraction (~ 67% of myoblast population) in response to bFGF gradients of 3.2 ng/mL/mm regardless of ECM identity. Thus, while ECM-bFGF crosstalk did impact chemoproliferation, it did not have a significant effect on chemokinesis or chemotaxis. These data suggest that the main physiological effect of bFGF on myoblast migration is chemokinesis and that changes in the surrounding ECM, resulting from aging and/or disease may impact muscle regeneration by altering myoblast migration and

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

    SciTech Connect

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

    2010-03-19

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

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

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

  7. Biomechanical strain vehicles for fibroblast-directed skeletal myoblast differentiation and myotube functionality in a novel coculture.

    PubMed

    Hicks, Michael R; Cao, Thanh V; Standley, Paul R

    2014-10-15

    Skeletal muscle functionality is governed by multiple stimuli, including cytokines and biomechanical strain. Fibroblasts embedded within muscle connective tissue respond to biomechanical strain by secreting cytokines that induce myoblast differentiation and, we hypothesize, regulate myotube function. A coculture was established to allow cross talk between fibroblasts in Bioflex wells and myoblasts on nondeformable coverslips situated above Bioflex wells. Cyclic short-duration strain (CSDS) modeling repetitive stress/injury, acyclic long-duration strain (ALDS) modeling manipulative therapy, and combined strain paradigms (CSDS + ALDS) were applied to fibroblasts. Nonstrained myoblasts in uniculture and coculture served as controls. After fibroblasts had induced myoblast differentiation, myotube contraction was assessed by perfusion of ACh (10(-11)-10(-3) M). CSDS-treated fibroblasts increased myotube contractile sensitivity vs. uniculture (P < 0.05). As contraction is dependent on ACh binding, expression and clustering of nicotinic ACh receptors (nAChRs) were measured. CSDS-treated fibroblasts increased nAChR expression (P < 0.05), which correlated with myotube contraction. ALDS-treated fibroblasts did not significantly affect contraction or nAChR expression. Agrin-treated myotubes were then used to design a computer algorithm to identify α-bungarotoxin-stained nAChR clusters. ALDS-treated fibroblasts increased nAChR clustering (P < 0.05), while CSDS-treated fibroblasts disrupted cluster formation. CSDS-treated fibroblasts produced nAChRs preferentially located in nonclustered regions (P < 0.05). Strain-activated fibroblasts mediate myotube differentiation with multiple functional phenotypes. Similar to muscle injury, CSDS-treated fibroblasts disrupted nAChR clusters and hypersensitized myotube contraction, while ALDS-treated fibroblasts aggregated nAChRs in large clusters, which may have important clinical implications. Cellular strategies aimed at improving muscle

  8. Impaired hypertrophy in myoblasts is improved with testosterone administration.

    PubMed

    Deane, Colleen S; Hughes, David C; Sculthorpe, Nicholas; Lewis, Mark P; Stewart, Claire E; Sharples, Adam P

    2013-11-01

    We investigated the ability of testosterone (T) to restore differentiation in multiple population doubled (PD) murine myoblasts, previously shown to have a reduced differentiation in monolayer and bioengineered skeletal muscle cultures vs. their parental controls (CON) (Sharples et al., 2011, 2012 [7,26]). Cells were exposed to low serum conditions in the presence or absence of T (100nM)±PI3K inhibitor (LY294002) for 72h and 7 days (early and late muscle differentiation respectively). Morphological analyses were performed to determine myotube number, diameter (μm) and myonuclear accretion as indices of differentiation and myotube hypertrophy. Changes in gene expression for myogenin, mTOR and myostatin were also performed. Myotube diameter in CON and PD cells increased from 17.32±2.56μm to 21.02±1.89μm and 14.58±2.66μm to 18.29±3.08μm (P≤0.05) respectively after 72h of T exposure. The increase was comparable in both PD (+25%) and CON cells (+21%) suggesting a similar intrinsic ability to respond to exogenous T administration. T treatment also significantly increased myonuclear accretion (% of myotubes expressing 5+ nuclei) in both cell types after 7 days exposure (P≤0.05). Addition of PI3K inhibitor (LY294002) in the presence of T attenuated these effects in myotube morphology (in both cell types) suggesting a role for the PI3K pathway in T stimulated hypertrophy. Finally, PD myoblasts showed reduced responsiveness to T stimulated mRNA expression of mTOR vs. CON cells and T also reduced myostatin expression in PD myoblasts only. The present study demonstrates testosterone administration improves hypertrophy in myoblasts that basally display impaired differentiation and hypertrophic capacity vs. their parental controls, the action of testosterone in this model was mediated by PI3K/Akt pathway. PMID:23714396

  9. Chronic Hyperinsulinemia Increases Myoblast Proliferation in Fetal Sheep Skeletal Muscle.

    PubMed

    Brown, Laura D; Wesolowski, Stephanie R; Kailey, Jenai; Bourque, Stephanie; Wilson, Averi; Andrews, Sasha E; Hay, William W; Rozance, Paul J

    2016-06-01

    Insulin is an important fetal growth factor. However, chronic experimental hyperinsulinemia in the fetus fails to accelerate linear and lean mass growth beyond normal rates. Mechanisms preventing accelerated lean mass accretion during hyperinsulinemia are unknown. To address potential mechanisms, late-gestation fetal sheep were infused with iv insulin and glucose to produce euglycemic hyperinsulinemia (INS) or saline for 7-9 days. Fetal substrate uptake and protein metabolic rates were measured. INS fetuses had 1.5-fold higher insulin concentrations (P < .0001) and equivalent glucose concentrations. INS fetuses had 20% more Pax7(+) nuclei in the biceps femoris, which indicates the potential for hyperinsulinemia to increase the number of myoblasts within late-gestation fetal skeletal muscle. Additionally, the percentage of Pax7(+) myoblasts that expressed Ki-67 was 1.3-fold higher and expression of myogenic regulatory factors was 50% lower in INS fetuses (MYF5 and MYOG [myogenin], P < .005), which indicates a shift toward myoblast proliferation over differentiation. There were no differences for fetal body, organ, or muscle weights, although INS placentas weighed 28% less (P < .05). Protein synthesis and accretion rates did not change in INS fetuses, nor did fiber muscle size. Essential amino acid concentrations were lower in the INS group (P < .05) except for tryptophan. Umbilical blood flow, net total amino acids, and O2 uptakes rates did not differ between groups. Arterial O2 content was 33% lower (P < .005) and norepinephrine was 100% higher in the INS fetuses (P < .01), all of which are factors that may counteract fetal protein accretion during hyperinsulinemia despite an increase in myoblast proliferation. PMID:27049667

  10. Vascular Endothelial Growth Factor Modulates Skeletal Myoblast Function

    PubMed Central

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

    2003-01-01

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

  11. Abnormal proliferation and spontaneous differentiation of myoblasts from a symptomatic female carrier of X-linked Emery-Dreifuss muscular dystrophy.

    PubMed

    Meinke, Peter; Schneiderat, Peter; Srsen, Vlastimil; Korfali, Nadia; Lê Thành, Phú; Cowan, Graeme J M; Cavanagh, David R; Wehnert, Manfred; Schirmer, Eric C; Walter, Maggie C

    2015-02-01

    Emery-Dreifuss muscular dystrophy (EDMD) is a neuromuscular disease characterized by early contractures, slowly progressive muscular weakness and life-threatening cardiac arrhythmia that can develop into cardiomyopathy. In X-linked EDMD (EDMD1), female carriers are usually unaffected. Here we present a clinical description and in vitro characterization of a mildly affected EDMD1 female carrying the heterozygous EMD mutation c.174_175delTT; p.Y59* that yields loss of protein. Muscle tissue sections and cultured patient myoblasts exhibited a mixed population of emerin-positive and -negative cells; thus uneven X-inactivation was excluded as causative. Patient blood cells were predominantly emerin-positive, but considerable nuclear lobulation was observed in non-granulocyte cells - a novel phenotype in EDMD. Both emerin-positive and emerin-negative myoblasts exhibited spontaneous differentiation in tissue culture, though emerin-negative myoblasts were more proliferative than emerin-positive cells. The preferential proliferation of emerin-negative myoblasts together with the high rate of spontaneous differentiation in both populations suggests that loss of functional satellite cells might be one underlying mechanism for disease pathology. This could also account for the slowly developing muscle phenotype. PMID:25454731

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

    PubMed

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

    2015-12-01

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

  13. CD36 is required for myoblast fusion during myogenic differentiation

    SciTech Connect

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

    2012-11-02

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

  14. Nitric oxide donors, sodium nitroprusside and S-nitroso-N-acetylpencillamine, stimulate myoblast proliferation in vitro

    NASA Technical Reports Server (NTRS)

    Ulibarri, J. A.; Mozdziak, P. E.; Schultz, E.; Cook, C.; Best, T. M.

    1999-01-01

    Nitric oxide (NO) is an inter- and intracellular messenger involved in a variety of physiologic and pathophysiologic conditions. The effect of two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) and their effect on myoblast proliferation was examined. Both donors stimulated an increase in myoblast cell number over a range (1-10 microM) of donor concentrations. However, 50 microM SNAP inhibited myoblast proliferation. Cell numbers from cultures treated with degraded 10 microM SNAP were equivalent to the control. Therefore, it appears NO can stimulate as well as inhibit myoblast proliferation.

  15. Phospho-tyrosine phosphatase inhibitor Bpv(Hopic) enhances C2C12 myoblast migration in vitro. Requirement of PI3K/AKT and MAPK/ERK pathways.

    PubMed

    Dimchev, Georgi A; Al-Shanti, Nasser; Stewart, Claire E

    2013-05-01

    Muscle progenitor cell migration is an important step in skeletal muscle myogenesis and regeneration. Migration is required for muscle precursors to reach the site of damage and for the alignment of myoblasts prior to their fusion, which ultimately contributes to muscle regeneration. Limited spreading and migration of donor myoblasts are reported problems of myoblast transfer therapy, a proposed therapeutic strategy for Duchenne Muscular Dystrophy, warranting further investigation into different approaches for improving the motility and homing of these cells. In this article, the effect of protein phospho-tyrosine phosphatase and PTEN inhibitor BpV(Hopic) on C2C12 myoblast migration and differentiation was investigated. Applying a wound healing migration model, it is reported that 1 μM BpV(Hopic) is capable of enhancing the migration of C2C12 myoblasts by approximately 40 % in the presence of myotube conditioned media, without significantly affecting their capacity to differentiate and fuse into multinucleated myotubes. Improved migration of myoblasts treated with 1 μM BpV(Hopic) was associated with activation of PI3K/AKT and MAPK/ERK pathways, while their inhibition with either LY294002 or UO126, respectively, resulted in a reduction of C2C12 migration back to control levels. These results propose that bisperoxovanadium compounds may be considered as potential tools for enhancing the migration of myoblasts, while not reducing their differentiation capacity and underpin the importance of PI3K/AKT and MAPK/ERK signalling for the process of myogenic progenitor migration. PMID:23553034

  16. Biological properties of human skeletal myoblasts genetically modified to simultaneously overexpress the pro-angiogenic factors vascular endothelial growth factor-A and fibroblast growth factor-4.

    PubMed

    Zimna, A; Janeczek, A; Rozwadowska, N; Fraczek, M; Kucharzewska, P; Rucinski, M; Mietkiewski, T; Kurpisz, M

    2014-04-01

    Myocardial infarction results in cardiomyocyte loss and may eventually lead to cardiac failure. Skeletal myoblast transplantation into the scar area may compensate for this observed cell loss by strengthening the weakened myocardium and inducing myogenesis. Moreover, skeletal myoblasts may serve as potential transgene carriers for the myocardium (i.e., delivering pro-angiogenic factors, which may potentially improve blood perfusion in infarcted heart). We examined the influence of the simultaneous overexpression of two potent pro-angiogenic factors, fibroblast growth factor-4 (FGF-4) and vascular endothelial growth factor (VEGF), on human primary myoblast proliferation, cell cycle, resistance to hypoxic stress conditions and myogenic gene expression, as well as the induction of pro-angiogenic activities. We used a bicistronic plasmid vector encoding two factors introduced via an efficient myoblast electroporation method. The levels of overexpressed proteins were assessed, and their functionality at capillary formation was evaluated. This combined approach led to a high level of non-viral transient overexpression of both pro-angiogenic proteins, which proved to be potent regulators of blood vessel development assayed in capillary formation tests. We demonstrated in in vitro conditions that the transfection of human skeletal myoblasts with both FGF-4 and VEGF did not affect their basic biological properties such as the cell cycle, proliferation or expression of myogenic lineage-specific genes, and the modified cells adapted to oxidative stress conditions. Overall, the results obtained suggest that the applied combined approach with the use of two pro-angiogenic genes overexpressed in skeletal muscle stem cells may be an interesting alternative for the effective therapy of myocardial infarction in animal models and/or prospective clinical trials. PMID:24781729

  17. [Transplantation of normal or genetically modified myoblasts for the treatment of hereditary or acquired diseases].

    PubMed

    Tremblay, J P; Vilquin, J T

    2001-01-01

    The clinical trials of myoblast transplantation in Duchenne Muscular Dystrophy (DMD) patients produced disappointing results. The main problems responsible for these poor results have since then been identified and partially resolved. One of them was related to the use of an inadequate immunosuppression and, since then, immunosuppression with FK506 has permitted successful myoblast transplantation not only in mice but also in monkeys. The requirement for a sustained immunosuppression may be eventually avoided by developing a state of tolerance to the allogeneic cells or by autologous transplantation of genetically corrected myoblasts or stem cells. The rapid death of 75-80% of the injected myoblasts during the first five days has also contributed to the limited success of the early trials. This death was due to an inflammatory reaction and has been compensated in animal experiments by the injection of a larger number of cells (30 millions per cc). Finally, the myoblasts migrated only 0.5 mm away from their site of injection. This problem is currently compensated in animal experiments by injecting the myoblasts at every mm. The number of injections required may eventually be reduced by transfecting myoblasts with one or several metalloproteinase genes. The very good results obtained during the last two years in primates permit us to undertake a new phase I clinical trial to verify that myoblast transplantation can lead to the formation of muscle fibers expressing normal dystrophin in muscles of DMD patients. PMID:11530497

  18. Facioscapulohumeral dystrophy myoblasts efficiently repair moderate levels of oxidative DNA damage.

    PubMed

    Bou Saada, Yara; Dib, Carla; Dmitriev, Petr; Hamade, Aline; Carnac, Gilles; Laoudj-Chenivesse, Dalila; Lipinski, Marc; Vassetzky, Yegor S

    2016-04-01

    Facioscapulohumeral dystrophy (FSHD) is a progressive muscular dystrophy linked to a deletion of a subset of D4Z4 macrosatellite repeats accompanied by a chromatin relaxation of the D4Z4 array on chromosome 4q. In vitro, FSHD primary myoblasts show altered expression of oxidative-related genes and are more susceptible to oxidative stress. Double homeobox 4 (DUX4) gene, encoded within each D4Z4 unit, is normally transcriptionally silenced but is found aberrantly expressed in skeletal muscles of FSHD patients. Its expression leads to a deregulation of DUX4 target genes including those implicated in redox balance. Here, we assessed DNA repair efficiency of oxidative DNA damage in FSHD myoblasts and DUX4-transfected myoblasts. We have shown that the DNA repair activity is altered neither in FSHD myoblasts nor in immortalized human myoblasts transiently expressing DUX4. DNA damage caused by moderate doses of an oxidant is efficiently repaired while FSHD myoblasts exposed for 24 h to high levels of oxidative stress accumulated more DNA damage than normal myoblasts, suggesting that FSHD myoblasts remain more vulnerable to oxidative stress at high doses of oxidants. PMID:26860865

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

    SciTech Connect

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

    2002-12-02

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

  20. Lkb1 deletion upregulates Pax7 expression through activating Notch signaling pathway in myoblasts.

    PubMed

    Shan, Tizhong; Zhang, Pengpeng; Xiong, Yan; Wang, Yizhen; Kuang, Shihuan

    2016-07-01

    Satellite cells play crucial roles in mediating the growth, maintenance, and repair of postnatal skeletal muscle. Activated satellite cells (myoblasts) can divide symmetrically or asymmetrically to generate progenies that self-renewal, proliferate or differentiate. Pax7 is a defining marker of quiescent and activated satellite cells, but not differentiated myoblast. We demonstrate here that deletion of Lkb1 upregulates Pax7 expression in myoblasts and inhibits asymmetric divisions that generate differentiating progenies. Furthermore, we find that Lkb1 activates the Notch signaling pathway, which subsequently increases Pax7 expression and promotes self-renewal and proliferation while inhibiting differentiation. Mechanistic studies reveal that Lkb1 regulates Notch activation through AMPK-mTOR pathway in myoblasts. Together, these results establish a key role of Lkb1 in regulating myoblast division and cell fates choices. PMID:27131604

  1. Prostaglandin E2 promotes proliferation of skeletal muscle myoblasts via EP4 receptor activation

    PubMed Central

    Mo, Chenglin; Zhao, Ruonan; Vallejo, Julian; Igwe, Orisa; Bonewald, Lynda; Wetmore, Lori; Brotto, Marco

    2015-01-01

    We recently demonstrated that conditioned media (CM) from osteocytes enhances myogenic differentiation of myoblasts, suggesting that signaling from bone may be important for skeletal muscle myogenesis. The effect of CM was closely mimicked by prostaglandin E2 (PGE2), a bioactive lipid mediator in various physiological or pathological conditions. PGE2 is secreted at high levels by osteocytes and such secretion is further enhanced under loading conditions. Although four types of receptors, EP1 to EP4, mediate PGE2 signaling, it is unknown whether these receptors play a role in myogenesis. Therefore, in this study, the expression of EPs in mouse primary myoblasts was characterized, followed by examination of their roles in myoblast proliferation by treating myoblasts with PGE2 or specific agonists. All four PGE2 receptor mRNAs were detectable by quantitative real-time PCR (qPCR), but only PGE2 and EP4 agonist CAY 10598 significantly enhance myoblast proliferation. EP1/EP3 agonist 17-phenyl trinor PGE2 (17-PT PGE2) and EP2 agonist butaprost did not have any significant effects. Moreover, treatment with EP4 antagonist L161,982 dose-dependently inhibited myoblast proliferation. These results were confirmed by cell cycle analysis and the gene expression of cell cycle regulators. Concomitant with the inhibition of myoblast proliferation, treatment with L161,982 significantly increased intracellular reactive oxygen species (ROS) levels. Cotreatment with antioxidant N-acetyl cysteine (NAC) or sodium ascorbate (SA) successfully reversed the inhibition of myoblast proliferation and ROS overproduction caused by L161,982. Therefore, PGE2 signaling via the EP4 receptor regulates myogenesis by promoting myoblast proliferation and blocking this receptor results in increased ROS production in myoblasts. PMID:25785867

  2. Prostaglandin E2 promotes proliferation of skeletal muscle myoblasts via EP4 receptor activation.

    PubMed

    Mo, Chenglin; Zhao, Ruonan; Vallejo, Julian; Igwe, Orisa; Bonewald, Lynda; Wetmore, Lori; Brotto, Marco

    2015-01-01

    We recently demonstrated that conditioned media (CM) from osteocytes enhances myogenic differentiation of myoblasts, suggesting that signaling from bone may be important for skeletal muscle myogenesis. The effect of CM was closely mimicked by prostaglandin E2 (PGE2), a bioactive lipid mediator in various physiological or pathological conditions. PGE2 is secreted at high levels by osteocytes and such secretion is further enhanced under loading conditions. Although four types of receptors, EP1 to EP4, mediate PGE2 signaling, it is unknown whether these receptors play a role in myogenesis. Therefore, in this study, the expression of EPs in mouse primary myoblasts was characterized, followed by examination of their roles in myoblast proliferation by treating myoblasts with PGE2 or specific agonists. All four PGE2 receptor mRNAs were detectable by quantitative real-time PCR (qPCR), but only PGE2 and EP4 agonist CAY 10598 significantly enhance myoblast proliferation. EP1/EP3 agonist 17-phenyl trinor PGE2 (17-PT PGE2) and EP2 agonist butaprost did not have any significant effects. Moreover, treatment with EP4 antagonist L161,982 dose-dependently inhibited myoblast proliferation. These results were confirmed by cell cycle analysis and the gene expression of cell cycle regulators. Concomitant with the inhibition of myoblast proliferation, treatment with L161,982 significantly increased intracellular reactive oxygen species (ROS) levels. Cotreatment with antioxidant N-acetyl cysteine (NAC) or sodium ascorbate (SA) successfully reversed the inhibition of myoblast proliferation and ROS overproduction caused by L161,982. Therefore, PGE2 signaling via the EP4 receptor regulates myogenesis by promoting myoblast proliferation and blocking this receptor results in increased ROS production in myoblasts. PMID:25785867

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

    PubMed

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

    2005-09-01

    During muscle development, the p75(NTR) is expressed transiently on myoblasts. The temporal expression pattern of the receptor raises the possibility that the receptor is influencing muscle development. To test this hypothesis, p75(NTR)-deficient mutant mice were tested for muscle strength by using a standard wire gripe strength test and were found to have significantly decreased strength relative to that of normal mice. When normal mybolasts were examined in vivo for expression of NGF receptors, p75(NTR) was detected on myoblasts but the high affinity NGF receptor, trk A, was not co-expressed with p75(NTR). In vitro, proliferating C2C12 and primary myoblasts co-expressed the p75(NTR) and MyoD, but immunofluorescent analysis of primary myoblasts and RT-PCR analysis of C2C12 mRNA revealed that myoblasts were devoid of trk A. In contrast to the cell death functions that characterize the p75(NTR) in neurons, p75(NTR)-positive primary and C2C12 myoblasts did not differentiate or undergo apoptosis in response to neurotrophins. Rather, myoblasts survived and even proliferated when grown at subconfluent densities in the presence of the neurotrophins. Furthermore, when myoblasts treated with NGF were lysed and immunoprecipitated with antibodies against phosphorylated I-kappaB and AKT, the cells contained increased levels of both phospho-proteins, both of which promote cell survival. By contrast, neurotrophin-treated myoblasts did not induce phosphorylation of Map Kinase p42/44 or p38, indicating the survival was not mediated by the trk A receptor. Taken together, the data indicate that the p75(NTR) mediates survival of myoblasts prior to differentiation and that the activity of this receptor during myogenesis is important for developing muscle. PMID:15754321

  4. Activation of nicotinic acetylcholine receptors increases the rate of fusion of cultured human myoblasts.

    PubMed Central

    Krause, R M; Hamann, M; Bader, C R; Liu, J H; Baroffio, A; Bernheim, L

    1995-01-01

    1. Fusion of myogenic cells is important for muscle growth and repair. The aim of this study was to examine the possible involvement of nicotinic acetylcholine receptors (nAChR) in the fusion process of myoblasts derived from postnatal human satellite cells. 2. Acetylcholine-activated currents (ACh currents) were characterized in pure preparations of freshly isolated satellite cells, proliferating myoblasts, myoblasts triggered to fuse and myotubes, using whole-cell and single-channel voltage clamp recordings. Also, the effect of cholinergic agonists on myoblast fusion was tested. 3. No nAChR were observed in freshly isolated satellite cells. nAChR were first observed in proliferating myoblasts, but ACh current densities increased markedly only just before fusion. At that time most mononucleated myoblasts had ACh current densities similar to those of myotubes. ACh channels had similar properties at all stages of myoblast maturation. 4. The fraction of myoblasts that did not fuse under fusion-promoting conditions had no ACh current and thus resembled freshly isolated satellite cells. 5. The rate of myoblast fusion was increased by carbachol, an effect antagonized by alpha-bungarotoxin, curare and decamethonium, but not by atropine, indicating that nAChR were involved. Even though a prolonged exposure to carbachol led to desensitization, a residual ACh current persisted after several days of exposure to the nicotinic agonist. 6. Our observations suggest that nAChR play a role in myoblast fusion and that part of this role is mediated by the flow of ions through open ACh channels. Images Figure 1 Figure 2 Figure 3 PMID:8788942

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

    PubMed

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

    2016-11-01

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

  6. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    SciTech Connect

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

    2010-09-03

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

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

    SciTech Connect

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

    2005-11-15

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

  8. S100B engages RAGE or bFGF/FGFR1 in myoblasts depending on its own concentration and myoblast density. Implications for muscle regeneration.

    PubMed

    Riuzzi, Francesca; Sorci, Guglielmo; Beccafico, Sara; Donato, Rosario

    2012-01-01

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

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

    PubMed Central

    Beccafico, Sara; Donato, Rosario

    2012-01-01

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

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

    PubMed Central

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

    2015-01-01

    Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates. PMID:26030912

  11. Biomechanical strain vehicles for fibroblast-directed skeletal myoblast differentiation and myotube functionality in a novel coculture

    PubMed Central

    Cao, Thanh V.; Standley, Paul R.

    2014-01-01

    Skeletal muscle functionality is governed by multiple stimuli, including cytokines and biomechanical strain. Fibroblasts embedded within muscle connective tissue respond to biomechanical strain by secreting cytokines that induce myoblast differentiation and, we hypothesize, regulate myotube function. A coculture was established to allow cross talk between fibroblasts in Bioflex wells and myoblasts on nondeformable coverslips situated above Bioflex wells. Cyclic short-duration strain (CSDS) modeling repetitive stress/injury, acyclic long-duration strain (ALDS) modeling manipulative therapy, and combined strain paradigms (CSDS + ALDS) were applied to fibroblasts. Nonstrained myoblasts in uniculture and coculture served as controls. After fibroblasts had induced myoblast differentiation, myotube contraction was assessed by perfusion of ACh (10−11–10−3 M). CSDS-treated fibroblasts increased myotube contractile sensitivity vs. uniculture (P < 0.05). As contraction is dependent on ACh binding, expression and clustering of nicotinic ACh receptors (nAChRs) were measured. CSDS-treated fibroblasts increased nAChR expression (P < 0.05), which correlated with myotube contraction. ALDS-treated fibroblasts did not significantly affect contraction or nAChR expression. Agrin-treated myotubes were then used to design a computer algorithm to identify α-bungarotoxin-stained nAChR clusters. ALDS-treated fibroblasts increased nAChR clustering (P < 0.05), while CSDS-treated fibroblasts disrupted cluster formation. CSDS-treated fibroblasts produced nAChRs preferentially located in nonclustered regions (P < 0.05). Strain-activated fibroblasts mediate myotube differentiation with multiple functional phenotypes. Similar to muscle injury, CSDS-treated fibroblasts disrupted nAChR clusters and hypersensitized myotube contraction, while ALDS-treated fibroblasts aggregated nAChRs in large clusters, which may have important clinical implications. Cellular strategies aimed at improving

  12. Induced differentiation of adipose-derived stromal cells into myoblasts.

    PubMed

    Wu, Guizhu; Zheng, Xiu; Jiang, Zhongqing; Wang, Jinhua; Song, Yanfeng

    2010-06-01

    This study aimed to induce the differentiation of isolated and purified adipose-derived stromal cells (ADSCs) into myoblasts, which may provide a new strategy for tissue engineering in patients with stress urinary incontinence (SUI). ADSCs, isolated and cultured ex vivo, were identified by flow cytometry and induced to differentiate into myoblasts in the presence of an induction solution consisting of DMEM supplemented with 5-azacytidine (5-aza), 5% FBS, and 5% horse serum. Cellular morphology was observed under an inverted microscope. Ultrastructural changes occurring during the differentiation were observed by transmission electron microscopy and confocal laser scanning microscopy. Cellular immunohistochemical staining was applied to determine the expression of desmin protein in cells with and without induced differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect mRNA and protein expression, respectively, of sarcomeric and desmin smooth muscle proteins. The results showed that ADSCs were mainly of a spindle or polygon shape. Flow cytometry analysis revealed that ADSCs did not express CD34, CD45, and CD106 but high levels of CD44 and CD90, which confirmed that the cultured cells were indeed ADSCs. After induction with a 5-aza-containing solution, morphological changes in ADSCs, including irregular cell size, were observed. Cells gradually changed from long spindles to polygons and star-shaped cells with microvilli on the cell surface. Many organelles were observed and the cytoplasm was found to contain many mitochondria, rough endoplasmic reticulum (rER), and myofilament-like structures. Cell immunohistochemical staining revealed different levels of desmin expression in each phase of the induction process, with the highest expression level found on day 28 of induction. RT-PCR and Western blot results confirmed significantly higher desmin gene expression in induced cells compared with control cells, but no

  13. Interleukin 17 inhibits myogenic and promotes osteogenic differentiation of C2C12 myoblasts by activating ERK1,2.

    PubMed

    Kocić, Jelena; Santibañez, Juan F; Krstić, Aleksandra; Mojsilović, Slavko; Dorđević, Ivana Okić; Trivanović, Drenka; Ilić, Vesna; Bugarski, Diana

    2012-04-01

    The present study evaluated the role of interleukin (IL) 17 in multilineage commitment of C2C12 myoblastic cells and investigated associated signaling pathways. The results concerning the effects on cell function showed that IL-17 inhibits the migration of C2C12 cells, while not affecting their proliferation. The data regarding the influence on differentiation demonstrated that IL-17 inhibits myogenic differentiation of C2C12 cells by down-regulating the myogenin mRNA level, myosin heavy chain expression and myotube formation, but promotes their osteogenic differentiation by up-regulating the Runt-related transcription factor 2 mRNA level, cyclooxygenase-2 expression and alkaline phosphatase activity. IL-17 exerted these effects by activating ERK1,2 mitogen activated protein kinase signaling pathway, which in turn regulated the expression of relevant genes and proteins to inhibit myogenic differentiation and induce osteogenic differentiation. Additional analysis showed that the induction of osteogenic differentiation by IL-17 is independent of BMP signaling. The results obtained demonstrate the potential of IL-17 not only to inhibit the myogenic differentiation of C2C12 myoblasts but also to convert their differentiation pathway into that of osteoblast lineage providing new insight into the capacities of IL-17 to modulate the differentiation commitment. PMID:22285818

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

    SciTech Connect

    Gao, Fei; Kishida, Tsunao; Ejima, Akika; Gojo, Satoshi; Mazda, Osam

    2013-02-08

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

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

    PubMed Central

    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. PMID:25733836

  16. Myoblasts transplanted into rat infarcted myocardium are functionally isolated from their host

    PubMed Central

    Léobon, Bertrand; Garcin, Isabelle; Menasché, Philippe; Vilquin, Jean-Thomas; Audinat, Etienne; Charpak, Serge

    2003-01-01

    Survival and differentiation of myogenic cells grafted into infarcted myocardium have raised the hope that cell transplantation becomes a new therapy for cardiovascular diseases. The approach was further supported by transplantation of skeletal myoblasts, which was shown to improve cardiac performance in several animal species. Despite the success of myoblast transplantation and its recent trial in human, the mechanism responsible for the functional improvement remains unclear. Here, we used intracellular recordings coupled to video and fluorescence microscopy to establish whether myoblasts, genetically labeled with enhanced GFP and transplanted into rat infarcted myocardium, retain excitable and contractile properties, and participate actively to cardiac function. Our results indicate that grafted myoblasts differentiate into peculiar hyperexcitable myotubes with a contractile activity fully independent of neighboring cardiomyocytes. We conclude that mechanisms other than electromechanical coupling between grafted and host cells are involved in the improvement of cardiac function. PMID:12805561

  17. [Dystrophin gene expression in patients with Duchenne muscular dystrophy after myoblast transplantation].

    PubMed

    Shishkin, S S; Terekhov, S M; Krokhina, T B; Shakhovskaia, N I; Podobedova, A N; Linnaia, G F; Tarasov, V I; Ovchinnikov, V I; Krakhmaleva, I N; Zakharov, S F; Ershova, E S; Limborskaia, S A; Pogoda, T V; Zotikov, E A; Kut'ina, R M; Tarksh, M A; Sukhorukov, V S; Gerasimova, N L

    2001-08-01

    Based on originally designed technique of myoblast cultivation and in accordance with the approved by the Russian Ministry of Health "one muscle treatment" protocol of myoblast transplantation to the Duchenne muscular dystrophy patients, the first in Russia clinical trial of this gene correction method was carried out. Immonologically related myoblast cultures (30 to 90 million cells per patient) were injected after all preliminary procedures into tibialis anterior muscles of four boys selected from a group of volunteer recipients (Duchenne muscular dystrophy patients) based on the analysis of a number of surface antigens in donor-recipient pairs. The condition of the patients remained satisfactory during the whole period of post-transplantation follow-up (from 6 months to 1.5 years). Six months after myoblast transplantation the presence of donor DNA or dystrophin synthesis was demonstrated in muscle biopsies of three out of four patients. This result confirms efficacy and safety of the procedure used. PMID:11642111

  18. Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells.

    PubMed

    Liang, Rui; Dong, Wei; Shen, Xiaopeng; Peng, Xiaoping; Aceves, Angie G; Liu, Yu

    2016-01-01

    Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases. PMID:27501221

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

    SciTech Connect

    Ding, Ke; Yang, Zhong; Xu, Jian-zhong; Liu, Wen-ying; Zeng, Qiang; Hou, Fang; Lin, Sen

    2015-09-10

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

  20. Restoration of anal sphincter function after myoblast cell therapy in incontinent rats.

    PubMed

    Bisson, Aurélie; Fréret, Manuel; Drouot, Laurent; Jean, Laetitia; Le Corre, Stéphanie; Gourcerol, Guillaume; Doucet, Christelle; Michot, Francis; Boyer, Olivier; Lamacz, Marek

    2015-01-01

    Fecal incontinence (FI) remains a socially isolating condition with profound impact on quality of life for which autologous myoblast cell therapy represents an attractive treatment option. We developed an animal model of FI and investigated the possibility of improving sphincter function by intrasphincteric injection of syngeneic myoblasts. Several types of anal cryoinjuries were evaluated on anesthetized Fischer rats receiving analgesics. The minimal lesion yielding sustainable anal sphincter deficiency was a 90° cryoinjury of the sphincter, repeated after a 24-h interval. Anal sphincter pressure was evaluated longitudinally by anorectal manometry under local electrostimulation. Myoblasts were prepared using a protocol mimicking a clinical-grade process and further transduced with a GFP-encoding lentiviral vector before intrasphincteric injection. Experimental groups were uninjured controls, cryoinjured + PBS, and cryoinjured + myoblasts (different doses or injection site). Myoblast injection was well tolerated. Transferred myoblasts expressing GFP integrated into the sphincter and differentiated in situ into dystrophin-positive mature myofibers. Posttreatment sphincter pressures increased over time. At day 60, pressures in the treated group were significantly higher than those of PBS-injected controls and not significantly different from those of normal rats. Longitudinal follow-up showed stability of the therapeutic effect on sphincter function over a period of 6 months. Intrasphincteric myoblast injections at the lesion borders were equally as effective as intralesion administration, but an injection opposite to the lesion was not. These results provide proof of principle for myoblast cell therapy to treat FI in a rat model. This strategy is currently being evaluated in humans in a randomized double-blind placebo-controlled clinical trial. PMID:24143883

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

    PubMed

    Poosala, Pornthida; Ichinose, Hirofumi; Kitaoka, Takuya

    2016-01-01

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

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

    PubMed

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

    2015-02-01

    Myoblast differentiation and fusion is a well-orchestrated multistep process that is essential for skeletal muscle development and regeneration. Phospholipase D1 (PLD1) has been implicated in the initiation of myoblast differentiation in vitro. However, whether PLD1 plays additional roles in myoblast fusion and exerts a function in myogenesis in vivo remains unknown. Here we show that PLD1 expression is up-regulated in myogenic cells during muscle regeneration after cardiotoxin injury and that genetic ablation of PLD1 results in delayed myofiber regeneration. Myoblasts derived from PLD1-null mice or treated with PLD1-specific inhibitor are unable to form mature myotubes, indicating defects in second-phase myoblast fusion. Concomitantly, the PLD1 product phosphatidic acid is transiently detected on the plasma membrane of differentiating myocytes, and its production is inhibited by PLD1 knockdown. Exogenous lysophosphatidylcholine, a key membrane lipid for fusion pore formation, partially rescues fusion defect resulting from PLD1 inhibition. Thus these studies demonstrate a role for PLD1 in myoblast fusion during myogenesis in which PLD1 facilitates the fusion of mononuclear myocytes with nascent myotubes. PMID:25428992

  3. Involvement of Transient Receptor Potential Cation Channel Vanilloid 1 (TRPV1) in Myoblast Fusion.

    PubMed

    Kurosaka, Mitsutoshi; Ogura, Yuji; Funabashi, Toshiya; Akema, Tatsuo

    2016-10-01

    The mechanisms that underlie the complex process of muscle regeneration after injury remain unknown. Transient receptor potential cation channel vanilloid 1 (TRPV1) is expressed in several cell types, including skeletal muscle, and is activated by high temperature and by certain molecules secreted during tissue inflammation. Severe inflammation and local temperature perturbations are induced during muscle regeneration, which suggests that TRPV1 might be activated and involved in the process. The aim of this study, was to clarify the role of TRPV1 in the myogenic potential of satellite cells responsible for muscle regeneration. We found that mRNA and protein levels of TRPV1 increased during regeneration after cardiotoxin (CTX)-induced muscle injury in mice. Using isolated mouse satellite cells (i.e., myoblasts), we observed that activation of TRPV1 by its agonist capsaicin (CAP) augmented myogenin protein levels. Whereas CAP did not alter myoblast proliferation, it facilitated myoblast fusion (evaluated using myonucleii number per myotube and fusion index). In contrast, suppression of TRPV1 by siRNA impaired myoblast fusion. Using mice, we also demonstrated that intramuscular injection of CAP facilitated muscle repair after CTX-induced muscle injury. Moreover, we showed that these roles of TRPV1 might be mediated by interleukin-4 and calcium signaling during myoblast fusion. Collectively, these results suggest that TRPV1 underlies normal myogenesis through promotion of myoblast fusion. J. Cell. Physiol. 231: 2275-2285, 2016. © 2016 Wiley Periodicals, Inc. PMID:26892397

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

    SciTech Connect

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

    2012-06-29

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

  5. Hepatocyte growth factor (HGF) signals through SHP2 to regulate primary mouse myoblast proliferation

    SciTech Connect

    Li, Ju; Reed, Sarah A.; Johnson, Sally E.

    2009-08-01

    Niche localized HGF plays an integral role in G{sub 0} exit and the return to mitotic activity of adult skeletal muscle satellite cells. HGF actions are regulated by MET initiated intracellular signaling events that include recruitment of SHP2, a protein tyrosine phosphatase. The importance of SHP2 in HGF-mediated signaling was examined in myoblasts and primary cultures of satellite cells. Myoblasts stably expressing SHP2 (23A2-SHP2) demonstrate increased proliferation rates by comparison to controls or myoblasts expressing a phosphatase-deficient SHP2 (23A2-SHP2DN). By comparison to 23A2 myoblasts, treatment of 23A2-SHP2 cells with HGF does not further increase proliferation rates and 23A2-SHP2DN myoblasts are unresponsive to HGF. Importantly, the effects of SHP2 are independent of downstream ERK1/2 activity as inclusion of PD98059 does not blunt the HGF-induced proliferative response. SHP2 function was further evaluated in primary satellite cell cultures. Ectopic expression of SHP2 in satellite cells tends to decrease proliferation rates and siSHP2 causes an increase the percentage of dividing myogenic cells. Interestingly, treatment of satellite cells with high concentrations of HGF (50 ng/ml) inhibits proliferation, which can be overcome by knockdown of SHP2. From these results, we conclude that HGF signals through SHP2 in myoblasts and satellite cells to directly alter proliferation rates.

  6. AG490 improves the survival of human myoblasts in vitro and in vivo.

    PubMed

    Gérard, Catherine; Dufour, Christine; Goudenege, Sébastien; Skuk, Daniel; Tremblay, Jacques P

    2012-01-01

    Cell therapies consist in transplanting healthy cells into a disabled tissue with the goal to repopulate it and restore its function at least partially. In muscular diseases, most of the time, myoblasts are chosen for their expansion capacity in culture. Nevertheless, cell transplantation has limitations, among them, death of the transplanted cells, during the days following the graft. One possibility to counteract this problem is to enhance the proliferation of the transplanted myoblasts before their fusion with the existing muscle fibers. AG490 is a specific inhibitor of janus tyrosine kinase 2 (JAK2). The hypothesis is to block myoblast differentiation with AG490, thus permitting their proliferation. The inhibition of myoblast fusion by AG490 was confirmed in this study by gene expression and with a myosin heavy chain staining (MyHC). Moreover, cell survival was estimated by flow cytometry. AG490 was found to protect myoblasts in vitro from apoptosis induced by H(2)O(2) or by preventing attachment of cells to their substrate. Finally, in an in vivo model of muscle regeneration, when AG490 was coinjected with the myoblasts their survival was increased by 45% at 5 days after their transplantation. PMID:22963730

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

    PubMed Central

    Poosala, Pornthida; Ichinose, Hirofumi; Kitaoka, Takuya

    2016-01-01

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

  8. TRPC3 cation channel plays an important role in proliferation and differentiation of skeletal muscle myoblasts

    PubMed Central

    Woo, Jin Seok; Cho, Chung-Hyun; Kim, Do Han

    2010-01-01

    During membrane depolarization associated with skeletal excitation-contraction (EC) coupling, dihydropyridine receptor [DHPR, a L-type Ca2+ channel in the transverse (t)-tubule membrane] undergoes conformational changes that are transmitted to ryanodine receptor 1 [RyR1, an internal Ca2+-release channel in the sarcoplasmic reticulum (SR) membrane] causing Ca2+ release from the SR. Canonical-type transient receptor potential cation channel 3 (TRPC3), an extracellular Ca2+-entry channel in the t-tubule and plasma membrane, is required for full-gain of skeletal EC coupling. To examine additional role(s) for TRPC3 in skeletal muscle other than mediation of EC coupling, in the present study, we created a stable myoblast line with reduced TRPC3 expression and without α1SDHPR (MDG/TRPC3 KD myoblast) by knock-down of TRPC3 in α1SDHPR-null muscular dysgenic (MDG) myoblasts using retrovirus-delivered small interference RNAs in order to eliminate any DHPR-associated EC coupling-related events. Unlike wild-type or α1SDHPR-null MDG myoblasts, MDG/TRPC3 KD myoblasts exhibited dramatic changes in cellular morphology (e.g., unusual expansion of both cell volume and the plasma membrane, and multi-nuclei) and failed to differentiate into myotubes possibly due to increased Ca2+ content in the SR. These results suggest that TRPC3 plays an important role in the maintenance of skeletal muscle myoblasts and myotubes. PMID:20644344

  9. Myoblast cytonemes mediate Wg signaling from the wing imaginal disc and Delta-Notch signaling to the air sac primordium

    PubMed Central

    Huang, Hai; Kornberg, Thomas B

    2015-01-01

    The flight muscles, dorsal air sacs, wing blades, and thoracic cuticle of the Drosophila adult function in concert, and their progenitor cells develop together in the wing imaginal disc. The wing disc orchestrates dorsal air sac development by producing decapentaplegic and fibroblast growth factor that travel via specific cytonemes in order to signal to the air sac primordium (ASP). Here, we report that cytonemes also link flight muscle progenitors (myoblasts) to disc cells and to the ASP, enabling myoblasts to relay signaling between the disc and the ASP. Frizzled (Fz)-containing myoblast cytonemes take up Wingless (Wg) from the disc, and Delta (Dl)-containing myoblast cytonemes contribute to Notch activation in the ASP. Wg signaling negatively regulates Dl expression in the myoblasts. These results reveal an essential role for cytonemes in Wg and Notch signaling and for a signal relay system in the myoblasts. DOI: http://dx.doi.org/10.7554/eLife.06114.001 PMID:25951303

  10. Mechanical strain applied to human fibroblasts differentially regulates skeletal myoblast differentiation.

    PubMed

    Hicks, Michael R; Cao, Thanh V; Campbell, David H; Standley, Paul R

    2012-08-01

    Cyclic short-duration stretches (CSDS) such as those resulting from repetitive motion strain increase the risk of musculoskeletal injury. Myofascial release is a common technique used by clinicians that applies an acyclic long-duration stretch (ALDS) to muscle fascia to repair injury. When subjected to mechanical strain, fibroblasts within muscle fascia secrete IL-6, which has been shown to induce myoblast differentiation, essential for muscle repair. We hypothesize that fibroblasts subjected to ALDS following CSDS induce myoblast differentiation through IL-6. Fibroblast conditioned media and fibroblast-myoblast cocultures were used to test fibroblasts' ability to induce myoblast differentiation. The coculture system applies strain to fibroblasts only but still allows for diffusion of potential differentiation mediators to unstrained myoblasts on coverslips. To determine the role of IL-6, we utilized myoblast unicultures ± IL-6 (0-100 ng/ml) and cocultures ± α-IL-6 (0-200 μg/ml). Untreated uniculture myoblasts served as a negative control. After 96 h, coverslips (n = 6-21) were microscopically analyzed and quantified by blinded observer for differentiation endpoints: myotubes per square millimeter (>3 nuclei/cell), nuclei/myotube, and fusion efficiency (%nuclei within myotubes). The presence of fibroblasts and fibroblast conditioned media significantly enhanced myotube number (P < 0.05). However, in coculture, CSDS applied to fibroblasts did not reproduce this effect. ALDS following CSDS increased myotube number by 78% and fusion efficiency by 96% vs. CSDS alone (P < 0.05). Fibroblasts in coculture increase IL-6 secretion; however, IL-6 secretion did not correlate with enhanced differentiation among strain groups. Exogenous IL-6 in myoblast uniculture failed to induce differentiation. However, α-IL-6 attenuated differentiation in all coculture groups (P < 0.05). Fibroblasts secrete soluble mediators that have profound effects on several measures of myoblast

  11. Mechanical strain applied to human fibroblasts differentially regulates skeletal myoblast differentiation

    PubMed Central

    Hicks, Michael R.; Cao, Thanh V.; Campbell, David H.

    2012-01-01

    Cyclic short-duration stretches (CSDS) such as those resulting from repetitive motion strain increase the risk of musculoskeletal injury. Myofascial release is a common technique used by clinicians that applies an acyclic long-duration stretch (ALDS) to muscle fascia to repair injury. When subjected to mechanical strain, fibroblasts within muscle fascia secrete IL-6, which has been shown to induce myoblast differentiation, essential for muscle repair. We hypothesize that fibroblasts subjected to ALDS following CSDS induce myoblast differentiation through IL-6. Fibroblast conditioned media and fibroblast-myoblast cocultures were used to test fibroblasts' ability to induce myoblast differentiation. The coculture system applies strain to fibroblasts only but still allows for diffusion of potential differentiation mediators to unstrained myoblasts on coverslips. To determine the role of IL-6, we utilized myoblast unicultures ± IL-6 (0–100 ng/ml) and cocultures ± α-IL-6 (0–200 μg/ml). Untreated uniculture myoblasts served as a negative control. After 96 h, coverslips (n = 6–21) were microscopically analyzed and quantified by blinded observer for differentiation endpoints: myotubes per square millimeter (>3 nuclei/cell), nuclei/myotube, and fusion efficiency (%nuclei within myotubes). The presence of fibroblasts and fibroblast conditioned media significantly enhanced myotube number (P < 0.05). However, in coculture, CSDS applied to fibroblasts did not reproduce this effect. ALDS following CSDS increased myotube number by 78% and fusion efficiency by 96% vs. CSDS alone (P < 0.05). Fibroblasts in coculture increase IL-6 secretion; however, IL-6 secretion did not correlate with enhanced differentiation among strain groups. Exogenous IL-6 in myoblast uniculture failed to induce differentiation. However, α-IL-6 attenuated differentiation in all coculture groups (P < 0.05). Fibroblasts secrete soluble mediators that have profound effects on several measures of

  12. A comparative study of magnetic-activated cell sorting, cytotoxicity and preplating for the purification of human myoblasts.

    PubMed

    Park, Yoon Ghil; Moon, Jae Ho; Kim, Jin

    2006-04-30

    Although cultured myoblast transplantation has been extensively studied as a gene complementation approach to muscular dystrophy treatment, clinical success has still been limited. The inability to adequately isolate and purify myoblasts presents a major limitation to the production of sufficient myoblasts for engrafting purposes. This study attempted to purify myoblasts from primary culture by magnetic-activated cell sorting (MACS), complement-mediated cytotoxicity, and a preplating technique. As a result of positive myoblasts selection by MACS, the average percentage of myoblasts in mixed culture was increased from 30.0% to 41.7%. We observed both myoblast lysis and fibroblast lysis after complement-mediated cytotoxicity. Enrichment of myoblasts in mixed culture was found to increase to 83.1% by using the preplating technique. In addition, higher purification (92.8%) was achieved by following the preplating technique with MACS. Thus, preplating in combination with magnetic-activated cell sorting allows for a rapid and effective isolation of myoblasts from human muscle tissue. PMID:16642545

  13. Thyroid Hormone Receptor α Plays an Essential Role in Male Skeletal Muscle Myoblast Proliferation, Differentiation, and Response to Injury.

    PubMed

    Milanesi, Anna; Lee, Jang-Won; Kim, Nam-Ho; Liu, Yan-Yun; Yang, An; Sedrakyan, Sargis; Kahng, Andrew; Cervantes, Vanessa; Tripuraneni, Nikita; Cheng, Sheue-yann; Perin, Laura; Brent, Gregory A

    2016-01-01

    Thyroid hormone plays an essential role in myogenesis, the process required for skeletal muscle development and repair, although the mechanisms have not been established. Skeletal muscle develops from the fusion of precursor myoblasts into myofibers. We have used the C2C12 skeletal muscle myoblast cell line, primary myoblasts, and mouse models of resistance to thyroid hormone (RTH) α and β, to determine the role of thyroid hormone in the regulation of myoblast differentiation. T3, which activates thyroid hormone receptor (TR) α and β, increased myoblast differentiation whereas GC1, a selective TRβ agonist, was minimally effective. Genetic approaches confirmed that TRα plays an important role in normal myoblast proliferation and differentiation and acts through the Wnt/β-catenin signaling pathway. Myoblasts with TRα knockdown, or derived from RTH-TRα PV (a frame-shift mutation) mice, displayed reduced proliferation and myogenic differentiation. Moreover, skeletal muscle from the TRα1PV mutant mouse had impaired in vivo regeneration after injury. RTH-TRβ PV mutant mouse model skeletal muscle and derived primary myoblasts did not have altered proliferation, myogenic differentiation, or response to injury when compared with control. In conclusion, TRα plays an essential role in myoblast homeostasis and provides a potential therapeutic target to enhance skeletal muscle regeneration. PMID:26451739

  14. Involvement of unconventional myosin VI in myoblast function and myotube formation.

    PubMed

    Karolczak, Justyna; Pavlyk, Iuliia; Majewski, Łukasz; Sobczak, Magdalena; Niewiadomski, Paweł; Rzhepetskyy, Yuriy; Sikorska, Agata; Nowak, Natalia; Pomorski, Paweł; Prószyński, Tomasz; Ehler, Elisabeth; Rędowicz, Maria Jolanta

    2015-07-01

    The important role of unconventional myosin VI (MVI) in skeletal and cardiac muscle has been recently postulated (Karolczak et al. in Histochem Cell Biol 139:873-885, 2013). Here, we addressed for the first time a role for this unique myosin motor in myogenic cells as well as during their differentiation into myotubes. During myoblast differentiation, the isoform expression pattern of MVI and its subcellular localization underwent changes. In undifferentiated myoblasts, MVI-stained puncti were seen throughout the cytoplasm and were in close proximity to actin filaments, Golgi apparatus, vinculin-, and talin-rich focal adhesion as well as endoplasmic reticulum. Colocalization of MVI with endoplasmic reticulum was enhanced during myotube formation, and differentiation-dependent association was also seen in sarcoplasmic reticulum of neonatal rat cardiomyocytes (NRCs). Moreover, we observed enrichment of MVI in myotube regions containing acetylcholine receptor-rich clusters, suggesting its involvement in the organization of the muscle postsynaptic machinery. Overexpression of the H246R MVI mutant (associated with hypertrophic cardiomyopathy) in myoblasts and NRCs caused the formation of abnormally large intracellular vesicles. MVI knockdown caused changes in myoblast morphology and inhibition of their migration. On the subcellular level, MVI-depleted myoblasts exhibited aberrations in the organization of actin cytoskeleton and adhesive structures as well as in integrity of Golgi apparatus and endoplasmic reticulum. Also, MVI depletion or overexpression of H246R mutant caused the formation of significantly wider or aberrant myotubes, respectively, indicative of involvement of MVI in myoblast differentiation. The presented results suggest an important role for MVI in myogenic cells and possibly in myoblast differentiation. PMID:25896210

  15. hHGF Overexpression in Myoblast Sheets Enhances Their Angiogenic Potential in Rat Chronic Heart Failure

    PubMed Central

    Siltanen, Antti; Kitabayashi, Katsukiyo; Lakkisto, Päivi; Mäkelä, Johanna; Pätilä, Tommi; Ono, Masamichi; Tikkanen, Ilkka; Sawa, Yoshiki; Kankuri, Esko; Harjula, Ari

    2011-01-01

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

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

    PubMed Central

    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

  17. SIRT3, a Mitochondrial NAD+-Dependent Deacetylase, Is Involved in the Regulation of Myoblast Differentiation

    PubMed Central

    Abdel Khalek, Waed; Cortade, Fabienne; Ollendorff, Vincent; Lapasset, Laure; Tintignac, Lionel

    2014-01-01

    Sirtuin 3 (SIRT3), one of the seven mammalian sirtuins, is a mitochondrial NAD+-dependent deacetylase known to control key metabolic pathways. SIRT3 deacetylases and activates a large number of mitochondrial enzymes involved in the respiratory chain, in ATP production, and in both the citric acid and urea cycles. We have previously shown that the regulation of myoblast differentiation is tightly linked to mitochondrial activity. Since SIRT3 modulates mitochondrial activity, we decide to address its role during myoblast differentiation. For this purpose, we first investigated the expression of endogenous SIRT3 during C2C12 myoblast differentiation. We further studied the impact of SIRT3 silencing on both the myogenic potential and the mitochondrial activity of C2C12 cells. We showed that SIRT3 protein expression peaked at the onset of myoblast differentiation. The inhibition of SIRT3 expression mediated by the stable integration of SIRT3 short inhibitory RNA (SIRT3shRNA) in C2C12 myoblasts, resulted in: 1) abrogation of terminal differentiation - as evidenced by a marked decrease in the myoblast fusion index and a significant reduction of Myogenin, MyoD, Sirtuin 1 and Troponin T protein expression - restored upon MyoD overexpression; 2) a decrease in peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and citrate synthase protein expression reflecting an alteration of mitochondrial density; and 3) an increased production of reactive oxygen species (ROS) mirrored by the decreased activity of manganese superoxide dismutase (MnSOD). Altogether our data demonstrate that SIRT3 mainly regulates myoblast differentiation via its influence on mitochondrial activity. PMID:25489948

  18. The MARVEL domain protein, Singles Bar, is required for progression past the pre-fusion complex stage of myoblast fusion

    PubMed Central

    Estrada, Beatriz; Maeland, Anne D.; Gisselbrecht, Stephen S.; Bloor, James W.; Brown, Nicholas H.; Michelson, Alan M.

    2007-01-01

    Summary Multinucleated myotubes develop by the sequential fusion of individual myoblasts. Using a convergence of genomic and classical genetic approaches, we have discovered a novel gene, singles bar (sing), that is essential for myoblast fusion. sing encodes a small multipass transmembrane protein containing a MARVEL domain, which is found in vertebrate proteins involved in processes such as tight junction formation and vesicle trafficking where—as in myoblast fusion—membrane apposition occurs. sing is expressed in both founder cells and fusion competent myoblasts preceding and during myoblast fusion. Examination of embryos injected with double-stranded sing RNA or embryos homozygous for ethane methyl sulfonate-induced sing alleles revealed an identical phenotype: replacement of multinucleated myofibers by groups of single, myosin-expressing myoblasts at a stage when formation of the mature muscle pattern is complete in wild-type embryos. Unfused sing mutant myoblasts form clusters, suggesting that early recognition and adhesion of these cells is unimpaired. To further investigate this phenotype, we undertook electron microscopic ultrastructural studies of fusing myoblasts in both sing and wild-type embryos. These experiments revealed that more sing mutant myoblasts than wild-type contain pre-fusion complexes, which are characterized by electron-dense vesicles paired on either side of the fusing plasma membranes. In contrast, embryos mutant for another muscle fusion gene, blown fuse (blow), have a normal number of such complexes. Together, these results lead to the hypothesis that sing acts at a step distinct from that of blow, and that sing is required on both founder cell and fusion-competent myoblast membranes to allow progression past the pre-fusion complex stage of myoblast fusion, possibly by mediating fusion of the electron-dense vesicles to the plasma membrane. PMID:17537424

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

    SciTech Connect

    Mitchell, Darrion L.; DiMario, Joseph X.

    2010-01-15

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

  20. Oxidative Stress and Plasma Membrane Repair in Single Myoblasts After Femtosecond Laser Photoporation.

    PubMed

    Duan, Xinxing; Chan, Kam Tai; Lee, Kenneth K H; Mak, Arthur F T

    2015-11-01

    Cell membranes are susceptible to biophysical damages. These biophysical damages often present themselves in challenging oxidative environments, such as in chronic inflammation. Here we report the damage evolution after single myoblasts were individually subjected to femtosecond (fs) laser photoporation on their plasma membranes under normal and oxidative conditions. A well-characterized tunable fs laser was coupled with a laser scanning confocal microscope. The post-damage wound evolution was documented by real-time imaging. The fs laser could generate a highly focused hole at a targeted site of the myoblast plasma membrane. The initial hole size depended on the laser dosage in terms of power and exposure duration. With the same laser power and irradiation duration, photoporation invoked bigger holes in the oxidative groups than in the control. Myoblasts showed difficulty in repairing holes with initial size beyond certain threshold. Within the threshold, holes could apparently be resealed within 100 s under the normal condition; while in oxidative condition, the resealing process could take 100-300 s. The hole-resealing capacity of myoblasts was compromised under oxidative stress particularly when the oxidative exposure was chronic. It is interesting to note that brief exposure to oxidative stress apparently could promote resealing in myoblasts after photoporation. PMID:26014361

  1. Combination of lipid metabolism alterations and their sensitivity to inflammatory cytokines in human lipin-1-deficient myoblasts.

    PubMed

    Michot, Caroline; Mamoune, Asmaa; Vamecq, Joseph; Viou, Mai Thao; Hsieh, Lu-Sheng; Testet, Eric; Lainé, Jeanne; Hubert, Laurence; Dessein, Anne-Frédérique; Fontaine, Monique; Ottolenghi, Chris; Fouillen, Laetitia; Nadra, Karim; Blanc, Etienne; Bastin, Jean; Candon, Sophie; Pende, Mario; Munnich, Arnold; Smahi, Asma; Djouadi, Fatima; Carman, George M; Romero, Norma; de Keyzer, Yves; de Lonlay, Pascale

    2013-12-01

    Lipin-1 deficiency is associated with massive rhabdomyolysis episodes in humans, precipitated by febrile illnesses. Despite well-known roles of lipin-1 in lipid biosynthesis and transcriptional regulation, the pathogenic mechanisms leading to rhabdomyolysis remain unknown. Here we show that primary myoblasts from lipin-1-deficient patients exhibit a dramatic decrease in LPIN1 expression and phosphatidic acid phosphatase 1 activity, and a significant accumulation of lipid droplets (LD). The expression levels of LPIN1-target genes [peroxisome proliferator-activated receptors delta and alpha (PPARδ, PPARα), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), acyl-coenzyme A dehydrogenase, very long (ACADVL), carnitine palmitoyltransferase IB and 2 (CPT1B and CPT2)] were not affected while lipin-2 protein level, a closely related member of the family, was increased. Microarray analysis of patients' myotubes identified 19 down-regulated and 51 up-regulated genes, indicating pleiotropic effects of lipin-1 deficiency. Special attention was paid to the up-regulated ACACB (acetyl-CoA carboxylase beta), a key enzyme in the fatty acid synthesis/oxidation balance. We demonstrated that overexpression of ACACB was associated with free fatty acid accumulation in patients' myoblasts whereas malonyl-carnitine (as a measure of malonyl-CoA) and CPT1 activity were in the normal range in basal conditions accordingly to the normal daily activity reported by the patients. Remarkably ACACB invalidation in patients' myoblasts decreased LD number and size while LPIN1 invalidation in controls induced LD accumulation. Further, pro-inflammatory treatments tumor necrosis factor alpha+Interleukin-1beta(TNF1α+IL-1ß) designed to mimic febrile illness, resulted in increased malonyl-carnitine levels, reduced CPT1 activity and enhanced LD accumulation, a phenomenon reversed by dexamethasone and TNFα or IL-1ß inhibitors. Our data suggest that the pathogenic mechanism

  2. Myostatin stimulates, not inihibits, C2C12 myoblast proliferation.

    PubMed

    Rodgers, Buel D; Wiedeback, Benjamin D; Hoversten, Knut E; Jackson, Melissa F; Walker, Ryan G; Thompson, Thomas B

    2014-03-01

    The immortal C2C12 cell line originates from dystrophic mouse thigh muscle and has been used to study the endocrine control of muscle cell growth, development, and function, including those actions regulated by myostatin. Previous studies suggest that high concentrations of recombinant myostatin generated in bacteria inhibit C2C12 proliferation and differentiation. Recombinant myostatin generated in eukaryotic systems similarly inhibits the proliferation of primary myosatellite cells, but consequently initiates, rather than inhibits, their differentiation and is bioactive at far lower concentrations. Our studies indicate that 2 different sources of recombinant myostatin made in eukaryotes stimulate, not inhibit, C2C12 proliferation. This effect occurred at different cell densities and serum concentrations and in the presence of IGF-I, a potent myoblast mitogen. This stimulatory effect was comparable to that obtained with TGFβ1, a related factor that also inhibits primary myosatellite cell proliferation. Attenuating the myostatin/activin (ie, Acvr2b) and TGFβ1 receptor signaling pathways with the Alk4/5 and Alk5 inhibitors, SB431542 and SB505142, respectively, similarly attenuated proliferation induced by serum, myostatin or TGFβ1 and in a dose-dependent manner. In serum-free medium, both myostatin and TGFβ1 stimulated Smad2 phosphorylation, but not that of Smad3, and a Smad3 inhibitor (SIS3) only inhibited proliferation in cells cultured in high serum. Thus, myostatin and TGFβ1 stimulate C2C12 proliferation primarily via Smad2. These results together question the physiological relevance of the C2C12 model and previous studies using recombinant myostatin generated in bacteria. They also support the alternative use of primary myosatellite cells and recombinant myostatin generated in eukaryotes. PMID:24424069

  3. Myomaker, Regulated by MYOD, MYOG and miR-140-3p, Promotes Chicken Myoblast Fusion.

    PubMed

    Luo, Wen; Li, Erxin; Nie, Qinghua; Zhang, Xiquan

    2015-01-01

    The fusion of myoblasts is an important step during skeletal muscle differentiation. A recent study in mice found that a transmembrane protein called Myomaker, which is specifically expressed in muscle, is critical for myoblast fusion. However, the cellular mechanism of its roles and the regulatory mechanism of its expression remain unclear. Chicken not only plays an important role in meat production but is also an ideal model organism for muscle development research. Here, we report that Myomaker is also essential for chicken myoblast fusion. Forced expression of Myomaker in chicken primary myoblasts promotes myoblast fusion, whereas knockdown of Myomaker by siRNA inhibits myoblast fusion. MYOD and MYOG, which belong to the family of myogenic regulatory factors, can bind to a conserved E-box located proximal to the Myomaker transcription start site and induce Myomaker transcription. Additionally, miR-140-3p can inhibit Myomaker expression and myoblast fusion, at least in part, by binding to the 3' UTR of Myomaker in vitro. These findings confirm the essential roles of Myomaker in avian myoblast fusion and show that MYOD, MYOG and miR-140-3p can regulate Myomaker expression. PMID:26540045

  4. KAT5-mediated SOX4 acetylation orchestrates chromatin remodeling during myoblast differentiation.

    PubMed

    Jang, S-M; Kim, J-W; Kim, C-H; An, J-H; Johnson, A; Song, P I; Rhee, S; Choi, K-H

    2015-01-01

    Transcription factor SOX4 has been implicated in skeletal myoblast differentiation through the regulation of Cald1 gene expression; however, the detailed molecular mechanism underlying this process is largely unknown. Here, we demonstrate that SOX4 acetylation at lysine 95 by KAT5 (also known as Tip60) is essential for Cald1 promoter activity at the onset of C2C12 myoblast differentiation. KAT5 chromodomain was found to facilitate SOX4 recruitment to the Cald1 promoter, which is involved in chromatin remodeling at the promoter. Chromatin occupancy analysis of SOX4, KAT5, and HDAC1 indicated that the expression of putative SOX4 target genes during C2C12 myoblast differentiation is specifically regulated by the molecular switching of the co-activator KAT5 and the co-repressor HDAC1 on SOX4 transcriptional activation. PMID:26291311

  5. Modulation of alignment and differentiation of skeletal myoblasts by submicron ridges/grooves surface structure.

    PubMed

    Wang, Peng-Yuan; Yu, Hung-Te; Tsai, Wei-Bor

    2010-06-01

    Alignment and fusion of myoblasts into parallel arrays of multinucleated myotubes are critical in skeletal muscle tissue engineering. It is well known that contact guidance by grooves/ridges structures induces myoblasts to align and to migrate along the anisotropic direction. In this study, two series of grooved substrata with different widths (450 and 900 nm) and different depths (100, 350, and 550 nm) were studied on their effects on myoblast adhesion, proliferation, and differentiation into myotubes. We found that C2C12 cells were aligned and elongated along the direction of grooves. Groove depth was more influential on cellular morphology, proliferation, and differentiation than groove width. While cell proliferation was retarded on the grooved surfaces especially on the substrate with 900/550 nm (width/depth), differentiation was also enhanced on the patterned surfaces compared to the flat control. Our results demonstrated the potential of grooved substrata with submicron scale in skeletal muscle tissue engineering. PMID:20148416

  6. KAT5-mediated SOX4 acetylation orchestrates chromatin remodeling during myoblast differentiation

    PubMed Central

    Jang, S-M; Kim, J-W; Kim, C-H; An, J-H; Johnson, A; Song, P I; Rhee, S; Choi, K-H

    2015-01-01

    Transcription factor SOX4 has been implicated in skeletal myoblast differentiation through the regulation of Cald1 gene expression; however, the detailed molecular mechanism underlying this process is largely unknown. Here, we demonstrate that SOX4 acetylation at lysine 95 by KAT5 (also known as Tip60) is essential for Cald1 promoter activity at the onset of C2C12 myoblast differentiation. KAT5 chromodomain was found to facilitate SOX4 recruitment to the Cald1 promoter, which is involved in chromatin remodeling at the promoter. Chromatin occupancy analysis of SOX4, KAT5, and HDAC1 indicated that the expression of putative SOX4 target genes during C2C12 myoblast differentiation is specifically regulated by the molecular switching of the co-activator KAT5 and the co-repressor HDAC1 on SOX4 transcriptional activation. PMID:26291311

  7. Injection of porous polycaprolactone beads containing autologous myoblasts in a dog model of fecal incontinence

    PubMed Central

    Kang, Sung-Bum; Lee, Hye Seung; Lim, Jae-Young; Oh, Se Heang; Kim, Sang Joon; Hong, Sa-Min; Jang, Je-Ho; Cho, Jeong-Eun; Lee, Sung-Min

    2013-01-01

    Purpose Few studies have examined whether bioengineering can improve fecal incontinence. This study designed to determine whether injection of porous polycaprolactone beads containing autologous myoblasts improves sphincter function in a dog model of fecal incontinence. Methods The anal sphincter of dogs was injured and the dogs were observed without and with (n = 5) the injection of porous polycaprolactone beads containing autologous myoblasts into the site of injury. Autologous myoblasts purified from the gastrocnemius muscles were transferred to the beads. Compound muscle action potentials (CMAP) of the pudendal nerve, anal sphincter pressure, and histopathology were determined 3 months after treatment. Results The amplitudes of the CMAP in the injured sphincter were significantly lower than those measured before injury (1.22 mV vs. 3.00 mV, P = 0.04). The amplitudes were not different between dogs with and without the injection of autologous myoblast beads (P = 0.49). Resting and squeezing pressures were higher in dogs treated with autologous myoblast beads (2.00 mmHg vs. 1.80 mmHg; 6.13 mmHg vs. 4.02 mmHg), although these differences were not significant in analyses of covariance adjusted for baseline values. The injection site was stained for smooth muscle actin, but showed evidence of foreign body inflammatory reactions. Conclusion This was the first study to examine whether bioengineering could improve fecal incontinence. Although the results did not show definite evidence that injection of autologous myoblast beads improves sphincter function, we found that the dog model was suitable and reliable for studying the effects of a potential treatment modality for fecal incontinence. PMID:23577316

  8. Amino acids downregulate the expression of several autophagy-related genes in rainbow trout myoblasts.

    PubMed

    Seiliez, Iban; Gabillard, Jean-Charles; Riflade, Marine; Sadoul, Bastien; Dias, Karine; Avérous, Julien; Tesseraud, Sophie; Skiba, Sandrine; Panserat, Stéphane

    2012-03-01

    Many fish species experience long periods of fasting often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as proteins of this tissue act as a major energy source. However, the molecular components involved in muscle protein degradation as well as the regulatory networks that control their function are still incompletely defined in fish. The present work aimed to characterize the response of the autophagy-lysosomal degradative pathway to nutrient and serum availability in primary culture of rainbow trout myoblasts. In this aim, 4-day-old cells were incubated in a serum and amino acid-rich medium (complete medium), a serum and amino acid-deprived medium (minimal medium) or a minimal medium plus amino acids, and both the transcription-independent short-term response and the transcription-dependent long-term response of the autophagy-lysosomal degradative pathway were analyzed. We report that serum and amino acids withdrawal is accompanied by a rapid increase of autophagosome formation but also by a slower induction of the expression of several autophagy-related genes (LC3B, gabarapl1, atg4b). We also showed that this latter response is controlled by amino acid (AA) availability and that both TOR-dependent and TOR-independent pathways are involved in this effect. Together these results suggest an important role for AA released by muscle proteolysis during the fasting period in regulating the subtle balance between using proteins as disposable furniture to provide energy, and conserving muscle through protein sparing. PMID:22252009

  9. Co-activation of nuclear factor-κB and myocardin/serum response factor conveys the hypertrophy signal of high insulin levels in cardiac myoblasts.

    PubMed

    Madonna, Rosalinda; Geng, Yong-Jian; Bolli, Roberto; Rokosh, Gregg; Ferdinandy, Peter; Patterson, Cam; De Caterina, Raffaele

    2014-07-11

    Hyperinsulinemia contributes to cardiac hypertrophy and heart failure in patients with the metabolic syndrome and type 2 diabetes. Here, high circulating levels of tumor necrosis factor (TNF)-α may synergize with insulin in signaling inflammation and cardiac hypertrophy. We tested whether high insulin affects activation of TNF-α-induced NF-κB and myocardin/serum response factor (SRF) to convey hypertrophy signaling in cardiac myoblasts. In canine cardiac myoblasts, treatment with high insulin (10(-8) to 10(-7) m) for 0-24 h increased insulin receptor substrate (IRS)-1 phosphorylation at Ser-307, decreased protein levels of chaperone-associated ubiquitin (Ub) E3 ligase C terminus of heat shock protein 70-interacting protein (CHIP), increased SRF activity, as well as β-myosin heavy chain (MHC) and myocardin expressions. Here siRNAs to myocardin or NF-κB, as well as CHIP overexpression prevented (while siRNA-mediated CHIP disruption potentiated) high insulin-induced SR element (SRE) activation and β-MHC expression. Insulin markedly potentiated TNF-α-induced NF-κB activation. Compared with insulin alone, insulin+TNF-α increased SRF/SRE binding and β-MHC expression, which was reversed by the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and by NF-κB silencing. In the hearts of db/db diabetic mice, in which Akt phosphorylation was decreased, p38MAPK, Akt1, and IRS-1 phosphorylation at Ser-307 were increased, together with myocardin expression as well as SRE and NF-κB activities. In response to high insulin, cardiac myoblasts increase the expression or the promyogenic transcription factors myocardin/SRF in a CHIP-dependent manner. Insulin potentiates TNF-α in inducing NF-κB and SRF/SRE activities. In hyperinsulinemic states, myocardin may act as a nuclear effector of insulin, promoting cardiac hypertrophy. PMID:24855642

  10. Myoblast transplantation between monozygotic twin girl carriers of Duchenne muscular dystrophy.

    PubMed

    Tremblay, J P; Bouchard, J P; Malouin, F; Théau, D; Cottrell, F; Collin, H; Rouche, A; Gilgenkrantz, S; Abbadi, N; Tremblay, M

    1993-01-01

    Monozygotic twin girls, both carriers of Duchenne muscular dystrophy, only one a severe symptomatic carrier and the other asymptomatic due to opposite lyonization, were studied. Myoblast clones were obtained from a muscle biopsy of the asymptomatic carrier. PCR amplification showed that most (94%) of these clones produced normal dystrophin mRNA. Roughly 704 million myoblasts were produced from 119 clones. These myoblasts were transplanted into the extensor carpi radialis (ECR) and in the biceps of one arm of the manifesting carrier while the other arm acted as the control. The strength of the patient was evaluated in a series of pre- and post-tests and a biopsy was obtained about 1 yr after the transplantation. The myoblast injections produced a significant force gain (12%-31%) in wrist extension but no force gain for elbow flexion. Muscle biopsies on the injected and control muscles obtained 1 yr after the injections showed only a small increase in the number of dystrophin positive fibers and the presence of numerous small type II fibers. The small beneficial effect of this transplantation cannot be attributed to immune problems, the donor and the recipient being identical twins, but may be due to a low level of spontaneous muscle regeneration. PMID:8186717

  11. Electrotransfection and lipofection show comparable efficiency for in vitro gene delivery of primary human myoblasts.

    PubMed

    Mars, Tomaz; Strazisar, Marusa; Mis, Katarina; Kotnik, Nejc; Pegan, Katarina; Lojk, Jasna; Grubic, Zoran; Pavlin, Mojca

    2015-04-01

    Transfection of primary human myoblasts offers the possibility to study mechanisms that are important for muscle regeneration and gene therapy of muscle disease. Cultured human myoblasts were selected here because muscle cells still proliferate at this developmental stage, which might have several advantages in gene therapy. Gene therapy is one of the most sought-after tools in modern medicine. Its progress is, however, limited due to the lack of suitable gene transfer techniques. To obtain better insight into the transfection potential of the presently used techniques, two non-viral transfection methods--lipofection and electroporation--were compared. The parameters that can influence transfection efficiency and cell viability were systematically approached and compared. Cultured myoblasts were transfected with the pEGFP-N1 plasmid either using Lipofectamine 2000 or with electroporation. Various combinations for the preparation of the lipoplexes and the electroporation media, and for the pulsing protocols, were tested and compared. Transfection efficiency and cell viability were inversely proportional for both approaches. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for lipofection, while for electroporation, RPMI medium and a pulsing protocol using eight pulses of 2 ms at E = 0.8 kV/cm proved to be the optimal combination. The transfection efficiencies for the optimal lipofection and optimal electrotransfection protocols were similar (32 vs. 32.5%, respectively). Both of these methods are effective for transfection of primary human myoblasts; however, electroporation might be advantageous for in vivo application to skeletal muscle. PMID:25534347

  12. Long-term evaluation of myoblast seeded patches implanted on infarcted rat hearts.

    PubMed

    Giraud, Marie-Noëlle; Flueckiger, Remy; Cook, Stéphane; Ayuni, Erick; Siepe, Matthias; Carrel, Thierry; Tevaearai, Hendrik

    2010-06-01

    Cell transplantation presents great potential for treatment of patients with severe heart failure. However, its clinical application was revealed to be more challenging than initially expected in experimental studies. Further investigations need to be undertaken to define the optimal treatment conditions. We previously reported on the epicardial implantation of a bio-engineered construct of skeletal myoblast-seeded polyurethane and its preventive effect on progression toward heart failure. In the present study, we present a long-term evaluation of this functional outcome. Left anterior descending coronary ligation was performed in female Lewis rats. Two weeks later, animals were treated with either epicardial implantation of biograft, acellular scaffold, sham operation, or direct intramyocardial skeletal myoblast injection. Functional assessments were performed with serial echocardiographies every 3 months and end point left ventricle pressure was assessed. Hearts were then harvested for histological examinations. Myocardial infarction induced a slow and progressive reduction in fractional shortening after 3 months. Progression toward heart failure was significantly prevented for up to 6 months after injection of myoblasts and for up to 9 months following biograft implantation. Nevertheless, this effect vanished after 12 months, with immunohistological examinations revealing an absence of the transplanted myoblasts within the scaffold. We demonstrated that tissue therapy is superior to cell therapy for stabilization of heart function. However, beneficial effects are transient. PMID:20482708

  13. Leucine and isoleucine reduce protein degradation in rainbow trout (Oncorhynchus mykiss) primary myoblast cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myogenic precursor cells were isolated from rainbow trout skeletal muscle and incubated in media containing 10% fetal bovine serum for 7 days, thereby differentiating into myoblasts. Rates of protein degradation were determined in response to minimal essential media (MEM) of various amino acid (AA)...

  14. HACD1, a regulator of membrane composition and fluidity, promotes myoblast fusion and skeletal muscle growth.

    PubMed

    Blondelle, Jordan; Ohno, Yusuke; Gache, Vincent; Guyot, Stéphane; Storck, Sébastien; Blanchard-Gutton, Nicolas; Barthélémy, Inès; Walmsley, Gemma; Rahier, Anaëlle; Gadin, Stéphanie; Maurer, Marie; Guillaud, Laurent; Prola, Alexandre; Ferry, Arnaud; Aubin-Houzelstein, Geneviève; Demarquoy, Jean; Relaix, Frédéric; Piercy, Richard J; Blot, Stéphane; Kihara, Akio; Tiret, Laurent; Pilot-Storck, Fanny

    2015-10-01

    The reduced diameter of skeletal myofibres is a hallmark of several congenital myopathies, yet the underlying cellular and molecular mechanisms remain elusive. In this study, we investigate the role of HACD1/PTPLA, which is involved in the elongation of the very long chain fatty acids, in muscle fibre formation. In humans and dogs, HACD1 deficiency leads to a congenital myopathy with fibre size disproportion associated with a generalized muscle weakness. Through analysis of HACD1-deficient Labradors, Hacd1-knockout mice, and Hacd1-deficient myoblasts, we provide evidence that HACD1 promotes myoblast fusion during muscle development and regeneration. We further demonstrate that in normal differentiating myoblasts, expression of the catalytically active HACD1 isoform, which is encoded by a muscle-enriched splice variant, yields decreased lysophosphatidylcholine content, a potent inhibitor of myoblast fusion, and increased concentrations of ≥ C18 and monounsaturated fatty acids of phospholipids. These lipid modifications correlate with a reduction in plasma membrane rigidity. In conclusion, we propose that fusion impairment constitutes a novel, non-exclusive pathological mechanism operating in congenital myopathies and reveal that HACD1 is a key regulator of a lipid-dependent muscle fibre growth mechanism. PMID:26160855

  15. Promotion of muscle regeneration by myoblast transplantation combined with the controlled and sustained release of bFGFcpr.

    PubMed

    Hagiwara, Koki; Chen, Guoping; Kawazoe, Naoki; Tabata, Yasuhiko; Komuro, Hiroaki

    2016-04-01

    Although myoblast transplantation is an attractive method for muscle regeneration, its efficiency remains limited. The efficacy of myoblast transplantation in combination with the controlled and sustained delivery of basic fibroblast growth factor (bFGF) was investigated. Defects of thigh muscle in Sprague-Dawley (SD) rats were created, and GFP-positive myoblasts were subsequently transplanted. The rats were divided into three groups. In control group 1 (C1) only myoblasts were transplanted, while in control group 2 (C2) myoblasts were introduced along with empty gelatin hydrogel microspheres. In the experimental group (Ex), myoblasts were transplanted along with bFGF incorporated into gelatin hydrogel microspheres. Four weeks after transplantation, GFP-positive myoblasts were found to be integrated into the recipient muscle and to contribute to muscle fibre regeneration in all groups. A significantly higher expression level of GFP in the Ex group demonstrated that the survival rate of transplanted myoblasts in Ex was remarkably improved compared with that in C1 and C2. Furthermore, myofibre regeneration, characterized by centralization of the nuclei, was markedly accelerated in Ex. The expression level of CD31 in Ex was higher than that in both C1 and C2, but the differences were not statistically significant. A significantly higher expression level of Myogenin and a lower expression level of MyoD1 were both observed in Ex after 4 weeks, suggesting the promotion of differentiation to myotubes. Our findings suggest that the controlled and sustained release of bFGF from gelatin hydrogel microspheres improves the survival rate of transplanted myoblasts and promotes muscle regeneration by facilitating myogenesis rather than angiogenesis. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23554408

  16. Increased Angiogenesis and Improved Left Ventricular Function after Transplantation of Myoblasts Lacking the MyoD Gene into Infarcted Myocardium

    PubMed Central

    Nakamura, Yasuhiro; Asakura, Yoko; Piras, Bryan A.; Hirai, Hiroyuki; Tastad, Christopher T.; Verma, Mayank; Christ, Amanda J.; Zhang, Jianyi; Yamazaki, Takanori; Yoshiyama, Minoru; Asakura, Atsushi

    2012-01-01

    Skeletal myoblast transplantation has therapeutic potential for repairing damaged heart. However, the optimal conditions for this transplantation are still unclear. Recently, we demonstrated that satellite cell-derived myoblasts lacking the MyoD gene (MyoD−/−), a master transcription factor for skeletal muscle myogenesis, display increased survival and engraftment compared to wild-type controls following transplantation into murine skeletal muscle. In this study, we compare cell survival between wild-type and MyoD−/− myoblasts after transplantation into infarcted heart. We demonstrate that MyoD−/− myoblasts display greater resistance to hypoxia, engraft with higher efficacy, and show a larger improvement in ejection fraction than wild-type controls. Following transplantation, the majority of MyoD−/− and wild-type myoblasts form skeletal muscle fibers while cardiomyocytes do not. Importantly, the transplantation of MyoD−/− myoblasts induces a high degree of angiogenesis in the area of injury. DNA microarray data demonstrate that paracrine angiogenic factors, such as stromal cell-derived factor-1 (SDF-1) and placental growth factor (PlGF), are up-regulated in MyoD−/− myoblasts. In addition, over-expression and gene knockdown experiments demonstrate that MyoD negatively regulates gene expression of these angiogenic factors. These results indicate that MyoD−/− myoblasts impart beneficial effects after transplantation into an infarcted heart, potentially due to the secretion of paracrine angiogenic factors and enhanced angiogenesis in the area of injury. Therefore, our data provide evidence that a genetically engineered myoblast cell type with suppressed MyoD function is useful for therapeutic stem cell transplantation. PMID:22848585

  17. BTB-Kelch protein Krp1 regulates proliferation and differentiation of myoblasts

    PubMed Central

    Paxton, Camille W.; Cosgrove, Ruth A.; Drozd, Anja C.; Wiggins, Emma L.; Woodhouse, Sam; Watson, Rachel A.; Spence, Heather J.; Ozanne, Brad W.

    2011-01-01

    The BTB-Kelch protein Krp1 is highly and specifically expressed in skeletal muscle, where it is proposed to have a role in myofibril formation. We observed significant upregulation of Krp1 in C2 cells early in myoblast differentiation, well before myofibrillogenesis. Krp1 has a role in cytoskeletal organization and cell motility; since myoblast migration and elongation/alignment are important events in early myogenesis, we hypothesized that Krp1 is involved with earlier regulation of differentiation. Krp1 protein levels were detectable by 24 h after induction of differentiation in C2 cells and were significantly upregulated by 48 h, i.e., following the onset myogenin expression and preceding myosin heavy chain (MHC) upregulation. Upregulation of Krp1 required a myogenic stimulus as signaling derived from increased myoblast cell density was insufficient to activate Krp1 expression. Examination of putative Krp1 proximal promoter regions revealed consensus E box elements associated with myogenic basic helix-loop-helix binding. The activity of a luciferase promoter-reporter construct encompassing this 2,000-bp region increased in differentiating C2 myoblasts and in C2 cells transfected with myogenin and/or MyoD. Knockdown of Krp1 via short hairpin RNA resulted in increased C2 cell number and proliferation rate as assessed by bromodeoxyuridine incorporation, whereas overexpression of Krp1-myc had the opposite effect; apoptosis was unchanged. No effects of changed Krp1 protein levels on cell migration were observed, either by scratch wound assay or live cell imaging. Paradoxically, both knockdown and overexpression of Krp1 inhibited myoblast differentiation assessed by expression of myogenin, MEF2C, MHC, and cell fusion. PMID:21368295

  18. Expression of human dystrophin following the transplantation of genetically modified mdx myoblasts.

    PubMed

    Moisset, P A; Gagnon, Y; Karpati, G; Tremblay, J P

    1998-10-01

    Transplantation of genetically modified autologous myoblasts has been proposed as a possible solution to avoid long-term use of immunosuppressive drugs. To determine the conditions to be used in this kind of approach for possible treatment of dystrophin deficiency, mdx myoblasts were infected at different multiplicities of infection (MOI or 0.01-1000) with an adenoviral vector containing a CMV promoter/enhancer driven 6.3 kb human dystrophin cDNA (minigene) and tested in vitro for transgene expression. In these cultures, dystrophin mRNA was found to be proportionate with increasing MOI. Primary myoblast cultures derived from transgenic mdx mice expressing beta-Gal under a muscle-specific promoter and showing high expression of the human mini-dystrophin transgene introduced by the adenoviral vector were grafted into anterior tibialis muscles of SCID mice. Ten and 24 days after transplantation, numerous muscle fibers expressing both human dystrophin and beta-Gal were detected throughout the mouse muscles by immunohistochemistry using an antibody specific for human dystrophin. The presence of the human mini-dystrophin mRNA was also detected by RT-PCR. These results demonstrate that three essential conditions in autologous myoblast transplantation can be achieved: (1) in vivo survival of at least some of the transduced myoblasts; (2) efficient fusion of these cells with the host muscle fibers; and (3) the high expression of the dystrophin transgene in situ. Furthermore, this article provides a novel RT-PCR-based technique to quantify the human dystrophin minigene expression in vitro and in vivo. PMID:9930339

  19. The synthesis and stability of cytoplasmic messenger RNA during myoblast differentiation in culture.

    PubMed

    Buckingham, M E; Caput, D; Cohen, A; Whalen, R G; Gros, F

    1974-04-01

    The synthesis of poly(A)-containing cytoplasmic RNA was examined in primary myoblast cultures prepared from skeletal muscle of fetal calves. After a period of cell division, these cells undergo fusion, with concomitant appearance of acetylcholine receptor and subsequent myosin synthesis. In the dividing myoblast there is a high level of messenger RNA synthesis, including a 26S RNA, the size of a putative messenger for the large subunit of myosin. In the transition period prior to fusion, there are quantitative changes in RNA synthesis. At this time, there is a pronounced production of 26S RNA, which diminishes during fusion. The possibility that 26S RNA is accumulated in the dividing myoblast was investigated by chase experiments. At fusion, there is a marked increase in the half-lives of a number of messenger RNA species, including 26 S, which increases from about 10 hr in the dividing cell to a value of more than 50 hr. The identity of the more rapidly turning over 26 S in the myoblasts, compared to that of the 26 S at fusion, was examined in terms of polysomal distribution, migration on gels, and hybridization with complementary DNA for the myosin message. The results of these analyses suggest that the 26S species are identical. Thus, it would appear that in a predetermined cell like the myoblast, the transition to the differentiated state of myotube that is synthesizing muscle specific proteins is effected by the stabilization of messenger already being actively transcribed: terminal differentiation, with respect to myosin synthesis, is preceded by the stabilization of 26S RNA. PMID:4524649

  20. Mechanical-Stretch of C2C12 Myoblasts Inhibits Expression of Toll-Like Receptor 3 (TLR3) and of Autoantigens Associated with Inflammatory Myopathies

    PubMed Central

    Liu, Xinghui; Adriouch, Sahil; Liao, Hua

    2013-01-01

    Recent studies in patients suffering from inflammatory autoimmune myopathies suggested that moderate exercise training improves or at least stabilizes muscle strength and function without inducing disease flares. However, the precise mechanisms involved in this beneficial effect have not been extensively studied. Here we used a model of in vitro stretched C2C12 myoblasts to investigate whether mechanical stretch could influence myoblast proliferation or the expression of proinflammatory genes. Our results demonstrated that cyclic mechanical stretch stimulated C2C12 cell cycling and early up-regulation of the molecules related to mechanical-stretch pathway in muscle (calmodulin, nNOS, MMP-2, HGF and c-Met). Unexpectedly, mechanical stretch also reduced the expression of TLR3 and of proteins known to represent autoantigens in inflammatory autoimmune myopathies (Mi-2, HRS, DNA-PKcs, U1-70). Interestingly, stimulation or inhibition of calmodulin, NOS, HGF or c-Met molecules in vitro affected the expression of autoantigens and TLR3 proteins confirming their role in the inhibition of autoantigens and TLR3 during mechanical stretch. Overall, this study demonstrates for the first time that mechanical stretch could be beneficial by reducing expression of muscle autoantigens and of pro-inflammatory TLR3 and may provide new insight to understand how resistance training can reduce the symptoms associated with myositis. PMID:24224022

  1. Proteomic Analysis of C2C12 Myoblast and Myotube Exosome-Like Vesicles: A New Paradigm for Myoblast-Myotube Cross Talk?

    PubMed Central

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

    2014-01-01

    Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. We found that these vesicles displayed the classical properties of exosomes isolated from other cell types containing components of the ESCRT machinery of the MVBs, as well as numerous tetraspanins. Specific muscle proteins were also identified confirming that ELV composition also reflects their muscle origin. Furthermore quantitative analysis revealed stage-preferred expression of 31 and 78 proteins in ELV-MB and ELV-MT respectively. We found that myotube-secreted ELVs, but not ELV-MB, reduced myoblast proliferation and induced differentiation, through, respectively, the down-regulation of Cyclin D1 and the up-regulation of myogenin. We also present evidence that proteins from ELV-MT can be incorporated into myoblasts by using the GFP protein as cargo within ELV-MT. Taken together, our data provide a useful database of proteins from C2C12-released ELVs throughout myogenesis and reveals the importance of exosome-like vesicles in skeletal muscle biology. PMID:24392111

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

    PubMed

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

    2014-01-01

    Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. We found that these vesicles displayed the classical properties of exosomes isolated from other cell types containing components of the ESCRT machinery of the MVBs, as well as numerous tetraspanins. Specific muscle proteins were also identified confirming that ELV composition also reflects their muscle origin. Furthermore quantitative analysis revealed stage-preferred expression of 31 and 78 proteins in ELV-MB and ELV-MT respectively. We found that myotube-secreted ELVs, but not ELV-MB, reduced myoblast proliferation and induced differentiation, through, respectively, the down-regulation of Cyclin D1 and the up-regulation of myogenin. We also present evidence that proteins from ELV-MT can be incorporated into myoblasts by using the GFP protein as cargo within ELV-MT. Taken together, our data provide a useful database of proteins from C2C12-released ELVs throughout myogenesis and reveals the importance of exosome-like vesicles in skeletal muscle biology. PMID:24392111

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

    PubMed

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

    2016-01-29

    Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) regulates postnatal myogenesis by alleviating myostatin activity, but the molecular mechanisms by which it regulates myogenesis are not fully understood. In this study, we investigate molecular mechanisms of PPARβ/δ in myoblast differentiation. C2C12 myoblasts treated with a PPARβ/δ agonist, GW0742 exhibit enhanced myotube formation and muscle-specific gene expression. GW0742 treatment dramatically activates promyogenic kinases, p38MAPK and Akt, in a dose-dependent manner. GW0742-stimulated myoblast differentiation is mediated by p38MAPK and Akt, since it failed to restore myoblast differentiation repressed by inhibition of p38MAPK and Akt. In addition, GW0742 treatment enhances MyoD-reporter activities. Consistently, overexpression of PPARβ/δ enhances myoblast differentiation accompanied by elevated activation of p38MAPK and Akt. Collectively, these results suggest that PPARβ/δ enhances myoblast differentiation through activation of promyogenic signaling pathways. PMID:26768366

  4. Kras activation in p53-deficient myoblasts results in high-grade sarcoma formation with impaired myogenic differentiation

    PubMed Central

    McKinnon, Timothy; Venier, Rosemarie; Dickson, Brendan C.; Kabaroff, Leah; Alkema, Manon; Chen, Li; Shern, Jack F.; Yohe, Marielle E.; Khan, Javed; Gladdy, Rebecca A.

    2015-01-01

    While genomic studies have improved our ability to classify sarcomas, the molecular mechanisms involved in the formation and progression of many sarcoma subtypes are unknown. To better understand developmental origins and genetic drivers involved in rhabdomyosarcomagenesis, we describe a novel sarcoma model system employing primary murine p53-deficient myoblasts that were isolated and lentivirally transduced with KrasG12D. Myoblast cell lines were characterized and subjected to proliferation, anchorage-independent growth and differentiation assays to assess the effects of transgenic KrasG12D expression. KrasG12D overexpression transformed p53−/− myoblasts as demonstrated by an increased anchorage-independent growth. Induction of differentiation in parental myoblasts resulted in activation of key myogenic regulators. In contrast, Kras-transduced myoblasts had impaired terminal differentiation. p53−/− myoblasts transformed by KrasG12D overexpression resulted in rapid, reproducible tumor formation following orthotopic injection into syngeneic host hindlimbs. Pathological analysis revealed high-grade sarcomas with myogenic differentiation based on the expression of muscle-specific markers, such as Myod1 and Myog. Gene expression patterns of murine sarcomas shared biological pathways with RMS gene sets as determined by gene set enrichment analysis (GSEA) and were 61% similar to human RMS as determined by metagene analysis. Thus, our novel model system is an effective means to model high-grade sarcomas along the RMS spectrum. PMID:25992772

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

    PubMed

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

    2012-09-01

    Myoblasts are muscle derived mesenchymal stem cell progenitors that have great potential for use in regenerative medicine, especially for cardiomyogenesis grafts and intracardiac cell transplantation. To utilise such cells for pre-clinical and clinical applications, and especially for personalized medicine, it is essential to generate a synchronised, homogenous, population of cells that display phenotypic and genotypic homogeneity within a population of cells. We demonstrate that the biomarker-free technique of dielectrophoresis (DEP) can be used to discriminate cells between stages of differentiation in the C2C12 myoblast multipotent mouse model. Terminally differentiated myotubes were separated from C2C12 myoblasts to better than 96% purity, a result validated by flow cytometry and Western blotting. To determine the extent to which cell membrane capacitance, rather than cell size, determined the DEP response of a cell, C2C12 myoblasts were co-cultured with GFP-expressing MRC-5 fibroblasts of comparable size distributions (mean diameter ∼10 μm). A DEP sorting efficiency greater than 98% was achieved for these two cell types, a result concluded to arise from the fibroblasts possessing a larger membrane capacitance than the myoblasts. It is currently assumed that differences in membrane capacitance primarily reflect differences in the extent of folding or surface features of the membrane. However, our finding by Raman spectroscopy that the fibroblast membranes contained a smaller proportion of saturated lipids than those of the myoblasts suggests that the membrane chemistry should also be taken into account. PMID:23940503

  6. Lipogenesis in myoblasts and its regulation of CTRP6 by AdipoR1/Erk/PPARγ signaling pathway.

    PubMed

    Wu, Wenjing; Sun, Yunmei; Zhao, Chen; Zhao, Cunzhen; Chen, Xiaochang; Wang, Guoqiang; Pang, Weijun; Yang, Gongshe

    2016-06-01

    The induced lipogenesis and its regulation in C2C12 myoblasts remain largely unclear. Here, we found that the cocktail method could significantly induce lipogenesis through regulating lipid metabolic genes and Erk1/2 phosphorylation in myoblasts. Meanwhile, the expression and secretion of CTRP6 were increased during ectopic lipogenesis. Moreover, CTRP6 knockdown down-regulated the levels of lipogenic genes and phosphorylated Erk1/2 (p-Erk1/2) in the early lipogenic stage, whereas up-regulated p-Erk1/2 in the terminal differentiation. Interestingly, the effect of CTRP6 siRNA was attenuated by U0126 (a special p-Erk1/2 inhibitor) in myoblasts. Furthermore, AdipoR1, not AdipoR2, was first identified as a receptor of CTRP6 during the process of mitotic clonal expansion. Collectively, we suggest that CTRP6 mediates the ectopic lipogenesis through AdipoR1/Erk/PPARγ signaling pathway in myoblasts. Our findings will shed light on the novel biological function of CTRP6 during myoblast lipogenesis and provide a hopeful direction of improving meat quality of domestic animal by lipogenic regulation in skeletal muscle myoblasts. PMID:27125977

  7. MiRNA let-7g regulates skeletal myoblast motility via Pinch-2.

    PubMed

    Boudoukha, S; Rivera Vargas, T; Dang, I; Kropp, J; Cuvellier, S; Gautreau, A; Polesskaya, A

    2014-05-01

    Post-transcriptional regulation of gene expression by RNA-binding proteins and by small non-coding RNAs plays an important role in cell biology. Our previous results show that in murine skeletal myoblasts, the expression of Pinch-2, a focal adhesion remodeling factor that regulates cell motility, is repressed by an RNA-binding protein IMP-2/Igf2bp2. We now show that the expression of Pinch-2 is also regulated by the miRNA let-7g. Let-7g and IMP-2 repress Pinch-2 expression independently of each other. A knock-down of let-7g leads to an increase in Pinch-2 expression, and to a decrease of cell motility, which can be reversed by a simultaneous knock-down of Pinch-2. We conclude that let-7g controls the motility of mouse myoblasts in cell culture by post-transcriptionally regulating the expression of Pinch-2. PMID:24613920

  8. Trbp Is Required for Differentiation of Myoblasts and Normal Regeneration of Skeletal Muscle.

    PubMed

    Ding, Jian; Nie, Mao; Liu, Jianming; Hu, Xiaoyun; Ma, Lixin; Deng, Zhong-Liang; Wang, Da-Zhi

    2016-01-01

    Global inactivation of Trbp, a regulator of miRNA pathways, resulted in developmental defects and postnatal lethality in mice. Recently, we showed that cardiac-specific deletion of Trbp caused heart failure. However, its functional role(s) in skeletal muscle has not been characterized. Using a conditional knockout model, we generated mice lacking Trbp in the skeletal muscle. Unexpectedly, skeletal muscle specific Trbp mutant mice appear to be phenotypically normal under normal physiological conditions. However, these mice exhibited impaired muscle regeneration and increased fibrosis in response to cardiotoxin-induced muscle injury, suggesting that Trbp is required for muscle repair. Using cultured myoblast cells we further showed that inhibition of Trbp repressed myoblast differentiation in vitro. The impaired myogenesis is associated with reduced expression of muscle-specific miRNAs, miR-1a and miR-133a. Together, our study demonstrated that Trbp participates in the regulation of muscle differentiation and regeneration. PMID:27159388

  9. Trbp Is Required for Differentiation of Myoblasts and Normal Regeneration of Skeletal Muscle

    PubMed Central

    Ding, Jian; Nie, Mao; Liu, Jianming; Hu, Xiaoyun; Ma, Lixin; Deng, Zhong-Liang; Wang, Da-Zhi

    2016-01-01

    Global inactivation of Trbp, a regulator of miRNA pathways, resulted in developmental defects and postnatal lethality in mice. Recently, we showed that cardiac-specific deletion of Trbp caused heart failure. However, its functional role(s) in skeletal muscle has not been characterized. Using a conditional knockout model, we generated mice lacking Trbp in the skeletal muscle. Unexpectedly, skeletal muscle specific Trbp mutant mice appear to be phenotypically normal under normal physiological conditions. However, these mice exhibited impaired muscle regeneration and increased fibrosis in response to cardiotoxin-induced muscle injury, suggesting that Trbp is required for muscle repair. Using cultured myoblast cells we further showed that inhibition of Trbp repressed myoblast differentiation in vitro. The impaired myogenesis is associated with reduced expression of muscle-specific miRNAs, miR-1a and miR-133a. Together, our study demonstrated that Trbp participates in the regulation of muscle differentiation and regeneration. PMID:27159388

  10. RNAi Screen Reveals Potentially Novel Roles of Cytokines in Myoblast Differentiation

    PubMed Central

    Ge, Yejing; Waldemer, Rachel J.; Nalluri, Ramakrishna; Nuzzi, Paul D.; Chen, Jie

    2013-01-01

    Cytokines are cell-secreted signaling molecules that modulate various cellular functions, with the best-characterized roles in immune responses. The expression of numerous cytokines in skeletal muscle tissues and muscle cells has been reported, but their function in skeletal myogenesis, the formation of skeletal muscle, has been largely underexplored. To systematically examine the potential roles of cytokines in skeletal myogenesis, we undertook an RNAi screen of 134 mouse cytokine genes for their involvement in the differentiation of C2C12 myoblasts. Our results have uncovered 29 cytokines as strong candidates for novel myogenic regulators, potentially conferring positive and negative regulation at distinct stages of myogenesis. These candidates represent a diverse collection of cytokine families, including interleukins, TNF-related factors, and chemokines. Our findings suggest the fundamental importance of cytokines in the cell-autonomous regulation of myoblast differentiation, and may facilitate future identification of novel therapeutic targets for improving muscle regeneration and growth in health and diseases. PMID:23844157

  11. Rab35 regulates cadherin-mediated adherens junction formation and myoblast fusion

    PubMed Central

    Charrasse, Sophie; Comunale, Franck; De Rossi, Sylvain; Echard, Arnaud; Gauthier-Rouvière, Cécile

    2013-01-01

    Cadherins are homophilic cell–cell adhesion molecules implicated in many fundamental processes, such as morphogenesis, cell growth, and differentiation. They accumulate at cell–cell contact sites and assemble into large macromolecular complexes named adherens junctions (AJs). Cadherin targeting and function are regulated by various cellular processes, many players of which remain to be uncovered. Here we identify the small GTPase Rab35 as a new regulator of cadherin trafficking and stabilization at cell–cell contacts in C2C12 myoblasts and HeLa cells. We find that Rab35 accumulates at cell–cell contacts in a cadherin-dependent manner. Knockdown of Rab35 or expression of a dominant-negative form of Rab35 impaired N- and M-cadherin recruitment to cell–cell contacts, their stabilization at the plasma membrane, and association with p120 catenin and led to their accumulation in transferrin-, clathrin-, and AP-2–positive intracellular vesicles. We also find that Rab35 function is required for PIP5KIγ accumulation at cell–cell contacts and phosphatidyl inositol 4,5-bisphosphate production, which is involved in cadherin stabilization at contact sites. Finally, we show that Rab35 regulates myoblast fusion, a major cellular process under the control of cadherin-dependent signaling. Taken together, these results reveal that Rab35 regulates cadherin-dependent AJ formation and myoblast fusion. PMID:23197472

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

    SciTech Connect

    Malone, Caroline M.P.; Domaschenz, Renae; Amagase, Yoko; Dunham, Ian; Murai, Kasumi; Jones, Philip H.

    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.

  13. The E3 ubiquitin ligase TRIM32 regulates myoblast proliferation by controlling turnover of NDRG2.

    PubMed

    Mokhonova, Ekaterina I; Avliyakulov, Nuraly K; Kramerova, Irina; Kudryashova, Elena; Haykinson, Michael J; Spencer, Melissa J

    2015-05-15

    Limb girdle muscular dystrophy 2H is caused by mutations in the gene encoding the E3 ubiquitin ligase, TRIM32. Previously, we generated and characterized a Trim32 knockout mouse (T32KO) that displays both neurogenic and myopathic features. The myopathy in these mice is attributable to impaired muscle growth, associated with satellite cell senescence and premature sarcopenia. This satellite cell senescence is due to accumulation of the SUMO ligase PIASy, a substrate of TRIM32. The goal of this investigation was to identify additional substrates of TRIM32 using 2D fluorescence difference gel electrophoresis (2D-DIGE) in order to further explore its role in skeletal muscle. Because TRIM32 is an E3 ubiquitin ligase, we reasoned that TRIM32's substrates would accumulate in its absence. 2D-DIGE identified 19 proteins that accumulate in muscles from the T32KO mouse. We focused on two of these proteins, NDRG2 and TRIM72, due to their putative roles in myoblast proliferation and myogenesis. Follow-up analysis confirmed that both proteins were ubiquitinated by TRIM32 in vitro; however, only NDRG2 accumulated in skeletal muscle and myoblasts in the absence of TRIM32. NDRG2 overexpression in myoblasts led to reduced cell proliferation and delayed cell cycle withdrawal during differentiation. Thus, we identified NDRG2 as a novel target for TRIM32; these findings further corroborate the hypothesis that TRIM32 is involved in control of myogenic cells proliferation and differentiation. PMID:25701873

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

    PubMed

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

    2016-08-01

    Infrared laser therapy is used for skeletal muscle repair based on its biostimulative effect on satellite cells. However, shortening of telomere length limits regenerative potential in satellite cells, which occurs after each cell division cycle. Also, laser therapy could be more effective on non-physiologic tissues. This study evaluated low-level infrared laser exposure effects on mRNA expression from muscle injury repair and telomere stabilization genes in myoblasts in normal and stressful conditions. Laser fluences were those used in clinical protocols. C2C12 myoblast cultures were exposed to low-level infrared laser (10, 35, and 70 J/cm(2)) in standard or normal (10 %) and reduced (2 %) fetal bovine serum concentrations; total RNA was extracted for mRNA expression evaluation from muscle injury repair (MyoD and Pax7) and chromosome stabilization (TRF1 and TRF2) genes by real time quantitative polymerization chain reaction. Data show that low-level infrared laser increases the expression of MyoD and Pax7 in 10 J/cm(2) fluence, TRF1 expression in all fluences, and TRF2 expression in 70 J/cm(2) fluence in both 10 and 2 % fetal bovine serum. Low-level infrared laser increases mRNA expression from genes related to muscle repair and telomere stabilization in myoblasts in standard or normal and stressful conditions. PMID:27220530

  15. Cofilin Phosphorylation Decreased by Serum-free Starvation with Low Glucose in the L6 Myoblasts.

    PubMed

    Kim, Mee-Young; Kim, Ju-Hyun; Lee, Jeong-Uk; Lee, Lim-Kyu; Yang, Seung-Min; Park, Byoung-Sun; Jeon, Hye-Joo; Lee, Won-Deok; Noh, Ji-Woong; Kwak, Taek-Yong; Jang, Sung-Ho; Lee, Tae-Hyun; Kim, Ju-Young; Kim, Tae-Whan; Kim, Bokyung; Kim, Junghwan

    2014-10-01

    [Purpose] Many studies have been using cell culture models of muscle cells with exogenous cytokines or glucocorticoids to mimic atrophy in in vivo and in vitro tests. However, the changes in the phosphorylation of atrophy-related cofilin are still poorly understood in starved skeletal muscle cells. In this study, we first examined whether or not phosphorylation of cofilin is altered in L6 myoblasts after 3, 6, 12, 24, 48, and 72 hours of serum-free starvation with low glucose. [Methods] We used Western blotting to exam protein expression and phosphorylation in atrophied L6 myoblasts. [Results] L6 cell sizes and numbers were diminished as a result of serum-free starvation in a time-dependent manner. Serum-free starvation for 3, 6, 12, 24, 48, and 72 hours significantly decreased the phosphorylation of cofilin, respectively. [Conclusion] These results suggest that starvation-induced atrophy may be in part related to changes in the phosphorylation of cofilin in L6 myoblasts. PMID:25364107

  16. The Mutual Interactions between Mesenchymal Stem Cells and Myoblasts in an Autologous Co-Culture Model

    PubMed Central

    Szczepanska, Izabela; Zarychta-Wisniewska, Weronika; Pajak, Beata; Bojarczuk, Kamil; Dybowski, Bartosz; Paczek, Leszek

    2016-01-01

    Both myoblasts and mesenchymal stem cells (MSC) take part in the muscle tissue regeneration and have been used as experimental cellular therapy in muscular disorders treatment. It is possible that co-transplantation approach could improve the efficacy of this treatment. However, the relations between those two cell types are not clearly defined. The aim of this study was to determine the reciprocal interactions between myoblasts and MSC in vitro in terms of the features important for the muscle regeneration process. Primary caprine muscle-derived cells (MDC) and bone marrow-derived MSC were analysed in autologous settings. We found that MSC contribute to myotubes formation by fusion with MDC when co-cultured directly, but do not acquire myogenic phenotype if exposed to MDC-derived soluble factors only. Experiments with exposure to hydrogen peroxide showed that MSC are significantly more resistant to oxidative stress than MDC, but a direct co-culture with MSC does not diminish the cytotoxic effect of H2O2 on MDC. Cell migration assay demonstrated that MSC possess significantly greater migration ability than MDC which is further enhanced by MDC-derived soluble factors, whereas the opposite effect was not found. MSC-derived soluble factors significantly enhanced the proliferation of MDC, whereas MDC inhibited the division rate of MSC. To conclude, presented results suggest that myogenic precursors and MSC support each other during muscle regeneration and therefore myoblasts-MSC co-transplantation could be an attractive approach in the treatment of muscular disorders. PMID:27551730

  17. Surface apposition and multiple cell contacts promote myoblast fusion in Drosophila flight muscles

    PubMed Central

    Dhanyasi, Nagaraju; Segal, Dagan; Shimoni, Eyal; Shinder, Vera

    2015-01-01

    Fusion of individual myoblasts to form multinucleated myofibers constitutes a widely conserved program for growth of the somatic musculature. We have used electron microscopy methods to study this key form of cell–cell fusion during development of the indirect flight muscles (IFMs) of Drosophila melanogaster. We find that IFM myoblast–myotube fusion proceeds in a stepwise fashion and is governed by apparent cross talk between transmembrane and cytoskeletal elements. Our analysis suggests that cell adhesion is necessary for bringing myoblasts to within a minimal distance from the myotubes. The branched actin polymerization machinery acts subsequently to promote tight apposition between the surfaces of the two cell types and formation of multiple sites of cell–cell contact, giving rise to nascent fusion pores whose expansion establishes full cytoplasmic continuity. Given the conserved features of IFM myogenesis, this sequence of cell interactions and membrane events and the mechanistic significance of cell adhesion elements and the actin-based cytoskeleton are likely to represent general principles of the myoblast fusion process. PMID:26459604

  18. Desmin Mutation in the C-Terminal Domain Impairs Traction Force Generation in Myoblasts.

    PubMed

    Charrier, Elisabeth E; Asnacios, Atef; Milloud, Rachel; De Mets, Richard; Balland, Martial; Delort, Florence; Cardoso, Olivier; Vicart, Patrick; Batonnet-Pichon, Sabrina; Hénon, Sylvie

    2016-01-19

    The cytoskeleton plays a key role in the ability of cells to both resist mechanical stress and generate force, but the precise involvement of intermediate filaments in these processes remains unclear. We focus here on desmin, a type III intermediate filament, which is specifically expressed in muscle cells and serves as a skeletal muscle differentiation marker. By using several complementary experimental techniques, we have investigated the impact of overexpressing desmin and expressing a mutant desmin on the passive and active mechanical properties of C2C12 myoblasts. We first show that the overexpression of wild-type-desmin increases the overall rigidity of the cells, whereas the expression of a mutated E413K desmin does not. This mutation in the desmin gene is one of those leading to desminopathies, a subgroup of myopathies associated with progressive muscular weakness that are characterized by the presence of desmin aggregates and a disorganization of sarcomeres. We show that the expression of this mutant desmin in C2C12 myoblasts induces desmin network disorganization, desmin aggregate formation, and a small decrease in the number and total length of stress fibers. We finally demonstrate that expression of the E413K mutant desmin also alters the traction forces generation of single myoblasts lacking organized sarcomeres. PMID:26789769

  19. Inhibition of Na{sup +} channel currents in rat myoblasts by 4-aminopyridine

    SciTech Connect

    Lu Boxun; Liu Linyun; Liao Lei; Zhang Zhihong; Mei Yanai . E-mail: yamei@fudan.edu.cn

    2005-09-15

    Our previous study revealed that 4-aminopyridine (4-AP), a specific blocker of A-type current, could also inhibit inward Na{sup +} currents (I {sub Na}) with a state-independent mechanism in rat cerebellar granule cells. In the present study, we report an inhibitory effect of 4-AP on voltage-gated and tetrodotoxin (TTX)-sensitive I {sub Na} recorded from cultured rat myoblasts. 4-AP inhibited I {sub Na} amplitude in a dose-dependent manner between the concentrations of 0.5 and 10 mM without significant alteration in the activation or inactivation kinetics of the channel. By comparison to the 4-AP-induced inhibitory effect on cerebellum neurons, the inhibitory effect on myoblasts was enhanced through repetitive pulse and inflected by changing frequency. Specifically, the lower the frequency of pulse, the higher the inhibition observed, suggesting that block manner is inversely use-dependent. Moreover, experiments adding 4-AP to the intracellular solution indicate that the inhibitory effects are localized inside the cell. Additionally, 4-AP significantly modifies the properties of steady-state activation and inactivation kinetics of the channel. Our data suggest that the K{sup +} channel blocker 4-AP inhibits both neuron and myoblast Na{sup +} channels via different mechanisms. These findings may also provide information regarding 4-AP-induced pharmacological and toxicological effects in clinical use and experimental research.

  20. ClipR-59 Interacts with Elmo2 and Modulates Myoblast Fusion*

    PubMed Central

    Sun, Yingmin; Ren, Wenying; Côté, Jean-François; Hinds, Philip W.; Hu, Xiaoxiang; Du, Keyong

    2015-01-01

    Recent studies using ClipR-59 knock-out mice implicated this protein in the regulation of muscle function. In this report, we have examined the role of ClipR-59 in muscle differentiation and found that ClipR-59 knockdown in C2C12 cells suppressed myoblast fusion. To elucidate the molecular mechanism whereby ClipR-59 regulates myoblast fusion, we carried out a yeast two-hybrid screen using ClipR-59 as the bait and identified Elmo2, a member of the Engulfment and cell motility protein family, as a novel ClipR-59-associated protein. We showed that the interaction between ClipR-59 and Elmo2 was mediated by the atypical PH domain of Elmo2 and the Glu-Pro-rich domain of ClipR-59 and regulated by Rho-GTPase. We have examined the impact of ClipR-59 on Elmo2 downstream signaling and found that interaction of ClipR-59 with Elmo2 enhanced Rac1 activation. Collectively, our studies demonstrate that formation of an Elmo2·ClipR-59 complex plays an important role in myoblast fusion. PMID:25572395

  1. MicroRNA-16 modulates macrophage polarization leading to improved insulin sensitivity in myoblasts.

    PubMed

    Talari, Malathi; Kapadia, Bandish; Kain, Vasundhara; Seshadri, Sriram; Prajapati, Bhumika; Rajput, Parth; Misra, Parimal; Parsa, Kishore V L

    2015-12-01

    Uncontrolled inflammation leads to several diseases such as insulin resistance, T2D and several types of cancers. The functional role of microRNAs in inflammation induced insulin resistance is poorly studied. MicroRNAs are post-transcriptional regulatory molecules which mediate diverse biological processes. We here show that miR-16 expression levels are down-regulated in different inflammatory conditions such as LPS/IFNγ or palmitate treated macrophages, palmitate exposed myoblasts and insulin responsive tissues of high sucrose diet induced insulin resistant rats. Importantly, forced expression of miR-16 in macrophages impaired the production of TNF-α, IL-6 and IFN-β leading to enhanced insulin stimulated glucose uptake in co-cultured skeletal myoblasts. Further, ectopic expression of miR-16 enhanced insulin stimulated glucose uptake in skeletal myoblasts via the up-regulation of GLUT4 and MEF2A, two key players involved in insulin stimulated glucose uptake. Collectively, our data highlight the important role of miR-16 in ameliorating inflammation induced insulin resistance. PMID:26453808

  2. Rigidity-patterned polyelectrolyte films to control myoblast cell adhesion and spatial organization

    PubMed Central

    Monge, Claire; Saha, Naresh; Boudou, Thomas; Pózos-Vásquez, Cuauhtemoc; Dulong, Virginie; Glinel, Karine; Picart, Catherine

    2014-01-01

    In vivo, cells are sensitive to the stiffness of their micro-environment and especially to the spatial organization of the stiffness. In vitro studies of this phenomenon can help to better understand the mechanisms of the cell response to spatial variations of the matrix stiffness. In this work, we design polelyelectrolyte multilayer films made of poly(L-lysine) and a photo-reactive hyaluronan derivative. These films can be photo-crosslinked through a photomask to create spatial patterns of rigidity. Quartz substrates incorporating a chromium mask are prepared to expose selectively the film to UV light (in a physiological buffer), without any direct contact between the photomask and the soft film. We show that these micropatterns are chemically homogeneous and flat, without any preferential adsorption of adhesive proteins. Three groups of pattern geometries differing by their shape (circles or lines), size (form 2 to 100 μm) or interspacing distance between the motifs are used to study the adhesion and spatial organization of myoblast cells. On large circular micropatterns, the cells form large assemblies that are confined to the stiffest parts. Conversely, when the size of the rigidity patterns is subcellular, the cells respond by forming protrusions. Finally, on linear micropatterns of rigidity, myoblasts align and their nuclei drastically elongate in specific conditions. These results pave the way for the study of the different steps of myoblast fusion in response to matrix rigidity in well-defined geometrical conditions. PMID:25100929

  3. The E3 ubiquitin ligase TRIM32 regulates myoblast proliferation by controlling turnover of NDRG2

    PubMed Central

    Mokhonova, Ekaterina I.; Avliyakulov, Nuraly K.; Kramerova, Irina; Kudryashova, Elena; Haykinson, Michael J.; Spencer, Melissa J.

    2015-01-01

    Limb girdle muscular dystrophy 2H is caused by mutations in the gene encoding the E3 ubiquitin ligase, TRIM32. Previously, we generated and characterized a Trim32 knockout mouse (T32KO) that displays both neurogenic and myopathic features. The myopathy in these mice is attributable to impaired muscle growth, associated with satellite cell senescence and premature sarcopenia. This satellite cell senescence is due to accumulation of the SUMO ligase PIASy, a substrate of TRIM32. The goal of this investigation was to identify additional substrates of TRIM32 using 2D fluorescence difference gel electrophoresis (2D-DIGE) in order to further explore its role in skeletal muscle. Because TRIM32 is an E3 ubiquitin ligase, we reasoned that TRIM32's substrates would accumulate in its absence. 2D-DIGE identified 19 proteins that accumulate in muscles from the T32KO mouse. We focused on two of these proteins, NDRG2 and TRIM72, due to their putative roles in myoblast proliferation and myogenesis. Follow-up analysis confirmed that both proteins were ubiquitinated by TRIM32 in vitro; however, only NDRG2 accumulated in skeletal muscle and myoblasts in the absence of TRIM32. NDRG2 overexpression in myoblasts led to reduced cell proliferation and delayed cell cycle withdrawal during differentiation. Thus, we identified NDRG2 as a novel target for TRIM32; these findings further corroborate the hypothesis that TRIM32 is involved in control of myogenic cells proliferation and differentiation. PMID:25701873

  4. The Mutual Interactions between Mesenchymal Stem Cells and Myoblasts in an Autologous Co-Culture Model.

    PubMed

    Kulesza, Agnieszka; Burdzinska, Anna; Szczepanska, Izabela; Zarychta-Wisniewska, Weronika; Pajak, Beata; Bojarczuk, Kamil; Dybowski, Bartosz; Paczek, Leszek

    2016-01-01

    Both myoblasts and mesenchymal stem cells (MSC) take part in the muscle tissue regeneration and have been used as experimental cellular therapy in muscular disorders treatment. It is possible that co-transplantation approach could improve the efficacy of this treatment. However, the relations between those two cell types are not clearly defined. The aim of this study was to determine the reciprocal interactions between myoblasts and MSC in vitro in terms of the features important for the muscle regeneration process. Primary caprine muscle-derived cells (MDC) and bone marrow-derived MSC were analysed in autologous settings. We found that MSC contribute to myotubes formation by fusion with MDC when co-cultured directly, but do not acquire myogenic phenotype if exposed to MDC-derived soluble factors only. Experiments with exposure to hydrogen peroxide showed that MSC are significantly more resistant to oxidative stress than MDC, but a direct co-culture with MSC does not diminish the cytotoxic effect of H2O2 on MDC. Cell migration assay demonstrated that MSC possess significantly greater migration ability than MDC which is further enhanced by MDC-derived soluble factors, whereas the opposite effect was not found. MSC-derived soluble factors significantly enhanced the proliferation of MDC, whereas MDC inhibited the division rate of MSC. To conclude, presented results suggest that myogenic precursors and MSC support each other during muscle regeneration and therefore myoblasts-MSC co-transplantation could be an attractive approach in the treatment of muscular disorders. PMID:27551730

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

    PubMed Central

    2012-01-01

    myoblasts treated with IL-1β or TNF-α, resulting in normal differentiation into myotubes. Studies in aged rats as a model of sarcopenia confirmed that this pro-inflammatory cytokine pathway identified is activated during aging. Conclusions In this study, we found an unexpected connection between cytokine and Activin signaling, revealing a new mechanism by which cytokines affect skeletal muscle, and establishing the physiologic relevance of this pathway in the impaired regeneration seen in sarcopenia. PMID:22313861

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

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

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

  7. [Construction of human growth hormone lentiviral vector and its expression in murine skeletal myoblasts].

    PubMed

    Liu, Xiang-Yang; Lu, Yong-Xin; Xu, Yu-Lan; Li, Xiao-Qing; Liu, Juan; Li, Ai-Hua; Luo, Ping; Wan, Jian-Ping

    2006-03-01

    The aim of this study is to construct a lentiviral vector encoding human growth hormone, and to achieve the long, efficient and stable expression in murine skeletal myoblasts. Primary skeletal myoblasts were isolated from Sprague-Dawley rats and cultured by enzymatic digestion. We tested them by Desmin immunohistochemistry stains and found their viability was up to 94% by Trypan blue. Human growth hormone (hGH) cDNA was subcloned into expression vector pLenti6/V5-D-TOPO to construct recombinant pLenti6/V5-hGH. The pLenti6/V5-hGH and the contructed pLenti6/V5-EGFP were transfected into murine skeletal myoblasts by the Lipofectamin 2000. Through counting by the Confocal Laser Scanning Microscope, we identified the transfection efficency. We added the blasticidin to the 6-well plate with lids and obtained stable myoblasts expressing hGH. The concentration of human growth hormone (hGH) in cell culture medium was detected by Radioimmunoassay (RIA). Polymerase Chain Reaction (PCR) and DNA sequence showed hGH cDNA had been correctly inserted into pLenti6/V5-D-TOPO vector. Bright green fluorescence of the transfected cells could be observed under the Confocal Laser Scanning Microscope after 24 h transfection with pLenti6/V5-EGFP plasmids, and the transfection rate reached 40%. The difference was distinct (P < 0.01) between the pLenti6/V5- hGH groups and control groups in the secretive level of human growth hormone. After 8 weeks, the expression of human growth hormone was still stable. Then, we validated the biological characterization of the rhGH by the enzyme-link immunosorbent assay (ELISA) of the Insulin-like growth factor I (IGF-1). These results demonstrate we have successfully constructed the recombinant pLenti6/V5-hGH plasmids and accomplished rhGH long, efficient and stable expression ectopic in skeletal muscle myoblasts. PMID:16607951

  8. Defective Regulation of MicroRNA Target Genes in Myoblasts from Facioscapulohumeral Dystrophy Patients*

    PubMed Central

    Dmitriev, Petr; Stankevicins, Luiza; Ansseau, Eugenie; Petrov, Andrei; Barat, Ana; Dessen, Philippe; Robert, Thomas; Turki, Ahmed; Lazar, Vladimir; Labourer, Emmanuel; Belayew, Alexandra; Carnac, Gilles; Laoudj-Chenivesse, Dalila; Lipinski, Marc; Vassetzky, Yegor S.

    2013-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant hereditary neuromuscular disorder linked to the deletion of an integral number of 3.3-kb-long macrosatellite repeats (D4Z4) within the subtelomeric region of chromosome 4q. Most genes identified in this region are overexpressed in FSHD myoblasts, including the double homeobox genes DUX4 and DUX4c. We have carried out a simultaneous miRNome/transcriptome analysis of FSHD and control primary myoblasts. Of 365 microRNAs (miRNAs) analyzed in this study, 29 were found to be differentially expressed between FSHD and normal myoblasts. Twenty-one microRNAs (miR-1, miR-7, miR-15a, miR-22, miR-30e, miR-32, miR-107, miR-133a, miR-133b, miR-139, miR-152, miR-206, miR-223, miR-302b, miR-331, miR-362, miR-365, miR-382, miR-496, miR-532, miR-654, and miR-660) were up-regulated, and eight were down-regulated (miR-15b, miR-20b, miR-21, miR-25, miR-100, miR-155, miR-345, and miR-594). Twelve of the miRNAs up-regulated in FHSD were also up-regulated in the cells ectopically expressing DUX4c, suggesting that this gene could regulate miRNA gene transcription. The myogenic miRNAs miR-1, miR-133a, miR-133b, and miR-206 were highly expressed in FSHD myoblasts, which nonetheless did not prematurely enter myogenic differentiation. This could be accounted for by the fact that in FSHD myoblasts, functionally important target genes, including cell cycle, DNA damage, and ubiquitination-related genes, escape myogenic microRNA-induced repression. PMID:24145033

  9. Titin and myosin, but not desmin, are linked during myofibrillogenesis in postmitotic mononucleated myoblasts.

    PubMed

    Hill, C S; Duran, S; Lin, Z X; Weber, K; Holtzer, H

    1986-12-01

    Monoclonal antibodies specific for the muscle protein titin have been used in conjunction with muscle-specific antibodies against myofibrillar myosin heavy chains (MHCs) and desmin to study myogenesis in cultured cells. Desmin synthesis is initiated in replicating presumptive myoblasts, whereas the synthesis of titin and MHC is initiated simultaneously in their progeny, the postmitotic, mononucleated myoblasts. Both titin and MHC are briefly localized to nonstriated and thereafter to definitively striated myofibrils. At no stage during myofibrillogenesis is either protein observed as part of a sequence of mini-sarcomeres. Titin antibodies bind to the A-I junction, MHC antibodies to the A bands in nascent, maturing, and mature myofibrils. In contrast, desmin remains distributed as longitudinal filaments until well after the definitive myofibrils have aligned laterally. This tight temporal and topographical linkage between titin and myosin is also observed in postmitotic, mononucleated myoblasts and multinucleated myotubes when myofibrillogenesis is perturbed with Colcemid or taxol. Colcemid induces elongating postmitotic mononucleated myoblasts and multinucleated myotubes to round up and form Colcemid myosacs. The myofibrils that emerge in these rounded cells are deployed in convoluted circles. The time required for their nonstriated myofibrils to transform into striated myofibrils is greatly protracted. Furthermore, as Colcemid induces immense desmin intermediate filament cables, the normal spatial relationships between emerging individual myofibrils is distorted. Despite these disturbances at all stages, the characteristic temporal and spatial relationship observed in normal myofibrils between titin and MHC is observed in myofibrils assembling in Colcemid-treated cells. Newly born postmitotic mononucleated myoblasts, or maturing myotubes, reared in taxol acquire a star-shaped configuration and are induced to assemble "pseudo-striated myofibrils." Pseudo

  10. Ghrelin stimulates myogenic differentiation in a mouse muscle satellite cell line and in primary cultures of bovine myoblasts.

    PubMed

    Montoya-Flores, D; Mora, O; Tamariz, E; González-Dávalos, L; González-Gallardo, A; Antaramian, A; Shimada, A; Varela-Echavarría, A; Romano-Muñoz, J L

    2012-08-01

    Ghrelin is an acylated hormone that influences food intake, energy metabolism and reproduction, among others. Ghrelin may also stimulate proliferating myoblast cell differentiation and multinucleated myotube fusion. The aim of this work was to assess the effect of human ghrelin (hGHRL) and human ghrelin fragment 1-18 (hGHRL1-18) on myoblast differentiation by means of mRNA expression and protein level. Two types of cells were tested, the cell line i28 obtained from mouse skeletal muscle and primary cultures of bovine myoblasts. Both ghrelin and its N-terminal fragment hGHRL1-18 were used at concentrations of 0, 0.01, 0.1, 1, 10 and 100 nm. Treatments were applied to pre-confluent cultures and were maintained for 4 days. We determined that between 0.1 and 100 nm, hGHRL and hGRHL1-18 had similar effects on myogenic differentiation of i28 cells (p < 0.01). On the other hand, only the higher concentrations (10 and 100 nm) of hGHRL stimulated bovine myoblast differentiation. These results could be attributed to the presence, in both i28 cells and in bovine myoblasts, of the mRNA for GHS-R1a and CD36 receptors. The use of ghrelin in livestock production is still questionable because of the limited effects shown in this study, and additional research is needed in this field. PMID:21777295

  11. Administration of a soluble activin type IIB receptor promotes the transplantation of human myoblasts in dystrophic mice.

    PubMed

    Fakhfakh, Raouia; Lee, Se-Jin; Tremblay, Jacques P

    2012-01-01

    Duchenne muscular dystrophy (DMD) is a recessive disease caused by a dystrophin gene mutation. Myoblast transplantation permits the introduction of the dystrophin gene into dystrophic muscle fibers. However, this strategy has so far produced limited results. Modulation of transforming growth factor-β (TGF-β) superfamily signaling promotes skeletal muscle differentiation and growth and myogenic regeneration. We investigated the possibility that the combination of TGF-β superfamily signaling inhibition with myoblast transplantation might be an effective therapeutic approach in dystrophin-deficient patients. In vitro, blocking myostatin and other ligands with a soluble form of the extracellular domain of the activin IIB receptor (ActRIIB/Fc) upregulated the expression of myogenic differentiation factors and increased human myoblast fusion. In vivo, systemic inhibition of activin IIB receptor signaling by delivery of ActRIIB/Fc increased the success of the myoblast transplantation. This effect was further increased by forcing the mice to swim weekly to induce cycles of muscle degeneration and regeneration. Treatment of dystrophic mice with ActRIIB/Fc led to increased body weight, increased skeletal muscle mass, and improved myoblast transplantation. Thus, ActRIIB/Fc represents an effective therapeutic strategy for muscular dystrophies, and its effects are enhanced when combined with muscle exercise. PMID:22449443

  12. Myoblast proliferation and syncytial fusion both depend on connexin43 function in transfected skeletal muscle primary cultures

    SciTech Connect

    Gorbe, Aniko; Krenacs, Tibor; Cook, Jeremy E.; Becker, David L. . E-mail: d.becker@ucl.ac.uk

    2007-04-01

    Muscles are formed by fusion of individual postmitotic myoblasts to form multinucleated syncytial myotubes. The process requires a well-coordinated transition from proliferation, through migratory alignment and cycle exit, to breakdown of apposed membranes. Connexin43 protein and cell-cycle inhibitor levels are correlated, and gap junction blockers can delay muscle regeneration, so a coordinating role for gap junctions has been proposed. Here, wild-type and dominant-negative connexin43 variants (wtCx43, dnCx43) were introduced into rat myoblasts in primary culture through pIRES-eGFP constructs that made transfected cells fluoresce. GFP-positive cells and vitally-stained nuclei were counted on successive days to reveal differences in proliferation, and myotubes were counted to reveal differences in fusion. Individual transfected cells were injected with Cascade Blue, which permeates gap junctions, mixed with FITC-dextran, which requires cytoplasmic continuity to enter neighbouring cells. Myoblasts transfected with wtCx43 showed more gap-junctional coupling than GFP-only controls, began fusion sooner as judged by the incidence of cytoplasmic coupling, and formed more myotubes. Myoblasts transfected with dnCx43 remained proliferative for longer than either GFP-only or wtCx43 myoblasts, showed less coupling, and underwent little fusion into myotubes. These results highlight the critical role of gap-junctional coupling in myotube formation.

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

    PubMed Central

    Law, Cindy; Cheung, Peter

    2015-01-01

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

  14. Characterization of a plasma membrane glycoprotein common to myoblasts, skeletal muscle satellite cells, and glia.

    PubMed

    Wakshull, E; Bayne, E K; Chiquet, M; Fambrough, D M

    1983-12-01

    A plasma membrane glycoprotein common to embryonic chick myoblasts and adult chicken skeletal muscle satellite cells is the antigen recognized by monoclonal antibody C3/1. Although traces of the same antigen are present on some muscle-derived fibroblasts, the density of antigenic sites on myoblasts and satellite cells is so high that these cell types can be identified in tissues by immunocytochemical techniques. The antigen is exposed on the surfaces of myogenic cells growing in tissue culture and can be solubilized with detergent. This and other criteria establish that the antigen is a plasma membrane protein. The antigen, purified by affinity techniques, consists of a single type of polypeptide chain which migrates as a relatively broad band of apparent molecular weight 38,000 Da in SDS-polyacrylamide gel electrophoresis. It has a very small sedimentation constant, suggesting that the solubilized form is either monomeric or dimeric. The concentration of antigenic sites increases during myogenesis in vitro; but during maturation the antigenic sites are lost from muscle fibers. Electron microscopic autoradiographic study of adult muscle labeled with iodinated monoclonal antibody demonstrated unequivocally that the antigenic sites in adult muscle are concentrated in the satellite cells. Although selective for myoblasts, immature myotubes and satellite cells in the myogenic lineage, the monoclonal antibody also binds at rather high levels to peripheral Schwann cells and teloglia, to some nonneuronal cells in cultures derived from embryonic spinal cord, to some glial elements of adult chicken brain, and to several cell types in the early embryo. PMID:6360753

  15. Functional improvement of damaged adult mouse muscle by implantation of primary myoblasts.

    PubMed Central

    Irintchev, A; Langer, M; Zweyer, M; Theisen, R; Wernig, A

    1997-01-01

    1. Myoblasts from expanded primary cultures were implanted into cryodamaged soleus muscles of adult BALB/c mice. One to four months later isometric tension recordings were performed in vitro, and the male donor cells implanted into female hosts were traced on histological sections using a Y-chromosome-specific probe. The muscles were either mildly or severely cryodamaged, which led to reductions in tetanic muscle force to 33% (n = 9 muscles, 9 animals) and 70% (n = 11) of normal, respectively. Reduced forces resulted from deficits in regeneration of muscle tissue as judged from the reduced desmin-positive cross-sectional areas (34 and 66% of control, respectively). 2. Implantation of 10(6) myogenic cells into severely cryodamaged muscles more than doubled muscle tetanic force (to 70% of normal, n = 14), as well as specific force (to 66% of normal). Absolute and relative amount of desmin-positive muscle cross-sectional areas were significantly increased indicating improved microarchitecture and less fibrosis. Newly formed muscle tissue was fully innervated since the tetanic forces resulting from direct and indirect (nerve-evoked) stimulation were equal. Endplates were found on numerous Y-positive muscle fibres. 3. As judged from their position under basal laminae of muscle fibres and the expression of M-cadherin, donor-derived cells contributed to the pool of satellite cells on small- and large-diameter muscle fibres. 4. Myoblast implantation after mild cryodamage and in undamaged muscles had little or no functional or structural effects; in both preparations only a few Y-positive muscle nuclei were detected. It is concluded that myoblasts from expanded primary cultures-unlike permanent cell lines-significantly contribute to muscle regeneration only when previous muscle damage is extensive and loss of host satellite cells is severe. Images Figure 1 Figure 2 Figure 3 PMID:9161990

  16. Comparative analysis of genetically engineered immunodeficient mouse strains as recipients for human myoblast transplantation.

    PubMed

    Silva-Barbosa, Suse D; Butler-Browne, Gillian S; Di Santo, James P; Mouly, Vincent

    2005-01-01

    The development of an optimized animal model for the in vivo analysis of human muscle cells remains an important goal in the search of therapy for muscular dystrophy. Here we examined the efficiency of human myoblast xenografts in three distinct immunodeficient mouse models. We found that different conditioning regimes used to provoke host muscle regeneration (i.e., cardiotoxin versus cryodamage) had a marked impact on xenograft success. Tibialis anterior muscle of Rag2-, Rag-/gammac-, and Rag-/gammac-/C5- mice was treated by cardiotoxin or cryodamage, submitted to enzymatic digestion, and analyzed by cytofluorometry to quantitate inflammatory cells. Human myoblasts were injected into pretreated muscles from immunodeficient recipients and the cell engraftment evaluated by immunocytochemistry, 4-8 weeks after transplantation. Donor cell differentiation and dispersion within the host muscles was also investigated. Host regeneration in cardiotoxin-treated mice was accompanied by a higher inflammatory cell infiltration when compared to that induced by cryodamage. Accordingly, when compared to the cardiotoxin group, more human myogenic cells were found after cryodamage. When the distinct immunodeficient mice were compared, we found that the alymphoid strain lacking the complement component C5 (Rag-/gammac-/C5- mice) was the most efficient host for human muscle xenografts, when compared with C5(+)Rag-/gammac- mice or Rag- mice. Our results demonstrate that cryolesion-conditioned muscles of Rag-/gammac-/C5- mice provide the best environment for long-term in vivo human myoblast differentiation, opening the way for a novel approach to study the pathophysiology of human muscle disorders. PMID:16285254

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

    PubMed Central

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

    2015-01-01

    Myoblast activation is a triggering event for muscle remodeling. We assessed the stimulatory effects of propolis, a beehive product, on myoblasts. After an 8 h treatment with 100 μg/mL of Brazilian propolis ethanol extract, expression of various chemokines, including CCL-2 and CCL-5, and cytokines, such as IL-6, increased. This propolis-induced cytokine production appears to depend on NF-κB activation, because the IKK inhibitor BMS-345541 repressed mRNA levels of CCL-2 by ~66%, CCL-5 by ~81%, and IL-6 by ~69% after propolis treatment. Supernatant from propolis-conditioned C2C12 cells upregulated RAW264 macrophage migration. The supernatant also stimulated RAW264 cells to produce angiogenic factors, including VEGF-A and MMP-12. Brazilian green propolis therefore causes myoblasts to secrete cytokines and chemokines, which might contribute to tissue remodeling of skeletal muscle. PMID:26604971

  18. Linker of nucleoskeleton and cytoskeleton (LINC) complex-mediated actin-dependent nuclear positioning orients centrosomes in migrating myoblasts

    PubMed Central

    Chang, Wakam; Antoku, Susumu; Östlund, Cecilia; Worman, Howard J; Gundersen, Gregg G

    2015-01-01

    Myoblast migration is essential for muscle development and repair; however, the factors that contribute to the polarity of migrating myoblasts are relatively unknown. We find that randomly migrating C2C12 myoblasts orient their centrosomes in the direction of migration. Using wounded monolayers, we further show that centrosome orientation is stimulated by the serum factor lysophosphatidic acid (LPA) and involves the rearward movement of the nucleus while the centrosome is maintained at the cell centroid. The rate of nuclear movement correlated with that of actin retrograde flow and both cytochalasin D and blebbistatin prevented nuclear movement and centrosome orientation. Actin-dependent rearward nuclear movement in fibroblasts is mediated by assembly of nuclear membrane nesprin-2G and SUN2 LINC complexes into transmembrane actin-associated nuclear (TAN) lines anchored by A-type lamins and emerin. In C2C12 myoblasts, depletion of nesprin-2G, SUN2 or lamin A/C prevented nuclear movement and endogenous nesprin-2G and a chimeric GFP-mini-nesprin-2G formed TAN lines during nuclear movement. Depleting nesprin-2G strongly interfered with directed cell migration and reduced the efficiency of myoblast fusion into multinucleated myotubes. Our results show that nuclear movement contributes to centrosome orientation and polarity for efficient migration and fusion of myoblasts. Given that mutations in the genes encoding A-type lamins, nesprin-2 and SUN2 cause Emery-Dreifuss muscular dystrophy and related myopathies, our results have implications for understanding the mechanism of disease pathogenesis. PMID:25587885

  19. Effect of injecting primary myoblasts versus putative muscle-derived stem cells on mass and force generation in mdx mice.

    PubMed

    Mueller, Gunhild M; O'Day, Terry; Watchko, Jon F; Ontell, Marcia

    2002-06-10

    It is well established that the injection of normal myoblasts or of muscle-derived stem cells (MDSCs) into the muscle of dystrophin-deficient mdx mice results in the incorporation of a number of donor myoblasts into the host muscle. However, the effect of the injected exogenous cells on mdx muscle mass and functional capacity has not been evaluated. This study evaluates the mass and functional capacity of the extensor digitorum longus (EDL) muscles of adult, male mdx mice that received intramuscular injections of primary myoblasts or of MDSCs (isolated by a preplating technique; Qu, Z., Balkir, L., van Deutekom, J.C., Robbins, P.D., Pruchnic, R., and Huard, J., J. Cell Biol. 1998;142:1257-1267) derived from normal mice. Evaluations were made 9 weeks after cell transplantation. Uninjected mdx EDL muscles have a mass 50% greater than that of age-matched C57BL/10J (normal) EDL muscles. Injections of either primary myoblasts or MDSCs have no effect on the mass of mdx EDL muscles. EDL muscles of mdx mice generate 43% more absolute twitch tension and 43% less specific tetanic tension then do EDL muscles of C57BL/10J mice. However, the absolute tetanic and specific twitch tension of mdx and C57BL/10J EDL muscles are similar. Injection of either primary myoblasts or MDSCs has no effect on the absolute or specific twitch and tetanic tensions of mdx muscle. Approximately 25% of the myofibers in mdx EDL muscles that received primary myoblasts react positively with antibody to dystrophin. There is no significant difference in the number of dystrophin-positive myofibers when MDSCs are injected. Regardless of the source of donor cells, dystrophin is limited to short distances (60-900 microm) along the length of the myofibers. This may, in part, explain the failure of cellular therapy to alter the contractile properties of murine dystrophic muscle. PMID:12067441

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

    PubMed

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

    1993-05-01

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

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

    PubMed Central

    1993-01-01

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

  2. The Paracrine Effect of Skeletal Myoblasts Is Cardioprotective Against Oxidative Stress and Involves EGFR-ErbB4 Signaling, Cystathionase, and the Unfolded Protein Response.

    PubMed

    Siltanen, Antti; Nuutila, Kristo; Imanishi, Yukiko; Uenaka, Hisazumi; Mäkelä, Johanna; Pätilä, Tommi; Vento, Antti; Miyagawa, Shigeru; Sawa, Yoshiki; Harjula, Ari; Kankuri, Esko

    2016-01-01

    Therapeutic effects of skeletal myoblast transplantation into the myocardium are mediated via paracrine factors. We investigated the ability of myoblast-derived soluble mediators to protect cardiomyocytes from oxidative stress. Fetal rat cardiac cells were treated with conditioned medium from cultures of myoblasts or cardiac fibroblasts, and oxidative stress was induced with H2O2. Myoblast-derived factors effectively prevented oxidative stress-induced cardiac cell death and loss of mitochondrial membrane potential. This protective effect was mediated via epidermal growth factor (EGF) receptor and c-Met signaling, and mimicked by neuregulin 1 but not EGF. Microarray analysis of cardiac cells treated with myoblast versus cardiac fibroblast-derived mediators revealed differential regulation of genes associated with antioxidative effects: cystathionine-γ-lyase (cst), xanthine oxidase, and thioredoxin-interacting protein as well as tribbles homolog 3 (trib3). Cardiac cell pretreatment with tunicamycin, an inducer of trib3, also protected them against H2O2-induced cell death. Epicardial transplantation of myoblast sheets in a rat model of acute myocardial infarction was used to evaluate the expression of CST and trib3 as markers of myoblasts' paracrine effect in vivo. Myoblast sheets induced expression of the CST as well as trib3 in infarcted myocardium. CST localized around blood vessels, suggesting smooth muscle cell localization. Our results provide a deeper molecular insight into the therapeutic mechanisms of myoblast-derived paracrine signaling in cardiac cells and suggest that myoblast transplantation therapy may prevent oxidative stress-induced cardiac deterioration and progression of heart failure. PMID:26021843

  3. Dynamics of the Skeletal Muscle Secretome during Myoblast Differentiation*

    PubMed Central

    Henningsen, Jeanette; Rigbolt, Kristoffer T. G.; Blagoev, Blagoy; Pedersen, Bente Klarlund; Kratchmarova, Irina

    2010-01-01

    During recent years, increased efforts have focused on elucidating the secretory function of skeletal muscle. Through secreted molecules, skeletal muscle affects local muscle biology in an auto/paracrine manner as well as having systemic effects on other tissues. Here we used a quantitative proteomics platform to investigate the factors secreted during the differentiation of murine C2C12 skeletal muscle cells. Using triple encoding stable isotope labeling by amino acids in cell culture, we compared the secretomes at three different time points of muscle differentiation and followed the dynamics of protein secretion. We identified and quantitatively analyzed 635 secreted proteins, including 35 growth factors, 40 cytokines, and 36 metallopeptidases. The extensive presence of these proteins that can act as potent signaling mediators to other cells and tissues strongly highlights the important role of the skeletal muscle as a prominent secretory organ. In addition to previously reported molecules, we identified many secreted proteins that have not previously been shown to be released from skeletal muscle cells nor shown to be differentially released during the process of myogenesis. We found 188 of these secreted proteins to be significantly regulated during the process of myogenesis. Comparative analyses of selected secreted proteins revealed little correlation between their mRNA and protein levels, indicating pronounced regulation by posttranscriptional mechanisms. Furthermore, analyses of the intracellular levels of members of the semaphorin family and their corresponding secretion dynamics demonstrated that the release of secreted proteins is tightly regulated by the secretory pathway, the stability of the protein, and/or the processing of secreted proteins. Finally, we provide 299 unique hydroxyproline sites mapping to 48 distinct secreted proteins and have discovered a novel hydroxyproline motif. PMID:20631206

  4. The Use of Platelet-Rich and Platelet-Poor Plasma to Enhance Differentiation of Skeletal Myoblasts

    PubMed Central

    Dragoo, Jason L.

    2016-01-01

    Objectives: Platelet-rich plasma (PRP) has been has been used to augment tissue repair and regeneration after musculoskeletal injury. However, there is increasing clinical evidence that PRP, and related blood products, do not show a consistent clinical effect. The purpose of this study is to compare the effects of non-neutrophil containing PRP (LP-PRP), modified LP-PRP (Mod LP-PRP) where TGF-β1 and myostatin (MSTN) were depleted, and platelet poor plasma (PPP) on human skeletal muscle myoblast (HSMM) differentiation. Our hypothesis was that LP-PRP would lead to myoblast proliferation, not differentiation, while modifications of PRP preparations will increase myoblast differentiation, which is necessary for skeletal muscle regeneration. Methods: Blood was simultaneously processed from eight healthy human donors to create LP-PRP, Mod-LP-PRP, PPP and second spin (ss) PRP and Mod-PRP groups. Mod-PRP was created using antibodies attached to sterile beads to remove TGF- β1 and MSTN. The biologics were then individually added to human skeletal muscle myoblasts (HSMM) and were analyzed over four days. Analysis for induction into myoblast proliferation and differentiation pathways included Western blot and RT-PCR, as well as confocal microscopy to assess for polynucleated myotubule formation. Results: LP-PRP treatment lead to increased myoblast proliferation compared to PPP (1.01 x 106 vs 5.1 x 105 cells), but showed no evidence differentiation into muscle cells either by myotubule formation or via inducing myosin heavy chain (MHC) RNA compared to negative controls (0.1x fold change; p>0.05). TGF- β1 and MSTN were successfully depleted in Mod-PRP, but this modification did little to improve myoblast differentiation (0.2x fold change MHC RNA vs control; p>0.05). Application of PPP to cultures induced myoblast differentiation that included visible multinucleated myotubule formation and MHC induction compared to negative controls (9.8x fold change; p<0.05). A second

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

    PubMed

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

    2010-08-01

    Recent studies suggest that the effects of VEGF-A, the prototype VEGF ligand, may extend to a variety of cell types other than endothelial cells. The expression of VEGF-A and its main receptors, Flt-1/VEGFR-1 and KDR/Flk-1/VEGFR-2, was indeed detected in several cell types, including cardiac myocytes and regenerating myotubes. In addition to its proangiogenic activity, evidence indicates that VEGF-A can sustain skeletal muscle regeneration by enhancing the survival and migration of myogenic cells and by promoting the growth of myogenic fibers. In this study, our aim was to investigate whether VEGF could protect skeletal muscle satellite cells from apoptotic cell death triggered by reactive oxygen species and to identify the main molecular mechanisms. C2C12 mouse myoblasts, cultured in vitro in the presence of exogenous VEGF or stably transfected with a plasmid vector expressing VEGF-A, were subjected to oxidative stress and analyzed for cell growth and survival, induction of apoptosis, and molecular signaling. The results of our study demonstrated that VEGF protects C2C12 myoblasts from apoptosis induced by oxidative or hypoxic-like stress. This protection did not correlate with the modulation of the expression of VEGF receptors, but is clearly linked to the phosphorylation of the KDR/Flk-1 receptor, the activation of NF-kappaB, and/or the overexpression of the antiapoptotic protein alphaB-crystallin. PMID:20441791

  6. Caveolin-3 inhibits growth signal in cardiac myoblasts in a Ca2+-dependent manner

    PubMed Central

    Fujita, Takayuki; Otsu, Kouji; Oshikawa, Jin; Hori, Hideaki; Kitamura, Hitoshi; Ito, Takaaki; Umemura, Satoshi; Minamisawa, Susumu; Ishikawa, Yoshihiro

    2006-01-01

    Caveolin, a major protein component of caveolae, directly interacts with multiple signaling molecules, such as Ras and growth factor receptors, and inhibits their function. However, the role of the second messenger system in mediating this inhibition by caveolin remains poorly understood. We examined the role of Ca2+ -dependent signal in caveloin-mediated growth inhibition using a rat cardiac myoblast cell line (H9C2), in which the expression of caveolin-3, the muscle specific subtype, can be induced using the LacSwitch system. Upon induction with IPTG and serum-starvation, the expression of caveolin-3 was increased by 3.3-fold relative to that of mock-induced cells. The recombinant caveolin-3 was localized to the same subcellular fraction as endogenous caveolin-3 after sucrose gradient purification. Angiotensin II enhanced ERK phosphorylation, but this enhancement was significantly decreased in caveolin-3-induced cells in comparison to that in mock-induced cells. Similarly, when cells were stimulated with fetal calf serum, DNA synthesis, as determined by [3H]-thymidine incorporation, was significantly decreased in caveolin-3-induced cells. When cells were treated with Ca2+ chelator (BAPTA and EGTA), however, this attenuation was blunted. Calphostin (PKC inhibitor), but not cyclosporine A treatment (calcineurin inhibitor), blunted this attenuation in caveolin-3 induced cells. Our findings suggest that caveolin exhibits growth inhibition in a Ca2+-dependent manner, most likely through PKC, in cardiac myoblasts. PMID:16563233

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

    SciTech Connect

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

    2014-09-26

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

  8. Elastase levels and activity are increased in dystrophic muscle and impair myoblast cell survival, proliferation and differentiation

    PubMed Central

    Arecco, N.; Clarke, C. J.; Jones, F. K.; Simpson, D. M.; Mason, D.; Beynon, R. J.; Pisconti, A.

    2016-01-01

    In Duchenne muscular dystrophy, progressive loss of muscle tissue is accompanied by fibrosis, chronic inflammation and reduced muscle regenerative capacity. Although much is known about the development of fibrosis and chronic inflammation in muscular dystrophy, less is known about how they are mechanistically linked to loss of muscle regenerative capacity. We have developed a proteomics method to discover dystrophy-associated changes in the muscle progenitor cell niche, which identified serine proteases, and especially neutrophil elastase, as candidates. We show that elastase activity is increased in dystrophic (mdx4cv) muscle and impairs myoblast survival in culture. While the effect of elastase on C2C12 cell survival correlates with the kinetics of elastase-mediated degradation of the substrate to which the cells adhere, the effect of elastase on satellite cell-derived primary myoblast growth and differentiation is substrate-independent and even more dramatic than the effect on C2C12 cells, suggesting a detrimental role for elastase on myogenesis in vivo. Additionally, elastase impairs differentiation of both primary and C2C12 myoblasts into myotubes. Our findings evidence the importance of neutrophil-mediated inflammation in muscular dystrophy and indicate elastase-mediated regulation of myoblast behaviour as a potential mechanism underlying loss of regenerative capacity in dystrophic muscle. PMID:27241590

  9. Elastase levels and activity are increased in dystrophic muscle and impair myoblast cell survival, proliferation and differentiation.

    PubMed

    Arecco, N; Clarke, C J; Jones, F K; Simpson, D M; Mason, D; Beynon, R J; Pisconti, A

    2016-01-01

    In Duchenne muscular dystrophy, progressive loss of muscle tissue is accompanied by fibrosis, chronic inflammation and reduced muscle regenerative capacity. Although much is known about the development of fibrosis and chronic inflammation in muscular dystrophy, less is known about how they are mechanistically linked to loss of muscle regenerative capacity. We have developed a proteomics method to discover dystrophy-associated changes in the muscle progenitor cell niche, which identified serine proteases, and especially neutrophil elastase, as candidates. We show that elastase activity is increased in dystrophic (mdx(4cv)) muscle and impairs myoblast survival in culture. While the effect of elastase on C2C12 cell survival correlates with the kinetics of elastase-mediated degradation of the substrate to which the cells adhere, the effect of elastase on satellite cell-derived primary myoblast growth and differentiation is substrate-independent and even more dramatic than the effect on C2C12 cells, suggesting a detrimental role for elastase on myogenesis in vivo. Additionally, elastase impairs differentiation of both primary and C2C12 myoblasts into myotubes. Our findings evidence the importance of neutrophil-mediated inflammation in muscular dystrophy and indicate elastase-mediated regulation of myoblast behaviour as a potential mechanism underlying loss of regenerative capacity in dystrophic muscle. PMID:27241590

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

    PubMed Central

    Gehmert, Sebastian; Nerlich, Michael; Gosau, Martin; Klein, Silvan; Schreml, Stephan; Prantl, Lukas

    2014-01-01

    Myostatin, a TGF-β family member, is associated with inhibition of muscle growth and differentiation and might interact with the IGF-1 signaling pathway. Since IGF-1 is secreted at a bioactive level by adipose tissue-derived mesenchymal stem cells (ASCs), these cells (ASCs) provide a therapeutic option for Duchenne Muscular Dystrophy (DMD). But the protective effect of stem cell secreted IGF-1 on myoblast under high level of myostatin remains unclear. In the present study murine myoblasts were exposed to myostatin under presence of ASCs conditioned medium and investigated for proliferation and apoptosis. The protective effect of IGF-1 was further examined by using IGF-1 neutralizing and receptor antibodies as well as gene silencing RNAi technology. MyoD expression was detected to identify impact of IGF-1 on myoblasts differentiation when exposed to myostatin. IGF-1 was accountable for 43.6% of the antiapoptotic impact and 48.8% for the proliferative effect of ASCs conditioned medium. Furthermore, IGF-1 restored mRNA and protein MyoD expression of myoblasts under risk. Beside fusion and transdifferentiation the beneficial effect of ASCs is mediated by paracrine secreted cytokines, particularly IGF-1. The present study underlines the potential of ASCs as a therapeutic option for Duchenne muscular dystrophy and other dystrophic muscle diseases. PMID:24575400

  11. E2F1-miR-20a-5p/20b-5p auto-regulatory feedback loop involved in myoblast proliferation and differentiation

    PubMed Central

    Luo, Wen; Li, Guihuan; Yi, Zhenhua; Nie, Qinghua; Zhang, Xiquan

    2016-01-01

    miR-17 family microRNAs (miRNAs) are crucial for embryo development, however, their role in muscle development is still unclear. miR-20a-5p and miR-20b-5p belong to the miR-17 family and are transcribed from the miR-17~92 and miR-106a~363 clusters respectively. In this study, we found that miR-20a-5p and miR-20b-5p promoted myoblast differentiation and repressed myoblast proliferation by directly binding the 3′ UTR of E2F transcription factor 1 (E2F1) mRNA. E2F1 is an important transcriptional factor for organism’s normal development. Overexpression of E2F1 in myoblasts promoted myoblast proliferation and inhibited myoblast differentiation. Conversely, E2F1 inhibition induced myoblast differentiation and repressed myoblast proliferation. Moreover, E2F1 can bind directly to promoters of the miR-17~92 and miR-106a~363 clusters and activate their transcription, and E2F1 protein expression is correlated with the expression of pri-miR-17~92 and pri-miR-106a~363 during myoblast differentiation. These results suggested an auto-regulatory feedback loop between E2F1 and miR-20a-5p/20b-5p, and indicated that miR-20a-5p, miR-20b-5p and E2F1 are involved in myoblast proliferation and differentiation through the auto-regulation between E2F1 and miR-20a-5p/20b-5p. These findings provide new insight into the mechanism of muscle differentiation, and further shed light on the understanding of muscle development and muscle diseases. PMID:27282946

  12. Morphological changes and spatial regulation of diacylglycerol kinase-zeta, syntrophins, and Rac1 during myoblast fusion.

    PubMed

    Abramovici, Hanan; Gee, Stephen H

    2007-07-01

    The fusion of mononuclear myoblasts into multinucleated myofibers is essential for the formation and growth of skeletal muscle. Myoblast fusion follows a well-defined sequence of cellular events, from initial recognition and adhesion, to alignment, and finally plasma membrane fusion. These processes depend upon coordinated remodeling of the actin cytoskeleton. Our recent studies suggest diacylglycerol kinase-zeta (DGK-zeta), an enzyme that metabolizes diacylglycerol to yield phosphatidic acid, plays an important role in actin reorganization. Here, we investigated whether DGK-zeta has a role in the fusion of cultured C2C12 myoblasts. We show that DGK-zeta and syntrophins, scaffold proteins of the dystrophin glycoprotein complex that bind directly to DGK-zeta, are spatially regulated during fusion. Both proteins accumulated with the GTPase Rac1 at sites where fine filopodia mediate the initial contact between myoblasts. In addition, DGK-zeta codistributed with the Ca(2+)-dependent cell adhesion molecule N-cadherin at nascent, but not previously established cell contacts. We provide evidence that C2 cells are pulled together at cell-cell junctions by N-cadherin-containing filopodia reminiscent of epithelial adhesion zippers, which guide the advance of lamellipodia from apposing cells. At later times, vesicles with properties of macropinosomes formed close to cell-cell junctions. Reconstruction of confocal optical sections showed these form dome-like protrusions from the dorsal surface of contacting cells. Collectively, these results suggest DGK-zeta and syntrophins play a role at multiple stages of the fusion process. Moreover, our findings provide a potential link between changes in the lipid content of the membrane bilayer and reorganization of the actin cytoskeleton during myoblast fusion. PMID:17410543

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

    PubMed Central

    Kim, Jeffrey; Carlson, Morgan E.; Watkins, Bruce A.

    2014-01-01

    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 25 μM of arachidonate (AA) or docosahexaenoate (DHA), 25 μM of EC [anandamide (AEA), 2-arachidonoylglycerol (2-AG), docosahexaenoylethanolamide (DHEA)], 1 μM of CB1 antagonist NESS0327, and CB2 inverse agonist 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. PMID:24711795

  14. Barx2 and Pax7 Regulate Axin2 Expression in Myoblasts by Interaction with β-Catenin and Chromatin Remodelling.

    PubMed

    Hulin, Julie-Ann; Nguyen, Thi Diem Tran; Cui, Shuang; Marri, Shashikanth; Yu, Ruth T; Downes, Michael; Evans, Ronald M; Makarenkova, Helen; Meech, Robyn

    2016-08-01

    Satellite cells are the resident stem cells of skeletal muscle; quiescent in adults until activated by injury to generate proliferating myoblasts. The canonical Wnt signalling pathway, mediated by T-cell factor/lymphoid enhancer factor (TCF/LEF) and β-catenin effector proteins, controls myoblast differentiation in vitro, and recent work suggests that timely termination of the Wnt/β-catenin signal is important for normal adult myogenesis. We recently identified the Barx2 and Pax7 homeobox proteins as novel components of the Wnt effector complex. Here, we examine molecular and epigenetic mechanisms by which Barx2 and Pax7 regulate the canonical Wnt target gene Axin2, which mediates critical feedback to terminate the transcriptional response to Wnt signals. Barx2 is recruited to the Axin2 gene via TCF/LEF binding sites, recruits β-catenin and the coactivator GRIP-1, and induces local H3K-acetylation. Barx2 also promotes nuclear localization of β-catenin. Conversely, Pax7 represses Axin2 promoter/intron activity and inhibits Barx2-mediated H3K-acetylation via the corepressor HDAC1. Wnt3a not only induces Barx2 mRNA, but also stabilises Barx2 protein in myoblasts; conversely, Wnt3a potently inhibits Pax7 protein expression. As Barx2 promotes myogenic differentiation and Pax7 suppresses it, this novel posttranscriptional regulation of Barx2 and Pax7 by Wnt3a may be involved in the specification of differentiation-competent and -incompetent myoblast populations. Finally, we propose a model for dual function of Barx2 downstream of Wnt signals: activation of myogenic target genes in association with canonical myogenic regulatory factors, and regulation of the negative feedback loop that limits the response of myoblasts to Wnt signals via direct interaction of Barx2 with the TCF/β-catenin complex. Stem Cells 2016;34:2169-2182. PMID:27144473

  15. Distinct genetic programs guide Drosophila circular and longitudinal visceral myoblast fusion

    PubMed Central

    2014-01-01

    Background The visceral musculature of Drosophila larvae comprises circular visceral muscles tightly interwoven with longitudinal visceral muscles. During myogenesis, the circular muscles arise by one-to-one fusion of a circular visceral founder cell (FC) with a visceral fusion-competent myoblast (FCM) from the trunk visceral mesoderm, and longitudinal muscles arise from FCs of the caudal visceral mesoderm. Longitudinal FCs migrate anteriorly under guidance of fibroblast growth factors during embryogenesis; it is proposed that they fuse with FCMs from the trunk visceral mesoderm to give rise to syncytia containing up to six nuclei. Results Using fluorescence in situ hybridization and immunochemical analyses, we investigated whether these fusion events during migration use the same molecular repertoire and cellular components as fusion-restricted myogenic adhesive structure (FuRMAS), the adhesive signaling center that mediates myoblast fusion in the somatic mesoderm. Longitudinal muscles were formed by the fusion of one FC with Sns-positive FCMs, and defects in FCM specification led to defects in longitudinal muscle formation. At the fusion sites, Duf/Kirre and the adaptor protein Rols7 accumulated in longitudinal FCs, and Blow and F-actin accumulated in FCMs. The accumulation of these four proteins at the fusion sites argues for FuRMAS-like adhesion and signaling centers. Longitudinal fusion was disturbed in rols and blow single, and scar wip double mutants. Mutants of wasp or its interaction partner wip had no defects in longitudinal fusion. Conclusions Our results indicated that all embryonic fusion events depend on the same cell-adhesion molecules, but that the need for Rols7 and regulators of F-actin distinctly differs. Rols7 was required for longitudinal visceral and somatic myoblast fusion but not for circular visceral fusion. Importantly, longitudinal fusion depended on Kette and SCAR/Wave but was independent of WASp-dependent Arp2/3 activation. Thus, the

  16. Transforming growth factor-beta1 upregulates myostatin expression in mouse C2C12 myoblasts.

    PubMed

    Budasz-Rwiderska, M; Jank, M; Motyl, T

    2005-06-01

    Myostatin (MSTN) and transforming growth factor-beta1 (TGF-beta1) belong to the same TGF-beta superfamily of proteins. They are involved in regulation of skeletal muscle growth and development as well as muscle catabolism. The aim of the present study was to investigate the relationship between MSTN and TGF-beta1 expression in proliferating and differentiating mouse C2C12 myoblasts cultured in normal and catabolic conditions and to evaluate the effect of exogenous TGF-beta1 as well as "knock down" of TGF-beta1 receptor type II on MSTN expression in proliferating and differentiating myogenic cells. The direct effect of TGF-beta1 on myostatin was also examined. Myostatin expression increased gradually with cell confluency in proliferating cultures, while the level of TGF-beta1, detected in the form of a 100 kDa small latent complex diminished. Myostatin expression was accompanied by a partial cell cycle arrest. Three forms of myostatin were found: a 52 kDa precursor, a 40 kDa latency associated propeptide, and a 26 kDa active peptide. A decrease in myostatin and TGF-beta1 levels was observed during the first three days of differentiation, which was subsequently followed by significant increase of their expression during next three to four days of differentiation. Catabolic state induced by dexamethasone significantly increased the level of all forms of myostatin as well as latent (100 kDa) and active (25 kDa) forms of TGF-beta1 in differentiating myoblasts in a dose dependent manner. Exogenous TGF-beta1 (2 ng/ml) significantly increased myostatin levels both in proliferating and differentiating C2C12 myoblasts, whereas silencing of the TGF-beta1 receptor II gene significantly lowered myostatin level in examined cells. The presented results indicate that TGF-beta1 may control myostatin-related regulation of myogenesis through up-regulation of myostatin, predominantly in the course of terminal differentiation and glucocorticoid-dependent catabolic stimulation. PMID

  17. Skeletal muscle Kv7 (KCNQ) channels in myoblast differentiation and proliferation

    SciTech Connect

    Roura-Ferrer, Meritxell; Sole, Laura; Martinez-Marmol, Ramon; Villalonga, Nuria; Felipe, Antonio

    2008-05-16

    Voltage-dependent K{sup +} channels (Kv) are involved in myocyte proliferation and differentiation by triggering changes in membrane potential and regulating cell volume. Since Kv7 channels may participate in these events, the purpose of this study was to investigate whether skeletal muscle Kv7.1 and Kv7.5 were involved during proliferation and myogenesis. Here we report that, while myotube formation did not regulate Kv7 channels, Kv7.5 was up-regulated during cell cycle progression. Although, Kv7.1 mRNA also increased during the G{sub 1}-phase, pharmacological evidence mainly involves Kv7.5 in myoblast growth. Our results indicate that the cell cycle-dependent expression of Kv7.5 is involved in skeletal muscle cell proliferation.

  18. Construction and Myogenic Differentiation of 3D Myoblast Tissues Fabricated by Fibronectin-Gelatin Nanofilm Coating

    PubMed Central

    Gribova, Varvara; Liu, Chen Yun; Nishiguchi, Akihiro; Matsusaki, Michiya; Boudou, Thomas; Picart, Catherine; Akashi, Mitsuru

    2016-01-01

    In this study, we used a recently developed approach of coating the cells with fibronectin-gelatin nanofilms to build 3D skeletal muscle tissue models. We constructed the microtissues from C2C12 myoblasts and subsequently differentiated them to form muscle-like tissue. The thickness of the constructs could be successfully controlled by altering the number of seeded cells. We were able to build up to ~ 76 µm thick 3D constructs that formed multinucleated myotubes. We also found that Rho-kinase inhibitor Y27632 improved myotube formation in thick constructs. Our approach makes it possible to rapidly form 3D muscle tissues and is promising for the in vitro construction of physiologically relevant human skeletal muscle tissue models. PMID:27125461

  19. Construction and myogenic differentiation of 3D myoblast tissues fabricated by fibronectin-gelatin nanofilm coating.

    PubMed

    Gribova, Varvara; Liu, Chun-Yen; Nishiguchi, Akihiro; Matsusaki, Michiya; Boudou, Thomas; Picart, Catherine; Akashi, Mitsuru

    2016-06-01

    In this study, we used a recently developed approach of coating the cells with fibronectin-gelatin nanofilms to build 3D skeletal muscle tissue models. We constructed the microtissues from C2C12 myoblasts and subsequently differentiated them to form muscle-like tissue. The thickness of the constructs could be successfully controlled by altering the number of seeded cells. We were able to build up to ∼76 μm thick 3D constructs that formed multinucleated myotubes. We also found that Rho-kinase inhibitor Y27632 improved myotube formation in thick constructs. Our approach makes it possible to rapidly form 3D muscle tissues and is promising for the in vitro construction of physiologically relevant human skeletal muscle tissue models. PMID:27125461

  20. Preparation of primary myogenic precursor cell/myoblast cultures from basal vertebrate lineages.

    PubMed

    Froehlich, Jacob Michael; Seiliez, Iban; Gabillard, Jean-Charles; Biga, Peggy R

    2014-01-01

    Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e. teleost fish) and full regeneration following appendage loss (i.e. urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystoma mexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream(1-4). PMID:24835774

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  3. Betaine supplement enhances skeletal muscle differentiation in murine myoblasts via IGF-1 signaling activation

    PubMed Central

    2013-01-01

    Background Betaine (BET) is a component of many foods, including spinach and wheat. It is an essential osmolyte and a source of methyl groups. Recent studies have hypothesized that BET might play a role in athletic performance. However, BET effects on skeletal muscle differentiation and hypertrophy are still poorly understood. Methods We examined BET action on neo myotubes maturation and on differentiation process, using C2C12 murine myoblastic cells. We used RT2-PCR array, Western blot and immunofluorescence analysis to study the BET effects on morphological features of C2C12 and on signaling pathways involved in muscle differentiation and hypertrophy. Results We performed a dose–response study, establishing that 10 mM BET was the dose able to stimulate morphological changes and hypertrophic process in neo myotubes. RT2-PCR array methodology was used to identify the expression profile of genes encoding proteins involved in IGF-1 pathway. A dose of 10 mM BET was found to promote IGF-1 receptor (IGF-1 R) expression. Western blot and immunofluorescence analysis, performed in neo myotubes, pointed out that 10 mM BET improved IGF-1 signaling, synthesis of Myosin Heavy Chain (MyHC) and neo myotubes length. In addition, we investigated BET role on myoblasts proliferation and differentiation. During proliferation, BET did not modify C2C12 proliferative rate, but promoted myogenic induction, enhancing MyoD protein content and cellular elongation. During differentiation, BET caused an increase of muscle-specific markers and IGF-1 R protein levels. Conclusions Our findings provide the first evidence that BET could promote muscle fibers differentiation and increase myotubes size by IGF-1 pathway activation, suggesting that BET might represent a possible new drug/integrator strategy, not only in sport performance but also in clinical conditions characterized by muscle function impairment. PMID:23870626

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

    PubMed

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

    2016-05-01

    Factor for adipocyte differentiation 24 (fad24) is a positive regulator of adipogenesis. We previously found that human fad24 is abundantly expressed in skeletal muscle. However, the function of fad24 in skeletal muscle remains largely unknown. Because skeletal muscle is a highly regenerative tissue, we focused on the function of fad24 in skeletal muscle regeneration. In this paper, we investigated the role of fad24 in the cell cycle re-entry of quiescent C2C12 myoblasts-mimicked satellite cells. The expression levels of fad24 and histone acetyltransferase binding to ORC1 (hbo1), a FAD24-interacting factor, were elevated at the early phase of the regeneration process in response to cardiotoxin-induced muscle injury. The knockdown of fad24 inhibited the proliferation of quiescent myoblasts, whereas fad24 knockdown did not affect differentiation. S phase entry following serum activation is abrogated by fad24 knockdown in quiescent cells. Furthermore, fad24 knockdown cells show a marked accumulation of p27(Kip1) protein. These results suggest that fad24 may have an important role in the S phase re-entry of quiescent C2C12 cells through the regulation of p27(Kip1) at the protein level. PMID:26902224

  5. Sirtuin 1 promotes the proliferation of C2C12 myoblast cells via the myostatin signaling pathway.

    PubMed

    Wang, Liang; Zhang, Ting; Xi, Yongyong; Yang, Cuili; Sun, Chengcao; Li, Dejia

    2016-08-01

    Accumulating evidence suggests that Sirtuin (Sirt)1 serves a significant role in proliferation and differentiation of myoblast cells; however the signaling mechanisms involved remain to be established. Myostatin (MSTN), a member of transforming growth factor‑β family, is an vital regulator of myoblast, fibroblast growth and differentiation. To determine if MSTN is involved in the regulation of myoblast cell proliferation by Sirt1, the present study administrated the Sirt1 activator resveratrol, inhibitor nicotinamide (NAM) and MSTN inhibitor SB431542 to C2C12 myoblast cells. It was demonstrated that the Sirt1 activator, resveratrol, repressed, whereas the Sirt1 inhibitor, NAM, enhanced C2C12 myoblast cells proliferation in a Sirt1‑dependent manner. SB431542 promoted the proliferation of C2C12 myoblast cells and reversed the inhibition effect of NAM on C2C12 myoblast cell proliferation. Additionally, resveratrol upregulated the mRNA expression of MyoD, but inhibited the expression of MSTN. Additionally, NAM significantly repressed the expression of MyoD and the phosphorylation of P107 (p‑P107), but enhanced the expression of MSTN and the protein expression of P107. SB431542 significantly mitigated the effect of NAM on the expression of MyoD, P107 and p‑P107. Taken together, these results indicated that Sirt1 promotes the proliferation of C2C12 myoblast cells via the MSTN signaling pathway. PMID:27279047

  6. Stem cell antigen-1 regulates the tempo of muscle repair through effects on proliferation of {alpha}7 integrin-expressing myoblasts

    SciTech Connect

    Epting, Conrad L.; Lopez, Javier E.; Pedersen, Anissa; Brown, Courtney; Spitz, Paul; Ursell, Philip C.; Bernstein, Harold S.

    2008-03-10

    Skeletal muscle repair occurs through a programmed series of events including myogenic precursor activation, myoblast proliferation, and differentiation into new myofibers. We previously identified a role for Stem cell antigen-1 (Sca-1) in myoblast proliferation and differentiation in vitro. We demonstrated that blocking Sca-1 expression resulted in sustained myoblast cell division. Others have since demonstrated that Sca-1-null myoblasts display a similar phenotype when cultured ex vivo. To test the importance of Sca-1 during myogenesis in vivo, we employed a myonecrotic injury model in Sca-1{sup -/-} and Sca-1{sup +/+} mice. Our results demonstrate that Sca-1{sup -/-} myoblasts exhibit a hyperproliferative response consisting of prolonged and accelerated cell division in response to injury. This leads to delayed myogenic differentiation and muscle repair. These data provide the first in vivo evidence for Sca-1 as a regulator of myoblast proliferation during muscle regeneration. These studies also suggest that the balance between myogenic precursor proliferation and differentiation is critical to normal muscle repair.

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

    PubMed

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

    2016-01-01

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

  8. Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR.

    PubMed

    Wang, Haohan; Li, Xinxin; Liu, Hehe; Sun, Lingli; Zhang, Rongping; Li, Liang; Wangding, Mincheng; Wang, Jiwen

    2016-03-01

    As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway. PMID:27007909

  9. Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR

    PubMed Central

    Wang, Haohan; Li, Xinxin; Liu, Hehe; Sun, Lingli; Zhang, Rongping; Li, Liang; Wangding, Mincheng; Wang, Jiwen

    2016-01-01

    Abstract As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway. PMID:27007909

  10. Genetic Evidence That Captured Retroviral Envelope syncytins Contribute to Myoblast Fusion and Muscle Sexual Dimorphism in Mice.

    PubMed

    Redelsperger, François; Raddi, Najat; Bacquin, Agathe; Vernochet, Cécile; Mariot, Virginie; Gache, Vincent; Blanchard-Gutton, Nicolas; Charrin, Stéphanie; Tiret, Laurent; Dumonceaux, Julie; Dupressoir, Anne; Heidmann, Thierry

    2016-09-01

    Syncytins are envelope genes from endogenous retroviruses, "captured" for a role in placentation. They mediate cell-cell fusion, resulting in the formation of a syncytium (the syncytiotrophoblast) at the fetomaternal interface. These genes have been found in all placental mammals in which they have been searched for. Cell-cell fusion is also pivotal for muscle fiber formation and repair, where the myotubes are formed from the fusion of mononucleated myoblasts into large multinucleated structures. Here we show, taking advantage of mice knocked out for syncytins, that these captured genes contribute to myoblast fusion, with a >20% reduction in muscle mass, mean muscle fiber area and number of nuclei per fiber in knocked out mice for one of the two murine syncytin genes. Remarkably, this reduction is only observed in males, which subsequently show muscle quantitative traits more similar to those of females. In addition, we show that syncytins also contribute to muscle repair after cardiotoxin-induced injury, with again a male-specific effect on the rate and extent of regeneration. Finally, ex vivo experiments carried out on murine myoblasts demonstrate the direct involvement of syncytins in fusion, with a >40% reduction in fusion index upon addition of siRNA against both syncytins. Importantly, similar effects are observed with primary myoblasts from sheep, dog and human, with a 20-40% reduction upon addition of siRNA against the corresponding syncytins. Altogether, these results show a direct contribution of the fusogenic syncytins to myogenesis, with a demonstrated male-dependence of the effect in mice, suggesting that these captured genes could be responsible for the muscle sexual dimorphism observed in placental mammals. PMID:27589388

  11. Space shuttle flight (STS-45) of L8 myoblast cells results in the isolation of a nonfusing cell line variant

    NASA Technical Reports Server (NTRS)

    Kulesh, D. A.; Anderson, L. H.; Wilson, B.; Otis, E. J.; Elgin, D. M.; Barker, M. J.; Mehm, W. J.; Kearney, G. P.

    1994-01-01

    Myoblast cell cultures have been widely employed in conventional (1g) studies of biological processes because characteristics of intact muscle can be readily observed in these cultured cells. We decided to investigate the effects of spaceflight on muscle by utilizing a well characterized myoblast cell line (L8 rat myoblasts) as cultured in the recently designed Space Tissue Loss Flight Module "A" (STL-A). The STL-A is a "state of the art," compact, fully contained, automated cell culture apparatus which replaces a single mid-deck locker on the Space Shuttle. The L8 cells were successfully flown in the STL-A on the Space Shuttle STS-45 mission. Upon return to earth, reculturing of these spaceflown L8 cells (L8SF) resulted in their unexpected failure to fuse and differentiate into myotubes. This inability of the L8SF cells to fuse was found to be a permanent phenotypic alteration. Scanning electron microscopic examination of L8SF cells growing at 1g on fibronectin-coated polypropylene fibers exhibited a strikingly different morphology as compared to control cells. In addition to their failure to fuse into myotubes, L8SF cells also piled up on top of each other. When assayed in fusion-promoting soft agar, L8SF cells gave rise to substantially more and larger colonies than did either preflight (L8AT) or ground control (L8GC) cells. All data to this point indicate that flying L8 rat myoblasts on the Space Shuttle for a duration of 7-10 d at subconfluent densities results in several permanent phenotypic alterations in these cells.

  12. siRNA delivery into cultured primary human myoblasts--optimization of electroporation parameters and theoretical analysis.

    PubMed

    Lojk, Jasna; Mis, Katarina; Pirkmajer, Sergej; Pavlin, Mojca

    2015-12-01

    Introduction of genetic material into muscle tissue has been extensively researched, including isolation and in vitro expansion of primary myoblasts as a potential source of cells for skeletal and heart muscle tissue engineering applications. In this study, we optimized the electroporation protocol for introduction of short interfering ribonucleic acid (siRNA) against messenger RNA for Hypoxia Inducible Factor 1α (HIF-1α) into cultured primary human myoblasts. We established optimal pulsing protocol for siRNA electro transfection, and theoretically analyzed the effect of electric field and pulse duration on silencing efficiency and electrophoretic displacement of siRNA. Silencing of HIF-1α was determined with quantitative polymerase chain reaction and Western Blot. The most efficient silencing (71% knockdown) was achieved with 8 × 2 ms pulses, E = 0.6 kV/cm. Viability was determined immediately, 1 h and 48 h after electroporation. In general, there was a trade-off between efficient silencing and preserved viability. Electric field and pulse duration are crucial parameters for silencing, since both increase membrane permeabilization and electrophoretic transfer of siRNA. Short-term viability showed immediate toxicity of pulses due to membrane damage, while indirect effects on cell proliferation were observed after 48 h. Presented results are important for faster optimization of electroporation parameters for ex vivo electrotransfer of short RNA molecules into primary human myoblasts. PMID:26388450

  13. Brg1 Controls the Expression of Pax7 to Promote Viability and Proliferation of Mouse Primary Myoblasts.

    PubMed

    Padilla-Benavides, Teresita; Nasipak, Brian T; Imbalzano, Anthony N

    2015-12-01

    Brg1 (Brahma-related gene 1) is a catalytic component of the evolutionarily conserved mammalian SWI/SNF ATP-dependent chromatin remodeling enzymes that disrupt histone-DNA contacts on the nucleosome. While the requirement for the SWI/SNF enzymes in cell differentiation has been extensively studied, its role in precursor cell proliferation and survival is not as well defined. Muscle satellite cells constitute the stem cell pool that sustains and regenerates myofibers in adult skeletal muscle. Here, we show that deletion of Brg1 in primary mouse myoblasts derived from muscle satellite cells cultured ex vivo leads to a cell proliferation defect and apoptosis. We determined that Brg1 regulates cell proliferation and survival by controlling chromatin remodeling and activating transcription at the Pax7 promoter, which is expressed during somite development and is required for controlling viability of the satellite cell population. Reintroduction of catalytically active Brg1 or of Pax7 into Brg1-deficient satellite cells rescued the apoptotic phenotype and restored proliferation. These data demonstrate that Brg1 functions as a positive regulator for cellular proliferation and survival of primary myoblasts. Therefore, the regulation of gene expression through Brg1-mediated chromatin remodeling is critical not just for skeletal muscle differentiation but for maintaining the myoblast population as well. PMID:26036967

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

    PubMed Central

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

    2016-01-01

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

  15. Proteasome activity or expression is not altered by activation of the heat shock transcription factor Hsf1 in cultured fibroblasts or myoblasts.

    PubMed

    Taylor, David M; Kabashi, Edor; Agar, Jeffrey N; Minotti, Sandra; Durham, Heather D

    2005-01-01

    Heat shock proteins (Hsps) with chaperoning function work together with the ubiquitin-proteasome pathway to prevent the accumulation of misfolded, potentially toxic proteins, as well as to control catabolism of the bulk of cytoplasmic, cellular protein. There is evidence for the involvement of both systems in neurodegenerative disease, and a therapeutic target is the heat shock transcription factor, Hsf1, which mediates upregulation of Hsps in response to cellular stress. The mechanisms regulating expression of proteasomal proteins in mammalian cells are less well defined. To assess any direct effect of Hsf1 on expression of proteasomal subunits and activity in mammalian cells, a plasmid encoding a constitutively active form of Hsf1 (Hsf1act) was expressed in mouse embryonic fibroblasts lacking Hsf1 and in cultured human myoblasts. Plasmid encoding an inactivatible form of Hsf1 (Hsf1inact) served as control. In cultures transfected with plasmid hsf1act, robust expression of the major stress-inducible Hsp, Hsp70, occurred but not in cultures transfected with hsf1inact. No significant changes in the level of expression of representative proteasomal proteins (structural [20Salpha], a nonpeptidase beta subunit [20Sbeta3], or 2 regulatory subunits [19S subunit 6b, 11 Salpha]) or in chymotrypsin-, trypsin-, and caspaselike activities of the proteasome were measured. Thus, stress-induced or pharmacological activation of Hsf1 in mammalian cells would upregulate Hsps but not directly affect expression or activity of proteasomes. PMID:16184768

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

    PubMed

    Khor, Shy Cian; Razak, Azraul Mumtazah; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum; Abdul Karim, Norwahidah; Makpol, Suzana

    2016-01-01

    Aging results in a loss of muscle mass and strength. Myoblasts play an important role in maintaining muscle mass through regenerative processes, which are impaired during aging. Vitamin E potentially ameliorates age-related phenotypes. Hence, this study aimed to determine the effects of the tocotrienol-rich fraction (TRF) and α-tocopherol (ATF) in protecting myoblasts from replicative senescence and promoting myogenic differentiation. Primary human myoblasts were cultured into young and senescent stages and were then treated with TRF or ATF for 24 h, followed by an analysis of cell proliferation, senescence biomarkers, cellular morphology and differentiation. Our data showed that replicative senescence impaired the normal regenerative processes of myoblasts, resulting in changes in cellular morphology, cell proliferation, senescence-associated β-galactosidase (SA-β-gal) expression, myogenic differentiation and myogenic regulatory factors (MRFs) expression. Treatment with both TRF and ATF was beneficial to senescent myoblasts in reclaiming the morphology of young cells, improved cell viability and decreased SA-β-gal expression. However, only TRF treatment increased BrdU incorporation in senescent myoblasts, as well as promoted myogenic differentiation through the modulation of MRFs at the mRNA and protein levels. MYOD1 and MYOG gene expression and myogenin protein expression were modulated in the early phases of myogenic differentiation. In conclusion, the tocotrienol-rich fraction is superior to α-tocopherol in ameliorating replicative senescence-related aberration and promoting differentiation via modulation of MRFs expression, indicating vitamin E potential in modulating replicative senescence of myoblasts. PMID:26885980

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

    PubMed Central

    Khor, Shy Cian; Razak, Azraul Mumtazah; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum; Abdul Karim, Norwahidah; Makpol, Suzana

    2016-01-01

    Aging results in a loss of muscle mass and strength. Myoblasts play an important role in maintaining muscle mass through regenerative processes, which are impaired during aging. Vitamin E potentially ameliorates age-related phenotypes. Hence, this study aimed to determine the effects of the tocotrienol-rich fraction (TRF) and α-tocopherol (ATF) in protecting myoblasts from replicative senescence and promoting myogenic differentiation. Primary human myoblasts were cultured into young and senescent stages and were then treated with TRF or ATF for 24 h, followed by an analysis of cell proliferation, senescence biomarkers, cellular morphology and differentiation. Our data showed that replicative senescence impaired the normal regenerative processes of myoblasts, resulting in changes in cellular morphology, cell proliferation, senescence-associated β-galactosidase (SA-β-gal) expression, myogenic differentiation and myogenic regulatory factors (MRFs) expression. Treatment with both TRF and ATF was beneficial to senescent myoblasts in reclaiming the morphology of young cells, improved cell viability and decreased SA-β-gal expression. However, only TRF treatment increased BrdU incorporation in senescent myoblasts, as well as promoted myogenic differentiation through the modulation of MRFs at the mRNA and protein levels. MYOD1 and MYOG gene expression and myogenin protein expression were modulated in the early phases of myogenic differentiation. In conclusion, the tocotrienol-rich fraction is superior to α-tocopherol in ameliorating replicative senescence-related aberration and promoting differentiation via modulation of MRFs expression, indicating vitamin E potential in modulating replicative senescence of myoblasts. PMID:26885980

  18. Photovoltaic surfaces enable clonal myoblastic cell release using visible light as external stimulation.

    PubMed

    Bhuyan, Mohammod Kabir; Rodriguez, Jorge; Tseng, Tzu-Liang Bill; Boland, Thomas

    2016-03-01

    Many new biomedical approaches to treating disease require the supply of cells delivered to an injured or diseased organ either individually, collectively as aggregates or sheets, or encapsulated with a scaffold. The collection of cells is accomplished by using enzymatic digestion witch suffer from the need to remove the enzymes after digestion. In addition, enzymatic methods are not applicable for all cells, cell aggregates, cell sheets or 3D structures. The objective of this study was to investigate the release of cultured cells from silicon based Photovoltaic (PV) surfaces using a light source as external stimulation. C2C12 myoblasts were cultured on the negative surface of a PV device and upon confluence they were exposed to light. The amount of released cells was quantified as a function light exposure. It was found that light exposure at 25 000 lux for one hour caused equivalent cell release from the PV surface than trypsination. The released cells are viable and can be re-cultured if needed. This mechanism may offer an alternative method to release excitable cells without using an enzymatic agent. This may be important for cell therapy if larger cell structures such as sheets need to be collected. PMID:26710125

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

    PubMed Central

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

    2016-01-01

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

  20. Creating Hierarchical Topographies on Fibrous Platforms Using Femtosecond Laser Ablation for Directing Myoblasts Behavior.

    PubMed

    Jun, Indong; Chung, Yong-Woo; Heo, Yun-Hoe; Han, Hyung-Seop; Park, Jimin; Jeong, Hongsoo; Lee, Hyunjung; Lee, Yu Bin; Kim, Yu-Chan; Seok, Hyun-Kwang; Shin, Heungsoo; Jeon, Hojeong

    2016-02-10

    Developing an artificial extracellular matrix that closely mimics the native tissue microenvironment is important for use as both a cell culture platform for controlling cell fate and an in vitro model system for investigating the role of the cellular microenvironment. Electrospinning, one of the methods for fabricating structures that mimic the native ECM, is a promising technique for creating fibrous platforms. It is well-known that align or randomly distributed electrospun fibers provide cellular contact guidance in a single pattern. However, native tissues have hierarchical structures, i.e., topographies on the micro- and nanoscales, rather than a single structure. Thus, we fabricated randomly distributed nanofibrous (720 ± 80 nm in diameter) platforms via a conventional electrospinning process, and then we generated microscale grooves using a femtosecond laser ablation process to develop engineered fibrous platforms with patterned hierarchical topographies. The engineered fibrous platforms can regulate cellular adhesive morphology, proliferation, and distinct distribution of focal adhesion proteins. Furthermore, confluent myoblasts cultured on the engineered fibrous platforms revealed that the direction of myotube assembly can be controlled. These results indicate that our engineered fibrous platforms may be useful tools in investigating the roles of nano- and microscale topographies in the communication between cells and ECM. PMID:26771693

  1. Chitooligomer-Immobilized Biointerfaces with Micropatterned Geometries for Unidirectional Alignment of Myoblast Cells.

    PubMed

    Poosala, Pornthida; Kitaoka, Takuya

    2016-01-01

    Skeletal muscle possesses a robust capacity to regenerate functional architectures with a unidirectional orientation. In this study, we successfully arranged skeletal myoblast (C2C12) cells along micropatterned gold strips on which chitohexaose was deposited via a vectorial chain immobilization approach. Hexa-N-acetyl-D-glucosamine (GlcNAc6) was site-selectively modified at its reducing end with thiosemicarbazide, then immobilized on a gold substrate in striped micropatterns via S-Au chemisorption. Gold micropatterns ranged from 100 to 1000 µm in width. Effects of patterning geometries on C2C12 cell alignment, morphology, and gene expression were investigated. Unidirectional alignment of C2C12 cells having GlcNAc6 receptors was clearly observed along the micropatterns. Decreasing striped pattern width increased cell attachment and proliferation, suggesting that the fixed GlcNAc6 and micropatterns impacted cell function. Possibly, interactions between nonreducing end groups of fixed GlcNAc6 and cell surface receptors initiated cellular alignment. Our technique for mimicking native tissue organization should advance applications in tissue engineering. PMID:26784249

  2. Chitooligomer-Immobilized Biointerfaces with Micropatterned Geometries for Unidirectional Alignment of Myoblast Cells

    PubMed Central

    Poosala, Pornthida; Kitaoka, Takuya

    2016-01-01

    Skeletal muscle possesses a robust capacity to regenerate functional architectures with a unidirectional orientation. In this study, we successfully arranged skeletal myoblast (C2C12) cells along micropatterned gold strips on which chitohexaose was deposited via a vectorial chain immobilization approach. Hexa-N-acetyl-d-glucosamine (GlcNAc6) was site-selectively modified at its reducing end with thiosemicarbazide, then immobilized on a gold substrate in striped micropatterns via S–Au chemisorption. Gold micropatterns ranged from 100 to 1000 µm in width. Effects of patterning geometries on C2C12 cell alignment, morphology, and gene expression were investigated. Unidirectional alignment of C2C12 cells having GlcNAc6 receptors was clearly observed along the micropatterns. Decreasing striped pattern width increased cell attachment and proliferation, suggesting that the fixed GlcNAc6 and micropatterns impacted cell function. Possibly, interactions between nonreducing end groups of fixed GlcNAc6 and cell surface receptors initiated cellular alignment. Our technique for mimicking native tissue organization should advance applications in tissue engineering. PMID:26784249

  3. The homeobox transcription factor Irxl1 negatively regulates MyoD expression and myoblast differentiation.

    PubMed

    Chuang, Han-Ni; Hsiao, Kuang-Ming; Chang, Hui-Yi; Wu, Chia-Chi; Pan, Huichin

    2014-07-01

    Irxl1/Mkx (Iroquois homeobox-like 1/Mohawk) encodes a member of the TALE subfamily of homeodomain proteins. It is expressed in multiple mesoderm-derived tissues and has recently been shown to regulate tendon differentiation during mouse embryonic development. Previously we showed that knockdown of Irxl1 in zebrafish caused a deficit in neural crest cells which consequently resulted in deformation of craniofacial muscles and arch cartilages. Here, we further demonstrate that loss of Irxl1 function results in deformed somites with disordered muscle fibers and myotendinous junctions. Because expression of myoD is increased in the somites of Irxl1 knockdown morphants, we test whether Irxl1 negatively regulates myoD expression. When stable C2C12 myoblasts overexpressing Irxl1/Mkx were induced to differentiate, myotube formation was inhibited and protein levels of myoD and myosin heavy chain were decreased accordingly. A series of deletion constructs of myoD promoter fragments were tested by luciferase reporter assays, which identified a promoter fragment that is necessary and sufficient for Irxl1-mediated repression. Direct interaction of Irxl1 and myoD promoter was subsequently elucidated by yeast one-hybrid assays, electrophoretic mobility shift assays and chromatin immunoprecipitation analysis. Furthermore, mouse Mkx also binds to and represses myoD promoter. These results indicate that Irxl1/Mkx can repress myoD expression through direct binding to its promoter and may thus play a negative regulatory role in muscle differentiation. PMID:24814716

  4. Reduced proliferative activity of primary POMGnT1-null myoblasts in vitro.

    PubMed

    Miyagoe-Suzuki, Yuko; Masubuchi, Nami; Miyamoto, Kaori; Wada, Michiko R; Yuasa, Shigeki; Saito, Fumiaki; Matsumura, Kiichiro; Kanesaki, Hironori; Kudo, Akira; Manya, Hiroshi; Endo, Tamao; Takeda, Shin'ichi

    2009-01-01

    Protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) is an enzyme that transfers N-acetylglucosamine to O-mannose of glycoproteins. Mutations of the POMGnT1 gene cause muscle-eye-brain (MEB) disease. To obtain a better understanding of the pathogenesis of MEB disease, we mutated the POMGnT1 gene in mice using a targeting technique. The mutant muscle showed aberrant glycosylation of alpha-DG, and alpha-DG from mutant muscle failed to bind laminin in a binding assay. POMGnT1(-/-) muscle showed minimal pathological changes with very low-serum creatine kinase levels, and had normally formed muscle basal lamina, but showed reduced muscle mass, reduced numbers of muscle fibers, and impaired muscle regeneration. Importantly, POMGnT1(-/-) satellite cells proliferated slowly, but efficiently differentiated into multinuclear myotubes in vitro. Transfer of a retrovirus vector-mediated POMGnT1 gene into POMGnT1(-/-) myoblasts completely restored the glycosylation of alpha-DG, but proliferation of the cells was not improved. Our results suggest that proper glycosylation of alpha-DG is important for maintenance of the proliferative activity of satellite cells in vivo. PMID:19114101

  5. Adult stem cells for cardiac repair: a choice between skeletal myoblasts and bone marrow stem cells.

    PubMed

    Ye, Lei; Haider, Husnain Kh; Sim, Eugene K W

    2006-01-01

    The real promise of a stem cell-based approach for cardiac regeneration and repair lies in the promotion of myogenesis and angiogenesis at the site of the cell graft to achieve both structural and functional benefits. Despite all of the progress and promise in this field, many unanswered questions remain; the answers to these questions will provide the much-needed breakthrough to harness the real benefits of cell therapy for the heart in the clinical perspective. One of the major issues is the choice of donor cell type for transplantation. Multiple cell types with varying potentials have been assessed for their ability to repopulate the infarcted myocardium; however, only the adult stem cells, that is, skeletal myoblasts (SkM) and bone marrow-derived stem cells (BMC), have been translated from the laboratory bench to clinical use. Which of these two cell types will provide the best option for clinical application in heart cell therapy remains arguable. With results pouring in from the long-term follow-ups of previously conducted phase I clinical studies, and with the onset of phase II clinical trials involving larger population of patients, transplantation of stem cells as a sole therapy without an adjunct conventional revascularization procedure will provide a deeper insight into the effectiveness of this approach. The present article discusses the pros and cons of using SkM and BMC individually or in combination for cardiac repair, and critically analyzes the progress made with each cell type. PMID:16380640

  6. MyoD-positive myoblasts are present in mature fetal organs lacking skeletal muscle

    PubMed Central

    Gerhart, Jacquelyn; Bast, Brian; Neely, Christine; Iem, Stephanie; Amegbe, Paula; Niewenhuis, Robert; Miklasz, Steven; Cheng, Pei Feng; George-Weinstein, Mindy

    2001-01-01

    The epiblast of the chick embryo gives rise to the ectoderm, mesoderm, and endoderm during gastrulation. Previous studies revealed that MyoD-positive cells were present throughout the epiblast, suggesting that skeletal muscle precursors would become incorporated into all three germ layers. The focus of the present study was to examine a variety of organs from the chicken fetus for the presence of myogenic cells. RT-PCR and in situ hybridizations demonstrated that MyoD-positive cells were present in the brain, lung, intestine, kidney, spleen, heart, and liver. When these organs were dissociated and placed in culture, a subpopulation of cells differentiated into skeletal muscle. The G8 antibody was used to label those cells that expressed MyoD in vivo and to follow their fate in vitro. Most, if not all, of the muscle that formed in culture arose from cells that expressed MyoD and G8 in vivo. Practically all of the G8-positive cells from the intestine differentiated after purification by FACS®. This population of ectopically located cells appears to be distinct from multipotential stem cells and myofibroblasts. They closely resemble quiescent, stably programmed skeletal myoblasts with the capacity to differentiate when placed in a permissive environment. PMID:11684706

  7. Gravitational force modulates G2/M phase exit in mechanically unloaded myoblasts

    PubMed Central

    Benavides Damm, Tatiana; Franco-Obregón, Alfredo; Egli, Marcel

    2013-01-01

    Prolonged spaceflight gives rise to muscle loss and reduced strength, a condition commonly referred to as space atrophy. During exposure to microgravity, skeletal muscle myoblasts are mechanically unloaded and respond with attenuated cell proliferation, slowed cell cycle progression, and modified protein expression. To elucidate the underlying mechanisms by which muscle mass declines in response to prolonged microgravity exposure, we grew C2C12 mouse muscle cells under conditions of simulated microgravity (SM) and analyzed their proliferative capacity, cell cycle progression, and cyclin B and D expression. We demonstrated that the retarded cell growth observed in SM was correlated with an approximate 16 h delay in G2/M phase progression, where cells accumulated specifically between the G2 checkpoint and the onset of anaphase, concomitantly with a positive expression for cyclin B. The effect was specific for gravitational mechanical unloading as cells grown under conditions of hypergravity (HG, 4 g) for similar durations of time exhibited normal proliferation and normal cell cycle progression. Our results show that SM and HG exert phenomenological distinct responses over cell cycle progression. The deficits of SM can be restored by terrestrial gravitational force, whereas the effects of HG are indistinguishable from the 1 g control. This suggests that the mechanotransduction apparatus of cells responds differently to mechanical unloading and loading. PMID:23974110

  8. Melatonin behavior in restoring chemical damaged C2C12 myoblasts.

    PubMed

    Salucci, Sara; Baldassarri, Valentina; Canonico, Barbara; Burattini, Sabrina; Battistelli, Michela; Guescini, Michele; Papa, Stefano; Stocchi, Vilberto; Falcieri, Elisabetta

    2016-06-01

    It is known that, besides a wide range of functions, melatonin provides protection against oxidative stress, thanks to its ability to act, directly, as a free radical scavenger and, indirectly, by stimulating antioxidant enzymes production and mitochondrial electron transport chain efficiency. Oxidative stress is one of the major players in initiating apoptotic cell death in skeletal muscle, as well as in other tissues. Apoptosis is essential for skeletal muscle development and homeostasis; nevertheless, its misregulation has been frequently observed in several myopathies, in sarcopenia, as well as in denervation and disuse. Melatonin activity was investigated in undifferentiated C2C12 skeletal muscle cells, after exposure to various apoptotic chemical triggers, chosen for their different mechanisms of action. Cells were pretreated with melatonin and then exposed to hydrogen peroxide, etoposide and staurosporine. Morphofunctional and molecular analyses show that in myoblasts melatonin prevents oxidative stress and apoptosis induced by chemicals following, at least in part, the mitochondria pathway. These results confirm melatonin ability to act as an antioxidant and antiapoptotic molecule in skeletal muscle cells, thus suggesting a possible therapeutic strategy for myopathies involving apoptosis misregulation. Microsc. Res. Tech. 79:532-540, 2016. © 2016 Wiley Periodicals, Inc. PMID:27059940

  9. Distinctive Effects of Cytochalasin B in Chick Primary Myoblasts and Fibroblasts

    PubMed Central

    de Andrade, Ivone Rosa; Costa, Manoel Luis; Mermelstein, Claudia

    2016-01-01

    Actin-based structures play fundamental roles in cellular functions. However it remains controversial how cells cope with the absence of F-actin structures. This report focuses on short- and long-term effects of cytochalasin B (CB) on actin-complexes in fibroblasts and myoblasts. Thirty min of CB treatment dispersed subplasma actin cortices, lamellipodia, ruffled membranes, stress fibers and adhesion plaques into actin patches in fibroblasts and muscle cells. In contrast, 72 hrs CB treatment showed distinct morphological effects. Fibroblasts became giant multinucleated-finger shaped with 5 to 10 protrusions, 3–8 μm in width, and >200 μm in length. They lacked cortical actin, stress fibers, adhesion plaques and ruffled membranes but contained immense lamelliopodia with abnormal adhesion plaque protein complexes. Muscle cells transformed into multinucleated globular-shaped but contained normal I-Z-I and A-bands, indicating that CB did not interfere with the assembly of myofibrils. Within 30 min after CB removal, finger-shaped fibroblasts returned to their original shape and actin-containing structures rapidly reappeared, whereas muscle cells respond slowly to form elongated myotubes following CB washout. The capacity to grow, complete several nuclear cycles, assemble intermediate filaments and microtubules without a morphologically recognizable actin cytoskeleton raises interesting issues related to the role of the actin compartments in eukaryotic cells. PMID:27119825

  10. Novel Endogenous, Insulin-Stimulated Akt2 Protein Interaction Partners in L6 Myoblasts

    PubMed Central

    Caruso, Michael; Zhang, Xiangmin; Ma, Danjun; Yang, Zhao; Qi, Yue; Yi, Zhengping

    2015-01-01

    Insulin resistance and Type 2 diabetes are marked by an aberrant response in the insulin signaling network. The phosphoinositide-dependent serine/threonine kinase, Akt2, plays a key role in insulin signaling and glucose uptake, most notably within skeletal muscle. Protein-protein interaction regulates the functional consequence of Akt2 and in turn, Akt2’s role in glucose uptake. However, only few insulin-responsive Akt2 interaction partners have been identified in skeletal muscle cells. In the present work, rat L6 myoblasts, a widely used insulin sensitive skeletal muscle cell line, were used to examine endogenous, insulin-stimulated Akt2 protein interaction partners. Akt2 co-immunoprecipitation was coupled with 1D-SDS-PAGE and fractions were analyzed by HPLC-ESI-MS/MS to reveal Akt2 protein-protein interactions. The pull-down assay displayed specificity for the Akt2 isoform; Akt1 and Akt3 unique peptides were not detected. A total of 49 were detected with a significantly increased (47) or decreased (2) association with Akt2 following insulin administration (n = 4; p<0.05). Multiple pathways were identified for the novel Akt2 interaction partners, such as the EIF2 and ubiquitination pathways. These data suggest that multiple new endogenous proteins may associate with Akt2 under basal as well as insulin-stimulated conditions, providing further insight into the insulin signaling network. Data are available via ProteomeXchange with identifier PXD002557. PMID:26465754

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

    PubMed

    Muratore, Massimo; Mitchell, Steve; Waterfall, Martin

    2013-09-01

    Multipotent progenitor cells have shown promise for use in biomedical applications and regenerative medicine. The implementation of such cells for clinical application requires a synchronized, phenotypically and/or genotypically, homogenous cell population. Here we have demonstrated the implementation of a biological tag-free dielectrophoretic device used for discrimination of multipotent myoblastic C2C12 model. The multipotent capabilities in differentiation, for these cells, diminishes with higher passage number, so for cultures above 70 passages only a small percentage of cells is able to differentiate into terminal myotubes. In this work we demonstrated that we could recover, above 96% purity, specific cell types from a mixed population of cells at high passage number without any biological tag using dielectrophoresis. The purity of the samples was confirmed by cytometric analysis using the cell specific marker embryonic myosin. To further investigate the dielectric properties of the cell plasma membrane we co-culture C2C12 with similar size, when in suspension, GFP-positive fibroblast as feeder layer. The level of separation between the cell types was above 98% purity which was confirmed by flow cytometry. These levels of separation are assumed to account for cell size and for the plasma membrane morphological differences between C2C12 and fibroblast unrelated to the stages of the cell cycle which was assessed by immunofluorescence staining. Plasma membrane conformational differences were further confirmed by scanning electron microscopy. PMID:23933253

  12. Establishment of a Novel Primary Human Skeletal Myoblast Cellular Model for Chikungunya Virus Infection and Pathogenesis

    PubMed Central

    Hussain, Khairunnisa’ Mohamed; Lee, Regina Ching Hua; Ng, Mary Mah-Lee; Chu, Justin Jang Hann

    2016-01-01

    Chikungunya virus (CHIKV) is a re-emerging arbovirus known to cause chronic myalgia and arthralgia and is now considered endemic in countries across Asia and Africa. The tissue tropism of CHIKV infection in humans remains, however, ill-defined. Due to the fact that myositis is commonly observed in most patients infected with CHIKV, we sought to develop a clinically relevant cellular model to better understand the pathogenesis of CHIKV infection. In this study, primary human skeletal muscle myoblasts (HSMM) were established as a novel human primary cell line that is highly permissive to CHIKV infection, with maximal amounts of infectious virions observed at 16 hours post infection. Genome-wide microarray profiling analyses were subsequently performed to identify and map genes that are differentially expressed upon CHIKV infection. Infection of HSMM cells with CHIKV resulted in altered expressions of host genes involved in skeletal- and muscular-associated disorders, innate immune responses, cellular growth and death, host metabolism and virus replication. Together, this study has shown the establishment of a clinically relevant primary human cell model that paves the way for the further analysis of host factors and their involvement in the various stages of CHIKV replication cycle and viral pathogenesis. PMID:26892458

  13. An NF-κB--EphrinA5-Dependent Communication between NG2(+) Interstitial Cells and Myoblasts Promotes Muscle Growth in Neonates.

    PubMed

    Gu, Jin-Mo; Wang, David J; Peterson, Jennifer M; Shintaku, Jonathan; Liyanarachchi, Sandya; Coppola, Vincenzo; Frakes, Ashley E; Kaspar, Brian K; Cornelison, Dawn D; Guttridge, Denis C

    2016-01-25

    Skeletal muscle growth immediately following birth is critical for proper body posture and locomotion. However, compared with embryogenesis and adulthood, the processes regulating the maturation of neonatal muscles is considerably less clear. Studies in the 1960s predicted that neonatal muscle growth results from nuclear accretion of myoblasts preferentially at the tips of myofibers. Remarkably, little information has been added since then to resolve how myoblasts migrate to the ends of fibers. Here, we provide insight into this process by revealing a unique NF-κB-dependent communication between NG2(+) interstitial cells and myoblasts. NF-κB in NG2(+) cells promotes myoblast migration to the tips of myofibers through cell-cell contact. This occurs through expression of ephrinA5 from NG2(+) cells, which we further deduce is an NF-κB target gene. Together, these results suggest that NF-κB plays an important role in the development of newborn muscles to ensure proper myoblast migration for fiber growth. PMID:26777211

  14. Downregulated microRNA-23b promotes BMP9-mediated osteogenesis in C2C12 myoblast cells by targeting Runx2

    PubMed Central

    CHEN, CHU; TANG, ZUCHUAN; SONG, QILING; YANG, MIN; SHI, QIONG; WENG, YAGUANG

    2016-01-01

    MicroRNAs are identified as negative regulators in gene expression through silencing gene expression at the post-transcriptional and translational levels. Bone morphogenetic protein 9 (BMP9) is the most effective in inducing osteogenesis in the BMP family, the members of which were originally identified as osteoinductive cytokines. In the current study, the role of miR-23b in the progression of BMP9-induced C2C12 myoblasts was investigated. The results indicated that miR-23b was significantly downregulated in C2C12 myoblasts induced by BMP9. Overexpression of miR-23b significantly inhibited osteogenesis in the C2C12 myoblasts. In addition, it was observed that Runx2 was negatively regulated by miR-23b at the post-transcriptional level, via a specific target site within the 3′UTR of Runx2. Knockdown of Runx2 promoted miR-23b-induced inhibition of osteogenesis in C2C12 myoblasts. The expression of Runx2 was observed to be frequently upregulated in osteoblast cell lines and inversely correlated with miR-23b expression. Thus, the results of the present study suggest that miR-23b inhibits BMP9-induced C2C12 myoblast osteogenesis via targeting of the Runx2 gene, acting as a suppressor. The current study contributes to the understanding of the functions of BMP9 in ossification. PMID:26820568

  15. Application of novel anodized titanium for enhanced recruitment of H9C2 cardiac myoblast

    PubMed Central

    Behjati, Mohaddeseh; Moradi, Iman; Kazemi, Mohammad

    2015-01-01

    Objective(s): Anodized treated titanium surfaces, have been proposed as potential surfaces with better cell attachment capacities. We have investigated the adhesion and proliferation properties of H9C2 cardiac myoblasts on anodized treated titanium surface. Materials and Methods: Surface topography and anodized tubules were examined by high-resolution scanning electron microscopy (SEM). Control and test substrates were inserted to the bottom of 24-well tissue culture plates. Culture media including H9C2 cells were loaded on the surface of substrate and control wells at the second passage. Evaluation of cell growth, proliferation, viability and surface cytotoxicity was performed using MTT test. After 48 hr, some samples were inspected by SEM. DAPI-staining was used to count attached cells. Results: MTT results for cells cultured on anodized titanium and unanodized titanium surfaces was equal to 1.56 and 0.55 fold change compared to tissue culture polystyrene (TCPS). The surface had no cytotoxic effects on cells. The average cell attachment to TCPS, unanodized and anodized titanium surface was 2497±40.16, 1250±20.11 and 4859.5±54.173, respectively. Cell adhesion to anodized titanium was showed 1.95 and 3.89 fold increase compared to TCPS and unanodized titanium, respectively (P<0.05). Conclusion: Anodized titanium surfaces can be potentially applied for enhanced recruitment of H9C2 cells. This unique property makes these inexpensive anodized surfaces as a candidate surface for attachment of cardiac cells and consequently for cardiac regeneration purposes. PMID:26526098

  16. Keap1 redox-dependent regulation of doxorubicin-induced oxidative stress response in cardiac myoblasts.

    PubMed

    Nordgren, Kendra K S; Wallace, Kendall B

    2014-01-01

    Doxorubicin (DOX) is a widely prescribed treatment for a broad scope of cancers, but clinical utility is limited by the cumulative, dose-dependent cardiomyopathy that occurs with repeated administration. DOX-induced cardiotoxicity is associated with the production of reactive oxygen species (ROS) and oxidation of lipids, DNA and proteins. A major cellular defense mechanism against such oxidative stress is activation of the Keap1/Nrf2-antioxidant response element (ARE) signaling pathway, which transcriptionally regulates expression of antioxidant genes such as Nqo1 and Gstp1. In the present study, we address the hypothesis that an initial event associated with DOX-induced oxidative stress is activation of the Keap1/Nrf2-dependent expression of antioxidant genes and that this is regulated through drug-induced changes in redox status of the Keap1 protein. Incubation of H9c2 rat cardiac myoblasts with DOX resulted in a time- and dose-dependent decrease in non-protein sulfhydryl groups. Associated with this was a near 2-fold increase in Nrf2 protein content and enhanced transcription of several of the Nrf2-regulated down-stream genes, including Gstp1, Ugt1a1, and Nqo1; the expression of Nfe2l2 (Nrf2) itself was unaltered. Furthermore, both the redox status and the total amount of Keap1 protein were significantly decreased by DOX, with the loss of Keap1 being due to both inhibited gene expression and increased autophagic, but not proteasomal, degradation. These findings identify the Keap1/Nrf2 pathway as a potentially important initial response to acute DOX-induced oxidative injury, with the primary regulatory events being the oxidation and autophagic degradation of the redox sensor Keap1 protein. PMID:24211725

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

    SciTech Connect

    Muratore, Massimo; Mitchell, Steve; Waterfall, Martin

    2013-09-06

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

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

    SciTech Connect

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

    2012-02-24

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

  19. Reprogramming of skeletal myoblasts for induction of pluripotency for tumor free cardiomyogenesis in the infarcted heart

    PubMed Central

    Ahmed, Rafeeq PH; Haider, Husnain Kh; Buccini, Stephanie; Li, Longhu; Jiang, Shujia; Ashraf, Muhammad

    2011-01-01

    Rationale Skeletal myoblasts (SMs) with inherent myogenic properties are better candidates for reprogramming to pluripotency. Objective To reprogram SMs to pluripotency and show that reprogrammed SMs (SiPs) express embryonic gene and microRNA profiles and transplantation of predifferentiated cardiac progenitors reduce tumor formation. Methods and Results The pMXs vector containing mouse cDNAs for Yamanaka’s quartet of stemness factors were used for transduction of SMs purified from male Oct4-GFP+ transgenic mouse. Three weeks later, GFP+ colonies of SiPS were isolated and propagated in vitro. SiPS were positive for alkaline phosphatase, expressed SSEA1 and displayed a panel of embryonic stem (ES) cell specific pluripotency markers. Embryoid body formation yielded beating cardiomyocyte-like cells which expressed early and late cardiac specific markers. SiPS also had embryonic microRNA profile which was altered during their cardiomyogenic differentiation. Noticeable abrogation of let-7 family and significant upregulation of miR-200a–c and miR-290 to 295 was observed in SiPS and SiPS derived cardiomyocytes respectively. In vivo studies in an experimental model of acute myocardial infarction showed extensive survival of SiPS and SiPS derived cardiomyocytes in mouse heart after transplantation. Our results from 4-week studies in DMEM without cells (group-1), SMs (group-2), SiPS (group-3) and SiPS derived cardiomyocytes (group-4) showed extensive myogenic integration of the transplanted cells in group-4 with attenuated infarct size and improved cardiac function without tumorgenesis. Conclusions Successful reprogramming was achieved in SMs with ES cell-like microRNA profile. Given the tumorgenic nature of SiPS, their pre-differentiation into cardiomyocytes would be important for tumor-free cardiogenesis in the heart. PMID:21566212

  20. Live imaging provides new insights on dynamic F-actin filopodia and differential endocytosis during myoblast fusion in Drosophila.

    PubMed

    Haralalka, Shruti; Shelton, Claude; Cartwright, Heather N; Guo, Fengli; Trimble, Rhonda; Kumar, Ram P; Abmayr, Susan M

    2014-01-01

    The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 pathways. In the present study, we utilize the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. We also observed differential endocytosis of cell surface components at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the

  1. The Hippo effector TAZ (WWTR1) transforms myoblasts and TAZ abundance is associated with reduced survival in embryonal rhabdomyosarcoma.

    PubMed

    Mohamed, Abdalla; Sun, Congshan; De Mello, Vanessa; Selfe, Joanna; Missiaglia, Edoardo; Shipley, Janet; Murray, Graeme I; Zammit, Pete S; Wackerhage, Henning

    2016-09-01

    The Hippo effector YAP has recently been identified as a potent driver of embryonal rhabdomyosarcoma (ERMS). Most reports suggest that the YAP paralogue TAZ (gene symbol WWTR1) functions as YAP but, in skeletal muscle, TAZ has been reported to promote myogenic differentiation, whereas YAP inhibits it. Here, we investigated whether TAZ is also a rhabdomyosarcoma oncogene or whether TAZ acts as a YAP antagonist. Immunostaining of rhabdomyosarcoma tissue microarrays revealed that TAZ is significantly associated with poor survival in ERMS. In 12% of fusion gene-negative rhabdomyosarcomas, the TAZ locus is gained, which is correlated with increased expression. Constitutively active TAZ S89A significantly increased proliferation of C2C12 myoblasts and, importantly, colony formation on soft agar, suggesting transformation. However, TAZ then switches to enhance myogenic differentiation in C2C12 myoblasts, unlike YAP. Conversely, lentiviral shRNA-mediated TAZ knockdown in human ERMS cells reduced proliferation and anchorage-independent growth. While TAZ S89A or YAP1 S127A similarly activated the 8XGTIIC-Luc Hippo reporter, only YAP1 S127A activated the Brachyury (T-box) reporter. Consistent with its oncogene function, TAZ S89A induced expression of the ERMS cancer stem cell gene Myf5 and the serine biosynthesis pathway (Phgdh, Psat1, Psph) in C2C12 myoblasts. Thus, TAZ is associated with poor survival in ERMS and could act as an oncogene in rhabdomyosarcoma. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. PMID:27184927

  2. Prevention of oxidative stress-induced apoptosis of C2C12 myoblasts by a Cichorium intybus root extract.

    PubMed

    Lee, Yong-Hyeon; Kim, Dae-Hyun; Kim, Yoon Suk; Kim, Tack-Joong

    2013-01-01

    Cell injury associated with reactive oxygen species (ROS) has been reported in various muscular disorders. We found that a Cichorium intybus (Cii) extract reduced H(2)O(2)-induced viability loss in C2C12 myoblasts, inhibited oxidative stress-induced apoptosis and increased intracellular heat shock protein 70 (Hsp 70) expression. Cii also inhibited the level of intracellular ceramide. These results indicate that Cii may prevent skeletal muscle atrophy by inducing the expression of Hsp 70 and inhibiting the level of ceramide. PMID:23391909

  3. Insulin-like growth factor-1 (IGF-1) promotes myoblast proliferation and skeletal muscle growth of embryonic chickens via the PI3K/Akt signalling pathway.

    PubMed

    Yu, Minli; Wang, Huan; Xu, Yali; Yu, Debing; Li, Dongfeng; Liu, Xiuhong; Du, Wenxing

    2015-08-01

    During embryonic development, IGF-1 fulfils crucial roles in skeletal myogenesis. However, the involvement of IGF-1-induced myoblast proliferation in muscle growth is still unclear. In the present study, we have characterised the role of IGF-1 in myoblast proliferation both in vitro and in vivo and have revealed novel details of how exogenous IGF-1 influences myogenic genes in chicken embryos. The results show that IGF-1 significantly induces the proliferation of cultured myoblasts in a dose-dependent manner. Additionally, the IGF-1 treatment significantly promoted myoblasts entering a new cell cycle and increasing the mRNA expression levels of cell cycle-dependent genes. However, these effects were inhibited by the PI3K inhibitor LY294002 and the Akt inhibitor KP372-1. These data indicated that the pro-proliferative effect of IGF-1 was mediated in response to the PI3K/Akt signalling pathway. Moreover, we also showed that exogenous IGF-1 stimulated myoblast proliferation in vivo. IGF-1 administration obviously promoted the incorporation of BrdU and remarkably increased the number of PAX7-positive cells in the skeletal muscle of chicken embryos. Administration of IGF-1 also significantly induced the upregulation of myogenic factors gene, the enhancement of c-Myc and the inhibition of myostatin (Mstn) expression. These findings demonstrate that IGF-1 has strong activity as a promoter of myoblast expansion and muscle fiber formation during early myogenesis. Therefore, this study offers insight into the mechanisms responsible for IGF-1-mediated stimulation of embryonic skeletal muscle development, which could have important implications for the improvement of chicken meat production. PMID:25808997

  4. PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase

    PubMed Central

    Madaro, Luca; Marrocco, Valeria; Fiore, Piera; Aulino, Paola; Smeriglio, Piera; Adamo, Sergio; Molinaro, Mario; Bouché, Marina

    2011-01-01

    Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKCθ, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKCθ is strongly up-regulated following freeze injury–induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKCθ knockout and muscle-specific PKCθ dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKCθ mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKCθ mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and β1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKCθ in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKCθ-null myoblasts. We thus propose that PKC

  5. Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells

    PubMed Central

    2014-01-01

    Background Via the hepatocyte growth factor receptor (Met), hepatocyte growth factor (HGF) exerts key roles involving skeletal muscle development and regeneration. Heparan sulfate proteoglycans (HSPGs) are critical modulators of HGF activity, but the role of specific HSPGs in HGF regulation is poorly understood. Glypican-1 is the only HSPG expressed in myoblasts that localize in lipid raft membrane domains, controlling cell responses to extracellular stimuli. We determined if glypican-1 in these domains is necessary to stabilize the HGF-Met signaling complex and myoblast response to HGF. Methods C2C12 myoblasts and a derived clone (C6) with low glypican-1 expression were used as an experimental model. The activation of Met, ERK1/2 and AKT in response to HGF was evaluated. The distribution of Met and its activated form in lipid raft domains, as well as its dependence on glypican-1, were characterized by sucrose density gradient fractionation in both cell types. Rescue experiments reexpressing glypican-1 or a chimeric glypican-1 fused to the transmembrane and cytoplasmic domains of mouse syndecan-1 or myoblast pretreatment with MβCD were conducted. In vitro and in vivo myoblast migration assays in response to HGF were also performed. Results Glypican-1 localization in membrane raft domains was required for a maximum cell response to HGF. It stabilized Met and HGF in lipid raft domains, forming a signaling complex where the active phospho-Met receptor was concentrated. Glypican-1 also stabilized CD44 in a HGF-dependent manner. In addition, glypican-1 was required for in vitro and in vivo HGF-dependent myoblast migration. Conclusions Glypican-1 is a regulator of HGF-dependent signaling via Met in lipid raft domains. PMID:24517345

  6. Disruption of GLUT1 glucose carrier trafficking in L6E9 and Sol8 myoblasts by the phosphatidylinositol 3-kinase inhibitor wortmannin.

    PubMed

    Kaliman, P; Viñals, F; Testar, X; Palacín, M; Zorzano, A

    1995-12-01

    In this study we have used wortmannin, a highly specific inhibitor of phosphatidylinositol (PI) 3-kinase, to assess the role of this enzyme on GLUT1 glucose carrier distribution and glucose transport activity in myoblasts from two skeletal-muscle cell lines, L6E9 and Sol8. As detected in L6E9 cells, myoblasts exhibited basal and insulin-stimulated PI 3-kinase activities. Incubation of intact myoblasts with wortmannin resulted in a marked inhibition of both basal and insulin-stimulated PI 3-kinase activities. L6E9 and Sol8 myoblasts showed basal and insulin-stimulated glucose transport activities, both of them inhibited by wortmannin in a dose-dependent manner (IC50 approximately 10-20 nM). Concomitantly, immunofluorescence analysis revealed that 1 h treatment with wortmannin led to a dramatic intracellular accumulation of GLUT1 carriers (the main glucose transporter expressed in L6E9 and Sol8 myoblasts) in both cell systems. The effect of wortmannin on GLUT1 cellular redistribution was independent of the presence of insulin. The cellular distribution of two structural plasma-membrane components such as beta 1-integrin or the alpha 1 subunit of the Na(+)-K(+)-ATPase were unaffected by wortmannin in both the absence and the presence of insulin. As a whole, our results indicate that PI 3-kinase is necessary to basal and insulin-stimulated glucose transport in L6E9 and Sol8 myoblasts. Moreover, immunofluorescence assays suggest that in both cellular models there is a constitutive GLUT 1 trafficking pathway (independent of insulin) that involves PI 3-kinase and which, when blocked, locks GLUT1 in a perinuclear compartment. PMID:8526858

  7. Disruption of GLUT1 glucose carrier trafficking in L6E9 and Sol8 myoblasts by the phosphatidylinositol 3-kinase inhibitor wortmannin.

    PubMed Central

    Kaliman, P; Viñals, F; Testar, X; Palacín, M; Zorzano, A

    1995-01-01

    In this study we have used wortmannin, a highly specific inhibitor of phosphatidylinositol (PI) 3-kinase, to assess the role of this enzyme on GLUT1 glucose carrier distribution and glucose transport activity in myoblasts from two skeletal-muscle cell lines, L6E9 and Sol8. As detected in L6E9 cells, myoblasts exhibited basal and insulin-stimulated PI 3-kinase activities. Incubation of intact myoblasts with wortmannin resulted in a marked inhibition of both basal and insulin-stimulated PI 3-kinase activities. L6E9 and Sol8 myoblasts showed basal and insulin-stimulated glucose transport activities, both of them inhibited by wortmannin in a dose-dependent manner (IC50 approximately 10-20 nM). Concomitantly, immunofluorescence analysis revealed that 1 h treatment with wortmannin led to a dramatic intracellular accumulation of GLUT1 carriers (the main glucose transporter expressed in L6E9 and Sol8 myoblasts) in both cell systems. The effect of wortmannin on GLUT1 cellular redistribution was independent of the presence of insulin. The cellular distribution of two structural plasma-membrane components such as beta 1-integrin or the alpha 1 subunit of the Na(+)-K(+)-ATPase were unaffected by wortmannin in both the absence and the presence of insulin. As a whole, our results indicate that PI 3-kinase is necessary to basal and insulin-stimulated glucose transport in L6E9 and Sol8 myoblasts. Moreover, immunofluorescence assays suggest that in both cellular models there is a constitutive GLUT 1 trafficking pathway (independent of insulin) that involves PI 3-kinase and which, when blocked, locks GLUT1 in a perinuclear compartment. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8526858

  8. Muscle-specific androgen receptor deletion shows limited actions in myoblasts but not in myofibers in different muscles in vivo.

    PubMed

    Rana, Kesha; Chiu, Maria W S; Russell, Patricia K; Skinner, Jarrod P; Lee, Nicole K L; Fam, Barbara C; Zajac, Jeffrey D; MacLean, Helen E

    2016-08-01

    The aim of this study was to investigate the direct muscle cell-mediated actions of androgens by comparing two different mouse lines. The cre-loxP system was used to delete the DNA-binding activity of the androgen receptor (AR) in mature myofibers (MCK mAR(ΔZF2)) in one model and the DNA-binding activity of the AR in both proliferating myoblasts and myofibers (α-actin mAR(ΔZF2)) in another model. We found that hind-limb muscle mass was normal in MCK mAR(ΔZF2) mice and that relative mass of only some hind-limb muscles was reduced in α-actin mAR(ΔZF2) mice. This suggests that myoblasts and myofibers are not the major cellular targets mediating the anabolic actions of androgens on male muscle during growth and development. Levator ani muscle mass was decreased in both mouse lines, demonstrating that there is a myofiber-specific effect in this unique androgen-dependent muscle. We found that the pattern of expression of genes including c-myc, Fzd4 and Igf2 is associated with androgen-dependent changes in muscle mass; therefore, these genes are likely to be mediators of anabolic actions of androgens. Further research is required to identify the major targets of androgen actions in muscle, which are likely to include indirect actions via other tissues. PMID:27402875

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

    SciTech Connect

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

    2012-03-02

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

  10. Proteome Profiling and Ultrastructural Characterization of the Human RCMH Cell Line: Myoblastic Properties and Suitability for Myopathological Studies.

    PubMed

    Kollipara, Laxmikanth; Buchkremer, Stephan; Weis, Joachim; Brauers, Eva; Hoss, Mareike; Rütten, Stephan; Caviedes, Pablo; Zahedi, René P; Roos, Andreas

    2016-03-01

    Studying (neuro)muscular disorders is a major topic in biomedicine with a demand for suitable model systems. Continuous cell culture (in vitro) systems have several technical advantages over in vivo systems and became widely used tools for discovering physiological/pathophysiological mechanisms in muscle. In particular, myoblast cell lines are suitable model systems to study complex biochemical adaptations occurring in skeletal muscle and cellular responses to altered genetic/environmental conditions. Whereas most in vitro studies use extensively characterized murine C2C12 cells, a comprehensive description of an equivalent human cell line, not genetically manipulated for immortalization, is lacking. Therefore, we characterized human immortal myoblastic RCMH cells using scanning (SEM) and transmission electron microscopy (TEM) and proteomics. Among more than 6200 identified proteins we confirm the known expression of proteins important for muscle function. Comparing the RCMH proteome with two well-defined nonskeletal muscle cells lines (HeLa, U2OS) revealed a considerable enrichment of proteins important for muscle function. SEM/TEM confirmed the presence of agglomerates of cytoskeletal components/intermediate filaments and a prominent rough ER. In conclusion, our results indicate RMCH as a suitable in vitro model for investigating muscle function-related processes such as mechanical stress burden and mechanotransduction, EC coupling, cytoskeleton, muscle cell metabolism and development, and (ER-associated) myopathic disorders. PMID:26781476

  11. Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method.

    PubMed

    Iyombe-Engembe, Jean-Paul; Ouellet, Dominique L; Barbeau, Xavier; Rousseau, Joël; Chapdelaine, Pierre; Lagüe, Patrick; Tremblay, Jacques P

    2016-01-01

    The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. We present here the CRISPR-induced deletion (CinDel), a new promising genome-editing technology to correct the DMD gene. This strategy is based on the use of two gRNAs targeting specifically exons that precede and follow the patient deletion in the DMD gene. This pair of gRNAs induced a precise large additional deletion leading to fusion of the targeted exons. Using an adequate pair of gRNAs, the deletion of parts of these exons and the intron separating them restored the DMD reading frame in 62% of the hybrid exons in vitro in DMD myoblasts and in vivo in electroporated hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted DYS protein was detected following the formation of myotubes by the unselected, treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene, this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides. PMID:26812655

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

    PubMed

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

    2016-01-01

    Thiazolidinedione (TZD), a specific peroxisome proliferator-activated receptor γ (PPARγ) agonist, was developed to control blood glucose in diabetes patients. However, several side effects were reported that increased the risk of heart failure. We used C2C12 myoblasts to investigate the role of PPARs and their transcriptional activity during myotube formation. The role of mechanical stretch during myogenesis was also explored by applying cyclic stretch to the differentiating C2C12 myoblasts with 10% strain deformation at 1 Hz. The myogenesis medium (MM), composed of Dulbecco's modified Eagle's medium with 2% horse serum, facilitated myotube formation with increased myosin heavy chain and α-smooth muscle actin (α-SMA) protein expression. The PPARγ protein and PPAR response element (PPRE) promoter activity decreased during MM induction. Cyclic stretch further facilitated the myogenesis in MM with increased α-SMA and decreased PPARγ protein expression and inhibited PPRE promoter activity. Adding a PPARγ agonist (TZD) to the MM stopped the myogenesis and restored the PPRE promoter activity, whereas a PPARγ antagonist (GW9662) significantly increased the myotube number and length. During the myogenesis induction, application of cyclic stretch rescued the inhibitory effects of TZD. These results provide novel perspectives for mechanical stretch to interplay and rescue the dysfunction of myogenesis with the involvement of PPARγ and its target drugs. PMID:27047938

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

    PubMed Central

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

    2016-01-01

    Thiazolidinedione (TZD), a specific peroxisome proliferator-activated receptor γ (PPARγ) agonist, was developed to control blood glucose in diabetes patients. However, several side effects were reported that increased the risk of heart failure. We used C2C12 myoblasts to investigate the role of PPARs and their transcriptional activity during myotube formation. The role of mechanical stretch during myogenesis was also explored by applying cyclic stretch to the differentiating C2C12 myoblasts with 10% strain deformation at 1 Hz. The myogenesis medium (MM), composed of Dulbecco’s modified Eagle’s medium with 2% horse serum, facilitated myotube formation with increased myosin heavy chain and α-smooth muscle actin (α-SMA) protein expression. The PPARγ protein and PPAR response element (PPRE) promoter activity decreased during MM induction. Cyclic stretch further facilitated the myogenesis in MM with increased α-SMA and decreased PPARγ protein expression and inhibited PPRE promoter activity. Adding a PPARγ agonist (TZD) to the MM stopped the myogenesis and restored the PPRE promoter activity, whereas a PPARγ antagonist (GW9662) significantly increased the myotube number and length. During the myogenesis induction, application of cyclic stretch rescued the inhibitory effects of TZD. These results provide novel perspectives for mechanical stretch to interplay and rescue the dysfunction of myogenesis with the involvement of PPARγ and its target drugs. PMID:27047938

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

    PubMed Central

    Waldemer-Streyer, R J; Chen, J

    2015-01-01

    Adult skeletal muscle tissue has a uniquely robust capacity for regeneration, which gradually declines with aging or is compromised in muscle diseases. The cellular mechanisms regulating adult myogenesis remain incompletely understood. Here we identify the cytokine tumor necrosis factor superfamily member 14 (Tnfsf14) as a positive regulator of myoblast differentiation in culture and muscle regeneration in vivo. We find that Tnfsf14, as well as its cognate receptors herpes virus entry mediator (HVEM) and lymphotoxin β receptor (LTβR), are expressed in both differentiating myocytes and regenerating myofibers. Depletion of Tnfsf14 or either receptor inhibits myoblast differentiation and promotes apoptosis. Our results also suggest that Tnfsf14 regulates myogenesis by supporting cell survival and maintaining a sufficient pool of cells for fusion. In addition, we show that Akt mediates the survival and myogenic function of Tnfsf14. Importantly, local knockdown of Tnfsf14 is found to impair injury-induced muscle regeneration in a mouse model, affirming an important physiological role for Tnfsf14 in myogenesis in vivo. Furthermore, we demonstrate that localized overexpression of Tnfsf14 potently enhances muscle regeneration, and that this regenerative capacity of Tnfsf14 is dependent on Akt signaling. Taken together, our findings reveal a novel regulator of skeletal myogenesis and implicate Tnfsf14 in future therapeutic development. PMID:26720335

  15. Reliable and versatile immortal muscle cell models from healthy and myotonic dystrophy type 1 primary human myoblasts.

    PubMed

    Pantic, Boris; Borgia, Doriana; Giunco, Silvia; Malena, Adriana; Kiyono, Tohru; Salvatori, Sergio; De Rossi, Anita; Giardina, Emiliano; Sangiuolo, Federica; Pegoraro, Elena; Vergani, Lodovica; Botta, Annalisa

    2016-03-01

    Primary human skeletal muscle cells (hSkMCs) are invaluable tools for deciphering the basic molecular mechanisms of muscle-related biological processes and pathological alterations. Nevertheless, their use is quite restricted due to poor availability, short life span and variable purity of the cells during in vitro culture. Here, we evaluate a recently published method of hSkMCs immortalization, relying on ectopic expression of cyclin D1 (CCND1), cyclin-dependent kinase 4 (CDK4) and telomerase (TERT) in myoblasts from healthy donors (n=3) and myotonic dystrophy type 1 (DM1) patients (n=2). The efficacy to maintain the myogenic and non-transformed phenotype, as well as the main pathogenetic hallmarks of DM1, has been assessed. Combined expression of the three genes i) maintained the CD56(NCAM)-positive myoblast population and differentiation potential; ii) preserved the non-transformed phenotype and iii) maintained the CTG repeat length, amount of nuclear foci and aberrant alternative splicing in immortal muscle cells. Moreover, immortal hSkMCs displayed attractive additional features such as structural maturation of sarcomeres, persistence of Pax7-positive cells during differentiation and complete disappearance of nuclear foci following (CAG)7 antisense oligonucleotide (ASO) treatment. Overall, the CCND1, CDK4 and TERT immortalization yields versatile, reliable and extremely useful human muscle cell models to investigate the basic molecular features of human muscle cell biology, to elucidate the molecular pathogenetic mechanisms and to test new therapeutic approaches for DM1 in vitro. PMID:26905645

  16. Keap1 redox-dependent regulation of doxorubicin-induced oxidative stress response in cardiac myoblasts

    SciTech Connect

    Nordgren, Kendra K.S. Wallace, Kendall B.

    2014-01-01

    Doxorubicin (DOX) is a widely prescribed treatment for a broad scope of cancers, but clinical utility is limited by the cumulative, dose-dependent cardiomyopathy that occurs with repeated administration. DOX-induced cardiotoxicity is associated with the production of reactive oxygen species (ROS) and oxidation of lipids, DNA and proteins. A major cellular defense mechanism against such oxidative stress is activation of the Keap1/Nrf2-antioxidant response element (ARE) signaling pathway, which transcriptionally regulates expression of antioxidant genes such as Nqo1 and Gstp1. In the present study, we address the hypothesis that an initial event associated with DOX-induced oxidative stress is activation of the Keap1/Nrf2-dependent expression of antioxidant genes and that this is regulated through drug-induced changes in redox status of the Keap1 protein. Incubation of H9c2 rat cardiac myoblasts with DOX resulted in a time- and dose-dependent decrease in non-protein sulfhydryl groups. Associated with this was a near 2-fold increase in Nrf2 protein content and enhanced transcription of several of the Nrf2-regulated down-stream genes, including Gstp1, Ugt1a1, and Nqo1; the expression of Nfe2l2 (Nrf2) itself was unaltered. Furthermore, both the redox status and the total amount of Keap1 protein were significantly decreased by DOX, with the loss of Keap1 being due to both inhibited gene expression and increased autophagic, but not proteasomal, degradation. These findings identify the Keap1/Nrf2 pathway as a potentially important initial response to acute DOX-induced oxidative injury, with the primary regulatory events being the oxidation and autophagic degradation of the redox sensor Keap1 protein. - Highlights: • DOX caused a ∼2-fold increase in Nrf2 protein content. • DOX enhanced transcription of several Nrf2-regulated down-stream genes. • Redox status and total amount of Keap1 protein were significantly decreased by DOX. • Loss of Keap1 protein was due to

  17. The cAMP response element binding protein (CREB) is activated by insulin-like growth factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast.

    PubMed

    Zuloaga, R; Fuentes, E N; Molina, A; Valdés, J A

    2013-10-18

    Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP3/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA-CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation. PMID:24064350

  18. Concordant but Varied Phenotypes among Duchenne Muscular Dystrophy Patient-Specific Myoblasts Derived using a Human iPSC-Based Model.

    PubMed

    Choi, In Young; Lim, HoTae; Estrellas, Kenneth; Mula, Jyothi; Cohen, Tatiana V; Zhang, Yuanfan; Donnelly, Christopher J; Richard, Jean-Philippe; Kim, Yong Jun; Kim, Hyesoo; Kazuki, Yasuhiro; Oshimura, Mitsuo; Li, Hongmei Lisa; Hotta, Akitsu; Rothstein, Jeffrey; Maragakis, Nicholas; Wagner, Kathryn R; Lee, Gabsang

    2016-06-01

    Duchenne muscular dystrophy (DMD) remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs). Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our "chemical-compound-based" strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFβ signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological "dual-SMAD" inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form "rescued" multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human "DMD-in-a-dish" model using hiPSC-based disease modeling. PMID:27239027

  19. Setdb1 Is Required for Myogenic Differentiation of C2C12 Myoblast Cells via Maintenance of MyoD Expression

    PubMed Central

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

    2015-01-01

    Setdb1, an H3-K9 specific histone methyltransferase, is associated with transcriptional silencing of euchromatic genes through chromatin modification. Functions of Setdb1 during development have been extensively studied in embryonic and mesenchymal stem cells as well as neurogenic progenitor cells. But the role of Sedtdb1 in myogenic differentiation remains unknown. In this study, we report that Setdb1 is required for myogenic potential of C2C12 myoblast cells through maintaining the expressions of MyoD and muscle-specific genes. We find that reduced Setdb1 expression in C2C12 myoblast cells severely delayed differentiation of C2C12 myoblast cells, whereas exogenous Setdb1 expression had little effect on. Gene expression profiling analysis using oligonucleotide micro-array and RNA-Seq technologies demonstrated that depletion of Setdb1 results in downregulation of MyoD as well as the components of muscle fiber in proliferating C2C12 cells. In addition, exogenous expression of MyoD reversed transcriptional repression of MyoD promoter-driven lucif-erase reporter by Setdb1 shRNA and rescued myogenic differentiation of C2C12 myoblast cells depleted of endogenous Setdb1. Taken together, these results provide new insights into how levels of key myogenic regulators are maintained prior to induction of differentiation. PMID:25715926

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

    SciTech Connect

    Zuloaga, R.; Fuentes, E.N.; Molina, A.; Valdés, J.A.

    2013-10-18

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

  1. Administration of insulin to newly hatched chicks improves growth performance via impairment of MyoD gene expression and enhancement of cell proliferation in chicken myoblasts.

    PubMed

    Sato, Kan; Aoki, Michiru; Kondo, Ryota; Matsushita, Kohichi; Akiba, Yukio; Kamada, Tosihiko

    2012-02-01

    The insulin/PI3K/Akt signaling pathway is strongly involved in the differentiation of C2C12 cells, as has been demonstrated by the addition of IGFs and insulin to culture media. In this study, we have characterized the role of insulin in chick myoblast proliferation and differentiation in vitro and in vivo, and have revealed novel details of how this exogenous hormone influences myogenic genes during differentiation. Chick myoblast cells cultured in differentiation medium (DMEM containing 2% FBS) supplemented with insulin exhibited a significant decrease in MyoD and myogenin mRNA expression after 12h of culture compared to cells cultured in differentiation media alone. MyoD and myogenin immunoreactive proteins in cells cultured in differentiation medium supplemented with insulin were quite low compared to those in control culture. Supplementation of the differentiation media containing insulin with LY294002 (a PI3K inhibitor) induced myoblast differentiation. A significant increase in MyoD and myogenin mRNA expression was observed in these cells after incubation for 12h, and the level of expression was similar to that of control cells incubated with differentiation media alone. The DNA content and the phosphor-Erk1/2 protein level were increased by the addition of insulin to the differentiation medium. These results suggest that insulin and its signaling pathway play an inhibitory role in chick myoblast differentiation. A high level of Pax7 mRNA was observed in the skeletal muscle of 3-day-old chicks administered insulin or tolbutamide at 1-day-of-age. In addition, body weight at 21 and 50 days-of-age was significantly greater for chickens administered insulin or tolbutamide at 1-day-of-age than for control chickens. These results detail not only species-specific differences in insulin action for myoblasts but also provide novel information that may be used for the improvement of chicken meat production. PMID:22172340

  2. Co-transplantation of plasmid-transfected myoblasts and myotubes into rat brains enables high levels of gene expression long-term

    NASA Technical Reports Server (NTRS)

    Jiao, S.; Williams, P.; Safda, N.; Schultz, E.; Wolff, J. A.

    1993-01-01

    We have previously proposed the use of primary muscle cells as a "platform," or "vehicle" for intracerebral transgene expression. Brain grafts of minced muscle, or cultured muscle cells persisted in rat brains for at least 6 mo without any decrease in graft size, or tumor formation. Stable, but moderate levels of intracerebral transgene expression were obtained by transplanting plasmid-transfected myotubes in culture. In the present study, high and stable levels of intracerebral transgene expression were achieved by the co-transplantation of plasmid-transfected myoblasts and myotubes in culture. Approximately 5 X 10(5) myoblasts and myotubes were transfected with 10 micrograms pRSVL plasmid DNA, and 30 micrograms Lipofectin (BRL), respectively. They were mixed together (total cell number was 1 million), and stereotactically injected into the caudate nucleus of an adult rat brain. The activity of luciferase, the product of transgene expression, was stable for at least 4 mo, and much higher than the levels in myotube grafts, or co-grafts of myoblasts and minced muscle. Presumably, the myotubes served as a framework on which the myoblasts can form myotubes. The sections of brains transplanted with co-graft of myoblasts, and myotubes transfected with pRSVLac-Z were stained immunofluorescently for beta-galactosidase activity. The muscle grafts contained beta-galactosidase positive myofibers 4 mo after transplantation. Such high and stable levels of in vivo expression after postnatal gene transfer have rarely been achieved. Primary muscle cells are useful vehicle for transgene expression in brains, and potentially valuable for gene therapy of degenerative neurological disorders.

  3. Long-Term Endurance Exercise in Humans Stimulates Cell Fusion of Myoblasts along with Fusogenic Endogenous Retroviral Genes In Vivo

    PubMed Central

    Suhr, Frank; Konou, Thierry M.; Tappe, Kim A.; Toigo, Marco; Jung, Hans H.; Henke, Christine; Steigleder, Ruth; Strissel, Pamela L.; Huebner, Hanna; Beckmann, Matthias W.; van der Keylen, Piet; Schoser, Benedikt; Schiffer, Thorsten; Frese, Laura; Bloch, Wilhelm; Strick, Reiner

    2015-01-01

    Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV) envelope genes due to their involvement in cell fusion of osteoclasts and syncytiotrophoblasts. Comparing muscle biopsies from post- with the pre-competitive seasons a significant 2.25-fold increase of myonuclei/mm fiber, a 2.38-fold decrease of fiber area/nucleus and a 3.1-fold decrease of satellite cells (SCs) occurred. We propose that during the pre-competitive season SC proliferation occurred following with increased cell fusion during the competitive season. Expression of twenty-two envelope genes of muscle biopsies demonstrated a significant increase of putative muscle-cell fusogenic genes Syncytin-1 and Syncytin-3, but also for the non-fusogenic erv3. Immunohistochemistry analyses showed that Syncytin-1 mainly localized to the sarcolemma of myofibers positive for myosin heavy-chain isotypes. Cellular receptors SLC1A4 and SLC1A5 of Syncytin-1 showed significant decrease of expression in post-competitive muscles compared with the pre-competitive season, but only SLC1A4 protein expression localized throughout the myofiber. Erv3 protein was strongly expressed throughout the myofiber, whereas envK1-7 localized to SC nuclei and myonuclei. Syncytin-1 transcription factors, PPARγ and RXRα, showed no protein expression in the myofiber, whereas the pCREB-Ser133 activator of Syncytin-1 was enriched to SC nuclei and myonuclei. Syncytin-1, Syncytin-3, SLC1A4 and PAX7 gene regulations along with MyoD1 and myogenin were verified during proliferating or actively-fusing human primary myoblast cell

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

    PubMed Central

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

    2016-01-01

    Conessine, a steroidal alkaloid isolated from Holarrhena floribunda, has anti-malarial activity and interacts with the histamine H3 receptor. However, the cellular effects of conessine are poorly understood. Accordingly, we evaluated the involvement of conessine in the regulation of autophagy. We searched natural compounds that modulate autophagy, and conessine was identified as an inhibitor of autophagic flux. Conessine treatment induced the formation of autophagosomes, and p62, an autophagic adapter, accumulated in the autophagosomes. Reactive oxygen species such as hydrogen peroxide (H2O2) result in muscle cell death by inducing excessive autophagic flux. Treatment with conessine inhibited H2O2-induced autophagic flux in C2C12 myoblast cells and also interfered with cell death. Our results indicate that conessine has the potential effect to inhibit muscle cell death by interfering with autophagic flux. PMID:27257813

  5. Role of Sulf1A in Wnt1- and Wnt6-induced growth regulation and myoblast hyper-elongation.

    PubMed

    Hitchins, L; Fletcher, F; Allen, S; Dhoot, G K

    2013-01-01

    Sulf1A expression, which is a characteristic of embryonic muscle, is undetectable in mature muscle fibres and quiescent satellite cells, but is re-activated in vivo upon injury and in vitro following activation of satellite cells. Sulf1A is known to enhance canonical Wnt signalling, and its association with Wnt1-induced satellite cell proliferation in vitro in the present study further confirmed this. However, exogenous Wnt6 decreased satellite cell proliferation but promoted the adoption of a hyper-elongated cell morphology in myoblasts on isolated single fibres in culture. Such Wnt6-induced cellular hyper-elongation and inhibition of proliferation was found to be dependent upon Sulf1A, as treatment with Sulf1A neutralising antibodies abolished both these effects. This indicates that Sulf1A can regulate Wnt6 signalling and cellular differentiation in skeletal muscle. PMID:23772371

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

    PubMed Central

    Janot, Mathilde; Audfray, Aymeric; Loriol, Céline; Germot, Agnès; Maftah, Abderrahman; Dupuy, Fabrice

    2009-01-01

    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. PMID:19843320

  7. Detection of Pancreatic Cancer-Induced Cachexia Using a Fluorescent Myoblast Reporter System and Analysis of Metabolite Abundance.

    PubMed

    Winnard, Paul T; Bharti, Santosh K; Penet, Marie-France; Marik, Radharani; Mironchik, Yelena; Wildes, Flonne; Maitra, Anirban; Bhujwalla, Zaver M

    2016-03-15

    The dire effects of cancer-induced cachexia undermine treatment and contribute to decreased survival rates. Therapeutic options for this syndrome are limited, and therefore efforts to identify signs of precachexia in cancer patients are necessary for early intervention. The applications of molecular and functional imaging that would enable a whole-body "holistic" approach to this problem may lead to new insights and advances for diagnosis and treatment of this syndrome. Here we have developed a myoblast optical reporter system with the purpose of identifying early cachectic events. We generated a myoblast cell line expressing a dual tdTomato:GFP construct that was grafted onto the muscle of mice-bearing human pancreatic cancer xenografts to provide noninvasive live imaging of events associated with cancer-induced cachexia (i.e., weight loss). Real-time optical imaging detected a strong tdTomato fluorescent signal from skeletal muscle grafts in mice with weight losses of only 1.2% to 2.7% and tumor burdens of only approximately 79 to 170 mm(3). Weight loss in cachectic animals was also associated with a depletion of lipid, cholesterol, valine, and alanine levels, which may provide informative biomarkers of cachexia. Taken together, our findings demonstrate the utility of a reporter system that is capable of tracking tumor-induced weight loss, an early marker of cachexia. Future studies incorporating resected tissue from human pancreatic ductal adenocarcinoma into a reporter-carrying mouse may be able to provide a risk assessment of cachexia, with possible implications for therapeutic development. PMID:26719527

  8. Automated High-Content Assay for Compounds Selectively Toxic to Trypanosoma cruzi in a Myoblastic Cell Line

    PubMed Central

    Alonso-Padilla, Julio; Cotillo, Ignacio; Presa, Jesús L.; Cantizani, Juan; Peña, Imanol; Bardera, Ana I.; Martín, Jose J.; Rodriguez, Ana

    2015-01-01

    Background Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease. Methodology/Principal Findings Genetically engineered parasitic strains are used for high throughput screening (HTS) of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6) and was validated against a series of known anti-trypanosomatid drugs. Conclusions/Significance We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite. PMID:25615687

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

    PubMed

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

    2012-01-01

    Nandrolone, an anabolic steroid, slows denervation atrophy of rat muscle, prevents denervation-induced nuclear accumulation of intracellular domain of the Notch receptor, and elevates expression of Numb. Numb acts as an inhibitor of Notch signaling and promotes myogenic differentiation of satellite cells. Turnover of Numb is regulated by mdm2, an E3 ubiquitin ligase. With these considerations in mind, we investigated the effects of nandrolone on the expression of Numb and mdm2 proteins and determined the effect of mdm2 on nandrolone-induced alterations in Numb protein in C2C12 myoblasts. When C2C12 cells were cultured in a medium favoring differentiation (Dulbecco modified Eagle medium containing 2% horse serum), nandrolone up-regulated Numb protein levels in a time-dependent manner and prolonged Numb protein half-life from 10 to 18 hours. In contrast, nandrolone reduced the expression of mdm2 protein. To determine whether the decreased mdm2 expression induced by nandrolone was responsible for the increased levels and prolonged half-life of Numb protein in this cell line, mdm2-small interfering RNA (siRNA) was employed to inhibit mdm2 expression. Compared to cells transfected with scrambled siRNA (negative control), transfection with mdm2-siRNA increased basal Numb protein expression but abolished the further increase in Numb protein levels by nandrolone. In addition, transfection of mdm2-siRNA mimicked the effect of nandrolone to prolong the half-life of Numb protein. Moreover, when C2C12 cells were forced to overexpress mdm2, there was a significant decline in the expression of both basal and inducible Numb protein. Our data suggest that nandrolone, by a novel mechanism for this agent in a muscle cell type, increases Numb protein levels in C2C12 myoblasts by stabilizing Numb protein against degradation, at least in part, via suppression of mdm2 expression. PMID:22700758

  10. A synthetic compound that potentiates bone morphogenetic protein-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype.

    PubMed

    Kato, Satoshi; Sangadala, Sreedhara; Tomita, Katsuro; Titus, Louisa; Boden, Scott D

    2011-03-01

    There is an urgent need to develop methods that lower costs of using recombinant human bone morphogenetic proteins (BMPs) to promote bone induction. In this study, we demonstrate the osteogenic effect of a low-molecular weight compound, SVAK-12, that potentiated the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. Here, we report a specific compound, SVAK-12, which was selected based on in silico screenings of small-molecule databases using the homology modeled interaction motif of Smurf1-WW2 domain. The enhancement of BMP-2 activity by SVAK-12 was characterized by evaluating a BMP-specific reporter activity and by monitoring the BMP-2-induced expression of mRNA for osteocalcin and alkaline phosphatase (ALP), which are widely accepted marker genes of osteoblast differentiation. Finally, we confirmed these results by also measuring the enhancement of BMP-2-induced activity of ALP. Smurf1 is an E3 ligase that targets osteogenic Smads for ubiquitin-mediated proteasomal degradation. Smurf1 is an interesting potential target to enhance bone formation based on the positive effects on bone of proteins that block Smurf1-binding to Smad targets or in Smurf1-/- knockout mice. Since Smads bind Smurf1 via its WW2 domain, we performed in silico screening to identify compounds that might interact with the Smurf1-WW2 domain. We recently reported the activity of a compound, SVAK-3. However, SVAK-3, while exhibiting BMP-potentiating activity, was not stable and thus warranted a new search for a more stable and efficacious compound among a selected group of candidates. In addition to being more stable, SVAK-12 exhibited a dose-dependent activity in inducing osteoblastic differentiation of myoblastic C2C12 cells even when multiple markers of the osteoblastic phenotype were parallelly monitored. PMID:21110071

  11. Reduction of myoblast differentiation following multiple population doublings in mouse C2 C12 cells: a model to investigate ageing?

    PubMed

    Sharples, Adam P; Al-Shanti, Nasser; Lewis, Mark P; Stewart, Claire E

    2011-12-01

    Ageing skeletal muscle displays declines in size, strength, and functional capacity. Given the acknowledged role that the systemic environment plays in reduced regeneration (Conboy et al. [2005] Nature 433: 760-764), the role of resident satellite cells (termed myoblasts upon activation) is relatively dismissed, where, multiple cellular divisions in-vivo throughout the lifespan could also impact on muscular deterioration. Using a model of multiple population doublings (MPD) in-vitro thus provided a system in which to investigate the direct impact of extensive cell duplications on muscle cell behavior. C(2) C(12) mouse skeletal myoblasts (CON) were used fresh or following 58 population doublings (MPD). As a result of multiple divisions, reduced morphological and biochemical (creatine kinase, CK) differentiation were observed. Furthermore, MPD cells had significantly increased cells in the S and decreased cells in the G1 phases of the cell cycle versus CON, following serum withdrawal. These results suggest continued cycling rather than G1 exit and thus reduced differentiation (myotube atrophy) occurs in MPD muscle cells. These changes were underpinned by significant reductions in transcript expression of: IGF-I and myogenic regulatory factors (myoD and myogenin) together with elevated IGFBP5. Signaling studies showed that decreased differentiation in MPD was associated with decreased phosphorylation of Akt, and with later increased phosphorylation of JNK1/2. Chemical inhibition of JNK1/2 (SP600125) in MPD cells increased IGF-I expression (non-significantly), however, did not enhance differentiation. This study provides a potential model and molecular mechanisms for deterioration in differentiation capacity in skeletal muscle cells as a consequence of multiple population doublings that would potentially contribute to the ageing process. PMID:21826704

  12. Exogenous connexin43-expressing autologous skeletal myoblasts ameliorate mechanical function and electrical activity of the rabbit heart after experimental infarction

    PubMed Central

    Antanavičiūtė, Ieva; Ereminienė, Eglė; Vysockas, Vaidas; Račkauskas, Mindaugas; Skipskis, Vilius; Rysevaitė, Kristina; Treinys, Rimantas; Benetis, Rimantas; Jurevičius, Jonas; Skeberdis, Vytenis A

    2015-01-01

    Acute myocardial infarction is one of the major causes of mortality worldwide. For regeneration of the rabbit heart after experimentally induced infarction we used autologous skeletal myoblasts (SMs) due to their high proliferative potential, resistance to ischaemia and absence of immunological and ethical concerns. The cells were characterized with muscle-specific and myogenic markers. Cell transplantation was performed by injection of cell suspension (0.5 ml) containing approximately 6 million myoblasts into the infarction zone. The animals were divided into four groups: (i) no injection; (ii) sham injected; (iii) injected with wild-type SMs; and (iv) injected with SMs expressing connexin43 fused with green fluorescent protein (Cx43EGFP). Left ventricular ejection fraction (LVEF) was evaluated by 2D echocardiography in vivo before infarction, when myocardium has stabilized after infarction, and 3 months after infarction. Electrical activity in the healthy and infarction zones of the heart was examined ex vivo in Langendorff-perfused hearts by optical mapping using di-4-ANEPPS, a potential sensitive fluorescent dye. We demonstrate that SMs in the coculture can couple electrically not only to abutted but also to remote acutely isolated allogenic cardiac myocytes through membranous tunnelling tubes. The beneficial effect of cellular therapy on LVEF and electrical activity was observed in the group of animals injected with Cx43EGFP-expressing SMs. L-type Ca2+ current amplitude was approximately fivefold smaller in the isolated SMs compared to healthy myocytes suggesting that limited recovery of LVEF may be related to inadequate expression or function of L-type Ca2+ channels in transplanted differentiating SMs. PMID:25529770

  13. IGF-1 induces IP3 -dependent calcium signal involved in the regulation of myostatin gene expression mediated by NFAT during myoblast differentiation.

    PubMed

    Valdés, Juan A; Flores, Sylvia; Fuentes, Eduardo N; Osorio-Fuentealba, Cesar; Jaimovich, Enrique; Molina, Alfredo

    2013-07-01

    Skeletal muscle differentiation is a complex and highly regulated process characterized by cell cycle arrest, which is associated with morphological changes including myoblast alignment, elongation, and fusion into multinucleated myotubes. This is a balanced process dynamically coordinated by positive and negative signals such as the insulin-like growth factor I (IGF-1) and myostatin (MSTN), respectively. In this study, we report that the stimulation of skeletal myoblasts during differentiation with IGF-1 induces a rapid and transient calcium increase from intracellular stores, which are principally mediated through the phospholipase C gamma (PLC γ)/inositol 1,4,5-triphosphate (IP3 )-dependent signaling pathways. This response was completely blocked when myoblasts were incubated with LY294002 or transfected with the dominant-negative p110 gamma, suggesting a fundamental role of phosphatidylinositol 3-kinase (PI3K) in PLCγ activation. Additionally, we show that calcium released via IP3 and induced by IGF-1 stimulates NFAT-dependent gene transcription and nuclear translocation of the GFP-labeled NFATc3 isoform. This activation was independent of extracellular calcium influx and calcium release mediated by ryanodine receptor (RyR). Finally, we examined mstn mRNA levels and mstn promoter activity in myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents and in reporter activity, which was inhibited by cyclosporin A, 11R-VIVIT, and by inhibitors of the PI3Kγ, PLCγ, and IP3 receptor. Our results strongly suggest that IGF-1 regulates myostatin transcription through the activation of the NFAT transcription factor in an IP3 /calcium-dependent manner. This is the first study to demonstrate a role of calcium-dependent signaling pathways in the mRNA expression of myostatin. PMID:23255067

  14. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

    SciTech Connect

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev; Renkawitz-Pohl, Renate

    2013-02-15

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity.

  15. Unexpected Distinct Roles of the Related Histone H3 Lysine 9 Methyltransferases G9a and G9a-Like Protein in Myoblasts.

    PubMed

    Battisti, Valentine; Pontis, Julien; Boyarchuk, Ekaterina; Fritsch, Lauriane; Robin, Philippe; Ait-Si-Ali, Slimane; Joliot, Véronique

    2016-06-01

    Lysine methyltransferases G9a and GLP (G9a-like protein) are highly homologous and form functional heterodimeric complexes that establish mono- and dimethylation on histone H3 lysine 9 (H3K9me1, H3K9me2) in euchromatin. Here, we describe unexpected distinct roles for G9a and GLP in skeletal muscle terminal differentiation. Indeed, gain- or loss-of-function assays in myoblasts showed, in agreement with previous reports, that G9a inhibits terminal differentiation. While GLP plays a more intricate role in muscle differentiation,in one hand, both GLP gain and loss of function inhibit late steps of differentiation; on the other hand, in contrast to G9a, GLP overexpression promotes abnormal precocious expression of muscle differentiation-specific genes already in proliferating myoblasts. In agreement, transcriptomic analysis indicates that G9a and GLP regulate different sets of genes. Thus, GLP, but not G9a, inhibits proteasome subunit-encoding genes expression, resulting in an inhibition of the proteasome activities. Subsequently, GLP, but not G9a, overexpression stabilizes MyoD that is likely to be responsible for muscle markers expression in proliferating myoblasts. PMID:27056598

  16. Change of hyaluronic acid synthesis during differentiation of myogenic cells and its relation to transformation of myoblasts by Rous sarcoma virus.

    PubMed

    Yoshimura, M

    1985-05-01

    Hyaluronic acid synthesis was examined in cultures of differentiating chick embryo muscle cells before, during and after fusion. Prior to fusion, hyaluronic acid was synthesized and secreted into the medium, but once fusion began this synthesis was reduced significantly. Synthesis then increased again after completion of fusion. Thus, production of hyaluronic acid was lowest at the time of or right before cell fusion. When myoblasts were transformed by Rous sarcoma virus (RSV), a higher amount of hyaluronic acid was synthesized, and cells were not able to fuse. The turnover rate of hyaluronic acid might be different between myotubes and RSV-transformed myoblasts. The addition of exogenous hyaluronic acid to myoblast cultures resulted in the partial inhibition of fusion. The effect was reversible because fusion took place after removal of the exogenous hyaluronic acid. These observations suggest that hyaluronic acid plays an important role in the differentiation of myogenic cells, and that elevated hyaluronic acid synthesis may partly be the reason for inhibition of myotube formation upon transformation by Rous sarcoma virus. PMID:2988797

  17. Mesoporous silica nanoparticle-based substrates for cell directed delivery of Notch signalling modulators to control myoblast differentiation

    NASA Astrophysics Data System (ADS)

    Böcking, Dominique; Wiltschka, Oliver; Niinimäki, Jenni; Shokry, Hussein; Brenner, Rolf; Lindén, Mika; Sahlgren, Cecilia

    2014-01-01

    Biochemical cues are critical to control stem cell function and can be utilized to develop smart biomaterials for stem cell engineering. The challenge is to deliver these cues in a restricted manner with spatial and temporal control. Here we have developed bilayer films of mesoporous silica nanoparticles for delayed cellular delivery of Notch modulators to promote muscle stem cell differentiation. We demonstrate that drug-loaded particles are internalized from the particle-covered surface, which allows for direct delivery of the drug into the cell and a delayed and confined drug release. Substrates of particles loaded with γ-secretase-inhibitors, which block the Notch signalling pathway, promoted efficient differentiation of myoblasts. The particle substrates were fully biocompatible and did not interfere with the inherent differentiation process. We further demonstrate that impregnating commercially available, biocompatible polymer scaffolds with MSNs allows for a free standing substrate for cell directed drug delivery.Biochemical cues are critical to control stem cell function and can be utilized to develop smart biomaterials for stem cell engineering. The challenge is to deliver these cues in a restricted manner with spatial and temporal control. Here we have developed bilayer films of mesoporous silica nanoparticles for delayed cellular delivery of Notch modulators to promote muscle stem cell differentiation. We demonstrate that drug-loaded particles are internalized from the particle-covered surface, which allows for direct delivery of the drug into the cell and a delayed and confined drug release. Substrates of particles loaded with γ-secretase-inhibitors, which block the Notch signalling pathway, promoted efficient differentiation of myoblasts. The particle substrates were fully biocompatible and did not interfere with the inherent differentiation process. We further demonstrate that impregnating commercially available, biocompatible polymer scaffolds with

  18. Paralogs of Atlantic salmon myoblast determination factor genes are distinctly regulated in proliferating and differentiating myogenic cells.

    PubMed

    Bower, Neil I; Johnston, Ian A

    2010-06-01

    The mRNA expression of myogenic regulatory factors, including myoD1 (myoblast determination factor) gene paralogs, and their regulation by amino acids and insulin-like growth factors were investigated in primary cell cultures isolated from fast myotomal muscle of Atlantic salmon (Salmo salar). The cell cycle and S phase were determined as 28.1 and 13.3 h, respectively, at 18 degrees C. Expression of myoD1b and myoD1c peaked at 8 days of culture in the initial proliferation phase and then declined more than sixfold as cells differentiated and was correlated with PCNA (proliferating cell nuclear antigen) expression (R = 0.88, P < 0.0001; R = 0.70, P < 0.0001). In contrast, myoD1a transcripts increased from 2 to 8 days and remained at elevated levels as myotubes were formed. mRNA levels of myoD1c were, on average, 3.1- and 5.7-fold higher than myoD1a and myoD1b, respectively. Depriving cells of amino acids and serum led to a rapid increase in pax7 and a decrease in myoD1c and PCNA expression, indicating a transition to a quiescent state. In contrast, amino acid replacement in starved cells produced significant increases in myoD1c (at 6 h), PCNA (at 12 h), and myoD1b (at 24 h) and decreases in pax7 expression as cells entered the cell cycle. Our results are consistent with temporally distinct patterns of myoD1c and myoD1b expression at the G(1) and S/G(2) phases of the cell cycle. Treatment of starved cells with insulin-like growth factor I or II did not alter expression of the myoD paralogs. It was concluded that, in vitro, amino acids alone are sufficient to stimulate expression of genes regulating myogenesis in myoblasts involving autocrine/paracrine pathways. The differential responses of myoD paralogs during myotube maturation and amino acid treatments suggest that myoD1b and myoD1c are primarily expressed in proliferating cells and myoD1a in differentiating cells, providing evidence for their subfunctionalization following whole genome and local duplications in

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

    PubMed

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

    2015-11-01

    The aim is to study the effects and underlying mechanisms of astragalus polysaccharide (APS) on the peroxide-induced injury in C2C12 myoblasts in vitro. Cell viability in the presence or absence of APS was detected by the methyl thiazolyl tetrazolium colorimetric assay. The autophagosomes were observed by electron microscopy to examine the influence of APS on autophagy caused by H2O2 in C2C12 cells, and the percentage of apoptosis cells was measured by flow cytometry. To further confirm the effect of H2O2 on C2C12 cells, the protein expression of LC3 and RARP, which are the markers of autophagy and apoptosis, respectively, was analyzed by Western blot, as well as the expression levels of p-p70S6K, p70S6K, Bcl-2, Bax, cyto-C, and Caspase-3, to reveal the underlying mechanisms. We observed multiple effects of APS on C2C12 functionality. APS treatment of C2C12 cells at 1 mg/mL reduced cell viability to less than 70 %, and analysis by electron microscopy revealed that APS also reduced the number of H2O2-induced autophagosome formation. Similarly, APS abated the H2O2-mediated increase in cell apoptosis, which was accompanied by the inhibition of LC3 II and RARP that are normally upregulated by H2O2. The expression of p-p70S6K and p70S6K, however, remained unchanged in C2C12 cells in the Control, H2O2 and H2O2 + APS groups. In addition, APS promoted the expression of protein Bcl-2 in H2O2-treated C2C12 cells, but did not change Bax, thus reducing the Bax/Bcl-2 ratio that in turn prevented the release of cytochrome c and the activation of caspase-3. APS inhibits the autophagy and apoptosis induced by peroxide injury in C2C12 myoblasts through two independent signaling pathways: the mTOR-independent pathway for the inhibition of autophagy, and the caspase-3-dependent pathway for the suppression of apoptosis. PMID:27352334

  20. WHAMY is a novel actin polymerase promoting myoblast fusion, macrophage cell motility and sensory organ development in Drosophila.

    PubMed

    Brinkmann, Klaus; Winterhoff, Moritz; Önel, Susanne-Filiz; Schultz, Jörg; Faix, Jan; Bogdan, Sven

    2016-02-01

    Wiskott-Aldrich syndrome proteins (WASPs) are nucleation-promoting factors (NPF) that differentially control the Arp2/3 complex. In Drosophila, three different family members, SCAR (also known as WAVE), WASP and WASH (also known as CG13176), have been analyzed so far. Here, we characterized WHAMY, the fourth Drosophila WASP family member. whamy originated from a wasp gene duplication and underwent a sub-neofunctionalization. Unlike WASP, we found that WHAMY specifically interacted with activated Rac1 through its two CRIB domains, which were sufficient for targeting WHAMY to lamellipodial and filopodial tips. Biochemical analyses showed that WHAMY promoted exceptionally fast actin filament elongation, although it did not activate the Arp2/3 complex. Loss- and gain-of-function studies revealed an important function of WHAMY in membrane protrusions and cell migration in macrophages. Genetic data further implied synergistic functions between WHAMY and WASP during morphogenesis. Double mutants were late-embryonic lethal and showed severe defects in myoblast fusion. Trans-heterozygous mutant animals showed strongly increased defects in sensory cell fate specification. Thus, WHAMY is a novel actin polymerase with an initial partitioning of ancestral WASP functions in development and subsequent acquisition of a new function in cell motility during evolution. PMID:26675239

  1. Small molecules dorsomorphin and LDN-193189 inhibit myostatin/GDF8 signaling and promote functional myoblast differentiation.

    PubMed

    Horbelt, Daniel; Boergermann, Jan H; Chaikuad, Apirat; Alfano, Ivan; Williams, Eleanor; Lukonin, Ilya; Timmel, Tobias; Bullock, Alex N; Knaus, Petra

    2015-02-01

    GDF8, or myostatin, is a member of the TGF-β superfamily of secreted polypeptide growth factors. GDF8 is a potent negative regulator of myogenesis both in vivo and in vitro. We found that GDF8 signaling was inhibited by the small molecule ATP competitive inhibitors dorsomorphin and LDN-193189. These compounds were previously shown to be potent inhibitors of BMP signaling by binding to the BMP type I receptors ALK1/2/3/6. We present the crystal structure of the type II receptor ActRIIA with dorsomorphin and demonstrate that dorsomorphin or LDN-193189 target GDF8 induced Smad2/3 signaling and repression of myogenic transcription factors. As a result, both inhibitors rescued myogenesis in myoblasts treated with GDF8. As revealed by quantitative live cell microscopy, treatment with dorsomorphin or LDN-193189 promoted the contractile activity of myotubular networks in vitro. We therefore suggest these inhibitors as suitable tools to promote functional myogenesis. PMID:25368322

  2. Comparative Proteomic Study of Fatty Acid-treated Myoblasts Reveals Role of Cox-2 in Palmitate-induced Insulin Resistance

    PubMed Central

    Chen, Xiulan; Xu, Shimeng; Wei, Shasha; Deng, Yaqin; Li, Yiran; Yang, Fuquan; Liu, Pingsheng

    2016-01-01

    Accumulated studies demonstrate that saturated fatty acids (FAs) such as palmitic acid (PA) inhibit insulin signaling in skeletal muscle cells and monounsaturated fatty acids such as oleic acid (OA) reverse the effect of PA on insulin signaling. The detailed molecular mechanism of these opposite effects remains elusive. Here we provide a comparative proteomic study of skeletal myoblast cell line C2C12 that were untreated or treated with PA, and PA plus OA. A total of 3437 proteins were quantified using SILAC in this study and 29 proteins fall into the pattern that OA reverses PA effect. Expression of some these proteins were verified using qRT-PCR and Western blot. The most significant change was cyclooxygenase-2 (Cox-2). In addition to whole cell comparative proteomic study, we also compared lipid droplet (LD)-associated proteins and identified that Cox-2 was one of three major altered proteins under the FA treatment. This finding was then confirmed using immunofluorescence. Finally, Cox-2 selective inhibitor, celecoxib protected cells from PA-reduced insulin signaling Akt phosphorylation. Together, these results not only provide a dataset of protein expression change in FA treatment but also suggest that Cox-2 and lipid droplets (LDs) are potential players in PA- and OA-mediated cellular processes. PMID:26899878

  3. Subnuclear localization and differentiation-dependent increased expression of DGK-zeta in C2C12 mouse myoblasts.

    PubMed

    Evangelisti, Camilla; Riccio, Massimo; Faenza, Irene; Zini, Nicoletta; Hozumi, Yasukazu; Goto, Kaoru; Cocco, L; Martelli, Alberto M

    2006-11-01

    Diacylglycerol kinases (DGKs) catalyze phosphorylation of diacylglycerol (DG) to yield phosphatidic acid (PA). Previous evidence has shown that the nucleus contains several DGK isoforms. In this study, we have analyzed the expression and subnuclear localization of DGK-zeta employing C2C12 mouse myoblasts. Immunocytochemistry coupled to confocal laser scanning microscopy showed that both endogenous and green fluorescent protein-tagged overexpressed DGK-zeta localized mostly to the nucleus. In contrast, overexpressed DGK-alpha, -beta, -delta, and -iota did not migrate to the nucleus. DGK-zeta was present in the nuclear speckle domains, as also revealed by immuno-electron microscopy analysis. Moreover, DGK-zeta co-localized and interacted with phosphoinositide-specific phospholipase Cbeta1 (PLCbeta1), that is involved in inositide-dependent signaling pathways important for the regulation of cell proliferation and differentiation. Furthermore, we report that DGK-zeta associated with nuclear matrix, the fundamental organizing principle of the nucleus where many cell functions take place, including DNA replication, gene expression, and protein phosphorylation. Nuclear DGK-zeta increased during myogenic differentiation of C2C12 cells, while DGK-zeta down-regulation by siRNA markedly impaired differentiation. Overall, our findings further support the importance of speckles and nuclear matrix in lipid-dependent signaling and suggest that nuclear DGK-zeta might play some fundamental role during myogenic differentiation of C2C12 cells. PMID:16897754

  4. Specific deletion of CMF1 nuclear localization domain causes incomplete cell cycle withdrawal and impaired differentiation in avian skeletal myoblasts

    SciTech Connect

    Dees, Ellen . E-mail: ellen.dees@vanderbilt.edu; Robertson, J. Brian; Zhu, Tianli; Bader, David

    2006-10-01

    CMF1 is a protein expressed in embryonic striated muscle with onset of expression preceding that of contractile proteins. Disruption of CMF1 in myoblasts disrupts muscle-specific protein expression. Preliminary studies indicate both nuclear and cytoplasmic distribution of CMF1 protein, suggesting functional roles in both cellular compartments. Here we examine the nuclear function of CMF1, using a newly characterized antibody generated against the CMF1 nuclear localization domain and a CMF1 nuclear localization domain-deleted stable myocyte line. The antibody demonstrates nuclear distribution of the CMF1 protein both in vivo and in cell lines, with clustering of CMF1 protein around chromatin during mitosis. In more differentiated myocytes, the protein shifts to the cytoplasm. The CMF1 NLS-deleted cell lines have markedly impaired capacity to differentiate. Specifically, these cells express less contractile protein than wild-type or full-length CMF1 stably transfected cells, and do not fuse properly into multinucleate syncytia with linear nuclear alignment. In response to low serum medium, a signal to differentiate, CMF1 NLS-deleted cells enter G0, but continue to express proliferation markers and will reenter the cell cycle when stimulated by restoring growth medium. These data suggest that CMF1 is involved in regulation the transition from proliferation to differentiation in embryonic muscle.

  5. Small Molecules Dorsomorphin and LDN-193189 Inhibit Myostatin/GDF8 Signaling and Promote Functional Myoblast Differentiation*

    PubMed Central

    Horbelt, Daniel; Boergermann, Jan H.; Chaikuad, Apirat; Alfano, Ivan; Williams, Eleanor; Lukonin, Ilya; Timmel, Tobias; Bullock, Alex N.; Knaus, Petra

    2015-01-01

    GDF8, or myostatin, is a member of the TGF-β superfamily of secreted polypeptide growth factors. GDF8 is a potent negative regulator of myogenesis both in vivo and in vitro. We found that GDF8 signaling was inhibited by the small molecule ATP competitive inhibitors dorsomorphin and LDN-193189. These compounds were previously shown to be potent inhibitors of BMP signaling by binding to the BMP type I receptors ALK1/2/3/6. We present the crystal structure of the type II receptor ActRIIA with dorsomorphin and demonstrate that dorsomorphin or LDN-193189 target GDF8 induced Smad2/3 signaling and repression of myogenic transcription factors. As a result, both inhibitors rescued myogenesis in myoblasts treated with GDF8. As revealed by quantitative live cell microscopy, treatment with dorsomorphin or LDN-193189 promoted the contractile activity of myotubular networks in vitro. We therefore suggest these inhibitors as suitable tools to promote functional myogenesis. PMID:25368322

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

    PubMed

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

    2013-01-01

    The myogenic differentiation of C2C12 myoblast cells is induced by the novel androgen receptor (AR) partial agonist, (17α,20E)-17,20-[(1-methoxyethylidene)bis-(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11), as well as by dihydrotestosterone (DHT). YK11 is a selective androgen receptor modulator (SARM), which activates AR without the N/C interaction. In this study, we further investigated the mechanism by which YK11 induces myogenic differentiation of C2C12 cells. The induction of key myogenic regulatory factors (MRFs), such as myogenic differentiation factor (MyoD), myogenic factor 5 (Myf5) and myogenin, was more significant in the presence of YK11 than in the presence of DHT. YK11 treatment of C2C12 cells, but not DHT, induced the expression of follistatin (Fst), and the YK11-mediated myogenic differentiation was reversed by anti-Fst antibody. These results suggest that the induction of Fst is important for the anabolic effect of YK11. PMID:23995658

  7. Modulation of Cell Cycle Progression by 5-Azacytidine Is Associated with Early Myogenesis Induction in Murine Myoblasts

    PubMed Central

    Montesano, Anna; Luzi, Livio; Senesi, Pamela; Terruzzi, Ileana

    2013-01-01

    Myogenesis is a multistep process, in which myoblasts withdraw from the cell cycle, cease to divide, elongate and fuse to form multinucleated myotubes. Cell cycle transition is controlled by a family of cyclin-dependent protein kinases (CDKs) regulated by association with cyclins, negative regulatory subunits and phosphorylation. Muscle differentiation is orchestrated by myogenic regulatory factors (MRFs), such as MyoD and Myf-5. DNA methylation is crucial in transcriptional control of genes involved in myogenesis. Previous work has indicated that treatment of fibroblasts with the DNA-demethylating agent 5-azacytidine (AZA) promotes MyoD expression. We studied the effects of AZA on cell cycle regulation and MRFs synthesis during myoblast proliferation and early myogenesis phases in C2C12 cells. During the proliferation phase, cells were incubated in growth medium with 5µM AZA (GMAZA) or without AZA (GM) for 24 hours. At 70% confluence, cells were kept in growth medium in order to spontaneously achieve differentiation or transferred to differentiation medium with 5μM AZA (DMAZA) or without AZA (DM) for 12 and 24 hours. Cells used as control were unstimulated. In the proliferation phase, AZA-treated cells seemed to lose their characteristic circular shape and become elongated. The presence of AZA resulted in significant increases in the protein contents of Cyclin-D (FC:1.23 GMAZA vs GM p≤0.05), p21 (FC: 1.23 GMAZA vs GM p≤0.05), Myf-5 (FC: 1.21 GMAZA vs GM p≤0.05) and MyoD (FC: 1.20 GMAZA vs GM p≤0.05). These results propose that AZA could inhibit cell proliferation. During 12 hours of differentiation, AZA decreased the downregulation of genes involved in cell cycle arrest and in restriction point (G1 and G1/S phase) and the expression of several cyclins, E2F Transcription Factors, cyclin-dependent kinase inhibitors, specific genes responsible of cell cycle negative regulation. During 24 hours of differentiation, AZA induced an increment in the protein

  8. Modulation of cell cycle progression by 5-azacytidine is associated with early myogenesis induction in murine myoblasts.

    PubMed

    Montesano, Anna; Luzi, Livio; Senesi, Pamela; Terruzzi, Ileana

    2013-01-01

    Myogenesis is a multistep process, in which myoblasts withdraw from the cell cycle, cease to divide, elongate and fuse to form multinucleated myotubes. Cell cycle transition is controlled by a family of cyclin-dependent protein kinases (CDKs) regulated by association with cyclins, negative regulatory subunits and phosphorylation. Muscle differentiation is orchestrated by myogenic regulatory factors (MRFs), such as MyoD and Myf-5. DNA methylation is crucial in transcriptional control of genes involved in myogenesis. Previous work has indicated that treatment of fibroblasts with the DNA-demethylating agent 5-azacytidine (AZA) promotes MyoD expression. We studied the effects of AZA on cell cycle regulation and MRFs synthesis during myoblast proliferation and early myogenesis phases in C2C12 cells. During the proliferation phase, cells were incubated in growth medium with 5µM AZA (GMAZA) or without AZA (GM) for 24 hours. At 70% confluence, cells were kept in growth medium in order to spontaneously achieve differentiation or transferred to differentiation medium with 5μM AZA (DMAZA) or without AZA (DM) for 12 and 24 hours. Cells used as control were unstimulated. In the proliferation phase, AZA-treated cells seemed to lose their characteristic circular shape and become elongated. The presence of AZA resulted in significant increases in the protein contents of Cyclin-D (FC:1.23 GMAZA vs GM p≤0.05), p21 (FC: 1.23 GMAZA vs GM p≤0.05), Myf-5 (FC: 1.21 GMAZA vs GM p≤0.05) and MyoD (FC: 1.20 GMAZA vs GM p≤0.05). These results propose that AZA could inhibit cell proliferation. During 12 hours of differentiation, AZA decreased the downregulation of genes involved in cell cycle arrest and in restriction point (G1 and G1/S phase) and the expression of several cyclins, E2F Transcription Factors, cyclin-dependent kinase inhibitors, specific genes responsible of cell cycle negative regulation. During 24 hours of differentiation, AZA induced an increment in the protein

  9. Alteration in Phospholipidome Profile of Myoblast H9c2 Cell Line in a Model of Myocardium Starvation and Ischemia.

    PubMed

    Sousa, Bebiana; Melo, Tânia; Campos, Ana; Moreira, Ana S P; Maciel, Elisabete; Domingues, Pedro; Carvalho, Rita Pereira; Rodrigues, Teresa Ribeiro; Girão, Henrique; Domingues, Maria Rosario M

    2016-10-01

    Myocardium infarction is one of the most deathly cardiovascular diseases. It is characterized by myocardium ischemia as a result of nutrients depletion and hypoxia. The cell can respond to this injury by autophagy or apoptosis, which determines the evolution and possible recovery of the myocardium infarction. Lipids play an important role in cardiovascular disease. However reports stating lipidome variations in cardiovascular disease are scarce and the role that lipids play in this pathological condition is not completely understood. The aim of this work was to identify changes in lipid profile of a myoblast H9c2 cell line under starvation and ischemia, to better understand and recognize new biomarkers for myocardial infarction. Lipidomic profile was evaluated by HILIC-LC-MS and GC-MS. Cardiac cells showed alterations in phosphatidylcholines PC (34:1) and PC (36:2), lysophosphatidylcholines lyso PC(16:0), lysoPC(18:1) and lysoPC(18:0), phosphatidylethanolamine PE (34:1), phosphatidylserine PS (36:1), phosphatidylinositol PI (36:2), PI (38:3) and PI (38:5), sphingomyelin SM (34:1) and cardiolipins CL(68:4), CL(72:5) and CL(74:7) in ischemia and/or starvation, in comparison with control. Specific differences observed only in starvation were decrease of SM (34:1) and FA (20:4), and increase of PS (36:1). Differences observed only in ischemia were decrease of PC (36:2), lyso PC (16:0) and FA (18:1) and simultaneous increase of FA (16:0), and FA (18:0). Interestingly, PC (34:1) increased in ischemia and decreased in starvation. In conclusion, our work suggests that lipids are potential markers for evaluation of cell fate, either cell death or recovery, which will be useful to improve diagnosis and prognostic of cardiovascular diseases. J. Cell. Physiol. 231: 2266-2274, 2016. © 2016 Wiley Periodicals, Inc. PMID:26887290

  10. Skeletal Myoblast Cell Sheet Implantation Ameliorates Both Systolic and Diastolic Cardiac Performance in Canine Dilated Cardiomyopathy Model

    PubMed Central

    Shirasaka, Tomonori; Miyagawa, Shigeru; Fukushima, Satsuki; Kawaguchi, Naomasa; Nakatani, Satoshi; Daimon, Takashi; Okita, Yutaka; Sawa, Yoshiki

    2016-01-01

    Background Improving both systolic and diastolic function may be the most important factor in treating heart failure. In this study, we hypothesized that cell-sheet transplantation could improve these function in the damaged heart. Methods We generated a dilated cardiomyopathy model in beagles by continuous ventricle pacing at 240 beats per minute. After 4 weeks, the beagles underwent skeletal myoblast cell sheet transplantation (SMCST) or a sham operation, and rapid ventricle pacing continued for an additional 4 weeks. Six of the e8 beagles treated by SMCST were still alive 4 weeks after the procedure. We evaluated SMCST's cardiotherapeutic effects by comparing beagles treated by SMCST with beagles that underwent a sham operation (control, n = 5). Results Diastolic function, as well as systolic function improved significantly in the SMCST group as compared with the sham group (control vs SMCST group, median [interquartile range]: E/E', 16 [0.9] vs 11 [1.0]; P < 0.001; tau, 47 [6.0] vs 36 [4.4] ms: P = 0.005. Ejection fraction, 22 (6.0) versus 46 (7.5) %, P < 0.001; end-systolic elastance, 2.5 (0.4) versus 8.2 (3.5) mm Hg/ml, P = 0.001). Histological examination revealed that the volume of collagen I and the collagen I/III ratio in the myocardium were significantly higher in the control than that in the SMCST group (collagen I, 6.0 [0.8] vs 2.6 [1.3]; P = 0.006; collagen I/III ratio, 4.8 [1.7] vs 1.2 [0.4]; P = 0.010). Conclusions The potential of SMCST to ameliorate both systolic and diastolic performance was proven. The SMCST may be an alternative therapy of conventional medical treatment in the dilated cardiomyopathy heart. PMID:26636739

  11. Folic acid promotes the myogenic differentiation of C2C12 murine myoblasts through the Akt signaling pathway.

    PubMed

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

    2015-10-01

    Folic acid is a water-soluble vitamin in the B-complex group, and an exogenous intake is required for health, growth and development. As a precursor to co-factors, folic acid is required for one-carbon donors in the synthesis of DNA bases and other essential biomolecules. A lack of dietary folic acid can lead to folic acid deficiency and can therefore result in several health problems, including macrocytic anemia, elevated plasma homocysteine levels, cardiovascular disease, birth defects, carcinogenesis, muscle weakness and difficulty in walking. Previous studies have indicated that folic acid exerts a positive effect on skeletal muscle functions. However, the precise role of folic acid in skeletal muscle cell differentiation remains poorly understood. Thus, in the present study, we examined the effects of folic acid on neo-myotube maturation and differentiation using C2C12 murine myoblasts. We found that folic acid promoted the formation of multinucleated myotubes, and increased the fusion index and creatine kinase (CK) activity in a concentration-dependent manner. In addition, western blot analysis revealed that the expression levels of the muscle-specific marker, myosin heavy chain (MyHC), as well as those of the myogenic regulatory factors (MRFs), MyoD and myogenin, were increased in the folic acid-treated myotubes during myogenic differentiation. Folic acid also promoted the activation of the Akt pathway, and this effect was inhibited by treatment of the C2C12 cells with LY294002 (Akt inhibitor). Blocking of the Akt pathway with a specific inhibitor revealed that it was necessary for mediating the stimulatory effects of folic acid on muscle cell differentiation and fusion. Taken together, our data suggest that folic acid promotes the differentiation of C2C12 cells through the activation of the Akt pathway. PMID:26310574

  12. The PARN Deadenylase Targets a Discrete Set of mRNAs for Decay and Regulates Cell Motility in Mouse Myoblasts

    PubMed Central

    Lee, Jerome E.; Lee, Ju Youn; Trembly, Jarrett; Wilusz, Jeffrey; Tian, Bin; Wilusz, Carol J.

    2012-01-01

    PARN is one of several deadenylase enzymes present in mammalian cells, and as such the contribution it makes to the regulation of gene expression is unclear. To address this, we performed global mRNA expression and half-life analysis on mouse myoblasts depleted of PARN. PARN knockdown resulted in the stabilization of 40 mRNAs, including that encoding the mRNA decay factor ZFP36L2. Additional experiments demonstrated that PARN knockdown induced an increase in Zfp36l2 poly(A) tail length as well as increased translation. The elements responsible for PARN-dependent regulation lie within the 3′ UTR of the mRNA. Surprisingly, changes in mRNA stability showed an inverse correlation with mRNA abundance; stabilized transcripts showed either no change or a decrease in mRNA abundance. Moreover, we found that stabilized mRNAs had reduced accumulation of pre–mRNA, consistent with lower transcription rates. This presents compelling evidence for the coupling of mRNA decay and transcription to buffer mRNA abundances. Although PARN knockdown altered decay of relatively few mRNAs, there was a much larger effect on global gene expression. Many of the mRNAs whose abundance was reduced by PARN knockdown encode factors required for cell migration and adhesion. The biological relevance of this observation was demonstrated by the fact that PARN KD cells migrate faster in wound-healing assays. Collectively, these data indicate that PARN modulates decay of a defined set of mRNAs in mammalian cells and implicate this deadenylase in coordinating control of genes required for cell movement. PMID:22956911

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

    PubMed Central

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

    2015-01-01

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

  14. Serotonin and Histamine Therapy Increases Tetanic Forces of Myoblasts, Reduces Muscle Injury, and Improves Grip Strength Performance of Dmdmdx Mice

    PubMed Central

    Lins, Jeremy; Lambert, Kristyn; Lazauski, Joan; Spaulding, James; McMichael, John

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a recessive X-linked fatal disorder caused by a mutation in the dystrophin gene. Although several therapeutic approaches have been studied, none has led to substantial long-term effects in patients. The aim of this study was to test a serotonin and histamine (S&H) combination on human skeletal myoblasts and Dmdmdx mice for its effects on muscle strength and injury. Normal human bioartificial muscles (BAMs) were treated, and muscle tetanic forces and muscle injury tests were performed using the MyoForce Analysis System. Dmdmdx mice, the murine model of DMD, were administered serotonin, histamine, or S&H combination twice daily for 6 weeks, and functional performance tests were conducted once a week. The S&H combination treatment caused significant increases in tetanic forces at all time points and concentrations tested as compared to the saline controls. Dose response of the BAMs to the treatment demonstrated a significant increase in force generation at all concentrations compared to the controls after 3 to 4 days of drug treatment. The highest 3 concentrations had a significant effect on lowering contractile-induced injury as measured by a reduction in the release of adenylate kinase. Histamine-only and S&H treatments improved grip strength of Dmdmdx mice, whereas serotonin-only treatment resulted in no significant improvement in muscle strength. The results of this study indicate that S&H therapy might be a promising new strategy for muscular dystrophies and that the mechanism should be further investigated. PMID:26740813

  15. NDRG2 promotes myoblast proliferation and caspase 3/7 activities during differentiation, and attenuates hydrogen peroxide – But not palmitate-induced toxicity

    PubMed Central

    Anderson, Kimberley J.; Russell, Aaron P.; Foletta, Victoria C.

    2015-01-01

    The function of the stress-responsive N-myc downstream-regulated gene 2 (NDRG2) in the control of myoblast growth, and the amino acids contributing to its function, are not well characterized. Here, we investigated the effect of increased NDRG2 levels on the proliferation, differentiation and apoptosis in skeletal muscle cells under basal and stress conditions. NDRG2 overexpression increased C2C12 myoblast proliferation and the expression of positive cell cycle regulators, cdk2, cyclin B and cyclin D, and phosphorylation of Rb, while the serine/threonine-deficient NDRG2, 3A-NDRG2, had less effect. The onset of differentiation was enhanced by NDRG2 as determined through the myogenic regulatory factor expression profiles and myocyte fusion index. However, the overall level of differentiation in myotubes was not different. While NDRG2 up-regulated caspase 3/7 activities during differentiation, no increase in apoptosis was measured by TUNEL assay or through cleavage of caspase 3 and PARP proteins. During H2O2 treatment to induce oxidative stress, NDRG2 helped protect against the loss of proliferation and ER stress as measured by GRP78 expression with 3A-NDRG2 displaying less protection. NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels. In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity. Our findings show that NDRG2 overexpression increases myoblast proliferation and caspase 3/7 activities without increasing overall differentiation. Furthermore, NDRG2 attenuates H2O2-induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells. PMID:26380811

  16. Transmembrane proteoglycans syndecan-2, 4, receptor candidates for the impact of HGF and FGF2 on semaphorin 3A expression in early-differentiated myoblasts.

    PubMed

    Do, Mai-Khoi Q; Shimizu, Naomi; Suzuki, Takahiro; Ohtsubo, Hideaki; Mizunoya, Wataru; Nakamura, Mako; Sawano, Shoko; Furuse, Mitsuhiro; Ikeuchi, Yoshihide; Anderson, Judy E; Tatsumi, Ryuichi

    2015-09-01

    Regenerative mechanisms that regulate intramuscular motor innervation are thought to reside in the spatiotemporal expression of axon-guidance molecules. Our previous studies proposed an unexplored role of resident myogenic stem cell (satellite cell)-derived myoblasts as a key presenter of a secreted neural chemorepellent semaphorin 3A (Sema3A); hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2) triggered its expression exclusively at the early differentiation phase. In order to advance this concept, the present study described that transmembrane heparan/chondroitin sulfate proteoglycans syndecan-2, 4 may be the plausible receptor candidates for HGF and FGF2 to signal Sema3A expression. Results showed that mRNA expression of syndecan-2, 4 was abundant (two magnitudes higher than syndecan-1, 3) in early-differentiated myoblasts and their in vitro knockdown diminished the HGF/FGF2-induced expression of Sema3A down to a baseline level. Pretreatment with heparitinase and chondroitinase ABC decreased the HGF and FGF2 responses, respectively, in non-knockdown cultures, supporting a possible model that HGF and FGF2 may bind to heparan and chondroitin sulfate chains of syndecan-2, 4 to signal Sema3A expression. The findings, therefore, extend our understanding that HGF/FGF2-syndecan-2, 4 association may stimulate a burst of Sema3A secretion by myoblasts recruited to the site of muscle injury; this would ensure a coordinated delay in the attachment of motoneuron terminals onto fibers early in muscle regeneration, and thus synchronize the recovery of muscle fiber integrity and the early resolution of inflammation after injury with reinnervation toward functional recovery. PMID:26381016

  17. PC4/Tis7/IFRD1 Stimulates Skeletal Muscle Regeneration and Is Involved in Myoblast Differentiation as a Regulator of MyoD and NF-κB*

    PubMed Central

    Micheli, Laura; Leonardi, Luca; Conti, Filippo; Maresca, Giovanna; Colazingari, Sandra; Mattei, Elisabetta; Lira, Sergio A.; Farioli-Vecchioli, Stefano; Caruso, Maurizia; Tirone, Felice

    2011-01-01

    In skeletal muscle cells, the PC4 (Tis7/Ifrd1) protein is known to function as a coactivator of MyoD by promoting the transcriptional activity of myocyte enhancer factor 2C (MEF2C). In this study, we show that up-regulation of PC4 in vivo in adult muscle significantly potentiates injury-induced regeneration by enhancing myogenesis. Conversely, we observe that PC4 silencing in myoblasts causes delayed exit from the cell cycle, accompanied by delayed differentiation, and we show that such an effect is MyoD-dependent. We provide evidence revealing a novel mechanism underlying the promyogenic actions of PC4, by which PC4 functions as a negative regulator of NF-κB, known to inhibit MyoD expression post-transcriptionally. In fact, up-regulation of PC4 in primary myoblasts induces the deacetylation, and hence the inactivation and nuclear export of NF-κB p65, in concomitance with induction of MyoD expression. On the contrary, PC4 silencing in myoblasts induces the acetylation and nuclear import of p65, in parallel with a decrease of MyoD levels. We also observe that PC4 potentiates the inhibition of NF-κB transcriptional activity mediated by histone deacetylases and that PC4 is able to form trimolecular complexes with p65 and HDAC3. This suggests that PC4 stimulates deacetylation of p65 by favoring the recruitment of HDAC3 to p65. As a whole, these results indicate that PC4 plays a role in muscle differentiation by controlling the MyoD pathway through multiple mechanisms, and as such, it positively regulates regenerative myogenesis. PMID:21127072

  18. Transmembrane proteoglycans syndecan-2, 4, receptor candidates for the impact of HGF and FGF2 on semaphorin 3A expression in early-differentiated myoblasts

    PubMed Central

    Do, Mai-Khoi Q; Shimizu, Naomi; Suzuki, Takahiro; Ohtsubo, Hideaki; Mizunoya, Wataru; Nakamura, Mako; Sawano, Shoko; Furuse, Mitsuhiro; Ikeuchi, Yoshihide; Anderson, Judy E; Tatsumi, Ryuichi

    2015-01-01

    Regenerative mechanisms that regulate intramuscular motor innervation are thought to reside in the spatiotemporal expression of axon-guidance molecules. Our previous studies proposed an unexplored role of resident myogenic stem cell (satellite cell)-derived myoblasts as a key presenter of a secreted neural chemorepellent semaphorin 3A (Sema3A); hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2) triggered its expression exclusively at the early differentiation phase. In order to advance this concept, the present study described that transmembrane heparan/chondroitin sulfate proteoglycans syndecan-2, 4 may be the plausible receptor candidates for HGF and FGF2 to signal Sema3A expression. Results showed that mRNA expression of syndecan-2, 4 was abundant (two magnitudes higher than syndecan-1, 3) in early-differentiated myoblasts and their in vitro knockdown diminished the HGF/FGF2-induced expression of Sema3A down to a baseline level. Pretreatment with heparitinase and chondroitinase ABC decreased the HGF and FGF2 responses, respectively, in non–knockdown cultures, supporting a possible model that HGF and FGF2 may bind to heparan and chondroitin sulfate chains of syndecan-2, 4 to signal Sema3A expression. The findings, therefore, extend our understanding that HGF/FGF2-syndecan-2, 4 association may stimulate a burst of Sema3A secretion by myoblasts recruited to the site of muscle injury; this would ensure a coordinated delay in the attachment of motoneuron terminals onto fibers early in muscle regeneration, and thus synchronize the recovery of muscle fiber integrity and the early resolution of inflammation after injury with reinnervation toward functional recovery. PMID:26381016

  19. Influence of PDGF-BB on Proliferation and Transition Through the MyoD-myogenin-MEF2A Expression Program During Myogenesis in Mouse C2 Myoblasts

    PubMed Central

    Yablonka-Reuveni, Zipora; Rivera, Anthony J.

    2014-01-01

    We have previously demonstrated that PDGF-BB enhances proliferation of C2 myoblasts. This has led us to examine whether the mitogenic influence of PDGF-BB in the C2 model correlates with modulation of specific steps associated with myogenic differentiation. C2 myoblasts transiting through these differentiation specific steps were monitored via immunocytochemistry. We show that the influence of PDGF on enhancing cell proliferation correlates with a delay in the emergence of cells positive for sarcomeric myosin. We further monitored the influence of PDGF-BB on differentiation steps preceding the emergence of myosin+ cells. We demonstrate that mononucleated C2 cells first express MyoD (MyoD+/myogenin− cells) and subsequently, myogenin. Cells negative for both MyoD and myogenin (the phenotype preceding the MyoD+ state) were present at all times in culture and comprised the majority, if not all, of the cells which responded mitogenically to PDGF. Additionally, the frequency of the MyoD+/myogenin+ cell phenotype was reduced in cultures receiving PDGF, suggesting that PDGF can modulate the transition of the cells into the myogenin+ state. We determined that many of the myogenin+ cells subsequently become MEF2A+ and this phenomenon is not influenced by PDGF-BB. FGF-2 also enhanced the proliferation of C2 myoblasts and suppressed the appearance of the myogenin+ cells, but did not influence the subsequent transition into the MEF2A+ state. The study raises the possibility that PDGF-BB and FGF-2 might delay the transition of the C2 cells into the MyoD+/myogenin+ state by depressing a paracrine signal that enhances differentiation. PMID:9401815

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

    SciTech Connect

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

    2011-01-15

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

  1. Characterization of the duck (Anas platyrhynchos) Rbm24 and Rbm38 genes and their expression profiles in myoblast and skeletal muscle tissues.

    PubMed

    Sun, Wenqiang; Hu, Yaodong; Xu, Hengyong; He, Hua; Han, Chunchun; Liu, Hehe; Wang, Jiwen; Li, Liang

    2016-08-01

    RNA-binding motif proteins 24 (Rbm24) and 38 (Rbm38) are known to regulate genes expression in a post-transcriptional way. However, it remains unclear about the similarities and differences between Rbm24 and Rbm38 in terms of their sequence characteristics and expression profiles during myoblast differentiation and skeletal muscle development. In this study, we found that the coding domain sequences (CDSs) of duck Rbm24 and Rbm38 consisted of 678 and 648 nucleotides, respectively. Both of them contain a conserved RNA-recognition motif (RRM). Phylogenetic analysis showed that duck Rbm24 and Rbm38 were clustered with other Aves, nevertheless, avian Rbm24 was clustered with mammalian and reptilian Rbm24; avian Rbm38 was clustered with amphibian and reptilian Rbm38. Real-time PCR results exhibited that during embryonic stage, Rbm24 and Rbm38 in leg and breast muscle increased to their peak (P<0.01) at same time. During postnatal stage, the peak of Rbm24 and Rbm38 in leg were found at W5, while the peak of them in breast was found at W6 (P<0.01). Moreover, a relative high value of Rbm24 and Rbm38 in leg was found at W1 and W3, respectively. Additionally, both Rbm24 and Rbm38 expressed at each stage of duck myoblast differentiation, however, Rbm24 shared similar expression profiles with MEF2C, and Rbm38 shared similar expression profiles with MyoG and MEF2A. Furthermore, hierarchical clustering results were consistent with the preceding findings. These results serve as a foundation for further investigations about similar and different effects of Rbm24 and Rbm38 on myoblast differentiation and skeletal muscle development. PMID:27040525

  2. Modulation of myoblast fusion by caveolin-3 in dystrophic skeletal muscle cells: implications for Duchenne muscular dystrophy and limb-girdle muscular dystrophy-1C.

    PubMed

    Volonte, Daniela; Peoples, Aaron J; Galbiati, Ferruccio

    2003-10-01

    Caveolae are vesicular invaginations of the plasma membrane. Caveolin-3 is the principal structural component of caveolae in skeletal muscle cells in vivo. We have recently generated caveolin-3 transgenic mice and demonstrated that overexpression of wild-type caveolin-3 in skeletal muscle fibers is sufficient to induce a Duchenne-like muscular dystrophy phenotype. In addition, we have shown that caveolin-3 null mice display mild muscle fiber degeneration and T-tubule system abnormalities. These data are consistent with the mild phenotype observed in Limb-girdle muscular dystrophy-1C (LGMD-1C) in humans, characterized by a approximately 95% reduction of caveolin-3 expression. Thus, caveolin-3 transgenic and null mice represent valid mouse models to study Duchenne muscular dystrophy (DMD) and LGMD-1C, respectively, in humans. Here, we derived conditionally immortalized precursor skeletal muscle cells from caveolin-3 transgenic and null mice. We show that overexpression of caveolin-3 inhibits myoblast fusion to multinucleated myotubes and lack of caveolin-3 enhances the fusion process. M-cadherin and microtubules have been proposed to mediate the fusion of myoblasts to myotubes. Interestingly, we show that M-cadherin is downregulated in caveolin-3 transgenic cells and upregulated in caveolin-3 null cells. For the first time, variations of M-cadherin expression have been linked to a muscular dystrophy phenotype. In addition, we demonstrate that microtubules are disorganized in caveolin-3 null myotubes, indicating the importance of the cytoskeleton network in mediating the phenotype observed in these cells. Taken together, these results propose caveolin-3 as a key player in myoblast fusion and suggest that defects of the fusion process may represent additional molecular mechanisms underlying the pathogenesis of DMD and LGMD-1C in humans. PMID:14517320

  3. The LIM domain protein nTRIP6 acts as a co-repressor for the transcription factor MEF2C in myoblasts

    PubMed Central

    Kemler, Denise; Dahley, Oliver; Roßwag, Sven; Litfin, Margarethe; Kassel, Olivier

    2016-01-01

    The transcription factor Myocyte enhancer factor 2C (MEF2C) plays a key role in the late differentiation of skeletal muscle progenitor cells, the so-called myoblasts. During myoblast differentiation, both MEF2C expression and transcriptional activity are regulated. We have reported that nTRIP6, the nuclear isoform of the focal adhesion LIM domain protein TRIP6, acts as an adaptor transcriptional co-activator for several transcription factors. It interacts with the promoter-bound transcription factors and consequently mediates the recruitment of other co-activators. Based on a described interaction between MEF2C and TRIP6 in a yeast-two-hybrid screen, we hypothesised a co-regulatory function of nTRIP6 for MEF2C. In proliferating myoblasts, nTRIP6 interacted with MEF2C and was recruited together with MEF2C to the MEF2-binding regions of the MEF2C target genes Myom2, Mb, Tnni2 and Des. Silencing nTRIP6 or preventing its interaction with MEF2C increased MEF2C transcriptional activity and increased the expression of these MEF2C target genes. Thus, nTRIP6 acts as a co-repressor for MEF2C. Mechanistically, nTRIP6 mediated the recruitment of the class IIa histone deacetylase HDAC5 to the MEF2C-bound promoters. In conclusion, our results unravel a transcriptional co-repressor function for nTRIP6. This adaptor co-regulator can thus exert either co-activator or co-repressor functions, depending on the transcription factor it interacts with. PMID:27292777

  4. Insulin-induced oxidative stress up-regulates heme oxygenase-1 via diverse signaling cascades in the C2 skeletal myoblast cell line.

    PubMed

    Aggeli, Ioanna-Katerina; Theofilatos, Dimitris; Beis, Isidoros; Gaitanaki, Catherine

    2011-04-01

    Impaired insulin sensitivity (insulin resistance) is a common denominator in many metabolic disorders, exerting pleiotropic effects on skeletal muscle, liver, and adipose tissue function. Heme oxygenase-1 (HOX-1), the rate-limiting enzyme in heme catabolism, has recently been shown to confer an antidiabetic effect while regulating cellular redox-buffering capacity. Therefore, in the present study, we probed into the mechanisms underlying the effect of insulin on HOX-1 in C2 skeletal myoblasts. Hence, insulin was found to suppress C2 myoblasts viability via stimulation of oxidative stress, with HOX-1 counteracting this action. Insulin induced HOX-1 expression in a time- and dose-dependent manner, an effect attenuated by selective inhibitors of ERK1/2 (PD98059), Src (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine), and c-Jun terminal kinases 1 and 2 (SP600125) pathways. Furthermore, nuclear factor-κB role in insulin-induced HOX-1 up-regulation was verified, with ERK1/2, Src, and c-Jun terminal kinases 1 and 2 mediating p65-nuclear factor-κB subunit phosphorylation. Overall, our novel findings highlight for the first time the transduction mechanisms mediating HOX-1 induction in insulin-treated C2 myoblasts. This effect was established to be cell type specific because insulin failed to promote HOX-1 expression in HepG2 hepatoma cells. Deciphering the signaling networks involved in insulin-stimulated HOX-1 up-regulation is of prominent significance because it may potentially contribute to elucidation of the mechanisms involved in associated metabolic pathologies. PMID:21325398

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

    SciTech Connect

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

    2011-04-29

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

  6. Transient exposure of human myoblasts to tumor necrosis factor-alpha inhibits serum and insulin-like growth factor-I stimulated protein synthesis.

    PubMed

    Frost, R A; Lang, C H; Gelato, M C

    1997-10-01

    Tumor necrosis factor-alpha (TNF-alpha) induces cachexia and is postulated to be responsible for muscle wasting in several pathophysiological conditions. The purpose of the present study was to investigate whether exposure of human myoblasts to TNF-alpha could directly inhibit the ability of serum or insulin-like growth factor I (IGF-I) to stimulate protein synthesis as assessed by the incorporation of [3H]phenylalanine into protein. Serum and IGF-I stimulated protein synthesis dose dependently. Half-maximal stimulation of protein synthesis occurred at 05% serum and 8 ng/ml of IGF-I, respectively. TNF-alpha inhibited IGF-I-stimulated protein synthesis in a dose-dependent manner. Additionally, as little as 2 ng/ml of TNF-alpha impaired the ability of IGF-I to stimulate protein synthesis by 33% and, at a dose of 100 ng/ml, TNF-alpha completely prevented the increase in protein synthesis induced by either serum or a maximally stimulating dose of IGF-I. Inhibition of protein synthesis was independent of whether TNF-alpha and growth factors were added to cells simultaneously or if the cells were pretreated with growth factors. Exposure ofmyoblasts to TNF-alpha for 10 min completely inhibited serum-induced stimulation of protein synthesis. TNF-alpha inhibited protein synthesis up to 48 h after addition of the cytokine. TNF-alpha also inhibited serum-stimulated protein synthesis in human myoblasts that were differentiated into myotubes. In contrast, exposure of myoblasts to TNF-alpha had no effect on IGF-I binding and failed to alter the ability of either IGF-I or serum to stimulate [3H]thymidine uptake. These data indicate that transient exposure of myoblasts or myotubes to TNF-alpha inhibits protein synthesis. Thus, the anabolic actions of IGF-I on muscle protein synthesis may be impaired during catabolic conditions in which TNF-alpha is over expressed. PMID:9322924

  7. Platelet-rich plasma, especially when combined with a TGF-β inhibitor promotes proliferation, viability and myogenic differentiation of myoblasts in vitro.

    PubMed

    Kelc, Robi; Trapecar, Martin; Gradisnik, Lidija; Rupnik, Marjan Slak; Vogrin, Matjaz

    2015-01-01

    Regeneration of skeletal muscle after injury is limited by scar formation, slow healing time and a high recurrence rate. A therapy based on platelet-rich plasma (PRP) has become a promising lead for tendon and ligament injuries in recent years, however concerns have been raised that PRP-derived TGF-β could contribute to fibrotic remodelling in skeletal muscle after injury. Due to the lack of scientific grounds for a PRP -based muscle regeneration therapy, we have designed a study using human myogenic progenitors and evaluated the potential of PRP alone and in combination with decorin (a TGF-β inhibitor), to alter myoblast proliferation, metabolic activity, cytokine profile and expression of myogenic regulatory factors (MRFs). Advanced imaging multicolor single-cell analysis enabled us to create a valuable picture on the ratio of quiescent, activated and terminally committed myoblasts in treated versus control cell populations. Finally high-resolution confocal microscopy validated the potential of PRP and decorin to stimulate the formation of polynucleated myotubules. PRP was shown to down-regulate fibrotic cytokines, increase cell viability and proliferation, enhance the expression of MRFs, and contribute to a significant myogenic shift during differentiation. When combined with decorin further synergistc effects were identified. These results suggest that PRP could not only prevent fibrosis but could also stimulate muscle commitment, especially when combined with a TGF-β inhibitor. PMID:25679956

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

    SciTech Connect

    Song, Young Joon; Lee, Hansol

    2010-02-15

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

  9. Sphingosine 1-Phosphate Induces Myoblast Differentiation through Cx43 Protein Expression: A Role for a Gap Junction-dependent and -independent Function

    PubMed Central

    Squecco, R.; Sassoli, C.; Nuti, F.; Martinesi, M.; Chellini, F.; Nosi, D.; Zecchi-Orlandini, S.; Francini, F.; Formigli, L.

    2006-01-01

    Although sphingosine 1-phosphate (S1P) has been considered a potent regulator of skeletal muscle biology, acting as a physiological anti-mitogenic and prodifferentiating agent, its downstream effectors are poorly known. In the present study, we provide experimental evidence for a novel mechanism by which S1P regulates skeletal muscle differentiation through the regulation of gap junctional protein connexin (Cx) 43. Indeed, the treatment with S1P greatly enhanced Cx43 expression and gap junctional intercellular communication during the early phases of myoblast differentiation, whereas the down-regulation of Cx43 by transfection with short interfering RNA blocked myogenesis elicited by S1P. Moreover, calcium and p38 MAPK-dependent pathways were required for S1P-induced increase in Cx43 expression. Interestingly, enforced expression of mutated Cx43Δ130–136 reduced gap junction communication and totally inhibited S1P-induced expression of the myogenic markers, myogenin, myosin heavy chain, caveolin-3, and myotube formation. Notably, in S1P-stimulated myoblasts, endogenous or wild-type Cx43 protein, but not the mutated form, coimmunoprecipitated and colocalized with F-actin and cortactin in a p38 MAPK-dependent manner. These data, together with the known role of actin remodeling in cell differentiation, strongly support the important contribution of gap junctional communication, Cx43 expression and Cx43/cytoskeleton interaction in skeletal myogenesis elicited by S1P. PMID:16957055

  10. Ethanol extract of Cyclolepis genistoides D. Don (palo azul) induces formation of myotubes, which involves differentiation of C2C12 myoblast cells.

    PubMed

    Sato, Hiromi; Funaki, Asami; Kimura, Yuki; Sumitomo, Mai; Yoshida, Hiroya; Fukata, Hideki; Ueno, Koichi

    2016-07-01

    In this study, we examined the cell differentiation effect of an ethanol extract of Cyclolepis genistoides D. Don, a herbaceous perennial belonging to the family Asteraceae (vernacular name: palo azul). Palo azul has numerous physiological effects that contribute to the prevention of metabolic syndromes, although the mechanism remains unclear. We previously suggested that palo azul has antidiabetic activity via an adipose differentiation effect. Here, we focused on whether palo azul promoted the differentiation of myoblasts. The mouse muscle myoblast cell line C2C12 was cultured and differentiated using horse serum with or without an ethanol extract of palo azul (12.5-200 μg/mL). Quantitative real-time polymerase chain reaction was performed to evaluate differentiation markers, including insulin-like growth factor-1 and myogenin. To evaluate myotube formation, myosin heavy-chain (MHC) expression and localization were detected by immunohistochemistry. Palo azul increased the expression of the differentiation markers. Furthermore, immunohistochemistry analysis revealed increased formation of MHC myotubes after palo azul treatment along with increased diameter and fusion indices of the myotubes. The expression level of MHC was also increased. In conclusion, palo azul may increase muscle mass in the body and improve insulin resistance conditions by facilitating the formation of myotubes by promoting myocyte differentiation. PMID:27262535

  11. Nanofiber Yarn/Hydrogel Core-Shell Scaffolds Mimicking Native Skeletal Muscle Tissue for Guiding 3D Myoblast Alignment, Elongation, and Differentiation.

    PubMed

    Wang, Ling; Wu, Yaobin; Guo, Baolin; Ma, Peter X

    2015-09-22

    Designing scaffolds that can mimic native skeletal muscle tissue and induce 3D cellular alignment and elongated myotube formation remains an ongoing challenge for skeletal muscle tissue engineering. Herein, we present a simple technique to generate core-shell composite scaffolds for mimicking native skeletal muscle structure, which comprise the aligned nanofiber yarn (NFY) core and the photocurable hydrogel shell. The aligned NFYs are prepared by the hybrid composition including poly(caprolactone), silk fibroin, and polyaniline via a developed dry-wet electrospinning method. A series of core-shell column and sheet composite scaffolds are ultimately obtained by encapsulating a piece and layers of aligned NFY cores within the hydrogel shell after photo-cross-linking. C2C12 myoblasts are seeded within the core-shell scaffolds, and the good biocompatibility of these scaffolds and their ability to induce 3D cellular alignment and elongation are successfully demonstrated. Furthermore, the 3D elongated myotube formation within core-shell scaffolds is also performed after long-term cultivation. These data suggest that these core-shell scaffolds combine the aligned NFY core that guides the myoblast alignment and differentiation and the hydrogel shell that provides a suitable 3D environment for nutrition exchange and mechanical protection to perform a great practical application for skeletal muscle regeneration. PMID:26280983

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

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

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

  13. DNA methylation analysis of human myoblasts during in vitro myogenic differentiation: de novo methylation of promoters of muscle-related genes and its involvement in transcriptional down-regulation

    PubMed Central

    Miyata, Kohei; Miyata, Tomoko; Nakabayashi, Kazuhiko; Okamura, Kohji; Naito, Masashi; Kawai, Tomoko; Takada, Shuji; Kato, Kiyoko; Miyamoto, Shingo; Hata, Kenichiro; Asahara, Hiroshi

    2015-01-01

    Although DNA methylation is considered to play an important role during myogenic differentiation, chronological alterations in DNA methylation and gene expression patterns in this process have been poorly understood. Using the Infinium HumanMethylation450 BeadChip array, we obtained a chronological profile of the genome-wide DNA methylation status in a human myoblast differentiation model, where myoblasts were cultured in low-serum medium to stimulate myogenic differentiation. As the differentiation of the myoblasts proceeded, their global DNA methylation level increased and their methylation patterns became more distinct from those of mesenchymal stem cells. Gene ontology analysis revealed that genes whose promoter region was hypermethylated upon myoblast differentiation were highly significantly enriched with muscle-related terms such as ‘muscle contraction’ and ‘muscle system process’. Sequence motif analysis identified 8-bp motifs somewhat similar to the binding motifs of ID4 and ZNF238 to be most significantly enriched in hypermethylated promoter regions. ID4 and ZNF238 have been shown to be critical transcriptional regulators of muscle-related genes during myogenic differentiation. An integrated analysis of DNA methylation and gene expression profiles revealed that de novo DNA methylation of non-CpG island (CGI) promoters was more often associated with transcriptional down-regulation than that of CGI promoters. These results strongly suggest the existence of an epigenetic mechanism in which DNA methylation modulates the functions of key transcriptional factors to coordinately regulate muscle-related genes during myogenic differentiation. PMID:25190712

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

    PubMed

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

    2015-10-01

    Recently, there has been considerable effort to develop suitable scaffolds for tissue engineering applications. Cell adhesion is a prerequisite for cells to survive. In nature, the extracellular matrix (ECM) plays this role. Therefore, an ideal scaffold should be structurally similar to the natural ECM and have biocompatibility and biodegradability. In addition, the scaffold should have biofunctionality, which provides the potent ability to enhance the cellular behaviors, such as adhesion, proliferation and differentiation. This study concentrates on fabricating cell-adhesive matrices composed of RGD peptide-displaying M13 bacteriophage (RGD-M13 phage) and poly(lactic-co-glycolic acid, PLGA) nanofibers. Long rod-shaped M13 bacteriophages are non-toxic and can express many desired proteins on their surface. A genetically engineered M13 phage was constructed to display RGD peptides on its surface. PLGA is a biodegradable polymer with excellent biocompatibility and suitable physicochemical property for adhesive matrices. In this study, RGD-M13 phage/PLGA hybrid nanofiber matrices were fabricated by electrospinning. The physicochemical properties of these matrices were characterized by scanning electron microscopy, atomic force microscopy, Raman spectroscopy, and contact angle measurement. In addition, the cellular behaviors, such as the initial attachment, proliferation and differentiation, were analyzed by a CCK-8 assay and immunofluorescence staining to evaluate the potential application of these matrices to tissue engineering scaffolds. The RGD-M13 phage/PLGA nanofiber matrices could enhance the cellular behaviors and promote the differentiation of C2C12 myoblasts. These results suggest that the RGD-M13 phage/PLGA nanofiber matrices are beneficial to myoblast differentiation and can serve as effective tissue engineering scaffolds. PMID:26726438

  15. Comparative transcriptomic analysis reveals the oncogenic fusion protein PAX3-FOXO1 globally alters mRNA and miRNA to enhance myoblast invasion

    PubMed Central

    Loupe, J M; Miller, P J; Bonner, B P; Maggi, E C; Vijayaraghavan, J; Crabtree, J S; Taylor, C M; Zabaleta, J; Hollenbach, A D

    2016-01-01

    Rhabdomyosarcoma, one of the most common childhood sarcomas, is comprised of two main subtypes, embryonal and alveolar (ARMS). ARMS, the more aggressive subtype, is primarily characterized by the t(2;13)(p35;p14) chromosomal translocation, which fuses two transcription factors, PAX3 and FOXO1 to generate the oncogenic fusion protein PAX3-FOXO1. Patients with PAX3-FOXO1-postitive tumors have a poor prognosis, in part due to the enhanced local invasive capacity of these cells, which leads to the increased metastatic potential for this tumor. Despite this knowledge, little is known about the role that the oncogenic fusion protein has in this increased invasive potential. In this report we use large-scale comparative transcriptomic analyses in physiologically relevant primary myoblasts to demonstrate that the presence of PAX3-FOXO1 is sufficient to alter the expression of 70 mRNA and 27 miRNA in a manner predicted to promote cellular invasion. In contrast the expression of PAX3 alters 60 mRNA and 23 miRNA in a manner predicted to inhibit invasion. We demonstrate that these alterations in mRNA and miRNA translate into changes in the invasive potential of primary myoblasts with PAX3-FOXO1 increasing invasion nearly 2-fold while PAX3 decreases invasion nearly 4-fold. Taken together, these results allow us to build off of previous reports and develop a more expansive molecular model by which the presence of PAX3-FOXO1 alters global gene regulatory networks to enhance the local invasiveness of cells. Further, the global nature of our observed changes highlights the fact that instead of focusing on a single-gene target, we must develop multi-faceted treatment regimens targeting multiple genes of a single oncogenic phenotype or multiple genes that target different oncogenic phenotypes for tumor progression. PMID:27454080

  16. Ectopic expression of cyclin D3 corrects differentiation of DM1 myoblasts through activation of RNA CUG-binding protein, CUGBP1

    SciTech Connect

    Salisbury, Elizabeth; Sakai, Keiko; Schoser, Benedikt; Huichalaf, Claudia; Schneider-Gold, Christiane; Nguyen, Heather; Wang, Gou-Li; Albrecht, Jeffrey H.; Timchenko, Lubov T.

    2008-07-01

    Differentiation of myocytes is impaired in patients with myotonic dystrophy type 1, DM1. CUG repeat binding protein, CUGBP1, is a key regulator of translation of proteins that are involved in muscle development and differentiation. In this paper, we present evidence that RNA-binding activity of CUGBP1 and its interactions with initiation translation complex eIF2 are differentially regulated during myogenesis by specific phosphorylation and that this regulation is altered in DM1. In normal myoblasts, Akt kinase phosphorylates CUGBP1 at Ser28 and increases interactions of CUGBP1 with cyclin D1 mRNA. During differentiation, CUGBP1 is phosphorylated by cyclinD3-cdk4/6 at Ser302, which increases CUGBP1 binding with p21 and C/EBP{beta} mRNAs. While cyclin D3 and cdk4 are elevated in normal myotubes; DM1 differentiating cells do not increase these proteins. In normal myotubes, CUGBP1 interacts with cyclin D3/cdk4/6 and eIF2; however, interactions of CUGBP1 with eIF2 are reduced in DM1 differentiating cells and correlate with impaired muscle differentiation in DM1. Ectopic expression of cyclin D3 in DM1 cells increases the CUGBP1-eIF2 complex, corrects expression of differentiation markers, myogenin and desmin, and enhances fusion of DM1 myoblasts. Thus, normalization of cyclin D3 might be a therapeutic approach to correct differentiation of skeletal muscle in DM1 patients.

  17. Comparative transcriptomic analysis reveals the oncogenic fusion protein PAX3-FOXO1 globally alters mRNA and miRNA to enhance myoblast invasion.

    PubMed

    Loupe, J M; Miller, P J; Bonner, B P; Maggi, E C; Vijayaraghavan, J; Crabtree, J S; Taylor, C M; Zabaleta, J; Hollenbach, A D

    2016-01-01

    Rhabdomyosarcoma, one of the most common childhood sarcomas, is comprised of two main subtypes, embryonal and alveolar (ARMS). ARMS, the more aggressive subtype, is primarily characterized by the t(2;13)(p35;p14) chromosomal translocation, which fuses two transcription factors, PAX3 and FOXO1 to generate the oncogenic fusion protein PAX3-FOXO1. Patients with PAX3-FOXO1-postitive tumors have a poor prognosis, in part due to the enhanced local invasive capacity of these cells, which leads to the increased metastatic potential for this tumor. Despite this knowledge, little is known about the role that the oncogenic fusion protein has in this increased invasive potential. In this report we use large-scale comparative transcriptomic analyses in physiologically relevant primary myoblasts to demonstrate that the presence of PAX3-FOXO1 is sufficient to alter the expression of 70 mRNA and 27 miRNA in a manner predicted to promote cellular invasion. In contrast the expression of PAX3 alters 60 mRNA and 23 miRNA in a manner predicted to inhibit invasion. We demonstrate that these alterations in mRNA and miRNA translate into changes in the invasive potential of primary myoblasts with PAX3-FOXO1 increasing invasion nearly 2-fold while PAX3 decreases invasion nearly 4-fold. Taken together, these results allow us to build off of previous reports and develop a more expansive molecular model by which the presence of PAX3-FOXO1 alters global gene regulatory networks to enhance the local invasiveness of cells. Further, the global nature of our observed changes highlights the fact that instead of focusing on a single-gene target, we must develop multi-faceted treatment regimens targeting multiple genes of a single oncogenic phenotype or multiple genes that target different oncogenic phenotypes for tumor progression. PMID:27454080

  18. Overexpression of ryanodine receptor type 1 enhances mitochondrial fragmentation and Ca2+-induced ATP production in cardiac H9c2 myoblasts

    PubMed Central

    O-Uchi, Jin; Jhun, Bong Sook; Hurst, Stephen; Bisetto, Sara; Gross, Polina; Chen, Ming; Kettlewell, Sarah; Park, Jongsun; Oyamada, Hideto; Smith, Godfrey L.; Murayama, Takashi

    2013-01-01

    Ca+ influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca2+ influx is mainly mediated by the mitochondrial Ca2+ uniporter (MCU). Growing evidence also indicates that mitochondrial Ca2+ influx mechanisms are regulated not solely by MCU but also by multiple channels/transporters. We have previously reported that skeletal muscle-type ryanodine receptor (RyR) type 1 (RyR1), which expressed at the mitochondrial inner membrane, serves as an additional Ca2+ uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca2+ influx mechanism is the dominant regulator of mitochondrial morphology/dynamics and energetics in cardiomyocytes. To investigate the role of mitochondrial RyR1 in the regulation of mitochondrial morphology/function in cardiac cells, RyR1 was transiently or stably overexpressed in cardiac H9c2 myoblasts. We found that overexpressed RyR1 was partially localized in mitochondria as observed using both immunoblots of mitochondrial fractionation and confocal microscopy, whereas RyR2, the main RyR isoform in the cardiac sarcoplasmic reticulum, did not show any expression at mitochondria. Interestingly, overexpression of RyR1 but not MCU or RyR2 resulted in mitochondrial fragmentation. These fragmented mitochondria showed bigger and sustained mitochondrial Ca2+ transients compared with basal tubular mitochondria. In addition, RyR1-overexpressing cells had a higher mitochondrial ATP concentration under basal conditions and showed more ATP production in response to cytosolic Ca2+ elevation compared with nontransfected cells as observed by a matrix-targeted ATP biosensor. These results indicate that RyR1 possesses a mitochondrial targeting/retention signal and modulates mitochondrial morphology and Ca2+-induced ATP production in cardiac H9c2 myoblasts. PMID:24124188

  19. Annexin A1 Deficiency does not Affect Myofiber Repair but Delays Regeneration of Injured Muscles

    PubMed Central

    Leikina, Evgenia; Defour, Aurelia; Melikov, Kamran; Van der Meulen, Jack H.; Nagaraju, Kanneboyina; Bhuvanendran, Shivaprasad; Gebert, Claudia; Pfeifer, Karl; Chernomordik, Leonid V.; Jaiswal, Jyoti K.

    2015-01-01

    Repair and regeneration of the injured skeletal myofiber involves fusion of intracellular vesicles with sarcolemma and fusion of the muscle progenitor cells respectively. In vitro experiments have identified involvement of Annexin A1 (Anx A1) in both these fusion processes. To determine if Anx A1 contributes to these processes during muscle repair in vivo, we have assessed muscle growth and repair in Anx A1-deficient mouse (AnxA1−/−). We found that the lack of Anx A1 does not affect the muscle size and repair of myofibers following focal sarcolemmal injury and lengthening contraction injury. However, the lack of Anx A1 delayed muscle regeneration after notexin-induced injury. This delay in muscle regeneration was not caused by a slowdown in proliferation and differentiation of satellite cells. Instead, lack of Anx A1 lowered the proportion of differentiating myoblasts that managed to fuse with the injured myofibers by days 5 and 7 after notexin injury as compared to the wild type (w.t.) mice. Despite this early slowdown in fusion of Anx A1−/− myoblasts, regeneration caught up at later times post injury. These results establish in vivo role of Anx A1 in cell fusion required for myofiber regeneration and not in intracellular vesicle fusion needed for repair of myofiber sarcolemma. PMID:26667898

  20. Affective Learning.

    ERIC Educational Resources Information Center

    Brown, Charles T.

    This paper addresses itself to the question, "What does feeling have to do with knowing?" Two movements in affective education are discussed which have come into focus in recent years and which attempt to define the relationship between knowing and feeling. The first, a conscious application of the role of arousal in learning, emphasizes arousal…

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

    SciTech Connect

    Hong, Gia-Ming

    2012-05-01

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

  2. [Affective dependency].

    PubMed

    Scantamburlo, G; Pitchot, W; Ansseau, M

    2013-01-01

    Affective dependency is characterized by emotional distress (insecure attachment) and dependency to another person with a low self-esteem and reassurance need. The paper proposes a reflection on the definition of emotional dependency and the confusion caused by various denominations. Overprotective and authoritarian parenting, cultural and socio-environmental factors may contribute to the development of dependent personality. Psychological epigenetic factors, such as early socio-emotional trauma could on neuronal circuits in prefronto-limbic regions that are essential for emotional behaviour.We also focus on the interrelations between dependent personality, domestic violence and addictions. The objective for the clinician is to propose a restoration of self-esteem and therapeutic strategies focused on autonomy. PMID:23888587

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

    PubMed Central

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

    2014-01-01

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

  4. Improved clinical course of autologous skeletal myoblast sheet (TCD-51073) transplantation when compared to a propensity score-matched cardiac resynchronization therapy population.

    PubMed

    Imamura, Teruhiko; Kinugawa, Koichiro; Sakata, Yasushi; Miyagawa, Shigeru; Sawa, Yoshiki; Yamazaki, Kenji; Ono, Minoru

    2016-03-01

    We recently reported a multi-center, single-arm, phase II study that evaluated the efficacy and safety of autologous skeletal myoblast sheet (TCD-51073) transplantation. The advantage of this procedure over a control group has not yet been analyzed. Seven patients with advanced heart failure due to ischemic etiology (TCD-51073 group, New York Heart Association (NYHA) class III; left ventricular ejection fraction (LVEF) <35 %) refractory to optimal medical and coronary revascularization therapy, received TCD-51073 at 3 study centers between 2012 and 2013 with a 2-year follow-up period. As previously reported, 112 patients received cardiac resynchronization therapy (CRT) with follow-up at the University of Tokyo Hospital between 2007 and 2014. Of them, 21 patients were selected for the control group by propensity score matching. No significant difference in baseline variables between the groups was observed. LVEF and NYHA class improved significantly in the TCD-51073 group during the 6-month study period (p < 0.05). During the 2-year follow-up, 7 patients (33 %) in the CRT group and no patient in the TCD-51073 group died due to cardiac disease or received VAD implantation (p = 0.128 by the log-rank test). In conclusion, transplantation of TCD-51073 is clinically advantageous in facilitating LV reverse remodeling, improving HF symptoms, and preventing cardiac death in patients with ischemic etiology when compared to background-matched patients receiving CRT. PMID:26267666

  5. Fine-Tuning of the Actin Cytoskeleton and Cell Adhesion During Drosophila Development by the Unconventional Guanine Nucleotide Exchange Factors Myoblast City and Sponge

    PubMed Central

    Biersmith, Bridget; Wang, Zong-Heng; Geisbrecht, Erika R.

    2015-01-01

    The evolutionarily conserved Dock proteins function as unconventional guanine nucleotide exchange factors (GEFs). Upon binding to engulfment and cell motility (ELMO) proteins, Dock–ELMO complexes activate the Rho family of small GTPases to mediate a diverse array of biological processes, including cell motility, apoptotic cell clearance, and axon guidance. Overlapping expression patterns and functional redundancy among the 11 vertebrate Dock family members, which are subdivided into four families (Dock A, B, C, and D), complicate genetic analysis. In both vertebrate and invertebrate systems, the actin dynamics regulator, Rac, is the target GTPase of the Dock-A subfamily. However, it remains unclear whether Rac or Rap1 are the in vivo downstream GTPases of the Dock-B subfamily. Drosophila melanogaster is an excellent genetic model organism for understanding Dock protein function as its genome encodes one ortholog per subfamily: Myoblast city (Mbc; Dock A) and Sponge (Spg; Dock B). Here we show that the roles of Spg and Mbc are not redundant in the Drosophila somatic muscle or the dorsal vessel. Moreover, we confirm the in vivo role of Mbc upstream of Rac and provide evidence that Spg functions in concert with Rap1, possibly to regulate aspects of cell adhesion. Together these data show that Mbc and Spg can have different downstream GTPase targets. Our findings predict that the ability to regulate downstream GTPases is dependent on cellular context and allows for the fine-tuning of actin cytoskeletal or cell adhesion events in biological processes that undergo cell morphogenesis. PMID:25908317

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

    PubMed

    Choi, Yung Hyun

    2016-09-01

    Preclinical Research The aim of the present study was to evaluate the effects of berberine hydrochloride (BBH), an isoquinoline alkaloid that can be isolated from a variety of herbs, on hydrogen peroxide (H2 O2 )-induced oxidative stress in C2C12 myoblasts and to investigate the molecular mechanisms involved in this process, especially the expression of the Nrf2/HO-1 pathway. BBH preconditioning attenuated H2 O2 -induced growth inhibition and DNA damage as well as apoptosis in C2C12 cells via suppression of the accumulation of intracellular reactive oxygen species (ROS). Treatment with BBHride alone effectively upregulated the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) and elevated HO-1 activity. However, the protective effects of BBH against H2 O2 -induced ROS generation and cell growth reduction were abolished by an HO-1 inhibitor. Moreover, BBH-mediated induction and activation of HO-1 were reduced by genetic silencing of Nrf2 using small interfering RNA (siRNA). In addition, the effects of BBH against H2 O2 -induced ROS accumulation and growth inhibition were abrogated in C2C12 cells transfected with Nrf2 siRNA. Therefore, the present study demonstrated that BBH could protect C2C12 cells against oxidative stress-induced injury and this effect involved activation of the Nrf2/HO-1 pathway. Drug Dev Res, 2016. © 2016 Wiley Periodicals, Inc. PMID:27535021

  7. Norepinephrine-induced apoptotic and hypertrophic responses in H9c2 cardiac myoblasts are characterized by different repertoire of reactive oxygen species generation

    PubMed Central

    Thakur, Anita; Alam, Md. Jahangir; Ajayakumar, MR; Ghaskadbi, Saroj; Sharma, Manish; Goswami, Shyamal K.

    2015-01-01

    Despite recent advances, the role of ROS in mediating hypertrophic and apoptotic responses in cardiac myocytes elicited by norepinephrine (NE) is rather poorly understood. We demonstrate through our experiments that H9c2 cardiac myoblasts treated with 2 µM NE (hypertrophic dose) generate DCFH-DA positive ROS only for 2 h; while those treated with 100 µM NE (apoptotic dose) sustains generation for 48 h, followed by apoptosis. Though the levels of DCFH fluorescence were comparable at early time points in the two treatment sets, its quenching by DPI, catalase and MnTmPyP suggested the existence of a different repertoire of ROS. Both doses of NE also induced moderate levels of H2O2 but with different kinetics. Sustained but intermittent generation of highly reactive species detectable by HPF was seen in both treatment sets but no peroxynitrite was generated in either conditions. Sustained generation of hydroxyl radicals with no appreciable differences were noticed in both treatment sets. Nevertheless, despite similar profile of ROS generation between the two conditions, extensive DNA damage as evident from the increase in 8-OH-dG content, formation of γ-H2AX and PARP cleavage was seen only in cells treated with the higher dose of NE. We therefore conclude that hypertrophic and apoptotic doses of NE generate distinct but comparable repertoire of ROS/RNS leading to two very distinct downstream responses. PMID:26070033

  8. The Myoblast C2C12 Transfected with Mutant Valosin-Containing Protein Exhibits Delayed Stress Granule Resolution on Oxidative Stress.

    PubMed

    Rodriguez-Ortiz, Carlos J; Flores, Julio C; Valenzuela, Joanna A; Rodriguez, Gema J; Zumkehr, Joannee; Tran, Diana N; Kimonis, Virginia E; Kitazawa, Masashi

    2016-06-01

    Valosin-containing protein (VCP) mutations cause inclusion body myopathy with Paget disease and frontotemporal dementia. However, the mechanisms by which mutant VCP triggers degeneration remain unknown. Here, we investigated the role of VCP in cellular stress and found that the oxidative stressor arsenite and heat shock-activated stress responses evident by T-intracellular antigen-1-positive granules in C2C12 myoblasts. Granules also contained phosphorylated transactive response DNA-binding protein 43, ubiquitin, microtubule-associated protein 1A/1B light chains 3, and lysosome-associated membrane protein 2. Mutant VCP produced more T-intracellular antigen-1-positive granules than wild-type in the postarsenite exposure period. Similar results were observed for other granule components, indicating that mutant VCP delayed clearance of stress granules. Furthermore, stress granule resolution was impaired on differentiated C2C12 cells expressing mutant VCP. To address whether mutant VCP triggers dysregulation of the stress granule pathway in vivo, we analyzed skeletal muscle of aged VCPR155H-knockin mice. We found significant increments in oxidated proteins but observed the stress granule markers RasGAP SH3-binding protein and phosphorylated eukaryotic translation initiation factor 2α unchanged. The mixed results indicate that mutant VCP together with aging lead to higher oxidative stress in skeletal muscle but were insufficient to disrupt the stress granule pathway. Our findings support that deficiencies in recovery from stressors may result in attenuated tolerance to stress that could trigger muscle degeneration. PMID:27106764

  9. Norepinephrine-induced apoptotic and hypertrophic responses in H9c2 cardiac myoblasts are characterized by different repertoire of reactive oxygen species generation.

    PubMed

    Thakur, Anita; Alam, Md Jahangir; Ajayakumar, M R; Ghaskadbi, Saroj; Sharma, Manish; Goswami, Shyamal K

    2015-08-01

    Despite recent advances, the role of ROS in mediating hypertrophic and apoptotic responses in cardiac myocytes elicited by norepinephrine (NE) is rather poorly understood. We demonstrate through our experiments that H9c2 cardiac myoblasts treated with 2 µM NE (hypertrophic dose) generate DCFH-DA positive ROS only for 2h; while those treated with 100 µM NE (apoptotic dose) sustains generation for 48 h, followed by apoptosis. Though the levels of DCFH fluorescence were comparable at early time points in the two treatment sets, its quenching by DPI, catalase and MnTmPyP suggested the existence of a different repertoire of ROS. Both doses of NE also induced moderate levels of H2O2 but with different kinetics. Sustained but intermittent generation of highly reactive species detectable by HPF was seen in both treatment sets but no peroxynitrite was generated in either conditions. Sustained generation of hydroxyl radicals with no appreciable differences were noticed in both treatment sets. Nevertheless, despite similar profile of ROS generation between the two conditions, extensive DNA damage as evident from the increase in 8-OH-dG content, formation of γ-H2AX and PARP cleavage was seen only in cells treated with the higher dose of NE. We therefore conclude that hypertrophic and apoptotic doses of NE generate distinct but comparable repertoire of ROS/RNS leading to two very distinct downstream responses. PMID:26070033

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1).

    PubMed

    Chen, Qiushi; Müller, Juliane S; Pang, Poh-Choo; Laval, Steve H; Haslam, Stuart M; Lochmüller, Hanns; Dell, Anne

    2015-01-01

    Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS) termed "limb-girdle CMS with tubular aggregates". CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS caused by mutations in DOK7, myopathy caused by mutations in MTND5, limb girdle muscular dystrophy type 2A (LGMD2A), and Pompe disease. A comparison of the relative abundances of bi-, tri-, and tetra-antennary N-glycans in each of the cell preparations revealed that all samples exhibited broadly similar levels of branching. Moreover, although some differences were observed in the relative abundances of some of the N-glycan constituents, these variations were modest and were not confined to the GFPT1 samples. Therefore, GFPT1 mutations in CMS patients do not appear to compromise global N-glycosylation in muscle cells. PMID:26501342

  12. Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1)

    PubMed Central

    Chen, Qiushi; Müller, Juliane S.; Pang, Poh-Choo; Laval, Steve H.; Haslam, Stuart M.; Lochmüller, Hanns; Dell, Anne

    2015-01-01

    Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS) termed “limb-girdle CMS with tubular aggregates”. CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS caused by mutations in DOK7, myopathy caused by mutations in MTND5, limb girdle muscular dystrophy type 2A (LGMD2A), and Pompe disease. A comparison of the relative abundances of bi-, tri-, and tetra-antennary N-glycans in each of the cell preparations revealed that all samples exhibited broadly similar levels of branching. Moreover, although some differences were observed in the relative abundances of some of the N-glycan constituents, these variations were modest and were not confined to the GFPT1 samples. Therefore, GFPT1 mutations in CMS patients do not appear to compromise global N-glycosylation in muscle cells. PMID:26501342

  13. Stimulatory effect of isoferulic acid on alpha1A-adrenoceptor to increase glucose uptake into cultured myoblast C2C12 cell of mice.

    PubMed

    Liu, I M; Tsai, C C; Lai, T Y; Cheng, J T

    2001-05-14

    In an attempt to elucidate the effect of isoferulic acid on alpha1-adrenoceptor (AR), the myoblast C2C12 cells of mice were employed to investigate the change of glucose uptake in the present study. Isoferulic acid enhanced the uptake of radioactive glucose into C2C12 cells in a concentration-dependent manner, which were abolished by pretreatment with prazosin. Effect of isoferulic acid on alpha1-AR was further characterized using the displacement of [3H]YM617 binding in C2C12 cells. The radioactive glucose uptake increasing action of isoferulic acid was abolished by tamsulosin or WB 4101 at concentration sufficient to block alpha1A-adrenoceptor (alpha1A-AR) but it was not modified by chlorethylclonidine (CEC) at the concentration sufficient to abolish alpha1B-AR. An activation of alpha1A-AR by isoferulic acid in C2C12 cells can thus be considered. Pharmacological inhibition of phospholipase C (PLC) by U73312 resulted in a concentration-dependent reduction of isoferulic acid-stimulated glucose uptake in C2C12 cells. This inhibition by U73112 was specific because the inactive congener, U73343, failed to modify the action of isoferulic acid. Also, chelerythrine and GF 109203X diminished the action of isoferulic acid at concentration sufficient to inhibit the activity of protein kinase C (PKC). The obtained data suggest that an activation of alpha1A-AR by isoferulic acid may increase the glucose uptake via PLC-PKC pathway in C2C12 cells. PMID:11474559

  14. Serotonin and Histamine Therapy Increases Tetanic Forces of Myoblasts, Reduces Muscle Injury, and Improves Grip Strength Performance of Dmd(mdx) Mice.

    PubMed

    Gurel, Volkan; Lins, Jeremy; Lambert, Kristyn; Lazauski, Joan; Spaulding, James; McMichael, John

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a recessive X-linked fatal disorder caused by a mutation in the dystrophin gene. Although several therapeutic approaches have been studied, none has led to substantial long-term effects in patients. The aim of this study was to test a serotonin and histamine (S&H) combination on human skeletal myoblasts and Dmd(mdx) mice for its effects on muscle strength and injury. Normal human bioartificial muscles (BAMs) were treated, and muscle tetanic forces and muscle injury tests were performed using the MyoForce Analysis System. Dmd(mdx) mice, the murine model of DMD, were administered serotonin, histamine, or S&H combination twice daily for 6 weeks, and functional performance tests were conducted once a week. The S&H combination treatment caused significant increases in tetanic forces at all time points and concentrations tested as compared to the saline controls. Dose response of the BAMs to the treatment demonstrated a significant increase in force generation at all concentrations compared to the controls after 3 to 4 days of drug treatment. The highest 3 concentrations had a significant effect on lowering contractile-induced injury as measured by a reduction in the release of adenylate kinase. Histamine-only and S&H treatments improved grip strength of Dmd(mdx) mice, whereas serotonin-only treatment resulted in no significant improvement in muscle strength. The results of this study indicate that S&H therapy might be a promising new strategy for muscular dystrophies and that the mechanism should be further investigated. PMID:26740813

  15. Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

    SciTech Connect

    Takegahara, Yuki; Yamanouchi, Keitaro Nakamura, Katsuyuki; Nakano, Shin-ichi; Nishihara, Masugi

    2014-05-15

    Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. - Highlights: • We examined the effects of pre- and mature adipocytes on myogenesis in vitro. • Preadipocytes and mature adipocytes affect myoblast fusion. • Preadipocytes play an important role in maintaining skeletal muscle mass. • Mature adipocytes lead to muscle deterioration observed in skeletal muscle pathologies.

  16. Effect of anthocyanidins on myogenic differentiation in induced and non-induced primary myoblasts from rainbow trout (Oncorhynchus mykiss).

    PubMed

    Villasante, Alejandro; Powell, Madison S; Murdoch, Gordon K; Overturf, Ken; Cain, Kenneth; Wacyk, Jurij; Hardy, Ronald W

    2016-01-01

    A study was conducted to test whether an anthocyanidin mixture (peonidin, cyanidin and pelargonidin chloride) modulates myogenesis in both induced and non-induced myogenic cells from juvenile rainbow trout (Oncorhynchus mykiss). We evaluated three different anthocyanidin concentrations (1×, 2.5× and 10×) at two sampling times (24 and 36h). To test for treatment effects, we analyzed the expression of myoD and pax7 as well as two target genes of the Notch signaling pathway, hey2 and her6. In induced myogenic cells, the lowest and middle anthocyanidin doses caused significantly greater expression of myoD after 24h of treatment compared to control. A significantly higher expression of pax7 in cells exposed to either anthocyanidin treatment during 36h compared was observed. Similarly, the pax7/myoD ratio was significantly lower in cells exposed to the lowest anthocyanidin doses during 24h compared to control. No significant effect of anthocyanidin treatments on the expression of hey2 and her6 at either sampling point was detected. In non-induced cells, we observed no effect of anthocyanidins on myoD expression and significant down-regulation on pax7 expression in cells exposed to either anthocyanidin mixture concentrations after 24 and 36h of treatment compared to control. Further, the pax7/myoD ratio was significantly lower in cells exposed to either anthocyanidin doses at both sampling time. In non-induced cells, the highest anthocyanidin dose provoked significantly greater expression of hey2 after 24h of treatment compared to control. We detected no such effect in non-induced cells exposed to the lowest and middle anthocyanidin doses during 24h of treatment. The expression of her6 was unaffected by anthocyanidin treatments at either sampling time or doses compared to control. Collectively, these findings provide evidence that anthocyanidins modulate specific components of the myogenic programming in fish, thereby potentially affecting somatic growth in fish fed

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

    SciTech Connect

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

    2008-04-01

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

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

    PubMed

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

    2015-08-14

    Transcription factors of the nuclear factor-kappa B (NF-κB) family play a pivotal role in inflammation, immunity and cell survival responses. Recent studies revealed that NF-κB also regulates the processes of muscle atrophy. NF-κB activity is regulated by various factors, including ankyrin repeat domain 2 (AnkrD2), which belongs to the muscle ankyrin repeat protein family. Another member of this family, AnkrD1 is also a transcriptional effector. The expression levels of AnkrD1 are highly upregulated in denervated skeletal muscle, suggesting an involvement of AnkrD1 in NF-κB mediated cellular responses to paralysis. However, the molecular mechanism underlying the interactive role of AnkrD1 in NF-κB mediated cellular responses is not well understood. In the current study, we examined the effect of AnkrD1 on NF-κB activity and determined the interactions between AnkrD1 expression and NF-κB signaling induced by TNFα in differentiating C2C12 myoblasts. TNFα upregulated AnkrD1 mRNA and protein levels. AnkrD1-siRNA significantly increased TNFα-induced transcriptional activation of NF-κB, whereas overexpression of AnkrD1 inhibited TNFα-induced NF-κB activity. Co-immunoprecipitation studies demonstrated that AnkrD1 was able to bind p50 subunit of NF-κB and vice versa. Finally, CHIP assays revealed that AnkrD1 bound chromatin at a NF-κB binding site in the AnrkD2 promoter and required NF-κB to do so. These results provide evidence of signaling integration between AnkrD1 and NF-κB pathways, and suggest a novel anti-inflammatory role of AnkrD1 through feedback inhibition of NF-κB transcriptional activity by which AnkrD1 modulates the balance between physiological and pathological inflammatory responses in skeletal muscle. PMID:26102030

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

    SciTech Connect

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

    2015-08-14

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

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

    PubMed

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

    2016-06-01

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

  1. Affective processing requires awareness.

    PubMed

    Lähteenmäki, Mikko; Hyönä, Jukka; Koivisto, Mika; Nummenmaa, Lauri

    2015-04-01

    Studies using backward masked emotional stimuli suggest that affective processing may occur outside visual awareness and imply primacy of affective over semantic processing, yet these experiments have not strictly controlled for the participants' awareness of the stimuli. Here we directly compared the primacy of affective versus semantic categorization of biologically relevant stimuli in 5 experiments (n = 178) using explicit (semantic and affective discrimination; Experiments 1-3) and implicit (semantic and affective priming; Experiments 4-5) measures. The same stimuli were used in semantic and affective tasks. Visual awareness was manipulated by varying exposure duration of the masked stimuli, and subjective level of stimulus awareness was measured after each trial using a 4-point perceptual awareness scale. When participants reported no awareness of the stimuli, semantic and affective categorization were at chance level and priming scores did not differ from zero. When participants were even partially aware of the stimuli, (a) both semantic and affective categorization could be performed above chance level with equal accuracy, (b) semantic categorization was faster than affective categorization, and (c) both semantic and affective priming were observed. Affective categorization speed was linearly dependent on semantic categorization speed, suggesting dependence of affective processing on semantic recognition. Manipulations of affective and semantic categorization tasks revealed a hierarchy of categorization operations beginning with basic-level semantic categorization and ending with superordinate level affective categorization. We conclude that both implicit and explicit affective and semantic categorization is dependent on visual awareness, and that affective recognition follows semantic categorization. PMID:25559654

  2. Affective Dynamics in Psychopathology

    PubMed Central

    Trull, Timothy J.; Lane, Sean P.; Koval, Peter; Ebner-Priemer, Ulrich W.

    2016-01-01

    We discuss three varieties of affective dynamics (affective instability, emotional inertia, and emotional differentiation). In each case, we suggest how these affective dynamics should be operationalized and measured in daily life using time-intensive methods, like ecological momentary assessment or ambulatory assessment, and recommend time-sensitive analyses that take into account not only the variability but also the temporal dependency of reports. Studies that explore how these affective dynamics are associated with psychological disorders and symptoms are reviewed, and we emphasize that these affective processes are within a nexus of other components of emotion regulation.

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

    PubMed

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

    2015-05-01

    This study was designed to examine the protective effects of the marine brown algae Petalonia binghamiae against oxidative stress-induced cellular damage and to elucidate the underlying mechanisms. P. binghamiae methanol extract (PBME) prevented hydrogen peroxide (H2O2)-induced growth inhibition and exhibited scavenging activity against intracellular reactive oxygen species (ROS) induced by H2O2 in mouse-derived C2C12 myoblasts. PBME also significantly attenuated H2O2-induced comet tail formation in a comet assay, histone γH2A.X phosphorylation, and annexin V-positive cells, suggesting that PBME prevented H2O2-induced cellular DNA damage and apoptotic cell death. Furthermore, PBME increased the levels of heme oxygenase-1 (HO-1), a potent antioxidant enzyme, associated with the induction of nuclear factor-erythroid 2 related factor 2 (Nrf2). However, zinc protoporphyrin IX, a HO-1 competitive inhibitor, significantly abolished the protective effects of PBME on H2O2-induced ROS generation, growth inhibition, and apoptosis. Collectively, these results demonstrate that PBME augments the antioxidant defense capacity through activation of the Nrf2/HO-1 pathway. PMID:25939035

  4. Myoblast differentiation of human mesenchymal stem cells on graphene oxide and electrospun graphene oxide-polymer composite fibrous meshes: importance of graphene oxide conductivity and dielectric constant on their biocompatibility.

    PubMed

    Chaudhuri, Biswadeep; Bhadra, Debabrata; Moroni, Lorenzo; Pramanik, Krishna

    2015-01-01

    Recently graphene and graphene based composites are emerging as better materials to fabricate scaffolds. Addition of graphene oxide (GO) nanoplatelets (GOnPs) in bioactive polymers was found to enhance its conductivity (σ) and, dielectric permittivity (ϵ) along with biocompatibility. In this paper, human cord blood derived mesenchymal stem cells (CB-hMSCs) were differentiated to skeletal muscle cells (hSkMCs) on spin coated thin GO sheets composed of GOnPs and on electrospun fibrous meshes of GO-PCL (poly-caprolactone) composite. Both substrates exhibited excellent myoblast differentiations and promoted self-alignedmyotubesformation similar to natural orientation. σ, ϵ, microstructural and vibration spectroscopic studies were carried out for the characterizations of GO sheet and the composite scaffolds. Significantly enhanced values of both σ and ϵ of the GO-PCL composite were considered to provide favourable cues for the formation of superior multinucleated myotubes on the electrospun meshes compared to those on thin GO sheets. The present results demonstrated that both substrates might be used as potential candidates for CB-hMSCs differentiation and proliferation for human skeletal muscle tissue regeneration. PMID:25691492

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

    PubMed Central

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

    2015-01-01

    This study was designed to examine the protective effects of the marine brown algae Petalonia binghamiae against oxidative stress-induced cellular damage and to elucidate the underlying mechanisms. P. binghamiae methanol extract (PBME) prevented hydrogen peroxide (H2O2)-induced growth inhibition and exhibited scavenging activity against intracellular reactive oxygen species (ROS) induced by H2O2 in mouse-derived C2C12 myoblasts. PBME also significantly attenuated H2O2-induced comet tail formation in a comet assay, histone γH2A.X phosphorylation, and annexin V-positive cells, suggesting that PBME prevented H2O2-induced cellular DNA damage and apoptotic cell death. Furthermore, PBME increased the levels of heme oxygenase-1 (HO-1), a potent antioxidant enzyme, associated with the induction of nuclear factor-erythroid 2 related factor 2 (Nrf2). However, zinc protoporphyrin IX, a HO-1 competitive inhibitor, significantly abolished the protective effects of PBME on H2O2-induced ROS generation, growth inhibition, and apoptosis. Collectively, these results demonstrate that PBME augments the antioxidant defense capacity through activation of the Nrf2/HO-1 pathway. PMID:25939035

  6. Assessing Student Affect

    ERIC Educational Resources Information Center

    Popham, W. James

    2009-01-01

    Student affect--the attitudes, interests, and values that students exhibit and acquire in school--can play a profoundly important role in students' postschool lives, possibly an even more significant role than that played by students' cognitive achievements. If student affect is so crucial, then why don't teachers assess it? One deterrent is that…

  7. Affective Involvement Instrument.

    ERIC Educational Resources Information Center

    Lemlech, Johanna K.

    1970-01-01

    The Affective Involvement Instrument (AII) describes and classifies affective involvement in the process of decision-making as it occurs during classroom activities such as role-playing or group discussions. The thirty-celled instrument behaviorizes the six processes involved in decision-making and combines them with the taxonomic levels of the…

  8. Affectional Patterns of Adolescents.

    ERIC Educational Resources Information Center

    O'Donnell, William J.

    1979-01-01

    This study sought to determine if there is a shift with age in affection (1) from parents to friends, (2) from one parent to the other, and (3) from same-sex to opposite-sex friends. Subjects, eighth graders and eleventh graders, completed the Measurement of Family Affective Structure. (Author)

  9. Establishment of clonal myogenic cell lines from severely affected dystrophic muscles - CDK4 maintains the myogenic population

    PubMed Central

    2011-01-01

    Background A hallmark of muscular dystrophies is the replacement of muscle by connective tissue. Muscle biopsies from patients severely affected with facioscapulohumeral muscular dystrophy (FSHD) may contain few myogenic cells. Because the chromosomal contraction at 4q35 linked to FSHD is thought to cause a defect within myogenic cells, it is important to study this particular cell type, rather than the fibroblasts and adipocytes of the endomysial fibrosis, to understand the mechanism leading to myopathy. Results We present a protocol to establish clonal myogenic cell lines from even severely dystrophic muscle that has been replaced mostly by fat, using overexpression of CDK4 and the catalytic component of telomerase (human telomerase reverse transcriptase; hTERT), and a subsequent cloning step. hTERT is necessary to compensate for telomere loss during in vitro cultivation, while CDK4 prevents a telomere-independent growth arrest affecting CD56+ myogenic cells, but not their CD56- counterpart, in vitro. Conclusions These immortal cell lines are valuable tools to reproducibly study the effect of the FSHD mutation within myoblasts isolated from muscles that have been severely affected by the disease, without the confounding influence of variable amounts of contaminating connective-tissue cells. PMID:21798090

  10. Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner.

    PubMed

    Takegahara, Yuki; Yamanouchi, Keitaro; Nakamura, Katsuyuki; Nakano, Shin-ichi; Nishihara, Masugi

    2014-05-15

    Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. PMID:24720912

  11. Interfering with the connection between the nucleus and the cytoskeleton affects nuclear rotation, mechanotransduction and myogenesis.

    PubMed

    Brosig, Michaela; Ferralli, Jacqueline; Gelman, Laurent; Chiquet, Matthias; Chiquet-Ehrismann, Ruth

    2010-10-01

    Mechanical stress controls a broad range of cellular functions. The cytoskeleton is physically connected to the extracellular matrix via integrin receptors, and to the nuclear lamina by the LINC complex that spans both nuclear membranes. We asked here how disruption of this direct link from the cytoskeleton to nuclear chromatin affects mechanotransduction. Fibroblasts grown on flexible silicone membranes reacted to cyclic stretch by nuclear rotation. This rotation was abolished by inhibition of actomyosin contraction as well as by overexpression of dominant-negative versions of nesprin or sun proteins that form the LINC complex. In an in vitro model of muscle differentiation, cyclic strain inhibits differentiation and induces proliferation of C2C12 myoblasts. Interference with the LINC complex in these cells abrogated their stretch-induced proliferation, while stretch increased p38 MAPK and NFkappaB phosphorylation and the transcript levels of myogenic transcription factors MyoD and myogenin. We found that the physical link from the cytoskeleton to the nuclear lamina is crucial for correct mechanotransduction, and that disruption of the LINC complex perturbs the mechanical control of cell differentiation. PMID:20621196

  12. [Affect and mimetic behavior].

    PubMed

    Zepf, S; Ullrich, B; Hartmann, S

    1998-05-01

    The relationship between facial expression and experienced affect presents many problems. The two diametrically opposed positions proposing solutions to this problem are exemplified using the conceptions of Mandler u. Izard. The underlying premises of both conceptions still prevail in various forms. The authors reject the concepts according to which facial expression is merely correlated to the affects (see Mandler 1975) as well as the view that facial expression controls the affects (see Izard 1977). The relationship between affect and facial expression is reexamined, subjecting it to a semiotic, essentially semantic analysis similar to the Ogden and Richards' language and meaning approach. This analysis involves a critical discussion of Scherer's attempt of a purely communicational interpretation using Bühler's organon model. In the author's approach, facial expression is seen not simply as a system of signals, but as a system of representative signs which signify the affects and refer to the emotive meaning of things for the subject. The authors develop the thesis that human beings are not born simply with the ability to speak, but also with the abstract possibility of performing facial expressions. This ability develops by way of coordinating patterns of expressions, which are presumably phylogenetically determined, with affects that take on a socially determined individual form, similar to language acquisition during socialisation. The authors discuss the methodological implications arising for studies investigating the affective meaning of facial expressions. PMID:9632951

  13. Compounds affecting cholesterol absorption

    NASA Technical Reports Server (NTRS)

    Hua, Duy H. (Inventor); Koo, Sung I. (Inventor); Noh, Sang K. (Inventor)

    2004-01-01

    A class of novel compounds is described for use in affecting lymphatic absorption of cholesterol. Compounds of particular interest are defined by Formula I: ##STR1## or a pharmaceutically acceptable salt thereof.

  14. Affective responses to dance.

    PubMed

    Christensen, Julia F; Pollick, Frank E; Lambrechts, Anna; Gomila, Antoni

    2016-07-01

    The objective of the present work was the characterization of mechanisms by which affective experiences are elicited in observers when watching dance movements. A total of 203 dance stimuli from a normed stimuli library were used in a series of independent experiments. The following measures were obtained: (i) subjective measures of 97 dance-naïve participants' affective responses (Likert scale ratings, interviews); and (ii) objective measures of the physical parameters of the stimuli (motion energy, luminance), and of the movements represented in the stimuli (roundedness, impressiveness). Results showed that (i) participants' ratings of felt and perceived affect differed, (ii) felt and perceived valence but not arousal ratings correlated with physical parameters of the stimuli (motion energy and luminance), (iii) roundedness in posture shape was related to the experience of more positive emotion than edgy shapes (1 of 3 assessed rounded shapes showed a clear effect on positiveness ratings while a second reached trend level significance), (iv) more impressive movements resulted in more positive affective responses, (v) dance triggered affective experiences through the imagery and autobiographical memories it elicited in some people, and (vi) the physical parameters of the video stimuli correlated only weakly and negatively with the aesthetics ratings of beauty, liking and interest. The novelty of the present approach was twofold; (i) the assessment of multiple affect-inducing mechanisms, and (ii) the use of one single normed stimulus set. The results from this approach lend support to both previous and present findings. Results are discussed with regards to current literature in the field of empirical aesthetics and affective neuroscience. PMID:27235953

  15. Drugs affecting glycosaminoglycan metabolism.

    PubMed

    Ghiselli, Giancarlo; Maccarana, Marco

    2016-07-01

    Glycosaminoglycans (GAGs) are charged polysaccharides ubiquitously present at the cell surface and in the extracellular matrix. GAGs are crucial for cellular homeostasis, and their metabolism is altered during pathological processes. However, little consideration has been given to the regulation of the GAG milieu through pharmacological interventions. In this review, we provide a classification of small molecules affecting GAG metabolism based on their mechanism of action. Furthermore, we present evidence to show that clinically approved drugs affect GAG metabolism and that this could contribute to their therapeutic benefit. PMID:27217160

  16. Affective Factors: Anxiety

    ERIC Educational Resources Information Center

    Tasnimi, Mahshad

    2009-01-01

    Affective factors seem to play a crucial role in success or failure in second language acquisition. Negative attitudes can reduce learners' motivation and harm language learning, while positive attitudes can do the reverse. Discovering students' attitudes about language will help both teacher and student in teaching learning process. Anxiety is…

  17. How Technology Affects Teaching.

    ERIC Educational Resources Information Center

    Wiske, Martha Stone; And Others

    This study presents composite profiles of teachers who were interviewed in order to assess how they are being affected by the challenges and opportunities presented by computer technology use. In-depth interviews were held with 76 teachers from 10 sites around the country, and the interview data were analyzed to identify themes and to construct…

  18. Factors affecting soil cohesion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil erodibility is a measure of a soil’s resistance against erosive forces and is affected by both intrinsic (or inherent) soil property and the extrinsic condition at the time erodibility measurement is made. Since soil erodibility is usually calculated from results obtained from erosion experimen...

  19. What Variables Affect Solubility?

    ERIC Educational Resources Information Center

    Baker, William P.; Leyva, Kathryn

    2003-01-01

    Helps middle school students understand the concept of solubility through hands-on experience with a variety of liquids and solids. As they explore factors that affect solubility and saturation, students gain content mastery and an understanding of the inquiry process. Also enables teachers to authentically assess student performance on several…

  20. How Body Affects Brain.

    PubMed

    Suzuki, Wendy A

    2016-08-01

    Studies show that physical exercise can affect a range of brain and cognitive functions. However, little is known about the peripheral signals that initiate these central changes. Moon et al. (2016) provide exciting new evidence that a novel myokine, cathepsin B (CTSB), released with exercise is associated with improved memory. PMID:27508865

  1. Food Affects Human Behavior.

    ERIC Educational Resources Information Center

    Kolata, Gina

    1982-01-01

    A conference on whether food and nutrients affect human behavior was held on November 9, 1982 at the Massachusetts Institute of Technology. Various research studies on this topic are reviewed, including the effects of food on brain biochemistry (particularly sleep) and effects of tryptophane as a pain reducer. (JN)

  2. Dynamic Synchronization of Teacher-Students Affection in Affective Instruction

    ERIC Educational Resources Information Center

    Zhang, Wenhai; Lu, Jiamei

    2011-01-01

    Based on Bower's affective network theory, the article links the dynamic analysis of affective factors in affective instruction, and presents affective instruction strategic of dynamic synchronization between teacher and students to implement the best ideal mood that promotes students' cognition and affection together. In the process of teaching,…

  3. Affective Processes and Academic Achievement.

    ERIC Educational Resources Information Center

    Feshbach, Norma Deitch; Feshbach, Seymour

    1987-01-01

    Data indicate that for girls, affective dispositional factors (empathy, depressive affectivity, aggression, and self-concept) are intimately linked to cognitive development and academic achievement. (PCB)

  4. Does Commuting Affect Health?

    PubMed

    Künn-Nelen, Annemarie

    2016-08-01

    This paper analyzes the relation between commuting time and health in the UK. I focus on four different types of health outcomes: subjective health measures, objective health measures, health behavior, and healthcare utilization. Fixed effect models are estimated with British Household Panel Survey data. I find that whereas objective health and health behavior are barely affected by commuting time, subjective health measures are clearly lower for people who commute longer. A longer commuting time is, moreover, related to more visits to the general practitioner. Effects turn out to be more pronounced for women and for commuters driving a car. For women, commuting time is also negatively related to regular exercise and positively to calling in sick. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26010157

  5. Factors Affecting Wound Healing

    PubMed Central

    Guo, S.; DiPietro, L.A.

    2010-01-01

    Wound healing, as a normal biological process in the human body, is achieved through four precisely and highly programmed phases: hemostasis, inflammation, proliferation, and remodeling. For a wound to heal successfully, all four phases must occur in the proper sequence and time frame. Many factors can interfere with one or more phases of this process, thus causing improper or impaired wound healing. This article reviews the recent literature on the most significant factors that affect cutaneous wound healing and the potential cellular and/or molecular mechanisms involved. The factors discussed include oxygenation, infection, age and sex hormones, stress, diabetes, obesity, medications, alcoholism, smoking, and nutrition. A better understanding of the influence of these factors on repair may lead to therapeutics that improve wound healing and resolve impaired wounds. PMID:20139336

  6. Pteridines and affective disorders.

    PubMed

    Hoekstra, R; Fekkes, D

    2002-06-01

    The pteridine tetrahydrobiopterin (BH4) is an essential cofactor in the biosynthesis of dopamine, (nor)epinephrine, serotonin and nitric oxide (NO). Furthermore, BH4 has a direct influence on release mechanisms of these neurotransmitters and on serotonin receptor binding activity immunology. The synthesis of BH4 is stimulated by interferon-gamma and hence there is a close relationship with the immune system HPA-axis. In animal experiments it was also found that the hypothalamus-pituitary-adrenal axis influences the pteridine metabolism. In clinical studies, so far, no evidence has been found for this relationship diseases. A congenital biopterin deficiency results in atypical phenylketonuria with severe neuropsychiatric symptoms. In several neurological diseases, such as Parkinson's disease, decreased levels of BH4 are found depression. Since 1984 there have been reports on decreased biopterin and increased neopterin levels in urine and plasma of depressed patients. Conflicting results have also been found, however, due probably to methodological problems therapy. Until now, oral administration of BH4 to depressed patients has been performed by two investigators, which resulted in mainly temporal clinical improvement discussion. Understanding of biochemical mechanisms in which pteridines are involved may contribute to our knowledge of the pathogenesis and treatment of affective disorders. This paper aims to provide an overview of the relevant literature and warrant for further research on this intriguing compound. PMID:26984153

  7. How Obesity Affects Tendons?

    PubMed

    Abate, Michele; Salini, Vincenzo; Andia, Isabel

    2016-01-01

    Several epidemiological and clinical observations have definitely demonstrated that obesity has harmful effects on tendons. The pathogenesis of tendon damage is multi-factorial. In addition to overload, attributable to the increased body weight, which significantly affects load-bearing tendons, systemic factors play a relevant role. Several bioactive peptides (chemerin, leptin, adiponectin and others) are released by adipocytes, and influence tendon structure by means of negative activities on mesenchymal cells. The ensuing systemic state of chronic, sub-clinic, low-grade inflammation can damage tendon structure. Metabolic disorders (diabetes, impaired glucose tolerance, and dislipidemia), frequently associated with visceral adiposity, are concurrent pathogenetic factors. Indeed, high glucose levels increase the formation of Advanced Glycation End-products, which in turn form stable covalent cross-links within collagen fibers, modifying their structure and functionality.Sport activities, so useful for preventing important cardiovascular complications, may be detrimental for tendons if they are submitted to intense acute or chronic overload. Therefore, two caution rules are mandatory: first, to engage in personalized soft training program, and secondly to follow regular check-up for tendon pathology. PMID:27535258

  8. Affective Incoherence: When Affective Concepts and Embodied Reactions Clash

    PubMed Central

    Centerbar, David B.; Clore, Gerald L.; Schnall, Simone; Garvin, Erika

    2008-01-01

    In five studies, we examined the effects on cognitive performance of coherence and incoherence between conceptual and experiential sources of affective information. The studies crossed the priming of happy and sad concepts with affective experiences. In different experiments, these included: approach or avoidance actions, happy or sad feelings, and happy or sad expressive behaviors. In all studies, coherence between affective concepts and affective experiences led to better recall of a story than affective incoherence. We suggested that the experience of such experiential affective cues serves as evidence of the appropriateness of affective concepts that come to mind. The results suggest that affective coherence has epistemic benefits, and that incoherence is costly, for cognitive performance. PMID:18361672

  9. Identifying Occupationally Specific Affective Behaviors.

    ERIC Educational Resources Information Center

    Pucel, David J.

    1993-01-01

    Data from two groups of cosmetology instructors (n=15) and two groups of machinist instructors (n=17) validated the Occupational Affective Behavior Analysis instrument as capable of identifying affective behaviors viewed as important to success in a given occupation. (SK)

  10. Drugs affecting the eye.

    PubMed

    Taylor, F

    1985-08-01

    This discussion reviews drugs that affect the eye, including antihyperglycemic agents; corticosteroids; antirheumatic drugs (quinolines, indomethacin, and allopurinol); psychiatric drugs (phenothiazine, thioridazine, and chlorpromazine); drugs used in cardiology (practolol, amiodarone, and digitalis gylcosides); drugs implicated in optic neuritis and atrophy, drugs with an anticholinergic action; oral contraceptives (OCs); and topical drugs and systemic effects. Refractive changes, either myopic or hypermetropic, can occur as a result of hyperglycemia, and variation in vision is sometimes a presenting symptom in diabetes mellitus. If it causes a change in the refraction, treatment of hyperglycemia almost always produces a temporary hypermetropia. A return to the original refractive state often takes weeks, sometimes months. There is some evidence that patients adequately treated with insulin improve more rapidly than those taking oral medication. Such patients always should be referred for opthalmological evaluation as other factors might be responsible, but it might not be possible to order the appropriate spectacle correction for some time. The most important ocular side effect of the systemic adiministration of corticosteroids is the formation of a posterior subcapsular cataract. Glaucoma also can result from corticosteroids, most often when they are applied topically. Corticosteroids have been implicated in the production of benign intracranial hypertension, which is paradoxical because they also are used in its treatment. The most important side effect of drugs such as chloroquine and hydroxychloroquine is an almost always irreversible maculopathy with resultant loss of central vision. Corneal and retinal changes similar to those caused by the quinolines have been reported with indomethacin, but there is some question about a cause and effect relationship. The National Registry of Drug Induced Ocular Side Effects in the US published 30 case histories of

  11. Individual difference variables, affective differentiation, and the structures of affect.

    PubMed

    Terracciano, Antonio; McCrae, Robert R; Hagemann, Dirk; Costa, Paul T

    2003-10-01

    Methodological arguments are usually invoked to explain variations in the structure of affect. Using self-rated affect from Italian samples (N=600), we show that individual difference variables related to affective differentiation can moderate the observed structure. Indices of circumplexity and congruence coefficients to the hypothesized target were used to quantify the observed structures. Results did not support the circumplex model as a universal structure. A circular structure with axes of activation and valence was approximated only among more affectively differentiated groups: students and respondents with high scores on Openness to Feelings and measures of negative emotionality. A different structure, with unipolar Positive Affect and Negative Affect factors, was observed among adults and respondents with low Openness to Feelings and negative emotionality. The observed structure of affect will depend in part on the nature of the sample studied. PMID:12932207

  12. Individual Difference Variables, Affective Differentiation, and the Structures of Affect

    PubMed Central

    Terracciano, Antonio; McCrae, Robert R.; Hagemann, Dirk; Costa, Paul T.

    2008-01-01

    Methodological arguments are usually invoked to explain variations in the structure of affect. Using self-rated affect from Italian samples (N = 600), we show that individual difference variables related to affective differentiation can moderate the observed structure. Indices of circumplexity (Browne, 1992) and congruence coefficients to the hypothesized target were used to quantify the observed structures. Results did not support the circumplex model as a universal structure. A circular structure with axes of activation and valence was approximated only among more affectively differentiated groups: students and respondents with high scores on Openness to Feelings and measures of negative emotionality. A different structure, with unipolar Positive Affect and Negative Affect factors, was observed among adults and respondents with low Openness to Feelings and negative emotionality. The observed structure of affect will depend in part on the nature of the sample studied. PMID:12932207

  13. Explorations of Affection and Aggression.

    ERIC Educational Resources Information Center

    Shuntich, Richard J.; Shapiro, Richard

    Considerable effort has been devoted to investigating various aspects of love and affection, but there have been few studies about direct expressions of affection. Relationships between gender composition of a dyad and the affection/aggression expressed by the dyad were examined as was the possibility of increasing the amount of affectionate…

  14. Affective Productions of Mathematical Experience

    ERIC Educational Resources Information Center

    Walshaw, Margaret; Brown, Tony

    2012-01-01

    In underscoring the affective elements of mathematics experience, we work with contemporary readings of the work of Spinoza on the politics of affect, to understand what is included in the cognitive repertoire of the Subject. We draw on those resources to tell a pedagogical tale about the relation between cognition and affect in settings of…

  15. Affective Induction and Creative Thinking

    ERIC Educational Resources Information Center

    Fernández-Abascal, Enrique G.; Díaz, María D. Martín

    2013-01-01

    Three studies explored the relation between affect and production of creative divergent thinking, assessed with the Torrance Tests of Creative Thinking (Figural TTCT). In the first study, general, positive, and negative affect, assessed with the Positive and Negative Affect Scale (PANAS) were compared with creative production. In the second study,…

  16. Thermal conditions experienced during differentiation affect metabolic and contractile phenotypes of mouse myotubes.

    PubMed

    Little, Alex G; Seebacher, Frank

    2016-09-01

    Central pathways regulate metabolic responses to cold in endotherms to maintain relatively stable internal core body temperatures. However, peripheral muscles routinely experience temperatures lower than core body temperature, so that it would be advantageous for peripheral tissues to respond to temperature changes independently from core body temperature regulation. Early developmental conditions can influence offspring phenotypes, and here we tested whether developing muscle can compensate locally for the effects of cold exposure independently from central regulation. Muscle myotubes originate from undifferentiated myoblasts that are laid down during embryogenesis. We show that in a murine myoblast cell line (C2C12), cold exposure (32°C) increased myoblast metabolic flux compared with 37°C control conditions. Importantly, myotubes that differentiated at 32°C compensated for the thermodynamic effects of low temperature by increasing metabolic rates, ATP production, and glycolytic flux. Myotube responses were also modulated by the temperatures experienced by "parent" myoblasts. Myotubes that differentiated under cold exposure increased activity of the AMP-stimulated protein kinase (AMPK), which may mediate metabolic changes in response cold exposure. Moreover, cold exposure shifted myosin heavy chains from slow to fast, presumably to overcome slower contractile speeds resulting from low temperatures. Adjusting thermal sensitivities locally in peripheral tissues complements central thermoregulation and permits animals to maintain function in cold environments. Muscle also plays a major metabolic role in adults, so that developmental responses to cold are likely to influence energy expenditure later in life. PMID:27385733

  17. Affect as a Psychological Primitive

    PubMed Central

    Barrett, Lisa Feldman; Bliss-Moreau, Eliza

    2009-01-01

    In this article, we discuss the hypothesis that affect is a fundamental, psychologically irreducible property of the human mind. We begin by presenting historical perspectives on the nature of affect. Next, we proceed with a more contemporary discussion of core affect as a basic property of the mind that is realized within a broadly distributed neuronal workspace. We then present the affective circumplex, a mathematical formalization for representing core affective states, and show that this model can be used to represent individual differences in core affective feelings that are linked to meaningful variation in emotional experience. Finally, we conclude by suggesting that core affect has psychological consequences that reach beyond the boundaries of emotion, to influence learning and consciousness. PMID:20552040

  18. Encountering science education's capacity to affect and be affected

    NASA Astrophysics Data System (ADS)

    Alsop, Steve

    2015-12-01

    What might science education learn from the recent affective turn in the humanities and social sciences? Framed as a response to Michalinos Zembylas's article, this essay draws from selected theorizing in affect theory, science education and science and technology studies, in pursuit of diverse and productive ways to talk of affect within science education. These discussions are framed by desires to transcend traditional epistemic boundaries and practices. The article concludes offering some associated ambiguities and tensions involved.

  19. Testing the Grandchildren's Received Affection Scale using Affection Exchange Theory.

    PubMed

    Mansson, Daniel H

    2013-04-01

    The purpose of this study was to test the Grandchildren's Received Affection Scale (GRAS) using Affection Exchange Theory (Floyd, 2006). In accordance with Affection Exchange Theory, it was hypothesized that grandchildren's scores on the Trait Affection Received Scale (i.e., the extent to which individuals by nature receive affection) would be related significantly and positively to their reports of received affection from their grandparents (i.e., their scores on the GRAS). Additionally, a research question was asked to explore if grandchildren's received affection from their grandparents is dependent on their grandparent's biological sex or lineage (i.e., maternal vs paternal). Thus, young adult grandchildren (N = 422) completed the GRAS and the Trait Affection Received Scale. The results of zero-order Pearson correlational analyses provided support for the hypothesis, whereas the results of MANOVAs tests only partially support extant grandparent-grandchild theory and research. These findings broaden the scope of Affection Exchange Theory and also bolster the GRAS's utility in future grandparent-grandchild affectionate communication research. PMID:23833883

  20. Positive affect and psychobiological processes

    PubMed Central

    Dockray, Samantha; Steptoe, Andrew

    2010-01-01

    Positive affect has been associated with favourable health outcomes, and it is likely that several biological processes mediate the effects of positive mood on physical health. There is converging evidence that positive affect activates the neuroendocrine, autonomic and immune systems in distinct and functionally meaningful ways. Cortisol, both total output and the awakening response, has consistently been shown to be lower among individuals with higher levels of positive affect. The beneficial effects of positive mood on cardiovascular function, including heart rate and blood pressure, and the immune system have also been described. The influence of positive affect on these psychobiological processes are independent of negative affect, suggesting that positive affect may have characteristic biological correlates. The duration and conceptualisation of positive affect may be important considerations in understanding how different biological systems are activated in association with positive affect. The association of positive affect and psychobiological processes has been established, and these biological correlates may be partly responsible for the protective effects of positive affect on health outcomes. PMID:20097225

  1. [Development of the affect system].

    PubMed

    Moser, U; Von Zeppelin, I

    1996-01-01

    The authors show that the development of the affect system commences with affects of an exclusively communicative nature. These regulate the relationship between subject and object. On a different plane they also provide information on the feeling of self deriving from the interaction. Affect is seen throughout as a special kind of information. One section of the article is given over to intensity regulation and early affect defenses. The development of cognitive processes leads to the integration of affect systems and cognitive structures. In the pre-conceptual concretistic phase, fantasies change the object relation in such a way as to make unpleasant affects disappear. Only at a later stage do fantasies acquire the capacity to deal with affects. Ultimately, the affect system is grounded on an invariant relationship feeling. On a variety of different levels it displays the features typical of situation theory and the theory of the representational world, thus making it possible to entertain complex object relations. In this process the various planes of the affect system are retained and practised. Finally, the authors discuss the consequences of their remarks for the understanding of psychic disturbances and the therapies brought to bear on them. PMID:8584745

  2. Infant Affect and Home Environment.

    ERIC Educational Resources Information Center

    Luster, Tom; And Others

    1993-01-01

    Examined data from National Longitudinal Survey of Youth to investigate relationship between infant affect and quality of home environment. Found that infant irritability was negatively correlated with quality of home environment in both low-risk and high-risk families. Infant positive affect was more strongly related to quality of care in…

  3. Current Research in Affective Development.

    ERIC Educational Resources Information Center

    Strayer, Janet

    1985-01-01

    Current research concerning affective development in infants and children is selectively reviewed. The focus of findings and discussion is on three general and related topics: (1) expression of emotion and affective interaction in infancy; (2) socialization and regulation of emotion; (3) comprehension of emotions and empathy with others by…

  4. Affect and Graphing Calculator Use

    ERIC Educational Resources Information Center

    McCulloch, Allison W.

    2011-01-01

    This article reports on a qualitative study of six high school calculus students designed to build an understanding about the affect associated with graphing calculator use in independent situations. DeBellis and Goldin's (2006) framework for affect as a representational system was used as a lens through which to understand the ways in which…

  5. Do You Measure Affective Behavior?

    ERIC Educational Resources Information Center

    Russell, Gene H.

    1978-01-01

    The affective domain, central to the learning process, cannot be ignored, regardless of difficulties involved in behavioral objective preparation and evaluation. A chart (available by mail) has been prepared to assist in the preparation and measurement of student behavior at levels of the affective doman defined by Krathwohl, et al. (DTT)

  6. Affect and Self-Regulation

    ERIC Educational Resources Information Center

    Malmivuori, Marja-Liisa

    2006-01-01

    This paper presents affect as an essential aspect of students' self-reflection and self-regulation. The introduced concepts of self-system and self-system process stress the importance of self-appraisals of personal competence and agency in affective responses and self-regulation in problem solving. Students are viewed as agents who constantly…

  7. Affect intensity and cardiac arousal.

    PubMed

    Blascovich, J; Brennan, K; Tomaka, J; Kelsey, R M; Hughes, P; Coad, M L; Adlin, R

    1992-07-01

    Relationships between affect intensity and basal, evoked, and perceived cardiac arousal were investigated in 3 experiments. Affect intensity was assessed using Larsen and Diener's (1987) Affect Intensity Measure (AIM). Cardiac arousal was evoked with exercise in the 1st study and with mental arithmetic in the 2nd and 3rd. Perceived cardiac arousal was measured under optimal conditions using a standard heartbeat discrimination procedure. Women as a group scored higher on the AIM. Affect intensity was unrelated to basal or evoked cardiac arousal and was negatively related to perceived cardiac arousal in all 3 studies. Data suggest that affect intensity, although unrelated to actual physiological arousal, is negatively related to the accuracy with which individuals perceive their own arousal. Results are discussed within the context of an expanded arousal-regulation model (Blascovich, 1990). PMID:1494983

  8. Intuition, Affect, and Peculiar Beliefs

    PubMed Central

    Boden, Matthew Tyler; Berenbaum, Howard; Topper, Maurice

    2012-01-01

    Research with college students has found that intuitive thinking (e.g., using hunches to ascribe meaning to experiences) and positive affect interactively predict ideas of reference and odd/magical beliefs. We investigated whether these results would generalize to a diverse community sample of adults that included individuals with elevated levels of peculiar perceptions and beliefs. We measured positive and negative affect and intuitive thinking through questionnaires, and peculiar beliefs (i.e., ideas of reference and odd/magical beliefs) through structured clinical interviews. We found that peculiar beliefs were associated with intuitive thinking and negative affect, but not positive affect. Furthermore, in no instance did the interaction of affect and intuitive thinking predict peculiar beliefs. These results suggest that there are important differences in the factors that contribute to peculiar beliefs between college students and clinically meaningful samples. PMID:22707815

  9. Startle modulation by affective faces

    PubMed Central

    Anokhin, Andrey P.; Golosheykin, Simon

    2009-01-01

    Startle reflex modulation by affective pictures is a well-established effect in human emotion research. However, much less is known about startle modulation by affective faces, despite the growing evidence that facial expressions robustly activate emotion-related brain circuits. In this study, acoustic startle probes were administered to 33 young adult participants (16 women) during the viewing of slides from the Pictures of Facial Affect set including neutral, happy, angry, and fearful faces. The effect of expression valence (happy, neutral, negative) on startle magnitude was highly significant (p<.001). Startle reflex was strongly potentiated by negative expressions (fearful and angry), however, no attenuation by happy faces was observed. A significant valence by gender interaction suggests stronger startle potentiation effects in females. These results demonstrate that affective facial expressions can produce significant modulation of the startle reflex. PMID:19833169

  10. On Patterns in Affective Media

    NASA Astrophysics Data System (ADS)

    ADAMATZKY, ANDREW

    In computational experiments with cellular automaton models of affective solutions, where chemical species represent happiness, anger, fear, confusion and sadness, we study phenomena of space time dynamic of emotions. We demonstrate feasibility of the affective solution paradigm in example of emotional abuse therapy. Results outlined in the present paper offer unconventional but promising technique to design, analyze and interpret spatio-temporal dynamic of mass moods in crowds.

  11. Family Intimacy and Affection: A Sociology of Positive Affect.

    ERIC Educational Resources Information Center

    Martinson, Floyd M.

    This paper deals with aspects of positive family affect in intimate family relationships such as: (1) the nuclear family of orientation, including the child-parent subgroup and the sibling subgroup; (2) the nuclear family of procreation, including the marital subgroup and parent-child subgroup; and (3) the dating relationship. Interpersonal…

  12. Behavioral Management: An Affective Approach. (Affective Education Trainers Manual).

    ERIC Educational Resources Information Center

    Heilman, John; Cole, Bob

    This manual provides a framework for training teachers who want to become more skilled in affective education. It is divided into three parts: teacher self-awareness, teacher-student interaction, and teacher-directed group activities. It is designed for use in a two-day workshop. Guidelines for discussions on expectations, responsibility,…

  13. [Poststroke-bipolar affective disorder].

    PubMed

    Bengesser, S A; Wurm, W E; Lackner, N; Birner, A; Reininghaus, B; Kapfhammer, H-P; Reininghaus, E

    2013-08-01

    A few weeks after suffering from a basal ganglia infarction (globus pallidus) with left-sided hemiplegia, a 23-year-old woman exhibited for the first time a pronounced mania with self-endangerment. The use of oral contraceptives was the only determinable risk factor. During the further course, the mother also developed a depressive disorder. Thus a certain genetic predisposition for affective disorders may be relevant, although this would not explain the outbreak by itself. An association between the right-sided basal ganglia infarction and the occurrence of a bipolar affective disorder has been described in the literature. Vascular or, respectively, inflammatory risk factors in synopsis with the aetiopathogenesis of bipolar affective disorders are also discussed in depth in this case report. PMID:23939559

  14. Factors Affecting the Tutoring Process.

    ERIC Educational Resources Information Center

    Hartman, Hope J.

    1990-01-01

    Analyzes factors internal to the tutor and tutee (i.e., cognition, metacognition, and affect) and external to them (e.g., teacher/tutor background knowledge, educational environment, content to be learned, socioeconomic status, family background, and cultural forces) that influence the tutoring process. Suggests a theoretical framework for…

  15. Motor Execution Affects Action Prediction

    ERIC Educational Resources Information Center

    Springer, Anne; Brandstadter, Simone; Liepelt, Roman; Birngruber, Teresa; Giese, Martin; Mechsner, Franz; Prinz, Wolfgang

    2011-01-01

    Previous studies provided evidence of the claim that the prediction of occluded action involves real-time simulation. We report two experiments that aimed to study how real-time simulation is affected by simultaneous action execution under conditions of full, partial or no overlap between observed and executed actions. This overlap was analysed by…

  16. Unconscious Affective Responses to Food.

    PubMed

    Sato, Wataru; Sawada, Reiko; Kubota, Yasutaka; Toichi, Motomi; Fushiki, Tohru

    2016-01-01

    Affective or hedonic responses to food are crucial for humans, both advantageously (e.g., enhancing survival) and disadvantageously (e.g., promoting overeating and lifestyle-related disease). Although previous psychological studies have reported evidence of unconscious cognitive and behavioral processing related to food, it remains unknown whether affective reactions to food can be triggered unconsciously and its relationship with daily eating behaviors. We investigated these issues by using the subliminal affective priming paradigm. Photographs of food or corresponding mosaic images were presented in the peripheral visual field for 33 ms. Target photos of faces with emotionally neutral expressions were then presented, and participants rated their preferences for the faces. Eating behaviors were also assessed using questionnaires. The food images, relative to the mosaics, increased participants' preference for subsequent target faces. Furthermore, the difference in the preference induced by food versus mosaic images was positively correlated with the tendency to engage in external eating. These results suggest that unconscious affective reactions are elicited by the sight of food and that these responses contribute to daily eating behaviors related to overeating. PMID:27501443

  17. On the Primacy of Affect.

    ERIC Educational Resources Information Center

    Zajonc, R. B.

    1984-01-01

    Reasserts view that there can be emotional or affective arousal without prior cognitive appraisal. Criticizes Lazarus's rejection of this view on the grounds that it presents no empirical evidence, is based on an arbitrary definition of emotion, and obliterates all distinctions between cognition, sensation, and perception. (CMG)

  18. Governmental Policies Affecting Community Colleges.

    ERIC Educational Resources Information Center

    Cohen, Arthur M.

    This document traces the influence of governmental policies on American community colleges, focusing on how different levels of government have affected the colleges at various stages of their development with respect to college organization and governance, finance, enrollment, and curriculum. The community college's main contribution has been to…

  19. Does Positive Affect Influence Health?

    ERIC Educational Resources Information Center

    Pressman, Sarah D.; Cohen, Sheldon

    2005-01-01

    This review highlights consistent patterns in the literature associating positive affect (PA) and physical health. However, it also raises serious conceptual and methodological reservations. Evidence suggests an association of trait PA and lower morbidity and of state and trait PA and decreased symptoms and pain. Trait PA is also associated with…

  20. Affective temperament and personal identity.

    PubMed

    Stanghellini, Giovanni; Rosfort, René

    2010-10-01

    The complex relationship between temperament and personal identity, and between these and mental disorders, is of critical interest to both philosophy and psychopathology. More than other living creatures, human beings are constituted and characterized by the interplay of their genotype and phenotype. There appears to be an explanatory gap between the almost perfect genetic identity and the individual differences among humans. One reason for this gap is that a human being is a person besides a physiological organism. We propose an outline of a theoretical model that might somewhat mitigate the explanatory discrepancies between physiological mechanisms and individual human emotional experience and behaviour. Arguing for the pervasive nature of human affectivity, i.e., for the assumption that human consciousness and behaviour is characterised by being permeated by affectivity; to envisage the dynamics of emotional experience, we make use of a three-levelled model of human personal identity that differentiates between factors that are simultaneously at work in the constitution of the individual human person: 1) core emotions, 2) affective temperament types/affective character traits, and 3) personhood. These levels are investigated separately in order to respect the methodological diversity among them (neuroscience, psychopathology, and philosophy), but they are eventually brought together in a hermeneutical account of human personhood. PMID:20236706

  1. Supersonic Wave Interference Affecting Stability

    NASA Technical Reports Server (NTRS)

    Love, Eugene S.

    1958-01-01

    Some of the significant interference fields that may affect stability of aircraft at supersonic speeds are briefly summarized. Illustrations and calculations are presented to indicate the importance of interference fields created by wings, bodies, wing-body combinations, jets, and nacelles.

  2. Aesthetics, Affect, and Educational Politics

    ERIC Educational Resources Information Center

    Means, Alex

    2011-01-01

    This essay explores aesthetics, affect, and educational politics through the thought of Gilles Deleuze and Jacques Ranciere. It contextualizes and contrasts the theoretical valences of their ethical and democratic projects through their shared critique of Kant. It then puts Ranciere's notion of dissensus to work by exploring it in relation to a…

  3. Affective Development in University Education

    ERIC Educational Resources Information Center

    Grootenboer, Peter

    2010-01-01

    There seems to be an increasing requirement for university courses and programs to develop students' affective qualities (beliefs, values, dispositions and attitudes). This study explored the ways academics determined what the desirable qualities were for their particular disciplines and the pedagogical strategies and approaches they used to…

  4. Depression Affects the Whole Family.

    ERIC Educational Resources Information Center

    Hojnar, Laura; Thomas, Dawn V.; Stillwell, Margaret; Bennett, Tess; Allison, Anita

    1997-01-01

    Discusses how expanding one's knowledge of depression can help in supporting Head Start families. Defines depression and lists symptoms of depression for adults and children of various ages, describes how parent's depression can affect child development and the family, and considers how Head Start and support agencies can support children and…

  5. Affective Experience in a Classroom.

    ERIC Educational Resources Information Center

    Anderson, Ariel; And Others

    This study introduces a novel methodology for examining affective experiences in the classroom. The general procedure is to make auditory and visual recordings of a particular child during a normal classroom period. That child is then immediately taken from the class and placed in a private room where the recordings are replayed. The student is…

  6. Unconscious Affective Responses to Food

    PubMed Central

    Sato, Wataru; Sawada, Reiko; Kubota, Yasutaka; Toichi, Motomi; Fushiki, Tohru

    2016-01-01

    Affective or hedonic responses to food are crucial for humans, both advantageously (e.g., enhancing survival) and disadvantageously (e.g., promoting overeating and lifestyle-related disease). Although previous psychological studies have reported evidence of unconscious cognitive and behavioral processing related to food, it remains unknown whether affective reactions to food can be triggered unconsciously and its relationship with daily eating behaviors. We investigated these issues by using the subliminal affective priming paradigm. Photographs of food or corresponding mosaic images were presented in the peripheral visual field for 33 ms. Target photos of faces with emotionally neutral expressions were then presented, and participants rated their preferences for the faces. Eating behaviors were also assessed using questionnaires. The food images, relative to the mosaics, increased participants’ preference for subsequent target faces. Furthermore, the difference in the preference induced by food versus mosaic images was positively correlated with the tendency to engage in external eating. These results suggest that unconscious affective reactions are elicited by the sight of food and that these responses contribute to daily eating behaviors related to overeating. PMID:27501443

  7. Affective Simon effects using facial expressions as affective stimuli.

    PubMed

    De Houwer, J; Hermans, D; Eelen, P

    1998-01-01

    Two experiments are reported in which facial expressions were presented and participants were asked to respond with the word POSITIVE or NEGATIVE on the basis of a relevant feature of the facial stimuli while ignoring the valence of the expression. Results showed that reaction times were influenced by the match between the valence of the facial expression and the valence of the correct response when the identity of the presented person had to be determined in order to select the correct response, but not when the gender of the presented person was relevant. The present experiments illustrate the flexibility of the affective Simon paradigm and provide a further demonstration of the generalizability of the affective Simon effect. PMID:9677856

  8. Bodily action penetrates affective perception

    PubMed Central

    Rigutti, Sara; Gerbino, Walter

    2016-01-01

    Fantoni & Gerbino (2014) showed that subtle postural shifts associated with reaching can have a strong hedonic impact and affect how actors experience facial expressions of emotion. Using a novel Motor Action Mood Induction Procedure (MAMIP), they found consistent congruency effects in participants who performed a facial emotion identification task after a sequence of visually-guided reaches: a face perceived as neutral in a baseline condition appeared slightly happy after comfortable actions and slightly angry after uncomfortable actions. However, skeptics about the penetrability of perception (Zeimbekis & Raftopoulos, 2015) would consider such evidence insufficient to demonstrate that observer’s internal states induced by action comfort/discomfort affect perception in a top-down fashion. The action-modulated mood might have produced a back-end memory effect capable of affecting post-perceptual and decision processing, but not front-end perception. Here, we present evidence that performing a facial emotion detection (not identification) task after MAMIP exhibits systematic mood-congruent sensitivity changes, rather than response bias changes attributable to cognitive set shifts; i.e., we show that observer’s internal states induced by bodily action can modulate affective perception. The detection threshold for happiness was lower after fifty comfortable than uncomfortable reaches; while the detection threshold for anger was lower after fifty uncomfortable than comfortable reaches. Action valence induced an overall sensitivity improvement in detecting subtle variations of congruent facial expressions (happiness after positive comfortable actions, anger after negative uncomfortable actions), in the absence of significant response bias shifts. Notably, both comfortable and uncomfortable reaches impact sensitivity in an approximately symmetric way relative to a baseline inaction condition. All of these constitute compelling evidence of a genuine top-down effect on

  9. Environmental issues affecting CCT development

    SciTech Connect

    Reidy, M.

    1997-12-31

    While no final legislative schedule has been set for the new Congress, two issues with strong environmental ramifications which are likely to affect the coal industry seem to top the list of closely watched debates in Washington -- the Environmental Protection Agency`s proposed new ozone and particulate matter standards and utility restructuring. The paper discusses the background of the proposed standards, public comment, the Congressional review of regulations, other legislative options, and utility restructuring.

  10. Affective cycling in thyroid disease

    SciTech Connect

    Tapp, A.

    1988-05-01

    Depression in an elderly man with primary recurrent unipolar depression responded to radioactive iodine treatment of a thyrotoxic nodule, without the addition of psychotropic medications. Two months later, manic symptoms developed concomitant with the termination of the hyperthyroid state secondary to the radioactive iodine treatment. Clinical implications of these findings in relation to the possible mechanism of action of thyroid hormones on affective cycling are discussed.

  11. Factors affecting maximal acid secretion

    PubMed Central

    Desai, H. G.

    1969-01-01

    The mechanisms by which different factors affect the maximal acid secretion of the stomach are discussed with particular reference to nationality, sex, age, body weight or lean body mass, procedural details, mode of calculation, the nature, dose and route of administration of a stimulus, the synergistic action of another stimulus, drugs, hormones, electrolyte levels, anaemia or deficiency of the iron-dependent enzyme system, vagal continuity and parietal cell mass. PMID:4898322

  12. Anticipation in bipolar affective disorder

    SciTech Connect

    McInnis, M.G.; McMahon, F.J.; Chase, G.A.; Simpson, S.G.; Ross, C.A.; DePaulo, J.R. Jr. )

    1993-08-01

    Anticipation refers to the increase in disease severity or decrease in age at onset in succeeding generations. This phenomenon, formerly ascribed to observation biases, correlates with the expansion of trinucleotide repeat sequences (TNRs) in some disorders. If present in bipolar affective disorder (BPAD), anticipation could provide clues to its genetic etiology. The authors compared age at onset and disease severity between two generations of 34 unilineal families ascertained for a genetic linkage study of BPAD. Life-table analyses showed a significant decrease in survival to first mania or depression from the first to the second generation (P <.001). Intergenerational pairwise comparisons showed both a significantly earlier age at onset (P < .001) and a significantly increased disease severity (P < .001) in the second generation. This difference was significant under each of four data-sampling schemes which excluded probands in the second generation. The second generation experienced onset 8.9-13.5 years earlier and illness 1.8-3.4 times more severe than did the first generation. In additional analyses, drug abuse, deaths of affected individuals prior to interview, decreased fertility, censoring of age at onset, and the cohort effect did not affect our results. The authors conclude that genetic anticipation occurs in this sample of unilineal BPAD families. These findings may implicate genes with expanding TNRs in the genetic etiology of BPAD. 24 refs., 1 fig., 1 tab.

  13. Anticipation in bipolar affective disorder.

    PubMed

    McInnis, M G; McMahon, F J; Chase, G A; Simpson, S G; Ross, C A; DePaulo, J R

    1993-08-01

    Anticipation refers to the increase in disease severity or decrease in age at onset in succeeding generations. This phenomenon, formerly ascribed to observation biases, correlates with the expansion of trinucleotide repeat sequences (TNRs) in some disorders. If present in bipolar affective disorder (BPAD), anticipation could provide clues to its genetic etiology. We compared age at onset and disease severity between two generations of 34 unilineal families ascertained for a genetic linkage study of BPAD. Life-table analyses showed a significant decrease in survival to first mania or depression from the first to the second generation (P < .001). Intergenerational pairwise comparisons showed both a significantly earlier age at onset (P < .001) and a significantly increased disease severity (P < .001) in the second generation. This difference was significant under each of four data-sampling schemes which excluded probands in the second generation. The second generation experienced onset 8.9-13.5 years earlier and illness 1.8-3.4 times more severe than did the first generation. In additional analyses, drug abuse, deaths of affected individuals prior to interview, decreased fertility, censoring of age at onset, and the cohort effect did not affect our results. We conclude that genetic anticipation occurs in this sample of unilineal BPAD families. These findings may implicate genes with expanding TNRs in the genetic etiology of BPAD. PMID:8328456

  14. Musical affect regulation in infancy.

    PubMed

    Trehub, Sandra E; Ghazban, Niusha; Corbeil, Mariève

    2015-03-01

    Adolescents and adults commonly use music for various forms of affect regulation, including relaxation, revitalization, distraction, and elicitation of pleasant memories. Mothers throughout the world also sing to their infants, with affect regulation as the principal goal. To date, the study of maternal singing has focused largely on its acoustic features and its consequences for infant attention. We describe recent laboratory research that explores the consequences of singing for infant affect regulation. Such work reveals that listening to recordings of play songs can maintain 6- to 9-month-old infants in a relatively contented or neutral state considerably longer than recordings of infant-directed or adult-directed speech. When 10-month-old infants fuss or cry and are highly aroused, mothers' multimodal singing is more effective than maternal speech at inducing recovery from such distress. Moreover, play songs are more effective than lullabies at reducing arousal in Western infants. We explore the implications of these findings along with possible practical applications. PMID:25773634

  15. Cortical Control of Affective Networks

    PubMed Central

    Kumar, Sunil; Black, Sherilynn J.; Hultman, Rainbo; Szabo, Steven T.; DeMaio, Kristine D.; Du, Jeanette; Katz, Brittany M.; Feng, Guoping; Covington, Herbert E.; Dzirasa, Kafui

    2013-01-01

    Transcranial magnetic stimulation and deep brain stimulation have emerged as therapeutic modalities for treatment refractory depression; however, little remains known regarding the circuitry that mediates the therapeutic effect of these approaches. Here we show that direct optogenetic stimulation of prefrontal cortex (PFC) descending projection neurons in mice engineered to express Chr2 in layer V pyramidal neurons (Thy1–Chr2 mice) models an antidepressant-like effect in mice subjected to a forced-swim test. Furthermore, we show that this PFC stimulation induces a long-lasting suppression of anxiety-like behavior (but not conditioned social avoidance) in socially stressed Thy1–Chr2 mice: an effect that is observed >10 d after the last stimulation. Finally, we use optogenetic stimulation and multicircuit recording techniques concurrently in Thy1–Chr2 mice to demonstrate that activation of cortical projection neurons entrains neural oscillatory activity and drives synchrony across limbic brain areas that regulate affect. Importantly, these neural oscillatory changes directly correlate with the temporally precise activation and suppression of limbic unit activity. Together, our findings show that the direct activation of cortical projection systems is sufficient to modulate activity across networks underlying affective regulation. They also suggest that optogenetic stimulation of cortical projection systems may serve as a viable therapeutic strategy for treating affective disorders. PMID:23325249

  16. The Affective Regulation of Cognitive Priming

    PubMed Central

    Storbeck, Justin; Clore, Gerald L.

    2008-01-01

    Semantic and affective priming are classic effects observed in cognitive and social psychology, respectively. We discovered that affect regulates such priming effects. In Experiment 1, positive and negative moods were induced prior to one of three priming tasks; evaluation, categorization, or lexical decision. As predicted, positive affect led to both affective priming (evaluation task) and semantic priming (category and lexical decision tasks). However, negative affect inhibited such effects. In Experiment 2, participants in their natural affective state completed the same priming tasks as in Experiment 1. As expected, affective priming (evaluation task) and category priming (categorization and lexical decision tasks) were observed in such resting affective states. Hence, we conclude that negative affect inhibits semantic and affective priming. These results support recent theoretical models, which suggest that positive affect promotes associations among strong and weak concepts, and that negative affect impairs such associations (Kuhl, 2000; Clore & Storbeck, 2006). PMID:18410195

  17. How commissioning affects community nursing.

    PubMed

    Cook, Jane; Horrocks, Susan; Gibbard, Emma; Harland, Lizanne; Wye, Lesley

    Community nurses have direct experience of how changes in the local health economy affect the quality of care patients receive, so it is important that they engage with commissioning to influence decisions made about the quality and direction of their service. This article seeks to demystify commissioning priorities by drawing on findings from a survey of Commissioning for Quality and Innovation indicators for community nursing conducted in England, 2014-15. The article focuses specifically on organisational goals, highlighting the impact of the Francis report and other NHS priorities on quality assessment in community nursing. PMID:26721091

  18. Does trade affect child health?

    PubMed

    Levine, David I; Rothman, Dov

    2006-05-01

    Frankel and Romer [Frankel, J., Romer, D., 1999. Does trade cause growth? American Economic Review 89 (3), 379-399] documented positive effects of geographically determined trade openness on economic growth. At the same time, critics fear that openness can lead to a "race to the bottom" that increases pollution and reduces government resources for investments in health and education. We use Frankel and Romer's gravity model of trade to examine how openness to trade affects children. Overall, we find little harm from trade, and potential benefits largely through slightly faster GDP growth. PMID:16303196

  19. Mood Swings: An Affective Interactive Art System

    NASA Astrophysics Data System (ADS)

    Bialoskorski, Leticia S. S.; Westerink, Joyce H. D. M.; van den Broek, Egon L.

    The progress in the field of affective computing enables the realization of affective consumer products, affective games, and affective art. This paper describes the affective interactive art system Mood Swings, which interprets and visualizes affect expressed by a person. Mood Swings is founded on the integration of a framework for affective movements and a color model. This enables Mood Swings to recognize affective movement characteristics as expressed by a person and display a color that matches the expressed emotion. With that, a unique interactive system is introduced, which can be considered as art, a game, or a combination of both.

  20. LITHIUM PROPHYLAXIS IN AFFECTIVE DISORDER

    PubMed Central

    Rao, A. Venkoba; Hariharasubramanian, N.; Devi, S. Parvathi; Sugumar, A.; Srinivasan, V.

    1982-01-01

    SUMMARY Out of 108 patients on the rolls in the Lithium clinic, Madurai Medical College and Govt. Rajaji Hospital, Madurai, India, 47 patients suffering from affective disorders receiving lithium continuously for more than three years were analysed with a view to study the recurrences. Thirteen suffered no relapses while on lithium while nineteen experienced them while on lithium. Four were free from recurrences after lithium was withdrawn- Seven defaulted but suffered recurrences while in four the drug was withdrawn and in both the groups remission was achieved with re-administration of lithium. The study reveals that lithium besides averting the recurrences can reduce the frequency, number, duration, intensity of episodes and improve the amenability to drugs. Among the symptoms, suicidal ideas and behaviour and insight were found to be influenced favourably by lithium. Among the factors that help favourable response to lithium were a positive family history of affective disorder, in the first degree relatives and lesser frequency and number of episodes in the pre-lithium period. A reappraisal of the natural history of the illness is called for in the light of lithium prophylaxis of manic depressive psychosis. PMID:21965880

  1. How decision reversibility affects motivation.

    PubMed

    Bullens, Lottie; van Harreveld, Frenk; Förster, Jens; Higgins, Tory E

    2014-04-01

    The present research examined how decision reversibility can affect motivation. On the basis of extant findings, it was suggested that 1 way it could affect motivation would be to strengthen different regulatory foci, with reversible decision making, compared to irreversible decision making, strengthening prevention-related motivation relatively more than promotion-related motivation. If so, then decision reversibility should have effects associated with the relative differences between prevention and promotion motivation. In 5 studies, we manipulated the reversibility of a decision and used different indicators of regulatory focus motivation to test these predictions. Specifically, Study 1 tested for differences in participants' preference for approach versus avoidance strategies toward a desired end state. In Study 2, we used speed and accuracy performance as indicators of participants' regulatory motivation, and in Study 3, we measured global versus local reaction time performance. In Study 4, we approached the research question in a different way, making use of the value-from-fit hypothesis (Higgins, 2000, 2002). We tested whether a fit between chronic regulatory focus and focus induced by the reversibility of the decision increased participants' subjective positive feelings about the decision outcome. Finally, in Study 5, we tested whether regulatory motivation, induced by decision reversibility, also influenced participants' preference in specific product features. The results generally support our hypothesis showing that, compared to irreversible decisions, reversible decisions strengthen a prevention focus more than a promotion focus. Implications for research on decision making are discussed. PMID:23815456

  2. Tardive dyskinesia in affective disorders.

    PubMed

    Kane, J M

    1999-01-01

    Soon after the introduction of antipsychotic drugs into clinical practice, these agents were observed to be capable of producing not only acute extrapyramidal ("parkinsonian") side effects, but also later occurring abnormal involuntary movements that came to be called tardive dyskinesia. Since antipsychotic drugs are used in a variety of conditions that include psychotic features, studies have attempted to determine whether specific diagnostic subgroups may experience different degrees of vulnerability to drug-induced movement disorders. This issue is important not only to inform clinical practice, but also to provide clues to pathophysiology. A number of studies suggest that patients with affective disorders are at greater risk for developing tardive dyskinesia (controlling, to the extent possible, for other relevant variables such as age, sex, length of treatment). Encouraging preliminary data with new antipsychotic drugs such as olanzapine suggest that the risk of tardive dyskinesia associated with long-term antipsychotic drug use may be substantially reduced. This would go a long way toward improving the benefit-to-risk ratio of antipsychotic drug treatment, particularly in patients with affective disorders. PMID:10192407

  3. Factors Affecting Medical Service Quality

    PubMed Central

    MOSADEGHRAD, Ali Mohammad

    2014-01-01

    Abstract Background A better understanding of factors influencing quality of medical service can pinpoint better strategies for quality assurance in medical services. This study aimed to identify factors affecting the quality of medical services provided by Iranian physicians. Methods Exploratory in-depth individual interviews were conducted with sixty-four physicians working in various medical institutions in Iran. Results Individual, organizational and environmental factors enhance or inhibit the quality of medical services. Quality of medical services depends on the personal factors of the physician and patient, and factors pertaining to the healthcare setting and the broader environment. Conclusion Differences in internal and external factors such as availability of resources, patient cooperation and collaboration among providers affect the quality of medical services and patient outcomes. Supportive leadership, proper planning, education and training and effective management of resources and processes improve the quality of medical services. This article contributes to healthcare theory and practice by developing a conceptual framework for understanding factors that influence medical services quality. PMID:26060745

  4. How anthropogenic noise affects foraging.

    PubMed

    Luo, Jinhong; Siemers, Björn M; Koselj, Klemen

    2015-09-01

    The influence of human activity on the biosphere is increasing. While direct damage (e.g. habitat destruction) is relatively well understood, many activities affect wildlife in less apparent ways. Here, we investigate how anthropogenic noise impairs foraging, which has direct consequences for animal survival and reproductive success. Noise can disturb foraging via several mechanisms that may operate simultaneously, and thus, their effects could not be disentangled hitherto. We developed a diagnostic framework that can be applied to identify the potential mechanisms of disturbance in any species capable of detecting the noise. We tested this framework using Daubenton's bats, which find prey by echolocation. We found that traffic noise reduced foraging efficiency in most bats. Unexpectedly, this effect was present even if the playback noise did not overlap in frequency with the prey echoes. Neither overlapping noise nor nonoverlapping noise influenced the search effort required for a successful prey capture. Hence, noise did not mask prey echoes or reduce the attention of bats. Instead, noise acted as an aversive stimulus that caused avoidance response, thereby reducing foraging efficiency. We conclude that conservation policies may seriously underestimate numbers of species affected and the multilevel effects on animal fitness, if the mechanisms of disturbance are not considered. PMID:26046451

  5. Bipolar Affective Disorder and Migraine

    PubMed Central

    Engmann, Birk

    2012-01-01

    This paper consists of a case history and an overview of the relationship, aetiology, and treatment of comorbid bipolar disorder migraine patients. A MEDLINE literature search was used. Terms for the search were bipolar disorder bipolar depression, mania, migraine, mood stabilizer. Bipolar disorder and migraine cooccur at a relatively high rate. Bipolar II patients seem to have a higher risk of comorbid migraine than bipolar I patients have. The literature on the common roots of migraine and bipolar disorder, including both genetic and neuropathological approaches, is broadly discussed. Moreover, bipolar disorder and migraine are often combined with a variety of other affective disorders, and, furthermore, behavioural factors also play a role in the origin and course of the diseases. Approach to treatment options is also difficult. Several papers point out possible remedies, for example, valproate, topiramate, which acts on both diseases, but no first-choice treatments have been agreed upon yet. PMID:22649454

  6. How competition affects evolutionary rescue

    PubMed Central

    Osmond, Matthew Miles; de Mazancourt, Claire

    2013-01-01

    Populations facing novel environments can persist by adapting. In nature, the ability to adapt and persist will depend on interactions between coexisting individuals. Here we use an adaptive dynamic model to assess how the potential for evolutionary rescue is affected by intra- and interspecific competition. Intraspecific competition (negative density-dependence) lowers abundance, which decreases the supply rate of beneficial mutations, hindering evolutionary rescue. On the other hand, interspecific competition can aid evolutionary rescue when it speeds adaptation by increasing the strength of selection. Our results clarify this point and give an additional requirement: competition must increase selection pressure enough to overcome the negative effect of reduced abundance. We therefore expect evolutionary rescue to be most likely in communities which facilitate rapid niche displacement. Our model, which aligns to previous quantitative and population genetic models in the absence of competition, provides a first analysis of when competitors should help or hinder evolutionary rescue. PMID:23209167

  7. Nutritional Factors Affecting Mental Health.

    PubMed

    Lim, So Young; Kim, Eun Jin; Kim, Arang; Lee, Hee Jae; Choi, Hyun Jin; Yang, Soo Jin

    2016-07-01

    Dietary intake and nutritional status of individuals are important factors affecting mental health and the development of psychiatric disorders. Majority of scientific evidence relating to mental health focuses on depression, cognitive function, and dementia, and limited evidence is available about other psychiatric disorders including schizophrenia. As life span of human being is increasing, the more the prevalence of mental disorders is, the more attention rises. Lists of suggested nutritional components that may be beneficial for mental health are omega-3 fatty acids, phospholipids, cholesterol, niacin, folate, vitamin B6, and vitamin B12. Saturated fat and simple sugar are considered detrimental to cognitive function. Evidence on the effect of cholesterol is conflicting; however, in general, blood cholesterol levels are negatively associated with the risk of depression. Collectively, the aims of this review are to introduce known nutritional factors for mental health, and to discuss recent issues of the nutritional impact on cognitive function and healthy brain aging. PMID:27482518

  8. Factors affecting gallbladder motility: drugs.

    PubMed

    Marzio, L

    2003-07-01

    Various drugs and medications that inhibit or stimulate gallbladder contraction and basal tone in humans are described. Active gallbladder contraction may be achieved using synthetic hormones such as cholecystokinin, caerulein and motilin, cholinomimetic drugs such as bethanecol, prostigmine, and erythromycin due to its motilin-like effect. Furthermore, cisapride and cholestyramine, may have some excitatory activity on the gallbladder muscle. Intravenous amino acids also induce gallbladder contraction through the release of cholecystokinin. Inhibition of gallbladder contraction induced by a meal, or reduction of the basal fasting tone may be achieved by using atropine and other cholinergics, and by inhibitory hormones such as somatostatin, the nitric acid releaser arginine, the calcium channel antagonist nifedipine, and progesterone. Other drugs such as trimebutine, loperamide and ondansetron may negatively affect gallbladder contraction. PMID:12974504

  9. Brain lesions affect penile reflexes.

    PubMed

    Monaghan, E P; Arjomand, J; Breedlove, S M

    1993-03-01

    Electrolytic lesions of several potential brain afferents to the spinal nucleus of the bulbocavernosus (SNB) affect the display of penile reflexes. Ablation of the median and pontine raphe areas significantly potentiates the expression of cups and flips. Animals with a bilateral lesion of the paraventricular nucleus of the hypothalamus have a shorter latency to the first erection but otherwise display normal reflex behavior. Although bilateral destruction of the lateral vestibular nucleus (LVN) completely eliminated penile reflex activity, it also caused significant motor impairment thus clouding conclusions concerning the normal role of the LVN in penile reflex behavior. These and other results support the hypothesis that these brain regions which project to the SNB region normally modulate spinal reflex behavior of the rat penis. PMID:8440513

  10. Nutritional Factors Affecting Mental Health

    PubMed Central

    Lim, So Young; Kim, Eun Jin; Kim, Arang; Lee, Hee Jae; Choi, Hyun Jin

    2016-01-01

    Dietary intake and nutritional status of individuals are important factors affecting mental health and the development of psychiatric disorders. Majority of scientific evidence relating to mental health focuses on depression, cognitive function, and dementia, and limited evidence is available about other psychiatric disorders including schizophrenia. As life span of human being is increasing, the more the prevalence of mental disorders is, the more attention rises. Lists of suggested nutritional components that may be beneficial for mental health are omega-3 fatty acids, phospholipids, cholesterol, niacin, folate, vitamin B6, and vitamin B12. Saturated fat and simple sugar are considered detrimental to cognitive function. Evidence on the effect of cholesterol is conflicting; however, in general, blood cholesterol levels are negatively associated with the risk of depression. Collectively, the aims of this review are to introduce known nutritional factors for mental health, and to discuss recent issues of the nutritional impact on cognitive function and healthy brain aging. PMID:27482518

  11. Does health affect portfolio choice?

    PubMed

    Love, David A; Smith, Paul A

    2010-12-01

    A number of recent studies find that poor health is empirically associated with a safer portfolio allocation. It is difficult to say, however, whether this relationship is truly causal. Both health status and portfolio choice are influenced by unobserved characteristics such as risk attitudes, impatience, information, and motivation, and these unobserved factors, if not adequately controlled for, can induce significant bias in the estimates of asset demand equations. Using the 1992-2006 waves of the Health and Retirement Study, we investigate how much of the connection between health and portfolio choice is causal and how much is due to the effects of unobserved heterogeneity. Accounting for unobserved heterogeneity with fixed effects and correlated random effects models, we find that health does not appear to significantly affect portfolio choice among single households. For married households, we find a small effect (about 2-3 percentage points) from being in the lowest of five self-reported health categories. PMID:19937612

  12. Factors affecting rotator cuff healing.

    PubMed

    Mall, Nathan A; Tanaka, Miho J; Choi, Luke S; Paletta, George A

    2014-05-01

    Several studies have noted that increasing age is a significant factor for diminished rotator cuff healing, while biomechanical studies have suggested the reason for this may be an inferior healing environment in older patients. Larger tears and fatty infiltration or atrophy negatively affect rotator cuff healing. Arthroscopic rotator cuff repair, double-row repairs, performing a concomitant acromioplasty, and the use of platelet-rich plasma (PRP) do not demonstrate an improvement in structural healing over mini-open rotator cuff repairs, single-row repairs, not performing an acromioplasty, or not using PRP. There is conflicting evidence to support postoperative rehabilitation protocols using early motion over immobilization following rotator cuff repair. PMID:24806015

  13. Interplanetary Disturbances Affecting Space Weather

    NASA Astrophysics Data System (ADS)

    Wimmer-Schweingruber, Robert F.

    2014-01-01

    The Sun somehow accelerates the solar wind, an incessant stream of plasma originating in coronal holes and some, as yet unidentified, regions. Occasionally, coronal, and possibly sub-photospheric structures, conspire to energize a spectacular eruption from the Sun which we call a coronal mass ejection (CME). These can leave the Sun at very high speeds and travel through the interplanetary medium, resulting in a large-scale disturbance of the ambient background plasma. These interplanetary CMEs (ICMEs) can drive shocks which in turn accelerate particles, but also have a distinct intrinsic magnetic structure which is capable of disturbing the Earth's magnetic field and causing significant geomagnetic effects. They also affect other planets, so they can and do contribute to space weather throughout the heliosphere. This paper presents a historical review of early space weather studies, a modern-day example, and discusses space weather throughout the heliosphere.

  14. Affective Dimensions of Intergroup Humiliation

    PubMed Central

    Leidner, Bernhard; Sheikh, Hammad; Ginges, Jeremy

    2012-01-01

    Despite the wealth of theoretical claims about the emotion of humiliation and its effect on human relations, there has been a lack of empirical research investigating what it means to experience humiliation. We studied the affective characteristics of humiliation, comparing the emotional experience of intergroup humiliation to two other emotions humiliation is often confused with: anger and shame. The defining characteristics of humiliation were low levels of guilt and high levels of other-directed outrage (like anger and unlike shame), and high levels of powerlessness (like shame and unlike anger). Reasons for the similarities and differences of humiliation with anger and shame are discussed in terms of perceptions of undeserved treatment and injustice. Implications for understanding the behavioral consequences of humiliation and future work investigating the role of humiliation in social life are discussed. PMID:23029499

  15. How Attention Affects Spatial Resolution

    PubMed Central

    Carrasco, Marisa; Barbot, Antoine

    2015-01-01

    We summarize and discuss a series of psychophysical studies on the effects of spatial covert attention on spatial resolution, our ability to discriminate fine patterns. Heightened resolution is beneficial in most, but not all, visual tasks. We show how endogenous attention (voluntary, goal driven) and exogenous attention (involuntary, stimulus driven) affect performance on a variety of tasks mediated by spatial resolution, such as visual search, crowding, acuity, and texture segmentation. Exogenous attention is an automatic mechanism that increases resolution regardless of whether it helps or hinders performance. In contrast, endogenous attention flexibly adjusts resolution to optimize performance according to task demands. We illustrate how psychophysical studies can reveal the underlying mechanisms of these effects and allow us to draw linking hypotheses with known neurophysiological effects of attention. PMID:25948640

  16. Rheumatoid arthritis affecting temporomandibular joint

    PubMed Central

    Sodhi, Amandeep; Naik, Shobha; Pai, Anuradha; Anuradha, Ardra

    2015-01-01

    Rheumatoid arthritis (RA) is a chronic, systemic, autoimmune inflammatory disorder that is characterized by joint inflammation, erosive properties and symmetric multiple joint involvement. Temporomandibular joint (TMJ) is very rare to be affected in the early phase of the disease, thus posing diagnostic challenges for the dentist. Conventional radiographs fail to show the early lesions due to its limitations. More recently cone-beam computed tomography (CBCT) has been found to diagnose the early degenerative changes of TMJ and hence aid in the diagnosis of the lesions more accurately. Our case highlights the involvement of TMJ in RA and the role of advanced imaging (CBCT) in diagnosing the bony changes in the early phase of the disease. PMID:25684928

  17. The affective shift model of work engagement.

    PubMed

    Bledow, Ronald; Schmitt, Antje; Frese, Michael; Kühnel, Jana

    2011-11-01

    On the basis of self-regulation theories, the authors develop an affective shift model of work engagement according to which work engagement emerges from the dynamic interplay of positive and negative affect. The affective shift model posits that negative affect is positively related to work engagement if negative affect is followed by positive affect. The authors applied experience sampling methodology to test the model. Data on affective events, mood, and work engagement was collected twice a day over 9 working days among 55 software developers. In support of the affective shift model, negative mood and negative events experienced in the morning of a working day were positively related to work engagement in the afternoon if positive mood in the time interval between morning and afternoon was high. Individual differences in positive affectivity moderated within-person relationships. The authors discuss how work engagement can be fostered through affect regulation. PMID:21766997

  18. Ash in fire affected ecosystems

    NASA Astrophysics Data System (ADS)

    Pereira, Paulo; Jordan, Antonio; Cerda, Artemi; Martin, Deborah

    2015-04-01

    Ash in fire affected ecosystems Ash lefts an important footprint in the ecosystems and has a key role in the immediate period after the fire (Bodi et al., 2014; Pereira et al., 2015). It is an important source of nutrients for plant recover (Pereira et al., 2014a), protects soil from erosion and controls soil hydrological process as runoff, infiltration and water repellency (Cerda and Doerr, 2008; Bodi et al., 2012, Pereira et al., 2014b). Despite the recognition of ash impact and contribution to ecosystems recuperation, it is assumed that we still have little knowledge about the implications of ash in fire affected areas. Regarding this situation we wanted to improve our knowledge in this field and understand the state of the research about fire ash around world. The special issue about "The role of ash in fire affected ecosystems" currently in publication in CATENA born from the necessity of joint efforts, identify research gaps, and discuss future cooperation in this interdisciplinary field. This is the first special issue about fire ash in the international literature. In total it will be published 10 papers focused in different aspects of the impacts of ash in fire affected ecosystems from several parts of the world: • Fire reconstruction using charcoal particles (Burjachs and Espositio, in press) • Ash slurries impact on rheological properties of Runoff (Burns and Gabet, in press) • Methods to analyse ash conductivity and sorbtivity in the laboratory and in the field (Balfour et al., in press) • Termogravimetric and hydrological properties of ash (Dlapa et al. in press) • Effects of ash cover in water infiltration (Leon et al., in press) • Impact of ash in volcanic soils (Dorta Almenar et al., in press; Escuday et al., in press) • Ash PAH and Chemical extracts (Silva et al., in press) • Microbiology (Barreiro et al., in press; Lombao et al., in press) We believe that this special issue will contribute importantly to the better understanding of

  19. Ash in fire affected ecosystems

    NASA Astrophysics Data System (ADS)

    Pereira, Paulo; Jordan, Antonio; Cerda, Artemi; Martin, Deborah

    2015-04-01

    Ash in fire affected ecosystems Ash lefts an important footprint in the ecosystems and has a key role in the immediate period after the fire (Bodi et al., 2014; Pereira et al., 2015). It is an important source of nutrients for plant recover (Pereira et al., 2014a), protects soil from erosion and controls soil hydrological process as runoff, infiltration and water repellency (Cerda and Doerr, 2008; Bodi et al., 2012, Pereira et al., 2014b). Despite the recognition of ash impact and contribution to ecosystems recuperation, it is assumed that we still have little knowledge about the implications of ash in fire affected areas. Regarding this situation we wanted to improve our knowledge in this field and understand the state of the research about fire ash around world. The special issue about "The role of ash in fire affected ecosystems" currently in publication in CATENA born from the necessity of joint efforts, identify research gaps, and discuss future cooperation in this interdisciplinary field. This is the first special issue about fire ash in the international literature. In total it will be published 10 papers focused in different aspects of the impacts of ash in fire affected ecosystems from several parts of the world: • Fire reconstruction using charcoal particles (Burjachs and Espositio, in press) • Ash slurries impact on rheological properties of Runoff (Burns and Gabet, in press) • Methods to analyse ash conductivity and sorbtivity in the laboratory and in the field (Balfour et al., in press) • Termogravimetric and hydrological properties of ash (Dlapa et al. in press) • Effects of ash cover in water infiltration (Leon et al., in press) • Impact of ash in volcanic soils (Dorta Almenar et al., in press; Escuday et al., in press) • Ash PAH and Chemical extracts (Silva et al., in press) • Microbiology (Barreiro et al., in press; Lombao et al., in press) We believe that this special issue will contribute importantly to the better understanding of

  20. Clinorotation affects soybean seedling morphology

    NASA Technical Reports Server (NTRS)

    Hilaire, Emmanuel; Guikema, James A.; Brown, Christopher S.

    1995-01-01

    Although spaceflight does not appear to significantly affect seed germination, it can influence subsequent plant growth. On STS-3 and SL-2, decreased growth (measured as plant length, fresh weight, and dry weight) was noted for pine, oat, and mung bean. In the CHROMEX-01 and 02 experiments with Haplopappus and in the CHROMEX-03 experiment with Arabidopsis, enhanced root growth was noted in the space-grown plants. In the CHROMEX-04 experiments with wheat, both leaf fresh weight and leaf area were diminished in the space-grown plants but there was no difference in total plant height (CS Brown, HG Levine, and AD Krikorian, unpublished data). These data suggest that microgravity impacts growth by whole plant partitioning of the assimilates. The objective of the present study was to determine the influence of clinorotation on the growth and the morphology of soybean seedlings grown in the Biological Research In Canister (BRIC) flight hardware. This experiment provided baseline data for a spaceflight experiment (BRIC-3) flown on STS-63 (February 3-11, 1995).

  1. Quantum Tunneling Affects Engine Performance.

    PubMed

    Som, Sibendu; Liu, Wei; Zhou, Dingyu D Y; Magnotti, Gina M; Sivaramakrishnan, Raghu; Longman, Douglas E; Skodje, Rex T; Davis, Michael J

    2013-06-20

    We study the role of individual reaction rates on engine performance, with an emphasis on the contribution of quantum tunneling. It is demonstrated that the effect of quantum tunneling corrections for the reaction HO2 + HO2 = H2O2 + O2 can have a noticeable impact on the performance of a high-fidelity model of a compression-ignition (e.g., diesel) engine, and that an accurate prediction of ignition delay time for the engine model requires an accurate estimation of the tunneling correction for this reaction. The three-dimensional model includes detailed descriptions of the chemistry of a surrogate for a biodiesel fuel, as well as all the features of the engine, such as the liquid fuel spray and turbulence. This study is part of a larger investigation of how the features of the dynamics and potential energy surfaces of key reactions, as well as their reaction rate uncertainties, affect engine performance, and results in these directions are also presented here. PMID:26283246

  2. Spatial layout affects speed discrimination

    NASA Technical Reports Server (NTRS)

    Verghese, P.; Stone, L. S.

    1997-01-01

    We address a surprising result in a previous study of speed discrimination with multiple moving gratings: discrimination thresholds decreased when the number of stimuli was increased, but remained unchanged when the area of a single stimulus was increased [Verghese & Stone (1995). Vision Research, 35, 2811-2823]. In this study, we manipulated the spatial- and phase relationship between multiple grating patches to determine their effect on speed discrimination thresholds. In a fusion experiment, we merged multiple stimulus patches, in stages, into a single patch. Thresholds increased as the patches were brought closer and their phase relationship was adjusted to be consistent with a single patch. Thresholds increased further still as these patches were fused into a single patch. In a fission experiment, we divided a single large patch into multiple patches by superimposing a cross with luminance equal to that of the background. Thresholds decreased as the large patch was divided into quadrants and decreased further as the quadrants were maximally separated. However, when the cross luminance was darker than the background, it was perceived as an occluder and thresholds, on average, were unchanged from that for the single large patch. A control experiment shows that the observed trend in discrimination thresholds is not due to the differences in perceived speed of the stimuli. These results suggest that the parsing of the visual image into entities affects the combination of speed information across space, and that each discrete entity effectively provides a single independent estimate of speed.

  3. Focus cues affect perceived depth

    PubMed Central

    Watt, Simon J.; Akeley, Kurt; Ernst, Marc O.; Banks, Martin S.

    2007-01-01

    Depth information from focus cues—accommodation and the gradient of retinal blur—is typically incorrect in three-dimensional (3-D) displays because the light comes from a planar display surface. If the visual system incorporates information from focus cues into its calculation of 3-D scene parameters, this could cause distortions in perceived depth even when the 2-D retinal images are geometrically correct. In Experiment 1 we measured the direct contribution of focus cues to perceived slant by varying independently the physical slant of the display surface and the slant of a simulated surface specified by binocular disparity (binocular viewing) or perspective/texture (monocular viewing). In the binocular condition, slant estimates were unaffected by display slant. In the monocular condition, display slant had a systematic effect on slant estimates. Estimates were consistent with a weighted average of slant from focus cues and slant from disparity/texture, where the cue weights are determined by the reliability of each cue. In Experiment 2, we examined whether focus cues also have an indirect effect on perceived slant via the distance estimate used in disparity scaling. We varied independently the simulated distance and the focal distance to a disparity-defined 3-D stimulus. Perceived slant was systematically affected by changes in focal distance. Accordingly, depth constancy (with respect to simulated distance) was significantly reduced when focal distance was held constant compared to when it varied appropriately with the simulated distance to the stimulus. The results of both experiments show that focus cues can contribute to estimates of 3-D scene parameters. Inappropriate focus cues in typical 3-D displays may therefore contribute to distortions in perceived space. PMID:16441189

  4. [Harmful practices affecting women's health].

    PubMed

    1990-07-01

    The harmful practices discussed in this article are based on case histories form the Central Maternity in Niamey, yet these practices universally affect women throughout Africa. Nutritional taboos are aimed at certain diseases such as measles, diarrhea, dysentery, malnutrition and anemia and consumption of foods rich in proteins and lipids are forbidden. Children are forbidden from eating eggs; pregnant women are forbidden from eating fruits and vegetables because of the fear of hemorrhaging from the sugar content in the fruit; camel meat is forbidden for fear of extending the pregnancy. Female circumcision, a dangerous practice, especially during childbirth, causes many medical problems that remain permanent. Adolescent pregnancy and marriages are practiced to avoid delinquency among children; yet such practices take place because of arranged marriages for a dowry to young men or to older rich men and these forced marriages to adolescents are the causes of increases in divorce, prostitution and desertion. These young marriages have serious consequences on the health status of the mother and the infant, often leading to maternal and infant death. The high level of fertility in Niger is a response to the social structure of the family. It is a patrilineal system that encourages women to have many children, especially sons. In Niger, pregnancy is surrounded by supernatural and mysterious forces, where a child is the intervention for ancestral spirits. In Islam a child is considered a "Gift of God". A woman is expected to work until the delivery of her baby otherwise she is jeered by her neighbors. During delivery women are not expected to cry or show any pain for fear of dishonoring her family irregardless of any medical compilations she faces. Women in Africa are exploited as free labor, deteriorate and age rapidly, are generally illiterate and are not protected under any laws. PMID:12342832

  5. A Multimodal Theory of Affect Diffusion.

    PubMed

    Peters, Kim; Kashima, Yoshihisa

    2015-09-01

    There is broad consensus in the literature that affect diffuses through social networks (such that a person may "acquire" or "catch" an affective state from his or her social contacts). It is further assumed that affect diffusion primarily occurs as the result of people's tendencies to synchronize their affective actions (such as smiles and frowns). However, as we show, there is a lack of clarity in the literature about the substrate and scope of affect diffusion. One consequence of this is a difficulty in distinguishing between affect diffusion and several other affective influence phenomena that look similar but have very different consequences. There is also a growing body of evidence that action synchrony is unlikely to be the only, or indeed the most important, pathway for affect diffusion. This paper has 2 key aims: (a) to craft a formal definition of affect diffusion that does justice to the core of the phenomenon while distinguishing it from other phenomena with which it is frequently confounded and (b) to advance a theory of the mechanisms of affect diffusion. This theory, which we call the multimodal theory of affect diffusion, identifies 3 parallel multimodal mechanisms that may act as routes for affect diffusion. It also provides a basis for novel predictions about the conditions under which affect is most likely to diffuse. PMID:26011791

  6. HIV Infection Seems to Affect Nervous System

    MedlinePlus

    ... fullstory_159344.html HIV Infection Seems to Affect Nervous System But symptoms tend to subside once antiretroviral drugs ... mild, it is clear that HIV affects the nervous system within days of infection," she said in a ...

  7. Factors Affecting Aerosol Radiative Forcing

    NASA Astrophysics Data System (ADS)

    Wang, Jingxu; Lin, Jintai; Ni, Ruijing

    2016-04-01

    Rapid industrial and economic growth has meant a large amount of aerosols in the atmosphere with strong radiative forcing (RF) upon the climate system. Over parts of the globe, the negative forcing of aerosols has overcompensated for the positive forcing of greenhouse gases. Aerosol RF is determined by emissions and various chemical-transport-radiative processes in the atmosphere, a multi-factor problem whose individual contributors have not been well quantified. In this study, we analyze the major factors affecting RF of secondary inorganic aerosols (SIOAs, including sulfate, nitrate and ammonium), primary organic aerosol (POA), and black carbon (BC). We analyze the RF of aerosols produced by 11 major regions across the globe, including but not limited to East Asia, Southeast Asia, South Asia, North America, and Western Europe. Factors analyzed include population size, per capita gross domestic production (GDP), emission intensity (i.e., emissions per unit GDP), chemical efficiency (i.e., mass per unit emissions) and radiative efficiency (i.e., RF per unit mass). We find that among the 11 regions, East Asia produces the largest emissions and aerosol RF, due to relatively high emission intensity and a tremendous population size. South Asia produce the second largest RF of SIOA and BC and the highest RF of POA, in part due to its highest chemical efficiency among all regions. Although Southeast Asia also has large emissions, its aerosol RF is alleviated by its lowest chemical efficiency. The chemical efficiency and radiative efficiency of BC produced by the Middle East-North Africa are the highest across the regions, whereas its RF is lowered by a small per capita GDP. Both North America and Western Europe have low emission intensity, compensating for the effects on RF of large population sizes and per capita GDP. There has been a momentum to transfer industries to Southeast Asia and South Asia, and such transition is expected to continue in the coming years. The

  8. Demographics, Affect, and Adolescents' Health Behaviors.

    ERIC Educational Resources Information Center

    Terre, Lisa; And Others

    1992-01-01

    Examined relationship between affect, demographics, and health-related lifestyle among 139 public high school students. Data analyses revealed distinctive demographic and affective correlates of different health behaviors. No one variable uniformly predicted adolescents' health behaviors. Demographics and affect showed differential relationships…

  9. Affective Priming with Associatively Acquired Valence

    ERIC Educational Resources Information Center

    Aguado, Luis; Pierna, Manuel; Saugar, Cristina

    2005-01-01

    Three experiments explored the effect of affectively congruent or incongruent primes on evaluation responses to positive or negative valenced targets (the "affective priming" effect). Experiment 1 replicated the basic affective priming effect with Spanish nouns: reaction time for evaluative responses (pleasant/unpleasant) were slower on…

  10. The Affective Experiences of Children during Mathematics.

    ERIC Educational Resources Information Center

    Prawat, Richard S.; Anderson, Ariel L. H.

    1994-01-01

    Stimulated recall was used to examine the affective experiences of (n=32) fourth and fifth graders engaged in mathematics seatwork. Students' affect was found to be primarily negative and achievement related. Anger was the most prevalent affective response. (42 references) (MKR)

  11. Empirical Testing of an Affective Learning Paradigm.

    ERIC Educational Resources Information Center

    Asmus, Edward P., Jr.

    1980-01-01

    This investigation of a college music course examined the effectiveness of a cyclical affective learning paradigm based on the premise that student affect toward a course of instruction will dictate, in part, cognitive performance. Results suggest that teachers would be better advised to concentrate on cognitive instruction than on affect.…

  12. Trait Affectivity and Nonreferred Adolescent Conduct Problems

    ERIC Educational Resources Information Center

    Loney, Bryan R.; Lima, Elizabeth N.; Butler, Melanie A.

    2006-01-01

    This study examined for profiles of positive trait affectivity (PA) and negative trait affectivity (NA) associated with adolescent conduct problems. Prior trait affectivity research has been relatively biased toward the assessment of adults and internalizing symptomatology. Consistent with recent developmental modeling of antisocial behavior, this…

  13. An Affect Control Theory of Technology

    ERIC Educational Resources Information Center

    Shank, Daniel B.

    2010-01-01

    Affect control theory is a theory of interaction that takes into account cultural meanings. Affect control research has previously considered interaction with technology, but there remains a lack of theorizing about inclusion of technology within the theory. This paper lays a foundation for an affect control theory of technology by addressing key…

  14. Dynamic Artificial Neural Networks with Affective Systems

    PubMed Central

    Schuman, Catherine D.; Birdwell, J. Douglas

    2013-01-01

    Artificial neural networks (ANNs) are processors that are trained to perform particular tasks. We couple a computational ANN with a simulated affective system in order to explore the interaction between the two. In particular, we design a simple affective system that adjusts the threshold values in the neurons of our ANN. The aim of this paper is to demonstrate that this simple affective system can control the firing rate of the ensemble of neurons in the ANN, as well as to explore the coupling between the affective system and the processes of long term potentiation (LTP) and long term depression (LTD), and the effect of the parameters of the affective system on its performance. We apply our networks with affective systems to a simple pole balancing example and briefly discuss the effect of affective systems on network performance. PMID:24303015

  15. Production of recombinant porcine IGF-binding protein-5 and its effect on proliferation of porcine embryonic myoblast cultures in the presence and absence of IGF-I and Long-R3-IGF-I.

    PubMed

    Pampusch, M S; Xi, G; Kamanga-Sollo, E; Loseth, K J; Hathaway, M R; Dayton, W R; White, M E

    2005-04-01

    investigate the role of IGFBP-5 in porcine myogenic cell proliferation and differentiation. The data provided here demonstrated that IGFBP-5 has the potential to affect proliferation of PEMCs during critical periods of in vitro muscle cell development and therefore may impact the capacity for ultimate postnatal muscle mass development in vivo. PMID:15817840

  16. Emotional task management: neural correlates of switching between affective and non-affective task-sets

    PubMed Central

    Reeck, Crystal

    2015-01-01

    Although task-switching has been investigated extensively, its interaction with emotionally salient task content remains unclear. Prioritized processing of affective stimulus content may enhance accessibility of affective task-sets and generate increased interference when switching between affective and non-affective task-sets. Previous research has demonstrated that more dominant task-sets experience greater switch costs, as they necessitate active inhibition during performance of less entrenched tasks. Extending this logic to the affective domain, the present experiment examined (a) whether affective task-sets are more dominant than non-affective ones, and (b) what neural mechanisms regulate affective task-sets, so that weaker, non-affective task-sets can be executed. While undergoing functional magnetic resonance imaging, participants categorized face stimuli according to either their gender (non-affective task) or their emotional expression (affective task). Behavioral results were consistent with the affective task dominance hypothesis: participants were slower to switch to the affective task, and cross-task interference was strongest when participants tried to switch from the affective to the non-affective task. These behavioral costs of controlling the affective task-set were mirrored in the activation of a right-lateralized frontostriatal network previously implicated in task-set updating and response inhibition. Connectivity between amygdala and right ventrolateral prefrontal cortex was especially pronounced during cross-task interference from affective features. PMID:25552571

  17. Audio-visual affective expression recognition

    NASA Astrophysics Data System (ADS)

    Huang, Thomas S.; Zeng, Zhihong

    2007-11-01

    Automatic affective expression recognition has attracted more and more attention of researchers from different disciplines, which will significantly contribute to a new paradigm for human computer interaction (affect-sensitive interfaces, socially intelligent environments) and advance the research in the affect-related fields including psychology, psychiatry, and education. Multimodal information integration is a process that enables human to assess affective states robustly and flexibly. In order to understand the richness and subtleness of human emotion behavior, the computer should be able to integrate information from multiple sensors. We introduce in this paper our efforts toward machine understanding of audio-visual affective behavior, based on both deliberate and spontaneous displays. Some promising methods are presented to integrate information from both audio and visual modalities. Our experiments show the advantage of audio-visual fusion in affective expression recognition over audio-only or visual-only approaches.

  18. Stronger suboptimal than optimal affective priming?

    PubMed

    Rotteveel, M; de Groot, P; Geutskens, A; Phaf, R H

    2001-12-01

    The finding of stronger affective priming in less conscious (suboptimal) conditions than in fully conscious (optimal) conditions (S. T. Murphy & R. B. Zajonc, 1993) is theoretically important because it contradicts notions that emotions are primarily reflected by conscious states. In 2 experiments, this pattern of results was obtained. Happy and angry faces were presented both optimally and suboptimally and were masked by unknown ideographs. In Experiment 1, instructions for the conscious and less conscious affective priming conditions were matched, and affective ratings of ideographs were determined. In Experiment 2, a more implicit affective measure (facial electromyography of musculus zygomaticus major and musculus corrugator supercilii) served as the dependent variable. Stronger suboptimal than optimal affective priming was found in both experiments. It is concluded that stronger suboptimal than optimal processing is characteristic for affective processing and that it can also be found when instructions are matched and when a more implicit measure is assessed. PMID:12901397

  19. Reliability Generalization: An Examination of the Positive Affect and Negative Affect Schedule

    ERIC Educational Resources Information Center

    Leue, Anja; Lange, Sebastian

    2011-01-01

    The assessment of positive affect (PA) and negative affect (NA) by means of the Positive Affect and Negative Affect Schedule has received a remarkable popularity in the social sciences. Using a meta-analytic tool--namely, reliability generalization (RG)--population reliability scores of both scales have been investigated on the basis of a random…

  20. The Affect Misattribution Procedure: hot or not?

    PubMed

    Blaison, Christophe; Imhoff, Roland; Hühnel, Isabell; Hess, Ursula; Banse, Rainer

    2012-04-01

    The Affect Misattribution Procedure (AMP; Payne, Cheng, Govorun, & Stewart, 2005) is an important tool in implicit social cognition research, but little is known about its underlying mechanisms. This paper investigates whether, as the name implies, affect-based processes really underlie the AMP. We used a modified AMP that enabled us to separate the influence of affective and nonaffective processes. In three studies, evidence for the implication of nonaffective processes was consistently found. In contrast, there was no evidence for affect-based processes. Thus, the AMP rather seems cold than hot. The generalizability of the results obtained with the modified AMP is discussed. PMID:22390705

  1. Affect regulation: holding, containing and mirroring.

    PubMed

    Pedersen, Signe Holm; Poulsen, Stig; Lunn, Susanne

    2014-10-01

    Gergely and colleagues' state that their "Social Biofeedback Theory of Parental Affect Mirroring" can be seen as a kind of operationalization of the classical psychoanalytic concepts of holding, containing and mirroring. This article examines to what extent the social biofeedback theory of parental affect mirroring may be understood as a specification of these concepts. It is argued that despite similarities at a descriptive level the concepts are embedded in theories with different ideas of subjectivity. Hence an understanding of the concept of affect regulation as a concretization and specification of the classical concepts dilutes the complexity of both the concept of affect regulation and of the classical concepts. PMID:25351730

  2. Self Concept: Reaching the Affective Child.

    ERIC Educational Resources Information Center

    Scher, Margaret E.

    This paper examines self-concept in children in order to learn and develop classroom strategies which will address the affective component of the primary school child. The introduction discusses the background of affective education, the history of educational movements, self-concept development, and the evaluation of self-concept through…

  3. 40 CFR 62.14102 - Affected facilities.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... burns homogeneous waste (such as automotive tires or used oil, but not including refuse-derived fuel... Requirements for Large Municipal Waste Combustors Constructed on or Before September 20, 1994 § 62.14102 Affected facilities. (a) The affected facility to which this subpart applies is each municipal...

  4. 40 CFR 62.14102 - Affected facilities.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... burns homogeneous waste (such as automotive tires or used oil, but not including refuse-derived fuel... Requirements for Large Municipal Waste Combustors Constructed on or Before September 20, 1994 § 62.14102 Affected facilities. (a) The affected facility to which this subpart applies is each municipal...

  5. Affective Scaffolds, Expressive Arts, and Cognition

    PubMed Central

    Maiese, Michelle

    2016-01-01

    Some theorists have argued that elements of the surrounding world play a crucial role in sustaining and amplifying both cognition and emotion. Such insights raise an interesting question about the relationship between cognitive and affective scaffolding: in addition to enabling the realization of specific affective states, can an affective niche also enable the realization of certain cognitive capacities? In order to gain a better understanding of this relationship between affective niches and cognition, I will examine the use of expressive arts in the context of psychotherapy and peacebuilding. In these settings, environmental resources and interpersonal scaffolds not only evoke emotion and encourage the adoption of particular bodily affective styles, but also support the development of capacities for self-awareness and interpersonal understanding. These affective scaffolds play a crucial role in therapy and peacebuilding, in fact, insofar as they facilitate the development of self-knowledge, enhance capacities associated with social cognition, and build positive rapport and trust among participants. I will argue that this is because affectivity is linked to the way that subjects frame and attend to their surroundings. Insofar as the regulation and modification of emotion goes hand in hand with opening up new interpretive frames and establishing new habits of mind, the creation of an affective niche can contribute significantly to various modes of cognition. PMID:27014164

  6. The Affective Regulation of Social Interaction

    ERIC Educational Resources Information Center

    Clore, Gerald L.; Pappas, Jesse

    2007-01-01

    The recent publication of David Heise's "Expressive Order" (2007) provides an occasion for discussing some of the key ideas in Affect Control Theory. The theory proposes that a few dimensions of affective meaning provide a common basis for interrelating personal identities and social actions. It holds that during interpersonal interactions, social…

  7. Affective Commitment among Student Affairs Professionals

    ERIC Educational Resources Information Center

    Boehman, Joseph

    2007-01-01

    Student affairs professionals in the United States were surveyed to determine the predictive value of overall job satisfaction, organizational support, organizational politics, and work/nonwork interaction on affective organizational commitment. Results indicate that a supportive work environment leads to increased affective attachment to the…

  8. Oklahoma Affective Education: A Resource Guide.

    ERIC Educational Resources Information Center

    Oklahoma City Public School System, OK.

    This resource guide is for elementary and secondary level educators who are interested in affective education and in formulating their own strategies for more meaningful learning experiences in their classrooms. The guide is based on the idea that affective learning is essential and that the best learning experiences are those in which the…

  9. Affective Learning: Environmental Ethics and Human Ecology

    ERIC Educational Resources Information Center

    Gough, Noel P.

    1977-01-01

    This discussion of home economics as a discipline which should focus on its affective foundations, covers the following areas: Affective context of home economics education, the adequacy of the home economics value complex for coping with environmental problems, and toward an acceptable environmental ethic. (SH)

  10. Affective Education for Gifted, Culturally Diverse Learners

    ERIC Educational Resources Information Center

    Baldwin, Alexinia

    2009-01-01

    Over the years, there has been an ongoing controversy about affective education. Some see it as an important element of good teaching, and some see it as fluff, diminishing academics, and playing into the "feel good" movement. While criticisms may be appropriate in some situations, affective education can play a fundamental role in other…

  11. 47 CFR 1.2003 - Applications affected.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 1 2010-10-01 2010-10-01 false Applications affected. 1.2003 Section 1.2003 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL PRACTICE AND PROCEDURE Implementation of the Anti-Drug Abuse Act of 1988 § 1.2003 Applications affected. The certification required by § 1.2002 must be...

  12. Does Attention Affect Visual Feature Integration?

    ERIC Educational Resources Information Center

    Prinzmetal, William; And Others

    This work investigates, first, whether the integration of color and shape information is affected by attending to the stimulus location, and second, whether attending to a stimulus location enhances the perceptual representation of the stimulus or merely affects decision processes. In three experiments, subjects were briefly presented with colored…

  13. Evaluation in the Affective Domain. NSPER: 76.

    ERIC Educational Resources Information Center

    Gephart, William J.; And Others

    The National Symposium for Professors of Educational Research for 1976 focused on two topics: the nature of affect, and principles and guidelines for measuring individual affect and learning environment. This document contains five major papers presented at the conference. The first paper contrasted the physiological and emotional concept of…

  14. Affective Priming with Auditory Speech Stimuli

    ERIC Educational Resources Information Center

    Degner, Juliane

    2011-01-01

    Four experiments explored the applicability of auditory stimulus presentation in affective priming tasks. In Experiment 1, it was found that standard affective priming effects occur when prime and target words are presented simultaneously via headphones similar to a dichotic listening procedure. In Experiment 2, stimulus onset asynchrony (SOA) was…

  15. Helping Crack-Affected Children Succeed.

    ERIC Educational Resources Information Center

    Waller, Mary Bellis

    1993-01-01

    Crack-affected children who experience early intervention can be mainstreamed successfully into regular classes. These children can be overwhelmed by stimuli and need stability, routine, and sameness in the intervention classroom. Teachers have discovered effective methods for working with crack-affected children. (16 references) (MLF)

  16. Summary of New Legislation Affecting Education, 1993.

    ERIC Educational Resources Information Center

    New York State Education Dept., Albany.

    This document summarizes major legislation, enacted by the 1993 New York State Legislature, that affected education and its related professions. Eight sections offer brief descriptions of legislation enacted in the following areas: laws affecting school districts generally; cultural education; state aid; taxation and financial administration;…

  17. 1989 Summary of New Legislation Affecting Education.

    ERIC Educational Resources Information Center

    New York State Education Dept., Albany. Office of the Counsel.

    This summary of major legislation enacted by the 1989 New York State Legislature affecting education and the professions is organized in eight sections: (1) laws affecting school districts generally; (2) state aid; (3) taxation and financial administration; (4) miscellaneous; (5) laws of local application; (6) higher education; (7) laws affecting…

  18. Affect and Engagement during Small Group Instruction

    ERIC Educational Resources Information Center

    Linnenbrink-Garcia, Lisa; Rogat, Toni Kempler; Koskey, Kristin L. K.

    2011-01-01

    Two studies (Study 1: n = 137; Study 2: n = 192) were conducted to investigate how upper-elementary students' affect during small group instruction related to their social-behavioral engagement during group work. A circumplex model of affect consisting of valence (positive, negative) and activation (high, low) was used to examine the relation of…

  19. Affective Outcomes of a World Geography Course

    ERIC Educational Resources Information Center

    Forsyth, Alfred S., Jr.; Maier, Joan N.

    2006-01-01

    Affective goals and objectives, rarely stated in geographic education standards, textbooks or course syllabi, include improving students' attitudes toward other people. World geography courses expose students to other parts of the world and to people different from themselves. Although affective goals may not be stated for such courses, could it…

  20. Nonverbal synchrony and affect in dyadic interactions

    PubMed Central

    Tschacher, Wolfgang; Rees, Georg M.; Ramseyer, Fabian

    2014-01-01

    In an experiment on dyadic social interaction, we invited participants to verbal interactions in cooperative, competitive, and ‘fun task’ conditions. We focused on the link between interactants’ affectivity and their nonverbal synchrony, and explored which further variables contributed to affectivity: interactants’ personality traits, sex, and the prescribed interaction tasks. Nonverbal synchrony was quantified by the coordination of interactants’ body movement, using an automated video-analysis algorithm (motion energy analysis). Traits were assessed with standard questionnaires of personality, attachment, interactional style, psychopathology, and interpersonal reactivity. We included 168 previously unacquainted individuals who were randomly allocated to same-sex dyads (84 females, 84 males, mean age 27.8 years). Dyads discussed four topics of general interest drawn from an urn of eight topics, and finally engaged in a fun interaction. Each interaction lasted 5 min. In between interactions, participants repeatedly assessed their affect. Using hierarchical linear modeling, we found moderate to strong effect sizes for synchrony to occur, especially in competitive and fun task conditions. Positive affect was associated positively with synchrony, negative affect was associated negatively. As for causal direction, data supported the interpretation that synchrony entailed affect rather than vice versa. The link between nonverbal synchrony and affect was strongest in female dyads. The findings extend previous reports of synchrony and mimicry associated with emotion in relationships and suggest a possible mechanism of the synchrony-affect correlation. PMID:25505435

  1. 40 CFR 1502.15 - Affected environment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 33 2011-07-01 2011-07-01 false Affected environment. 1502.15 Section 1502.15 Protection of Environment COUNCIL ON ENVIRONMENTAL QUALITY ENVIRONMENTAL IMPACT STATEMENT § 1502.15 Affected environment. The environmental impact statement shall succinctly describe...

  2. 40 CFR 1502.15 - Affected environment.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 32 2010-07-01 2010-07-01 false Affected environment. 1502.15 Section 1502.15 Protection of Environment COUNCIL ON ENVIRONMENTAL QUALITY ENVIRONMENTAL IMPACT STATEMENT § 1502.15 Affected environment. The environmental impact statement shall succinctly describe...

  3. 40 CFR 1502.15 - Affected environment.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 34 2012-07-01 2012-07-01 false Affected environment. 1502.15 Section 1502.15 Protection of Environment COUNCIL ON ENVIRONMENTAL QUALITY ENVIRONMENTAL IMPACT STATEMENT § 1502.15 Affected environment. The environmental impact statement shall succinctly describe...

  4. 40 CFR 1502.15 - Affected environment.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 34 2013-07-01 2013-07-01 false Affected environment. 1502.15 Section 1502.15 Protection of Environment COUNCIL ON ENVIRONMENTAL QUALITY ENVIRONMENTAL IMPACT STATEMENT § 1502.15 Affected environment. The environmental impact statement shall succinctly describe...

  5. 40 CFR 1502.15 - Affected environment.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 33 2014-07-01 2014-07-01 false Affected environment. 1502.15 Section 1502.15 Protection of Environment COUNCIL ON ENVIRONMENTAL QUALITY ENVIRONMENTAL IMPACT STATEMENT § 1502.15 Affected environment. The environmental impact statement shall succinctly describe...

  6. Affective Scaffolds, Expressive Arts, and Cognition.

    PubMed

    Maiese, Michelle

    2016-01-01

    Some theorists have argued that elements of the surrounding world play a crucial role in sustaining and amplifying both cognition and emotion. Such insights raise an interesting question about the relationship between cognitive and affective scaffolding: in addition to enabling the realization of specific affective states, can an affective niche also enable the realization of certain cognitive capacities? In order to gain a better understanding of this relationship between affective niches and cognition, I will examine the use of expressive arts in the context of psychotherapy and peacebuilding. In these settings, environmental resources and interpersonal scaffolds not only evoke emotion and encourage the adoption of particular bodily affective styles, but also support the development of capacities for self-awareness and interpersonal understanding. These affective scaffolds play a crucial role in therapy and peacebuilding, in fact, insofar as they facilitate the development of self-knowledge, enhance capacities associated with social cognition, and build positive rapport and trust among participants. I will argue that this is because affectivity is linked to the way that subjects frame and attend to their surroundings. Insofar as the regulation and modification of emotion goes hand in hand with opening up new interpretive frames and establishing new habits of mind, the creation of an affective niche can contribute significantly to various modes of cognition. PMID:27014164

  7. Affect in the "Communicative" Classroom: A Model.

    ERIC Educational Resources Information Center

    Acton, William

    Recent research on affective variables and classroom second language learning suggests that: (1) affective variables are context-sensitive in at least two ways; (2) attitudes are contagious, and the general attitude of students can be influenced from various directions; (3) research in pragmatics, discourse analysis, and communicative functions…

  8. Affect and Problem Solving: Two Theoretical Perspectives.

    ERIC Educational Resources Information Center

    McLeod, Douglas B.

    Cognitive factors related to problem solving have been explored, but affective factors also play an important role in the teaching of mathematical problem solving. This paper outlines the theories of George Mandler and Bernard Weiner, providing a useful background for research related to affect and problem solving. Data related to the two theories…

  9. Affective Evaluation Techniques in School Health Education.

    ERIC Educational Resources Information Center

    Sutherland, Mary S.

    1981-01-01

    Ways in which affective measurement and evaluation techniques could be used for health instruction include: (1) determining student behavioral information and assisting in the removal of barriers to smooth classroom functioning; (2) attitude measurement; and (3) evaluation of classroom learning. Several types of affective measurement techniques…

  10. Do School Facilities Affect Academic Outcomes?

    ERIC Educational Resources Information Center

    Schneider, Mark

    This review explores which facility attributes affect academic outcomes the most and in what manner and degree. The research is examined in six categories: indoor air quality, ventilation, and thermal comfort; lighting; acoustics; building age and quality; school size; and class size. The review concludes that school facilities affect learning.…

  11. Trait Affect and Job Search Outcomes

    ERIC Educational Resources Information Center

    Cote, Stephane; Saks, Alan M.; Zikic, Jelena

    2006-01-01

    The present study examines the role of trait affect in job search. One hundred and twenty-three university students completed measures of positive and negative affectivity, conscientiousness, job search self-efficacy, job search clarity, and job search intensity during their last year of school while on the job market. At the end of the school…

  12. Priming Effects for Affective vs. Neutral Faces

    ERIC Educational Resources Information Center

    Burton, Leslie A.; Rabin, Laura; Wyatt, Gwinne; Frohlich, Jonathan; Vardy, Susan B.; Dimitri, Diana

    2005-01-01

    Affective and Neutral Tasks (faces with negative or neutral content, with different lighting and orientation) requiring reaction time judgments of poser identity were administered to 32 participants. Speed and accuracy were better for the Affective than Neutral Task, consistent with literature suggesting facilitation of performance by affective…

  13. Affective Arousal as Information: How Affective Arousal Influences Judgments, Learning, and Memory

    PubMed Central

    Storbeck, Justin; Clore, Gerald L.

    2014-01-01

    The affect-as-information framework posits that affect is embodied information about value and importance. The valence dimension of affect provides evaluative information about stimulus objects, which plays a role in judgment and decisionmaking. Affect can also provide evaluative information about one's own cognitions and response inclinations, information that guides thinking and reasoning. In particular, positive affect often promotes, and negative affect inhibits, accessible responses or dominant modes of thinking. Affect thus moderates many of the textbook phenomena in cognitive psychology. In the current review, we suggest additionally that the arousal dimension of affect amplifies reactions, leading to intensified evaluations, increased reliance on particular styles of learning, and enhanced long-term memory for events. We conclude that whereas valenced affective cues serve as information about value, the arousal dimension provides information about urgency or importance. PMID:25067943

  14. Affective Arousal as Information: How Affective Arousal Influences Judgments, Learning, and Memory.

    PubMed

    Storbeck, Justin; Clore, Gerald L

    2008-09-01

    The affect-as-information framework posits that affect is embodied information about value and importance. The valence dimension of affect provides evaluative information about stimulus objects, which plays a role in judgment and decisionmaking. Affect can also provide evaluative information about one's own cognitions and response inclinations, information that guides thinking and reasoning. In particular, positive affect often promotes, and negative affect inhibits, accessible responses or dominant modes of thinking. Affect thus moderates many of the textbook phenomena in cognitive psychology. In the current review, we suggest additionally that the arousal dimension of affect amplifies reactions, leading to intensified evaluations, increased reliance on particular styles of learning, and enhanced long-term memory for events. We conclude that whereas valenced affective cues serve as information about value, the arousal dimension provides information about urgency or importance. PMID:25067943

  15. The Affective Regulation of Social Interaction*

    PubMed Central

    Clore, Gerald L.; Pappas, Jesse

    2008-01-01

    The recent publication of David Heise’s Expressive Order (2007) provides an occasion for discussing some of the key ideas in Affect Control Theory. The theory proposes that a few dimensions of affective meaning provide a common basis for interrelating personal identities and social actions. It holds that during interpersonal interactions, social behavior is continually regulated to maintain an affective tone compatible with whatever social roles or identities define the situation. We outline the intellectual history of the proposed dimensions and of the idea that each social action invites an action from the other that has a particular location along these dimensions. We also relate these ideas to the Affect-as-Information hypothesis, an approach that often guides research in psychology on the role of affect in regulating judgment and thought. PMID:18461152

  16. Human cerebral response to animal affective vocalizations

    PubMed Central

    Belin, Pascal; Fecteau, Shirley; Charest, Ian; Nicastro, Nicholas; Hauser, Marc D; Armony, Jorge L

    2007-01-01

    It is presently unknown whether our response to affective vocalizations is specific to those generated by humans or more universal, triggered by emotionally matched vocalizations generated by other species. Here, we used functional magnetic resonance imaging in normal participants to measure cerebral activity during auditory stimulation with affectively valenced animal vocalizations, some familiar (cats) and others not (rhesus monkeys). Positively versus negatively valenced vocalizations from cats and monkeys elicited different cerebral responses despite the participants' inability to differentiate the valence of these animal vocalizations by overt behavioural responses. Moreover, the comparison with human non-speech affective vocalizations revealed a common response to the valence in orbitofrontal cortex, a key component on the limbic system. These findings suggest that the neural mechanisms involved in processing human affective vocalizations may be recruited by heterospecific affective vocalizations at an unconscious level, supporting claims of shared emotional systems across species. PMID:18077254

  17. Embodied affectivity: on moving and being moved

    PubMed Central

    Fuchs, Thomas; Koch, Sabine C.

    2014-01-01

    There is a growing body of research indicating that bodily sensation and behavior strongly influences one's emotional reaction toward certain situations or objects. On this background, a framework model of embodied affectivity1 is suggested: we regard emotions as resulting from the circular interaction between affective qualities or affordances in the environment and the subject's bodily resonance, be it in the form of sensations, postures, expressive movements or movement tendencies. Motion and emotion are thus intrinsically connected: one is moved by movement (perception; impression; affection2) and moved to move (action; expression; e-motion). Through its resonance, the body functions as a medium of emotional perception: it colors or charges self-experience and the environment with affective valences while it remains itself in the background of one's own awareness. This model is then applied to emotional social understanding or interaffectivity which is regarded as an intertwinement of two cycles of embodied affectivity, thus continuously modifying each partner's affective affordances and bodily resonance. We conclude with considerations of how embodied affectivity is altered in psychopathology and can be addressed in psychotherapy of the embodied self. PMID:24936191

  18. Embodied affectivity: on moving and being moved.

    PubMed

    Fuchs, Thomas; Koch, Sabine C

    2014-01-01

    There is a growing body of research indicating that bodily sensation and behavior strongly influences one's emotional reaction toward certain situations or objects. On this background, a framework model of embodied affectivity is suggested: we regard emotions as resulting from the circular interaction between affective qualities or affordances in the environment and the subject's bodily resonance, be it in the form of sensations, postures, expressive movements or movement tendencies. Motion and emotion are thus intrinsically connected: one is moved by movement (perception; impression; affection) and moved to move (action; expression; e-motion). Through its resonance, the body functions as a medium of emotional perception: it colors or charges self-experience and the environment with affective valences while it remains itself in the background of one's own awareness. This model is then applied to emotional social understanding or interaffectivity which is regarded as an intertwinement of two cycles of embodied affectivity, thus continuously modifying each partner's affective affordances and bodily resonance. We conclude with considerations of how embodied affectivity is altered in psychopathology and can be addressed in psychotherapy of the embodied self. PMID:24936191

  19. Affective brain–computer music interfacing

    NASA Astrophysics Data System (ADS)

    Daly, Ian; Williams, Duncan; Kirke, Alexis; Weaver, James; Malik, Asad; Hwang, Faustina; Miranda, Eduardo; Nasuto, Slawomir J.

    2016-08-01

    Objective. We aim to develop and evaluate an affective brain–computer music interface (aBCMI) for modulating the affective states of its users. Approach. An aBCMI is constructed to detect a user's current affective state and attempt to modulate it in order to achieve specific objectives (for example, making the user calmer or happier) by playing music which is generated according to a specific affective target by an algorithmic music composition system and a case-based reasoning system. The system is trained and tested in a longitudinal study on a population of eight healthy participants, with each participant returning for multiple sessions. Main results. The final online aBCMI is able to detect its users current affective states with classification accuracies of up to 65% (3 class, p\\lt 0.01) and modulate its user's affective states significantly above chance level (p\\lt 0.05). Significance. Our system represents one of the first demonstrations of an online aBCMI that is able to accurately detect and respond to user's affective states. Possible applications include use in music therapy and entertainment.

  20. Implicit Processing of Visual Emotions Is Affected by Sound-Induced Affective States and Individual Affective Traits

    PubMed Central

    Quarto, Tiziana; Blasi, Giuseppe; Pallesen, Karen Johanne; Bertolino, Alessandro; Brattico, Elvira

    2014-01-01

    The ability to recognize emotions contained in facial expressions are affected by both affective traits and states and varies widely between individuals. While affective traits are stable in time, affective states can be regulated more rapidly by environmental stimuli, such as music, that indirectly modulate the brain state. Here, we tested whether a relaxing or irritating sound environment affects implicit processing of facial expressions. Moreover, we investigated whether and how individual traits of anxiety and emotional control interact with this process. 32 healthy subjects performed an implicit emotion processing task (presented to subjects as a gender discrimination task) while the sound environment was defined either by a) a therapeutic music sequence (MusiCure), b) a noise sequence or c) silence. Individual changes in mood were sampled before and after the task by a computerized questionnaire. Additionally, emotional control and trait anxiety were assessed in a separate session by paper and pencil questionnaires. Results showed a better mood after the MusiCure condition compared with the other experimental conditions and faster responses to happy faces during MusiCure compared with angry faces during Noise. Moreover, individuals with higher trait anxiety were faster in performing the implicit emotion processing task during MusiCure compared with Silence. These findings suggest that sound-induced affective states are associated with differential responses to angry and happy emotional faces at an implicit stage of processing, and that a relaxing sound environment facilitates the implicit emotional processing in anxious individuals. PMID:25072162

  1. Implicit processing of visual emotions is affected by sound-induced affective states and individual affective traits.

    PubMed

    Quarto, Tiziana; Blasi, Giuseppe; Pallesen, Karen Johanne; Bertolino, Alessandro; Brattico, Elvira

    2014-01-01

    The ability to recognize emotions contained in facial expressions are affected by both affective traits and states and varies widely between individuals. While affective traits are stable in time, affective states can be regulated more rapidly by environmental stimuli, such as music, that indirectly modulate the brain state. Here, we tested whether a relaxing or irritating sound environment affects implicit processing of facial expressions. Moreover, we investigated whether and how individual traits of anxiety and emotional control interact with this process. 32 healthy subjects performed an implicit emotion processing task (presented to subjects as a gender discrimination task) while the sound environment was defined either by a) a therapeutic music sequence (MusiCure), b) a noise sequence or c) silence. Individual changes in mood were sampled before and after the task by a computerized questionnaire. Additionally, emotional control and trait anxiety were assessed in a separate session by paper and pencil questionnaires. Results showed a better mood after the MusiCure condition compared with the other experimental conditions and faster responses to happy faces during MusiCure compared with angry faces during Noise. Moreover, individuals with higher trait anxiety were faster in performing the implicit emotion processing task during MusiCure compared with Silence. These findings suggest that sound-induced affective states are associated with differential responses to angry and happy emotional faces at an implicit stage of processing, and that a relaxing sound environment facilitates the implicit emotional processing in anxious individuals. PMID:25072162

  2. How Coriolis meter design affects field performance

    SciTech Connect

    Levien, A.; Dudiak, A.

    1995-12-31

    Although many possibilities exist for the design of Coriolis flowmeters, a common set of fundamental physical principles affect practical meter design. Design criteria such as tube geometry, alloy section, operating frequencies, stress levels, and tubing wall thickness have varying impacts on meter performance. Additionally, field conditions such as changing temperature, pressure, pipeline stress and vibration affect measurement performance. The challenge created in Coriolis flow meter design is to maximize the sensitivity of the meter Coriolis forces, while minimizing the impact of outside environmental influences. Data are presented on the physical principles that affect Coriolis flowmeters, and how the various aspects of meter design influence field performance.

  3. [Affective touch according the nurse's perspective].

    PubMed

    Dias, Andréa Basílio; Oliveira, Leonor; Dias, Denise Gamio; Santana, Maria da Glória

    2008-01-01

    This study aimed at investigating the conception of nurses about the affective touch as a tool for care promotion and to identify its meanings in nursing care and the moment when it is used as care instrument. Qualitative research differentiating two ways to touch: instrumental touch (objective care) and the affective touch (subjective care). For data collection a semi-structured interview was used, with three assistance nurses of two hospitals in the South Region of Brazil. Through data analysis, the affective touch was demonstrated to be restrictedly, during the invasive procedures, in despite considering it effective instrument to establish empathy. PMID:18982224

  4. Affect as Information in Persuasion: A Model of Affect Identification and Discounting

    PubMed Central

    Albarracín, Dolores; Kumkale, G. Tarcan

    2016-01-01

    Three studies examined the implications of a model of affect as information in persuasion. According to this model, extraneous affect may have an influence when message recipients exert moderate amounts of thought, because they identify their affective reactions as potential criteria but fail to discount them as irrelevant. However, message recipients may not use affect as information when they deem affect irrelevant or when they do not identify their affective reactions at all. Consistent with this curvilinear prediction, recipients of a message that either favored or opposed comprehensive exams used affect as a basis for attitudes in situations that elicited moderate thought. Affect, however, had no influence on attitudes in conditions that elicited either large or small amounts of thought. PMID:12635909

  5. Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts.

    PubMed

    Le Bihan, Marie-Catherine; Barrio-Hernandez, Inigo; Mortensen, Tenna Pavia; Henningsen, Jeanette; Jensen, Søren Skov; Bigot, Anne; Blagoev, Blagoy; Butler-Browne, Gillian; Kratchmarova, Irina

    2015-08-01

    Muscle stem cells, or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although they have been extensively studied, the key regulatory steps and the complex temporal protein dynamics accompanying the differentiation of primary human muscle cells remain poorly understood. Here, we demonstrate the advantages of applying a MS-based quantitative approach, stable isotope labeling by amino acids in cell culture (SILAC), for studying human myogenesis in vitro and characterize the fine-tuned changes in protein expression underlying the dramatic phenotypic conversion of primary mononucleated human muscle cells during in vitro differentiation to form multinucleated myotubes. Using an exclusively optimized triple encoding SILAC procedure, we generated dynamic expression profiles during the course of myogenic differentiation and quantified 2240 proteins, 243 of which were regulated. These changes in protein expression occurred in sequential waves and underlined vast reprogramming in key processes governing cell fate decisions, i.e., cell cycle withdrawal, RNA metabolism, cell adhesion, proteolysis, and cytoskeletal organization. In silico transcription factor target analysis demonstrated that the observed dynamic changes in the proteome could be attributed to a cascade of transcriptional events involving key myogenic regulatory factors as well as additional regulators not yet known to act on muscle differentiation. In addition, we created of a dynamic map of the developing myofibril, providing valuable insights into the formation and maturation of the contractile apparatus in vitro. Finally, our SILAC-based quantitative approach offered the possibility to follow the expression profiles of several muscle disease-associated proteins simultaneously and therefore could be a valuable resource for future studies investigating pathogenesis of degenerative muscle disorders as well as assessing new therapeutic strategies. PMID:26074025

  6. Examining an Affective Aggression Framework: Weapon and Temperature Effects on Aggressive Thoughts, Affect, and Attitudes.

    ERIC Educational Resources Information Center

    Anderson, Craig A.; And Others

    1996-01-01

    A general framework for studying affective aggression, integrating many insights from previous models, is presented. New research examining effects of extreme temperature and photos of guns on arousal, cognition, and affect is presented. Hostile cognition was assessed using automatic priming tasks (i.e., Stroop interference). Hostile affect was…

  7. How State Laws Affect Regional Media Services.

    ERIC Educational Resources Information Center

    Vick, Nancy Harper

    1978-01-01

    Discusses ways in which state legislation affects such regional media service administrative units as (1) the state education agency, (2) regional educational service agencies, and (3) educational cooperative centers. (CMV)

  8. CDC Vital Signs: Hospital Actions Affect Breastfeeding

    MedlinePlus

    ... Read the MMWR Science Clips Hospital Actions Affect Breastfeeding Language: English Español (Spanish) Recommend on Facebook Tweet ... in many US hospitals do not fully support breastfeeding. Some of the Ten Steps on which hospitals ...

  9. Cognitive and Affective Control in Insomnia

    PubMed Central

    Schmidt, Ralph E.; Harvey, Allison G.; Van der Linden, Martial

    2011-01-01

    Insomnia is a prevalent disabling chronic disorder. The aim of this paper is fourfold: (a) to review evidence suggesting that dysfunctional forms of cognitive control, such as thought suppression, worry, rumination, and imagery control, are associated with sleep disturbance; (b) to review a new budding field of scientific investigation – the role of dysfunctional affect control in sleep disturbance, such as problems with down-regulating negative and positive affective states; (c) to review evidence that sleep disturbance can impair next-day affect control; and (d) to outline, on the basis of the reviewed evidence, how the repetitive-thought literature and the affective science literature can be combined to further understanding of, and intervention for, insomnia. PMID:22162971

  10. Affective forecasting and the Big Five

    PubMed Central

    Hoerger, Michael; Quirk, Stuart W.

    2011-01-01

    Recent studies on affective forecasting clarify that the emotional reactions people anticipate often differ markedly from those they actually experience in response to affective stimuli and events. However, core personality differences in affective forecasting have received limited attention, despite their potential relevance to choice behavior. In the present study, 226 college undergraduates rated their anticipated and experienced reactions to the emotionally-evocative event of Valentine’s Day and completed a measure of the Big Five personality traits – neuroticism, extraversion, openness to experience, agreeableness, and conscientiousness – and their facet scales. Neuroticism and extraversion were associated with baseline mood, experienced emotional reactions, and anticipated emotional reactions. The present findings hold implications for the study of individual differences in affective forecasting, personality theory, and interventions research. PMID:22021944

  11. Smoking Affects You | Smokefree.gov

    Cancer.gov

    */ 18 Ways Smoking Affects Your Health Smoking harms nearly every organ of the body. Some of these harmful effects are immediate. Find out the health effects of smoking and what happens to your body when you quit.

  12. Gasoline Composition Regulations Affecting LUST Sites

    EPA Science Inventory

    Passage of the Clean Air Act Amendments in 1990 imposed requirements on gasoline composition in the United States. Impacts to ground water are affected by the provisions that required oxygenated additives and limited benzene concentration. Reformulated and oxygenated gasoline w...

  13. Legal Issues Affecting Libraries and Librarians.

    ERIC Educational Resources Information Center

    Mika, Joseph J.; Shuman, Bruce A.

    1988-01-01

    Review of employment laws affecting libraries includes definitions of qualified personnel, laws pertaining to medical and honesty testing, affirmative action, and sexual harassment. Laws covering crime within libraries are also discussed, as well as problems with existing legislation. (CLB)

  14. Will Stress during Pregnancy Affect My Baby?

    MedlinePlus

    ... Research Information Clinical Trials Resources and Publications Will stress during pregnancy affect my baby? Skip sharing on ... health care provider during your prenatal visits. Posttraumatic Stress Disorder (PTSD) and Pregnancy PTSD is a more ...

  15. Hold it! Memory affects attentional dwell time.

    PubMed

    Parks, Emily L; Hopfinger, Joseph B

    2008-12-01

    The allocation of attention, including the initial orienting and the subsequent dwell time, is affected by several bottom-up and top-down factors. How item memory affects these processes, however, remains unclear. Here, we investigated whether item memory affects attentional dwell time by using a modified version of the attentional blink (AB) paradigm. Across four experiments, our results revealed that the AB was significantly affected by memory status (novel vs. old), but critically, this effect depended on the ongoing memory context. Specifically, items that were unique in terms of memory status demanded more resources, as measured by a protracted AB. The present findings suggest that a more comprehensive understanding of memory's effects on attention can be obtained by accounting for an item's memorial context, as well as its individual item memory strength. Our results provide new evidence that item memory and memory context play a significant role in the temporal allocation of attention. PMID:19001579

  16. 28 CFR 80.5 - Affected parties.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) FOREIGN CORRUPT PRACTICES ACT OPINION PROCEDURE § 80.5 Affected parties. An FCPA Opinion shall have no application to any party which does not join in the request for the opinion....

  17. 28 CFR 80.5 - Affected parties.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) FOREIGN CORRUPT PRACTICES ACT OPINION PROCEDURE § 80.5 Affected parties. An FCPA Opinion shall have no application to any party which does not join in the request for the opinion....

  18. 28 CFR 80.5 - Affected parties.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) FOREIGN CORRUPT PRACTICES ACT OPINION PROCEDURE § 80.5 Affected parties. An FCPA Opinion shall have no application to any party which does not join in the request for the opinion....

  19. 28 CFR 80.5 - Affected parties.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) FOREIGN CORRUPT PRACTICES ACT OPINION PROCEDURE § 80.5 Affected parties. An FCPA Opinion shall have no application to any party which does not join in the request for the opinion....

  20. 28 CFR 80.5 - Affected parties.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) FOREIGN CORRUPT PRACTICES ACT OPINION PROCEDURE § 80.5 Affected parties. An FCPA Opinion shall have no application to any party which does not join in the request for the opinion....