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Sample records for c2c12 myoblast differentiation

  1. Silencing myotubularin related protein 7 enhances proliferation and early differentiation of C2C12 myoblast.

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    Yuan, Zhuning; Chen, Yaosheng; Zhang, Xumeng; Zhou, Xingyu; Li, Mingsen; Chen, Hu; Wu, Ming; Zhang, Ying; Mo, Delin

    2017-03-11

    Myotubularin related protein 7 (MTMR7) is a key member of the highly conserved myotubularin related proteins (MTMRs) family, which has phosphatase activity. MTMR7 was increased during myoblast differentiation and exhibited high expression level at primary fibers formation stages in pigs. This suggests that MTMR7 may be involved in myogenesis. In our study, we investigated the roles of MTMR7 on proliferation and differentiation of C2C12 myoblasts. Knocking down MTMR7 not only enhanced myoblast early differentiation via altering the expression of Myf5, but also promoted myoblast proliferation through increasing cyclinA2 expression. The improved proliferation capacity was related to the increased phosphorylation of AKT. Taken together, our research demonstrates that MTMR7 plays an important role in proliferation and early differentiation of C2C12 myoblast.

  2. Notch pathway activation contributes to inhibition of C2C12 myoblast differentiation by ethanol.

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    Michelle A Arya

    Full Text Available The loss of muscle mass in alcoholic myopathy may reflect alcohol inhibition of myogenic cell differentiation into myotubes. Here, using a high content imaging system we show that ethanol inhibits C2C12 myoblast differentiation by reducing myogenic fusion, creating smaller and less complex myotubes compared with controls. Ethanol administration during C2C12 differentiation reduced MyoD and myogenin expression, and microarray analysis identified ethanol activation of the Notch signaling pathway target genes Hes1 and Hey1. A reporter plasmid regulated by the Hes1 proximal promoter was activated by alcohol treatment in C2C12 cells. Treatment of differentiating C2C12 cells with a gamma secretase inhibitor (GSI abrogated induction of Hes1. On a morphological level GSI treatment completely rescued myogenic fusion defects and partially restored other myotube parameters in response to alcohol. We conclude that alcohol inhibits C2C12 myoblast differentiation and the inhibition of myogenic fusion is mediated by Notch pathway activation.

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

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

    2014-09-26

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

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

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    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

  5. Graphene-Based Patterning and Differentiation of C2C12 Myoblasts

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    Bajaj, Piyush; Rivera, Jose A; Marchwiany, Daniel

    2014-01-01

    This study aims at generating highly aligned functional myotubes using graphene as the underlying scaffold. Graphene not only supports the growth of C2C12 muscle cells but also enhances its differentiation and leads to spontaneous patterning of myotubes....

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

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    Jeffrey eKim

    2014-03-01

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

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

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    Dupuy Fabrice

    2009-10-01

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

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

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

    2011-01-01

    to specific receptors on the cell surface. We showed that mechanical stimuli promote an increase of FAK phosphorylation. In order to further shed light in the process of myoblast induced differentiation by mechanical stimuli, we performed a proteomic analysis. Thirteen proteins were found to be affected...... by mechanical stimulation including Galectin-1, Annexin III, and RhoGDI. In this study we demonstrate how the combination of this method of mechanical stimuli and proteomic analysis can be a powerful tool to detect proteins that are potentially interacting in biochemical pathways or complex cellular mechanisms...... during the process of myoblast differentiation. We determined an increase in expression and changes in cellular localization of Galectin-1, in mechanically stimulated myoblasts. A potential involvement of Galectin-1 in myoblast differentiation is presented....

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

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

    2016-01-01

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

  10. miR-143-3p促进C2C12成肌细胞分化%miR-143-3p Is Implicated in C2C12 Myoblasts Differentiation

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    云青; 吴国芳; 魏欢; 庞卫军; 杨公社; 沈清武

    2013-01-01

    MicroRNAs (miRNAs) are small non-coding RNA that play important roles in skeletal muscle development.To explore the function of miR-143-3p in the differentiation of C2C12 myoblasts,we detected miR-143-3p levels by real-time PCR in different mouse tissues,as well as C2C12 myoblasts during myogenesis.After the trasfection of miR-143-3p mimics and inhibitor in C2C12 myoblasts,the expression of myogenic regulatory factor MyoG and myogenic marker gene MyHC were detected by realtime PCR and Western blotting.The myotubule formation was detected by immunofluorescent staining.The results showed that miR-143-3p was ubiquitously expressed in various tissues and was upregulated during cell differentiation.The differentiation of C2C12 myoblasts was promoted with miR-143-3p overexpression as significant upregulation of MyoG and MyHC,and increased number of myotubules.The inhibitor of miR-143-3p significantly repressed cell differentiation.Interestingly,the transfection of miR-143-3p mimics had little effect on the expression of MyHCs.Our data suggested that miR-143-3p might be involved during the myogeneis of C2C12 myoblasts,but not directly impact MyHC expression.%MicroRNAs (miRNAs)是一类小非编码RNA,近年研究发现其在骨骼肌发育调控中发挥重要作用.为探明miR-143-3p在C2C12成肌细胞分化中的调控作用,采用real-time PCR检测了miR-143-3p在小鼠各组织及C2C12成肌细胞分化过程中的表达;使用miR-143-3p的模拟物和特异性抑制剂分别处理细胞,采用real-time PCR和Western印迹分别检测成肌因子MyoG和成肌标志基因MyHC mRNA和蛋白水平的变化;用免疫荧光染色的方法观察肌管的形成.结果显示,miR-143-3p在小鼠各组织中均有表达,并且随着细胞分化表达量逐渐增加;C2C12成肌细胞过表达miR-143-3p,与对照组相比,成肌调控因子MyoG和成肌标志基因MyHC的mRNA和蛋白表达均显著升高,肌管数量明显增多;抑制剂处理结果显示,细胞分

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

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    Long Jia

    2013-12-01

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

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

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    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.

  13. 组蛋白乙酰化/去乙酰化失衡对C2C12肌原细胞成肌分化的影响%The Effects of Imbalance Between Histone Acetylation and Deacetylation on C2C12 Myoblasts Differentiation

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    李一飞; 华益民; 方婕; 王川; 詹雅兰; 朱琦; 母得志; 周开宇

    2014-01-01

    Objective To explore the effects of histone acetylation and deacetylation on C2C12 myoblasts differentiation.Methods Based on the differentiation of C2C12 myoblasts into myotubes using high glucose dulbecco′s modified eagle medium (DMEM)containing 2% horse serum invitro,valproic acid (VPA)was given to C2C12 myoblasts during differentiation with different concentrations,which contained 1 mmol/L VPA,2 mmol/L VPA,4 mmol/L VPA and 8 mmol/L VPA in the final concentrations with 2%horse serum and high glucose DMEM.So that this experiment was scheduled into 6 groups as control group (contain 10% fetal bovine serum in growth medium),horse serum induced differentiation group,1 mmol/L VPA group,2 mmol/L VPA group,4 mmol/L VPA group and 8 mmol/L VPA group according to different growth medium.There were 6 samples in each group.The myotube differentiation rate were compared in different concentration VPA groups and horse serum induced differentiation group.Besides,mRNA and protein expression levels of muscle-related proteins (including Myosin, Troponin I-SS, myogenic differentiation 1)and histone deacetylases (HDAC,including HDAC1,2,3)were also evaluated with real time polymerase chain reaction (RT-PCR)and Western blotting.The mRNA and protein expression levels of them were compared and analyzed.Results ①The mRNA and protein expression levels of muscle-related proteins of horse serum induced differentiation group were significantly higher than those of control group, and the differences were statistically significant (P 0.05 ).② The myotube differentiation rate in every concentration VPA group compared with horse serum induced differentiation group were significantly lower,and the differences were statistically significant (P0.05)。②各浓度 VPA 组肌小管分化率分别较马血清诱导分化组显著下降,且差异有统计学意义(P<0.05)。③4 mmol/L VPA组及8 mmol/L VPA组肌相关蛋白及 HDAC mRNA和蛋白表达水平分别较马血清诱导分化组

  14. First intron of nestin gene regulates its expression during C2C12 myoblast ifferentiation

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    Hua Zhong; Zhigang Jin; Yongfeng Chen; Ting Zhang; Wei Bian; Xing Cui; Naihe Jing

    2008-01-01

    Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China Nestin is an intermediate filament protein expressed in neural progenitor cells and in developing skeletal muscle. Nestin has been widely used as a neural progenitor cell marker. It is well established that the specific expression of the nestin gene in neural progenitor cells is conferred by the neural-specific enhancer located in the second intron of the nestin gene. However, the transcriptional mechanism of nestin expression in developing muscle is still unclear. In this study, we identified a muscle cell-specific enhancer in the first intron of mouse nestin gene in mouse myoblast C2C12 cells.We localized the core enhancer activity to the 291-661 region of the first intron, and showed that the two E-boxes in the core enhancer region were important for enhancer activity in differentiating C2C12 cells. We also showed that MyoD protein was involved in the regulation of nestin expression in the myogenic differentiation of C2C12 cells.

  15. Lrrc75b is a novel negative regulator of C2C12 myogenic differentiation

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    Zhong, Yuechun; Zou, Liyi; Wang, Zonggui; Pan, Yaqiong; Dai, Zhong; Liu, Xinguang; Cui, Liao; Zuo, Changqing

    2016-01-01

    Many transcription factors and signaling molecules involved in the guidance of myogenic differentiation have been investigated in previous studies. However, the precise molecular mechanisms of myogenic differentiation remain largely unknown. In the present study, by performing a meta-analysis of C2C12 myogenic differentiation microarray data, we found that leucine-rich repeat-containing 75B (Lrrc75b), also known as AI646023, a molecule of unknown biological function, was downregulated during C2C12 myogenic differentiation. The knockdown of Lrrc75b using specific siRNA in C2C12 myoblasts markedly enhanced the expression of muscle-specific myogenin and increased myoblast fusion and the myotube diameter. By contrast, the adenovirus-mediated overexpression of Lrrc75b in C2C12 cells markedly inhibited myoblast differentiation accompanied by a decrease in myogenin expression. In addition, the phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) was suppressed in the cells in which Lrrc75b was silenced. Taken together, our results demonstrate that Lrrc75b is a novel suppressor of C2C12 myogenic differentiation by modulating myogenin and Erk1/2 signaling. PMID:27633041

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

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

    2014-01-01

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

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

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

    2017-02-01

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

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

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    Qiangling Zhang

    2015-08-01

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

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

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

    2015-01-01

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

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

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

    2015-09-30

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

  1. Recombinant myostatin reduces highly expressed microRNAs in differentiating C2C12 cells

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    Zachary A. Graham

    2017-03-01

    Full Text Available Myostatin is small glycopeptide that is produced and secreted by skeletal muscle. It is a potent negative regulator of muscle growth that has been associated with conditions of frailty. In C2C12 cells, myostatin limits cell differentiation. Myostatin acts through activin receptor IIB, activin receptor-like kinase (ALK and Smad transcription factors. microRNAs (miRNA are short, 22 base pair nucleotides that bind to the 3′ UTR of target mRNA to repress translation or reduce mRNA stability. In the present study, expression in differentiating C2C12 cells of the myomiRs miR-1 and 133a were down-regulated following treatment with 1 µg of recombinant myostatin at 1 d post-induction of differentiation while all myomiRs (miR-1, 133a/b and 206 were upregulated by SB431542, a potent ALK4/5/7 inhibitor which reduces Smad2 signaling, at 1 d and all, with the exception of miR-206, were upregulated by SB431542 at 3 d. The expression of the muscle-enriched miR-486 was greater following treatment with SB431542 but not altered by myostatin. Other highly expressed miRNAs in skeletal muscle, miR-23a/b and 145, were altered only at 1 d post-induction of differentiation. miR-27b responded differently to treatments at 1 d, where it was upregulated, as compared to 3 d, where it was downregulated. Neither myostatin nor SB431542 altered cell size or cell morphology. The data indicate that myostatin represses myomiR expression in differentiating C2C12 cells and that inhibition of Smad signaling with SB431542 can result in large changes in highly expressed miRNAs in differentiating myoblasts.

  2. Truncated Human LMP-1 Triggers Differentiation of C2C12 Cells to an Osteoblastic Phenotype in vitro

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

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

    Science.gov (United States)

    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

  4. Induced differentiation of C2C12 to osteoblast via adenovirus-mediated Cbfa1 in vitro%体外诱导C2C12细胞向成骨细胞的分化

    Institute of Scientific and Technical Information of China (English)

    张勇; 杨彤涛; 胡运生; 廖博; 文艳华; 范清宇

    2013-01-01

    目的 成骨细胞特异性转录因子a1(core binding factor a1,Cbfa1)通过调节生长因子和骨特异性细胞外基质蛋白的基因表达而参与成骨细胞的分化和骨发育过程.文中构建成Cbfa1,以腺病毒载体转染成肌细胞C2C12,为种子细胞构建组织工程化骨.方法 体外培养小鼠成肌细胞C2C12,用重组腺病毒质粒pAd-IL-31介导Cbfa1/Osf2基因瞬时转染小鼠成肌C2C12细胞,Western blot检测Cbfa1蛋白表达.结果 Cbfa1蛋白表达、碱性磷酸酶(alkaline phosphatase,ALP)活性测定、骨钙素(osteocalcin,OCN)分泌量以及茜素红染色感染组明显高于对照组.结论 成肌细胞C2C12可以作为种子细胞构建组织工程化骨.%Objective Osteoblast core binding factor a 1 ( Cbfal) plays a role in osteoblast differentiation and development by regulating the gene of growth factor and extracellular matrix proteins . Recombinant adenovirus vector mediated Cbfa 1 was transferred to myoblast C2C12 to construct the tissue-engineered bone. Methods The myoblast C2C12 was cultured in vitro, and then transiently transfected with recombinant adenovirus vector pAd -IL-31 mediated-Cbfal/Osf2. Western blot was used to detect the expression of Cbfal. Results Compared with the control group , the expression of Cbfal, activity of alkaline phosphtase (ALP) , secretory volume of osteocalcin (OCN) and staining via alizarin bordeaux were higher in the transfection group . Conclusion Myoblast C2C12 acts as a seed cell for constructing tissue -engineered bone.

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

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    Hyunju Kim

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

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

    Science.gov (United States)

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

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-15

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-05-01

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

  9. Histamine H3 receptor inhibited electrically evoked cytoplasmic calcium in differentiated skeletal C2C12 myoblasts%组胺 H3受体降低电激发收缩的小鼠成肌细胞胞浆中钙离子浓度

    Institute of Scientific and Technical Information of China (English)

    齐麟; 冯晓; 陈燕; 薛瑞; 张凤; 王素云; 孙素珂; 建国

    2015-01-01

    目的:探讨组胺H3受体(H3R)在小鼠成肌细胞C2C12成肌分化过程及分化后的横纹肌细胞中的表达和可能发挥的作用。方法:诱导C2C12细胞成肌分化,测量H3R和分化晚期标志物肌球蛋白重链mRNA和蛋白的表达;分化过程中加入H3R拮抗剂ciproxifan,测量分化早期标志物desmin、中期标志物myogenin和肌球蛋白重链mRNA的表达。 Fluo-4结合剂标记分化后的横纹肌胞内钙离子,测量双极交流电200 mA刺激下,H3R激动剂甲基组胺(RMeHA)对胞浆中钙离子浓度的影响。结果:H3R和肌球蛋白重链在成肌分化过程中表达量逐渐增加。 Ciproxifan在成肌分化过程中对3种分化标志物mRNA的表达与对照组相比无差异( P>0.05)。 RMeHA在浓度10 nmol/L~100μmol/L刺激细胞5~20 min,可呈钟形降低因交流电引起的肌浆钙离子浓度的升高( P<0.05),其中RMeHA 100 nmol/L在10 min和20 min对电刺激细胞中Ca2+的抑制百分率最高。相同浓度的RMeHA在20 min和10 min时对Ca2+的抑制率比其在5 min时高(P<0.05)。结论:H3R可能在成肌分化过程中的作用不大,而在分化成熟细胞中可以降低电刺激引起的胞浆钙离子浓度的升高。%AIM:To explore the expression and possible function of histamine H3 receptor (H3R) in striated myogenesis and the differentiated C2C12 cells.METHODS: H3R and myogenesis late marker myosin heavy chain (MHC) were detected at mRNA and protein levels during C2C12 myogenesis.H3R antagonist ciproxifan was added and the expression of the myogenesis early marker desmin, intermediate markers myogenin and MHC was detected.Differentia-ted myoblasts were loaded with Fluo-4 calcium indicator dye and the effect of R-( a)-methylhistamine ( RMeHA) on the cy-toplasmic calcium concentration was determined under the 200 mA electrical stimulation.RESULTS: The expression of H3R and MHC was increased during myogenesis

  10. Expression of the myodystrophic R453W mutation of lamin A in C2C12 myoblasts causes promoter-specific and global epigenetic defects.

    Science.gov (United States)

    Håkelien, Anne-Mari; Delbarre, Erwan; Gaustad, Kristine G; Buendia, Brigitte; Collas, Philippe

    2008-05-01

    Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is characterized by muscle wasting and is caused by mutations in the LMNA gene encoding A-type lamins. Overexpression of the EDMD lamin A R453W mutation in C2C12 myoblasts impairs myogenic differentiation. We show here the influence of stable expression of the R453W and of the Dunnigan-type partial lipodystrophy R482W mutation of lamin A in C2C12 cells on transcription and epigenetic regulation of the myogenin (Myog) gene and on global chromatin organization. Expression of R453W-, but not R482W-lamin A, impairs activation of Myog and maintains a repressive chromatin state on the Myog promoter upon induction of differentiation, marked by H3 lysine (K) 9 dimethylation and failure to hypertrimethylate H3K4. Cells expressing WT-LaA also fail to hypertrimethylate H3K4. No defect occurs at the level of Myog promoter DNA methylation in any of the clones. Expression of R453W-lamin A and to a lesser extent R482W-lamin A in undifferentiated C2C12 cells redistributes H3K9me3 from pericentric heterochromatin. R453W-lamin A also elicits a redistribution of H3K27me3 from inactive X (Xi) and partial decondensation of Xi, but maintains Xist expression and coating of Xi, indicating that Xi remains inactivated. Our results argue that gene-specific and genome-wide chromatin rearrangements may constitute a molecular basis for laminopathies.

  11. Expression of Basic Fibroblast Growth Factor Results in the Decrease of Myostatin mRNA in Murine C2C12 Myoblasts

    Institute of Scientific and Technical Information of China (English)

    Hua-Zhong LIU; Qing LI; Xing-Yuan YANG; Lin LIU; Lei LIU; Xiao-Rong AN; Yong-Fu CHEN

    2006-01-01

    During the development and regeneration of skeletal muscle, many growth factors, such as basic fibroblast growth factor (bFGF, FGF-2) and myostatin, have been shown to play regulating roles.bFGF contributes to promote proliferation and to inhibit differentiation of skeletal muscle, whereas myostatin plays a series of contrasting roles. In order to elucidate whether the expression of bFGF has any relationship with the expression of myostatin in skeletal muscle cells, we constructed a eukaryotic expression vector for the expression of exogenous bFGF in murine C2C12 myoblasts. Quantitative RT-PCR assays indicated that with the increase of the expression of exogenous bFGF gene, the expression of endogenous myostatin gene was suppressed at mRNA level and protein level.

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

    Directory of Open Access Journals (Sweden)

    Poussard S

    2015-02-01

    Full Text Available Sylvie Poussard,1,2 Marion Decossas,1,2 Olivier Le Bihan,1,2 Stéphane Mornet,3 Grégoire Naudin,1,2 Olivier Lambert1,2 1Institute of Chemistry and Biology of Membranes and Nanoobjects, University of Bordeaux, UMR5248, Pessac, France; 2Institute of Chemistry and Biology of Membranes and Nanoobjects, Centre National de la Recherche Scientifique, Institute of Chemistry and Biology of Membranes and Nanoobjects, UMR5248, Pessac, France; 3ICMCB, Institut de Chimie de la Matière Condensée de Bordeaux, CNRS UPR9048, Université de Bordeaux, Pessac, France Abstract: 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. Keywords: silica, nanoparticle, muscle, cell encapsulation, transmission electron microscopy, apoptosis

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

    Directory of Open Access Journals (Sweden)

    Ji Sook Kang

    2014-12-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2008-11-01

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

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

    Directory of Open Access Journals (Sweden)

    G Ciofani

    2010-04-01

    Full Text Available G Ciofani1, L Ricotti1, S Danti2,3, S Moscato4, C Nesti2, D D’Alessandro2,4, D Dinucci5, F Chiellini5, A Pietrabissa3, M Petrini2,3, A Menciassi1,61Scuola Superiore Sant’Anna, Pisa, Italy; 2CUCCS-RRMR, Center for the Clinical Use of Stem Cells – Regional Network of Regenerative Medicine, 3Department of Oncology, Transplants and Advanced Technologies, 4Department of Human Morphology and Applied Biology, University of Pisa, Pisa, Italy; 5Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental Applications (BIOlab, UdR INSTM, Department of Chemistry and Industrial Chemistry, University of Pisa, San Piero a Grado, Italy; 6Italian Institute of Technology, Genova, ItalyAbstract: Boron nitride nanotubes (BNNTs have generated considerable interest within the scientific community by virtue of their unique physical properties, which can be exploited in the biomedical field. In the present in vitro study, we investigated the interactions of poly-L-lysine-coated BNNTs with C2C12 cells, as a model of muscle cells, in terms of cytocompatibility and BNNT internalization. The latter was performed using both confocal and transmission electron microscopy. Finally, we investigated myoblast differentiation in the presence of BNNTs, evaluating the protein synthesis of differentiating cells, myotube formation, and expression of some constitutive myoblastic markers, such as MyoD and Cx43, by reverse transcription – polymerase chain reaction and Western blot analysis. We demonstrated that BNNTs are highly internalized by C2C12 cells, with neither adversely affecting C2C12 myoblast viability nor significantly interfering with myotube formation.Keywords: boron nitride nanotubes, C2C12 cells, cytocompatibility, up-take, differentiation, MyoD, connexin 43

  16. Spatial segregation of BMP/Smad signaling affects osteoblast differentiation in C2C12 cells.

    Directory of Open Access Journals (Sweden)

    Eva Heining

    Full Text Available BACKGROUND: Bone morphogenetic proteins (BMPs are involved in a plethora of cellular processes in embryonic development and adult tissue homeostasis. Signaling specificity is achieved by dynamic processes involving BMP receptor oligomerization and endocytosis. This allows for spatiotemporal control of Smad dependent and non-Smad pathways. In this study, we investigate the spatiotemporal regulation within the BMP-induced Smad transcriptional pathway. METHODOLOGY/PRINCIPAL FINDINGS: Here we discriminate between Smad signaling events that are dynamin-dependent (i.e., require an intact endocytic pathway and dynamin-independent. Inhibition of dynamin-dependent endocytosis in fluorescence microscopy and fractionation studies revealed a delay in Smad1/5/8 phosphorylation and nuclear translocation after BMP-2 stimulation of C2C12 cells. Using whole genome microarray and qPCR analysis, we identified two classes of BMP-2 induced genes that are differentially affected by inhibition of endocytosis. Thus, BMP-2 induced gene expression of Id1, Id3, Dlx2 and Hey1 is endocytosis-dependent, whereas BMP-2 induced expression of Id2, Dlx3, Zbtb2 and Krt16 is endocytosis-independent. Furthermore, we demonstrate that short term inhibition of endocytosis interferes with osteoblast differentiation as measured by alkaline phosphatase (ALP production and qPCR analysis of osteoblast marker gene expression. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that dynamin-dependent endocytosis is crucial for the concise spatial activation of the BMP-2 induced signaling cascade. Inhibition of endocytic processes during BMP-2 stimulation leads to altered Smad1/5/8 signaling kinetics and results in differential target gene expression. We show that interfering with the BMP-2 induced transcriptional network by endocytosis inhibition results in an attenuation of osteoblast differentiation. This implies that selective sensitivity of gene expression to endocytosis provides an

  17. 1α,25(OH)2D3-dependent modulation of Akt in proliferating and differentiating C2C12 skeletal muscle cells.

    Science.gov (United States)

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

    2012-04-01

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

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

    Science.gov (United States)

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

    2006-02-15

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

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

    Directory of Open Access Journals (Sweden)

    Felicia Carotenuto

    2016-11-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-14

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

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

    Science.gov (United States)

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

    2015-03-20

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

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

    Directory of Open Access Journals (Sweden)

    Elena Barbieri

    2016-01-01

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

  3. 人参皂苷Rg1对体外培养C2C12成肌细胞凋亡的影响%Effect of ginsenoside Rg1 during serum-deprivation induced apoptosis in C2C12 myoblasts cultured in vitro

    Institute of Scientific and Technical Information of China (English)

    叶东明; 余磊; 王乐禹; 邱小忠; 欧阳钧

    2011-01-01

    Objective: To investigate the effect and possible mechanism of ginsenoside Rgl during serum-deprivation induced apoptosis in C2C12 myoblasts cultured in vitro. Methods: The effect of different concentrations of ginsenoside Rgl during the cell apoptosis was assessed by MTT assay, Hoechst 33258-PI double staining and RT-PCR analysis. Results: After 48 h treatment, various doses of ginsenoside Rgl increased cell viability in serum-deprived C2C12 myoblasts using MTT assay. Hoechst 33258-PI double staining showed that the rate of apoptosis cells significantly decreased after being treated by ginsenoside Rgl. RT-PCR showed that ginsenoside Rgl caused the downregulation of pro-apoptotic caspase-3, Bax and AIF genes, while caused the up-regulation of anti-apoptotic Bcl-2 gene. Conclusion: Ginsenoside Rgl can protect the serum-deprived apoptosis in C2C12 myoblasts.%目的:研究人参皂苷Rg1对体外无血清诱导培养的C2C12成肌细胞凋亡的影响及其可能机制.方法:采用MTT法、人参皂苷Rg1处理48 h后hoechst 33258-PI染色,以及RT-PCR方法观察不同浓度人参皂苷Rg1对C2C12成肌细胞凋亡的影响.结果:MTT法结果显示人参皂苷Rg1处理48 h后可抑制C2C12成肌细胞凋亡;Hoechst 33258-PI染色可见C2C12成肌细胞凋亡率人参皂苷处理前后差异有统计学意义,人参皂苷处理后C2C12成肌细胞凋亡率显著下降;RT-PCR法结果显示人参皂苷Rg1可抑制Caspase-3、Bax和AIF mRNA表达,并能诱导Bcl-2 mRNA表达.结论:人参皂苷Rg1对C2C12成肌细胞凋亡具有保护作用.

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

    DEFF Research Database (Denmark)

    Grossi, Alberto; Lawson, Moira Ann

    Abstract Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation and cell differentiation. A. Grossi, M. A. Lawson; Department of Food Science, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark The process of muscle...... documented and has been shown to affect transcription of specific gene sequences, protein synthesis, the immune system and increase in Ca2+ influx. The past 10 years has seen a dramatic increase in the understanding of how proteolytic enzymes such as calpains can affect the growth of muscle. In vivo studies...... have shown that m-calpain is necessary for myoblast fusion leading to the formation of muscle fibers and that inhibition of this enzyme restricts myotube formation. Whether there is a link between stretchor load induced signaling and m-calpain expression and activation is not known. Using a magnetic...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-08-08

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

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

    Directory of Open Access Journals (Sweden)

    Friedmann Theodore

    2009-08-01

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

  7. Transcription activation of myostatin by trichostatin A in differentiated C2C12 myocytes via ASK1-MKK3/4/6-JNK and p38 mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Han, Der-Sheng; Huang, Hsiang-Po; Wang, Tyng-Guey; Hung, Meng-Yu; Ke, Jia-Yu; Chang, Kuei-Ting; Chang, Hsin-Yu; Ho, Yu-Ping; Hsieh, Wei-Yuan; Yang, Wei-Shiung

    2010-10-15

    Myostatin is a negative regulator of skeletal muscle mass. The pathways employed in modulating myostatin gene expression are scarcely known. We aimed to determine the signaling pathway of myostatin induction by a histone deacetylase (HDAC) inhibitor-trichostatin A (TSA) in differentiated C(2)C(12) myocytes. TSA increased myostatin mRNA expression up to 40-fold after treatment for 24 h, and induced myostatin promoter activity up to 3.8-fold. Pretreatment with actinomycin D reduced the TSA-induced myostatin mRNA by 93%, suggesting TSA-induced myostatin expression mainly at the transcriptional level. Pretreatment with p38 MAPK (SB203580) and JNK (SP600125) inhibitors, but not ERK (PD98059) inhibitor, blocked TSA-induced myostatin expression, respectively, by 72% and 43%. Knockdown of p38 MAPK by RNAi inhibited the TSA-induced myostatin expression by 77% in C(2)C(12) myoblasts. The protein levels of phosphorylated p38 MAPK, JNK, but not ERK, increased with TSA treatment in differentiated C(2)C(12) cells. Direct activation of p38 MAPK and JNK by anisomycin in the absence of TSA increased myostatin mRNA by fourfold. The phosphorylated form of the kinase MKK3/4/6 and ASK1, upstream cascades of p38 MAPK and JNK, also increased with TSA treatment. We concluded that the induction of myostatin by TSA treatment in differentiated C(2)C(12) cells is in part through ASK1-MKK3/6-p38 MAPK and ASK1-MKK4-JNK signaling pathways. Activation of p38 MAPK and JNK axis is necessary, but not sufficient for TSA-induced myostatin expression.

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

    Science.gov (United States)

    Choi, Yung Hyun

    2016-04-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-15

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

  11. Chromium Picolinate did not Effect on the Proliferation and Differentiation of Myoblasts

    Directory of Open Access Journals (Sweden)

    M. C. Tsa

    2007-01-01

    Full Text Available This experiment is conducted in vitro to investigate trivalent chromium picolinate affects the proliferation and differentiation of myoblasts. A myoblasts cell line (C2C12 from rats was used in the experiment. These were randomly divided into the control group, the Pic group (50ppb picolinate and the CrPic group (50ppb chromium picolinate. The differentiation of myoblasts reveals that the number of differentiated myotubes, creatine kinase (CK activity and the aldolase (ALB activity do not differ among the three groups (P > 0.05. The activity of hexokinase in the CrPic and Pic groups clearly exceeds that in the control group (P 0.05. Myoblast proliferation was the same across the three groups (P > 0.05, and the quantity of DNA in the control group exceeded that in the Pic group (P < 0.05. The experiment indicated that 200ppb chromium picolinate did not influence the proliferation and differentiation of myoblasts.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-01-17

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

  13. Induction of Bone Matrix Protein Expression by Native Bone Matrix Proteins in C2C12 Culture

    Institute of Scientific and Technical Information of China (English)

    ZHEN-MING HU; SEAN A. F. PEEL; STEPHEN K. C. HO; GEORGE K. B. SANDOR; CAMERON M. L. CLOKIE

    2009-01-01

    Objective To study the expression of bone matrix protein (BMP) induced by bovine bone morphogenetic proteins (BMPs) in vitro. Methods Type I collagen, osteopontin (OPN), osteonectin (ON), osteocalcin (OC), and bone sialoprotein (BSP) were detected by immunohistochemistry in C2C12 cultured from day 1 to day 28. Results The signaling of bone matrix protein expression became weaker except for type I collagen, OC and BSP after 5 days. Fourteen days after culture, the positive signaling of type I collagen, OPN, ON, OC, and BSP was gradually declined, and could be detected significantly as compared with that of the negative control on day 28. BMP assay showed that the Ikaline phosphatase (ALP) activity was higher in C2C12 culture than in the control during the 14-day culture. Also, total protein and DNA significantly increased during the 14-day culture. High levels of ALP were seen in preosteoblasts and osteoblsts in vivo and in differentiating ostcoblasts in vitro. ALP was well recognized as a marker reflecting osteoblastic activity. Conclusion Native bovine BMP induces conversion of myoblasts into osteoblasts, produces type 1 collagen, and plays significantly role in osteoinduction and bone matrix mineralization of C2C12 in vitro.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    reorganization due to the activity of the ubiquitous proteolytic enzymes, calpains, has been reported. Whether there is a link between stretch- or load-induced signaling and calpain expression and activation is not known. Using a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have...... demonstrated that mechanical stimulation via laminin receptors leads to an increase in m-calpain expression, but no increase in the expression of other calpain isoforms. Our study revealed that after a short period of stimulation, m-calpain relocates into focal adhesion complexes and is followed by a breakdown...... of specific focal adhesion proteins previously identified as substrates for this enzyme. We show that stimulation also leads to an increase in calpain activity in these cells. These data support the pivotal role for m-calpain in the control of muscle precursor cell differentiation and thus strengthen the idea...

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

    Directory of Open Access Journals (Sweden)

    Alexander Kleger

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2015-05-01

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

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

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    Isma Mian

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

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

    Science.gov (United States)

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

    2013-02-01

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

  2. Replication of prions in differentiated muscle cells.

    Science.gov (United States)

    Herbst, Allen; Aiken, Judd M; McKenzie, Debbie

    2014-01-01

    We have demonstrated that prions accumulate to high levels in non-proliferative C2C12 myotubes. C2C12 cells replicate as myoblasts but can be differentiated into myotubes. Earlier studies indicated that C2C12 myoblasts are not competent for prion replication. (1) We confirmed that observation and demonstrated, for the first time, that while replicative myoblasts do not accumulate PrP(Sc), differentiated post-mitotic myotube cultures replicate prions robustly. Here we extend our observations and describe the implication and utility of this system for replicating prions.

  3. Effect of cortisol on calpains in the C2C12 and 3T3-L1 cells.

    Science.gov (United States)

    Muthuraman, Pandurangan; Ravikumar, Sambandam; Muthuviveganandavel, Veerappan; Kim, Jongpil

    2014-03-01

    The present study was carried out to understand the effect of cortisol on calpain system in the C2C12 and 3T3-L1 adipocyte cells under co-culture system. Cells were co-cultured by using transwell inserts with a 0.4 μm porous membrane to separate C2C12 and 3T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates. Ten microgram per milliliter of cortisol was added to the medium. Following treatment for 3 days, the cells in the lower well were harvested for analysis. Calpains such as μ-calpain, m-calpain, and calpastatin were selected for the analysis. RT-PCR results indicated the significant increase in the mRNA expression of μ-calpain, m-calpain, and calpastatin. In addition, the confocal microscopical investigation indicated the cortisol treatment increases calpain expression in the C2C12 and 3T3-L1 cells. Taking all these together, cortisol treatment with co-culture system shows most reliable status of calpains expression in the cells, which is quite distinct from one-dimensional monocultured cells.

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

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    Jun Myoung Park

    2015-03-01

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

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

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    Masaki Horie

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

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

    Science.gov (United States)

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

    2017-01-01

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

  7. Lignan compounds and 4,4'-dihydroxybiphenyl protect C2C12 cells against damage from oxidative stress.

    Science.gov (United States)

    Yoshikawa, Ayumu; Saito, Yumiko; Maruyama, Kei

    2006-05-26

    Lignan compounds are known to have various biological activities, especially antioxidative effects. We investigated whether lignan compounds show antioxidative activity in myoblast C2C12 cells. Among 14 lignan compounds investigated, two lignans containing two phenolic functional groups, namely Gomisin J and GR-12, prevented hydrogen peroxide (H(2)O(2))-induced cell death. A simple compound, 4,4'-dihydroxybiphenyl, which was found to be a common component of Gomisin J and GR-12, also largely prevented H(2)O(2)-induced cell death and almost completely prevented H(2)O(2)-induced increases in p38 MAPK phosphorylation. Our present results provide a useful in vitro system for clarifying the molecular mechanisms of lignan-mediated antioxidative effects and evaluating lead molecules toward the development of therapeutic drugs.

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

    Science.gov (United States)

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

    2012-10-01

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

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

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    Mi Young Song

    2015-01-01

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

  10. Dynamics of the skeletal muscle secretome during myoblast differentiation

    DEFF Research Database (Denmark)

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

    2010-01-01

    of 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...... 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...

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

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    Manuel Antonio Riquelme

    2015-05-01

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

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

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    Gábor Oláh

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

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

    DEFF Research Database (Denmark)

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

    Abstract Mechanical Stimulation of C2C12 Cells Increases m-calpain Expression and Activity, Focal Adhesion Plaque Degradation and Cell Fusion A. Grossi, A. H. Karlsson, M. A. Lawson; Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark...... to the activity of ubiquitous proteolytic enzymes known as calpains has been reported. Whether there is a link between stretch- or load induced signaling and calpain expression and activation is not known. Using a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have demonstrated...... that mechanical stimulation via laminin receptors leads to an increase in m-calpain expression, but no increase in the expression of other calpain isoforms. Our study revealed that after a short period of stimulation, m-calpain relocates into focal adhesion complexes and is followed by a breakdown of specific...

  14. Functional aspects of dexamethasone upregulated nicotinic acetylcholine receptors in C2C12 myotubes

    NARCIS (Netherlands)

    Maestrone, E; Lagostena, L; Henning, RH; DenHertog, A; Nobile, M

    1995-01-01

    Three days of treatment with the glucocorticoid dexamethasone (1 nM-mu M) induced a concentration-dependent up-regulation of muscle nicotinic acetylcholine receptor (nAChR) in C2C12 mouse myotubes (EC(50)=10+/-7.3 nM), as assessed by [H-3]alpha-BuTx binding. The maximum increase in binding amounted

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-08-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-06-08

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

  17. Syncytin-1 in differentiating human myoblasts

    DEFF Research Database (Denmark)

    Bjerregard, Bolette; Ziomkiewicz, Iwona; Schulz, Alexander;

    2014-01-01

    and to myotubes. Weaker staining was present over intracellular vesicles and tubules. Caveolin-3 was detected in the sarcolemma and in vesicles and tubules in a subset of myoblasts and myotubes. The strongest staining occurred in multinucleated myotubes. Wide-field fluorescence microscopy indicated a partial...

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

    Energy Technology Data Exchange (ETDEWEB)

    Rovetta, Francesca [Unit of Biotechnologies, Department of Molecular and Translational Medicine, University of Brescia, Brescia I-25123 (Italy); Interuniversity Institute of Myology (IIM) (Italy); Stacchiotti, Alessandra [Institute of Human Anatomy, Department of Clinical and Experimental Sciences, University of Brescia, Brescia I-25123 (Italy); Faggi, Fiorella [Unit of Biotechnologies, Department of Molecular and Translational Medicine, University of Brescia, Brescia I-25123 (Italy); Interuniversity Institute of Myology (IIM) (Italy); Catalani, Simona; Apostoli, Pietro [Unit of Occupational Health and Industrial Hygiene, Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia, Brescia I-25123 (Italy); Fanzani, Alessandro, E-mail: fanzani@med.unibs.it [Unit of Biotechnologies, Department of Molecular and Translational Medicine, University of Brescia, Brescia I-25123 (Italy); Interuniversity Institute of Myology (IIM) (Italy); Aleo, Maria Francesca, E-mail: aleo@med.unibs.it [Unit of Biotechnologies, Department of Molecular and Translational Medicine, University of Brescia, Brescia I-25123 (Italy); Interuniversity Institute of Myology (IIM) (Italy)

    2013-09-01

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

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

    Science.gov (United States)

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

    2014-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Chunzi Liang

    2014-01-01

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

  1. Induced Differentiation of Adipose-derived Stromal Cells into Myoblasts

    Institute of Scientific and Technical Information of China (English)

    吴桂珠; 郑秀; 江忠清; 王金华; 宋岩峰

    2010-01-01

    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 i...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2002-12-02

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

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

    Directory of Open Access Journals (Sweden)

    Allen Herbst

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-03-22

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Miyake, Masato; Hayashi, Shinichiro; Iwasaki, Shunsuke; Chao, Guozheng; Takahashi, Hideyuki; Watanabe, Kouichi; Ohwada, Shyuichi; Aso, Hisashi [Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-Ku, Sendai 981-8555 (Japan); Yamaguchi, Takahiro, E-mail: ty1010@bios.tohoku.ac.jp [Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-Ku, Sendai 981-8555 (Japan)

    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. MicroRNA-17-92 regulates myoblast proliferation and differentiation by targeting the ENH1/Id1 signaling axis

    Science.gov (United States)

    Qiu, H; Liu, N; Luo, L; Zhong, J; Tang, Z; Kang, K; Qu, J; Peng, W; Liu, L; Li, L; Gou, D

    2016-01-01

    Myogenesis is an important biological process that occurs during both skeletal muscle regeneration and postnatal growth. Growing evidence points to the critical role of microRNAs (miRNAs) in myogenesis. Our analysis of miRNA expression patterns reveal that miRNAs of miR-17-92 cluster are dramatically downregulated in C2C12 cells after myogenesis stimulation, are strongly induced in mouse skeletal muscle after injury and decrease steadily thereafter and are downregulated with age in skeletal muscle during mouse and porcine postnatal growth. However, their roles in muscle developmental processes remain elusive. We show that the miR-17-92 cluster promotes mouse myoblast proliferation but inhibits myotube formation. miR-17, -20a and -92a target the actin-associated protein enigma homolog 1 (ENH1). The silencing of ENH1 increased the nuclear accumulation of the inhibitor of differentiation 1 (Id1) and represses myogenic differentiation. Furthermore, the injection of adenovirus expressing miR-20a into the tibialia anterior muscle downregulates ENH1 and delays regeneration. In addition, the downregulation of miR-17-92 during myogenesis is transcriptionally regulated by E2F1. Overall, our results reveal a E2F1/miR-17-92/ENH1/Id1 regulatory axis during myogenesis. PMID:27315298

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

    Science.gov (United States)

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

    2014-07-15

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

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

    Directory of Open Access Journals (Sweden)

    Julie eMcLean

    2014-12-01

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

  9. Multiple AMPK activators inhibit L-Carnitine uptake in C2C12 skeletal muscle myotubes.

    Science.gov (United States)

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

    2017-03-15

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

  10. Effect of dehydroepiandrosterone on insulin action and development of insulin-induced resistance in C2C12 muscle cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Dehydroepiandrosterone (DHEA), a precursor of androgens and estrogens, has been demonstrated to have effect of preventing insulin resistance and development of diabetes mellitus. Administration of testosterone appears to induce a marked insulin resistance. How these two hormones affect insulin resistance through regulation of sensitivity of tissues to insulin deserves further studies. Here, the effects of DHEA and testosterone on response to insulin in C2C12 muscle cells are analyzed. After 24 h of DHEA (10-6 mol/L) treatment, C2C12 cells showed an increased insulin- stimulated glucose uptake and enhanced activities of glycogen synthase (GS), phosphofructokinase (PFK) and pyruvate dehydrogenase (PDH), whereas testosterone gave the opposite effects. Incubation of C2C12 cells with high-dose insulin (5×10-7 mol/L) for 24 hours decreased their sensitivity to insulin and led to a state of resistance as assessed on insulin-stimulated glucose uptake and activities of GS, PFK and PDH. Addition of DHEA to insulin-resistant C2C12 cells could reverse the response of these cells to high-dose insulin, but testosterone could further impair insulin sensitivity in insulin-resistant C2C12 cells. These results suggest that the two hormones may influence the development or inhibition of insulin-resistance in type 2 diabetes through regulating glucose uptake, glycogenesis and glycolysis to some extent.

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

    Science.gov (United States)

    Pandurangan, Muthuraman; Kim, Doo Hwan

    2015-11-01

    The present study aimed to investigate the effect of ZnO nanoparticles on alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) enzyme expressions in C2C12 cells. ZnO nanoparticles are widely used in the several cosmetic lotions and other biomedical products. Several studies report on ZnO nanoparticle mediated cytotoxicity. However, there are no reports on the effect of ZnO nanoparticles on ALT, AST, ALP and LDH enzyme expressions in C2C12 cells. A cytotoxicity assay was carried out to determine the effect of ZnO nanoparticles (1-5 mg/ml) on C2C12 cell viability at 48 and 72 h. ZnO nanoparticles increased ALT, AST, ALP and LDH enzyme mRNA expression and their activities in C2C12 cells. In conclusion, the present study showed that ZnO nanoparticles increased these enzyme activities and its mRNA expression in C2C12 cells in a dose-dependent manner.

  12. Biocompatibility of Sylgard184 coated with different matrix materials and C2C12 cells%不同基质材料修饰的Sylgard184与C2C12细胞的相容性

    Institute of Scientific and Technical Information of China (English)

    王齐; 廖华; 秦建强; 余磊; 邱小忠; 于巧莲; 艾鹤英

    2009-01-01

    目的 筛选能提高硅酮橡胶弹性体(Sylgard184)与C2C12相容性的理想基质材料. 方法Sylgard184双组分以10:1的比例均匀混合,倒入6孔板的其中4孔,室温下静置固化,其余2孔做为空白对照培养组(A组);固化后的Sylgard184表面依次经过以下处理:I型胶原包被(B组)、层黏连蛋白包被(C组)、多聚赖氨酸包被(D组);未经包被(E组),每组共6个样本.在不同基质材料修饰的Sylgard 184表面培养C2C12细胞,利用倒置显微镜观察5组C2C12细胞的增殖、分化状态,流式细胞术(FCM)检测增殖培养48h后C2C12细胞的分裂增殖情况,RT-PCR检测增殖和分化培养48h后C2C12细胞内MyoD、myogenin mRNA的表达.结果 Sylgard184材料存在细胞毒性,E组接种的C2C12细胞在24h内全部漂浮死亡;D组的大多数细胞出现死亡,仅少数贴壁存活;而B、C两组材料包被后明显减少Syhgard 184的毒性,增强其表面与C2C12细胞的相容性,且C组细胞处于合成期的百分比以及增殖期的MyoD和分化期Myogenin基因mRNA的表达水平均显著高于A、B两组(P<0.05). 结论 经层黏连蛋白包被后的Sylgard184表面更有利于C2C12细胞的增殖及分化活性的表达.

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

    Science.gov (United States)

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

    2017-01-01

    Dysregulation of lipid metabolism in skeletal muscle is involved in the development of insulin resistance. Mutations in lipin-1, a key lipid metabolism regulator leads to significant systemic insulin resistance in fld mice. However, the function of lipin-1 on lipid metabolism and insulin sensitivity in skeletal muscle is still unclear. Herein we demonstrated that downregulation of lipin-1 in C2C12 myotubes by siRNA transfection suppressed insulin action, characterized by reduced insulin stimulated Akt phosphorylation and glucose uptake. Correspondingly, decreased lipin-1 expression was observed in palmitate-induced insulin resistance in C2C12 myotubes, suggested that lipin-1 might play a role in the etiology of insulin resistance in skeletal muscle. The insulin resistance induced by lipin-1 downregulation was related to the disturbance of lipid homeostasis. Lipin-1 silencing reduced intracellular DAG and TAG levels, but elevated ceramide accumulation in C2C12 myotubes. Moreover, the impaired insulin stimulated Akt phosphorylation and glucose uptake caused by lipin-1 silencing could be blocked by the pretreatment with SPT inhibitor myriocin, ceramide synthase inhibitor FB1, or PP2A inhibitor okadaic acid, suggested that the increased ceramide accumulation might be responsible for the development of insulin resistance induced by lipin-1 silencing in C2C12 myotubes. Meanwhile, decreased lipin-1 expression also impaired mitochondrial function in C2C12 myotubes. Therefore, our study suggests that lipin-1 plays an important role in lipid metabolism and downregulation of lipin-1 induces insulin resistance by increasing intracellular ceramide accumulation in C2C12 myotubes. These results offer a molecular insight into the role of lipin-1 in the development of insulin resistance in skeletal muscle. PMID:28123341

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

    Science.gov (United States)

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

    2017-04-01

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

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

    Science.gov (United States)

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

    2013-12-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  17. Substrate stiffness affects skeletal myoblast differentiation in vitro

    Science.gov (United States)

    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.

  18. Establishment and Identification of a Stable Human ASB12-Expressed C2C12 Cell Line%稳定表达人ASB12的C2C12细胞系的建立及鉴定

    Institute of Scientific and Technical Information of China (English)

    文斗斗; 周军媚; 赵明一; 胡维新; 吴秀山; 王跃群

    2012-01-01

    The human ASB12 (Homo sapiens ankyrin repeat and SOCS box containing 12) protein contains five ANK (ankyrin repeat sequence) domains and a SOCS (suppressor of cytokine signaling) box domain, belonging to the ASBs family. It was reported that ASB12 especially expressed in skeletal and cardiac muscles of adult tissues, which suggested that ASB12 closely associated with skeleton muscle development. To construct a stable ASB12-expressed C2C12 cell line, the fusion expression plasmid pCMV-tag2B-ASB12, which was identified by enzyme digestion and sequencing analysis, was transfected into C2C12 cell by cationic polymer. After screening culture by G418, the expression of ASB12 was detected by immunofluorescfence, RT-PCR and Western-blotting. The C2C12 cell line that expressing ASB12 stably was established successfully, which provide a cell model for studying the molecular function of ASB12 in skeleton muscle development.%ASB12 (homo sapiens ankyrin repeat and SOCS box containing 12)蛋白含有5个ANK (ankyrin repeat sequence)序列和一个保守的SOCS (suppressor of cytokine signaling)盒结构域,是ASBs (human ankyrin repeat and SOCS box containing protein family,ASB family)家族的成员.人类ASB12基因在成体心肌和骨骼肌组织中特异表达,是成肌分化的候选基因.利用阳离子聚合物转染技术将重组表达质粒pCMV-tag2B-ASB 12转染小鼠骨骼肌细胞系C2C12细胞,通过G418筛选、免疫荧光检测、RT-PCR分析、Western blotting检测建立了稳定表达ASB12的细胞系C2C12-ASB12,为研究ASB12在骨骼肌发育及其相关功能提供有用的细胞研究模型.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Cui Hao

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-02-08

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

  2. Construction of highly organized three-dimensional muscle tissue induced by C2C12 cells in vitro%C2C12细胞诱导构建三维骨骼肌组织

    Institute of Scientific and Technical Information of China (English)

    王齐; 廖华; 秦建强; 余磊; 艾鹤英; 汪海仪; 邱小忠

    2010-01-01

    目的 利用修饰并铸型后的Sylgard 184凹槽与C2C12细胞复合培养、诱导分化,获取三维极性骨骼肌组织. 方法 Sylgard 184双组分以10∶1的比例均匀混合并倒板,室温下静置固化并对其表面压槽铸型,Hank液冲洗凹槽,Matrigel和胶原的混合液均匀铺被凹槽底部,置生物安全柜待细胞基质自然干燥、紫外线照射消毒1h以上时接种C2C12细胞悬液,细胞增殖约80%汇合时改用分化培养基进行分化诱导,倒置显微镜下观察肌管的分化状态, RT-PCR方法检测肌管内myogenin和desmin基因mRNAs的表达,免疫荧光检测生肌转录因子myogenin和desmin蛋白的表达,扫描电镜观察肌管形态和肌管间的连接. 结果 C2C12细胞在Sylgard 184弹性体铸型压槽中培养分化7d后,倒置显微镜下可见肌管呈极性分化,且肌管之间融合紧密;21d后,扫描电镜检测可见肌管之间排列紧密且相互重叠,形成膜样结构,厚度可达0.15mm,具有三维性;RT-PCR、免疫荧光检测证实极性分化肌管内具有myogenin和desmin的阳性表达. 结论 修饰并铸型的Sylgard 184凹槽具有一定的方向引导效应,能促进C2C12细胞分化形成多核肌管,且肌管呈极性重叠排列,形成三维极性骨骼肌组织结构.

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

    Science.gov (United States)

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

    2015-10-01

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

  4. MicroRNA-494, upregulated by tumor necrosis factor-α, desensitizes insulin effect in C2C12 muscle cells.

    Directory of Open Access Journals (Sweden)

    Hyunjoo Lee

    Full Text Available Chronic inflammation is fundamental for the induction of insulin resistance in the muscle tissue of vertebrates. Although several miRNAs are thought to be involved in the development of insulin resistance, the role of miRNAs in the association between inflammation and insulin resistance in muscle tissue is poorly understood. Herein, we investigated the aberrant expression of miRNAs by conducting miRNA microarray analysis of TNF-α-treated mouse C2C12 myotubes. We identified two miRNAs that were upregulated and six that were downregulated by a >1.5-fold change compared to normal cells. Among the findings, qRT-PCR analysis confirmed that miR-494 is consistently upregulated by TNF-α-induced inflammation. Overexpression of miR-494 in CHO(IR/IRS1 and C2C12 myoblasts suppressed insulin action by down-regulating phosphorylations of GSK-3α/β, AS160 and p70S6K, downstream of Akt. Moreover, overexpression of miR-494 did not regulate TNF-α-mediated inflammation . Among genes bearing the seed site for miR-494, RT-PCR analysis showed that the expression of Stxbp5, an inhibitor of glucose transport, was downregulated following miR-494 inhibition. In contrast, the expression of PTEN decreased in the cells analyzed, thus showing that both positive and negative regulators of insulin action may be simultaneously controlled by miR-494. To investigate the overall effect of miR-494 on insulin signaling, we performed a PCR array analysis containing 84 genes related to the insulin signaling pathway, and we observed that 25% of genes were downregulated (P<0.05 and 11% were upregulated (P<0.05. These results confirm that miR-494 might contribute to insulin sensitivity by positive and negative regulation of the expression of diverse genes. Of note, PCR array data showed downregulation of Slc2A4, a coding gene for Glut4. Altogether, the present study concludes that the upregulation of miR-494 expression by TNF-α-mediated inflammation exacerbates insulin resistance

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

    Directory of Open Access Journals (Sweden)

    Lipinski M.

    2011-10-01

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

  6. ACTIVATION OF THE PHOSPHOLIPASE-C PATHWAY BY ATP IS MEDIATED EXCLUSIVELY THROUGH NUCLEOTIDE TYPE P2-PURINOCEPTORS IN C2C12 MYOTUBES

    NARCIS (Netherlands)

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

    1993-01-01

    1 The presence of a nucleotide receptor and a discrete ATP-sensitive receptor on C2C12 myotubes has been shown by electrophysiological experiments. In this study, the ATP-sensitive receptors of C2C12 myotubes were further characterized by measuring the formation of inositol(1,4,5)trisphosphate (Ins(

  7. Slowing Down Differentiation of Engrafted Human Myoblasts Into Immunodeficient Mice Correlates With Increased Proliferation and Migration

    Science.gov (United States)

    Riederer, Ingo; Negroni, Elisa; Bencze, Maximilien; Wolff, Annie; Aamiri, Ahmed; Di Santo, James P; Silva-Barbosa, Suse D.; Butler-Browne, Gillian; Savino, Wilson; Mouly, Vincent

    2012-01-01

    We have used a model of xenotransplantation in which human myoblasts were transplanted intramuscularly into immunodeficient Rag2-/-γC-/- mice, in order to investigate the kinetics of proliferation and differentiation of the transplanted cells. After injection, most of the human myoblasts had already differentiated by day 5. This differentiation correlated with reduction in proliferation and limited migration of the donor cells within the regenerating muscle. These results suggest that the precocious differentiation, already detected at 3 days postinjection, is a limiting factor for both the migration from the injection site and the participation of the donor cells to muscle regeneration. When we stimulated in vivo proliferation of human myoblasts, transplanting them in a serum-containing medium, we observed 5 days post-transplantation a delay of myogenic differentiation and an increase in cell numbers, which colonized a much larger area within the recipient's muscle. Importantly, these myoblasts maintained their ability to differentiate, since we found higher numbers of myofibers seen 1 month postengraftment, as compared to controls. Conceptually, these data suggest that in experimental myoblast transplantation, any intervention upon the donor cells and/or the recipient's microenvironment aimed at enhancing proliferation and migration should be done before differentiation of the implanted cells, e.g., day 3 postengraftment. PMID:21934656

  8. Slowing down differentiation of engrafted human myoblasts into immunodeficient mice correlates with increased proliferation and migration.

    Science.gov (United States)

    Riederer, Ingo; Negroni, Elisa; Bencze, Maximilien; Wolff, Annie; Aamiri, Ahmed; Di Santo, James P; Silva-Barbosa, Suse D; Butler-Browne, Gillian; Savino, Wilson; Mouly, Vincent

    2012-01-01

    We have used a model of xenotransplantation in which human myoblasts were transplanted intramuscularly into immunodeficient Rag2(-/-)γC(-/-) mice, in order to investigate the kinetics of proliferation and differentiation of the transplanted cells. After injection, most of the human myoblasts had already differentiated by day 5. This differentiation correlated with reduction in proliferation and limited migration of the donor cells within the regenerating muscle. These results suggest that the precocious differentiation, already detected at 3 days postinjection, is a limiting factor for both the migration from the injection site and the participation of the donor cells to muscle regeneration. When we stimulated in vivo proliferation of human myoblasts, transplanting them in a serum-containing medium, we observed 5 days post-transplantation a delay of myogenic differentiation and an increase in cell numbers, which colonized a much larger area within the recipient's muscle. Importantly, these myoblasts maintained their ability to differentiate, since we found higher numbers of myofibers seen 1 month postengraftment, as compared to controls. Conceptually, these data suggest that in experimental myoblast transplantation, any intervention upon the donor cells and/or the recipient's microenvironment aimed at enhancing proliferation and migration should be done before differentiation of the implanted cells, e.g., day 3 postengraftment.

  9. Cytotoxic and apoptotic effects of scorpion Leiurus quinquestriatus venom on 293T and C2C12 eukaryotic cell lines

    Directory of Open Access Journals (Sweden)

    M. A. A. Omran

    2003-01-01

    Full Text Available Scorpion venom toxicity is of major concern due to its influence on human activities and public health. The cytotoxicity and apoptosis induced by scorpion L. quinquestriatus venom on two established eukaryotic cell lines (293T and C2C12 were analyzed. Both cultured cell lines were incubated with varying doses (10, 20, and 50 µg/ml of scorpion venom in serum free medium (SFM for 0.5, 1, 2, 4, and 8 hours at 37°C. The percentage of total lactate dehydrogenase (LDH released in the culture during venom incubation was used as an index of cell damage. Control culture was treated with an equal amount of SFM. Cell injury was recognized morphologically and apoptosis was researched by a Fluorescing Apoptosis Detection System using the principle of TUNEL (TdT-mediated dUTP Nick-End Labelling assay and confirmed by another assay concerning nuclear DNA staining with DAPI stain. Cytotoxicity was remarkable and cell survival highly reduced at the highest tested concentration (50 µg/ml. These effects were rapid and observed within 30 minutes. The apparent initial damage to the nucleus and lysis of the plasmalemma and/or organelle membranes, which was evident by a significant increase in cytosolic LDH release, suggested that this toxin acts at the membrane level. The morphological changes that occurred in apoptotic cells include condensation and compartmentalization of nuclear and cytoplasmic materials into structurally preserved membrane-bound fragments or blebs. The cytotoxic effects are dose and time dependent and cell death by apoptosis was more characteristic of 293T cells than C2C12 cells. The apoptotic effects were more prominent and clear in the early stages of toxicity, while other forms of cell damage such as swelling, rupture, and/or necrosis occurred at later stages.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-07-17

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

  11. Nuclei of non-muscle cells bind centrosome proteins upon fusion with differentiating myoblasts.

    Directory of Open Access Journals (Sweden)

    Xavier Fant

    Full Text Available BACKGROUND: In differentiating myoblasts, the microtubule network is reorganized from a centrosome-bound, radial array into parallel fibres, aligned along the long axis of the cell. Concomitantly, proteins of the centrosome relocalize from the pericentriolar material to the outer surface of the nucleus. The mechanisms that govern this relocalization are largely unknown. METHODOLOGY: In this study, we perform experiments in vitro and in cell culture indicating that microtubule nucleation at the centrosome is reduced during myoblast differentiation, while nucleation at the nuclear surface increases. We show in heterologous cell fusion experiments, between cultures of differentiating mouse myoblasts and human cells of non-muscular origin, that nuclei from non-muscle cells recruit centrosome proteins once fused with the differentiating myoblasts. This recruitment still occurs in the presence of cycloheximide and thus appears to be independent of new protein biosynthesis. CONCLUSIONS: Altogether, our data suggest that nuclei of undifferentiated cells have the dormant potential to bind centrosome proteins, and that this potential becomes activated during myoblast differentiation.

  12. Changes in mitochondrial reactive oxygen species synthesis during differentiation of skeletal muscle cells.

    Science.gov (United States)

    Malinska, Dominika; Kudin, Alexei P; Bejtka, Malgorzata; Kunz, Wolfram S

    2012-01-01

    Myogenesis is accompanied by an intensive metabolic remodeling. We investigated the mitochondrial reactive oxygen species (ROS) generation at different levels of skeletal muscle differentiation: in C2C12 myoblasts, in C2C12 myotubes and in adult mouse skeletal muscle. Differentiation was accompanied by an increase in mitochondrial content and respiratory chain activity. The detected ROS production levels correlated with mitochondrial content, being the lowest in the myoblasts. Unlike the adult skeletal muscle, myoblast ROS production was significantly stimulated by the complex I inhibitor rotenone. Our results show that mitochondria are an important ROS source in skeletal muscle cells. The substantial changes in mitochondrial ROS synthesis during skeletal muscle differentiation can be explained by intensive bioenergetic remodeling.

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

    Science.gov (United States)

    Straadt, Ida K; Young, Jette F; Petersen, Bent O; Duus, Jens Ø; Gregersen, Niels; Bross, Peter; Oksbjerg, Niels; Bertram, Hanne C

    2010-06-01

    In the present study, the metabolic effects of heat and anoxic stress in myotubes from the mouse cell line C2C12 were investigated by using a combination of (13)C, (1)H, and (31)P nuclear magnetic resonance (NMR) spectroscopy and enrichment with [(13)C]-glucose. Both the (13)C and the (1)H NMR spectra showed reduced levels of the amino acids alanine, glutamate, and aspartate after heat or anoxic stress. The decreases were smallest at 42 degrees C, larger at 45 degrees C, and most pronounced after anoxic conditions. In addition, in both the (1)H and the (31)P NMR spectra, decreases in the high-energy phosphate compounds adenosine triphosphate and phosphocreatine with increasing severity of stress were identified. At anoxic conditions, an increase in (13)C-labeled lactate and appearance of glycerol-3-phosphate were observed. Accumulation of lactate and glycerol-3-phosphate is in agreement with a shift to anaerobic metabolism due to inhibition of the aerobic pathway in the mitochondria. Conversely, lower levels of unlabeled ((12)C) lactate were apparent at increasing severity of stress, which indicate that lactate is released from the myotubes to the medium. In conclusion, the metabolites identified in the present study may be useful markers for identifying severity of stress in muscles.

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

    KAUST Repository

    Ballarino, Monica

    2014-12-15

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

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

    Science.gov (United States)

    Gluck, George; Gilbert, Richard; Ortiz, Christine

    2002-03-01

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

  16. Differential regulation of iPLA2beta splice variants by in vitro ischemia in C2C12 myotubes

    DEFF Research Database (Denmark)

    Poulsen, K. A.; Kolko, M.; Lambert, I. H.

    2006-01-01

    in mice. Using PCR-cloning we identified a PCR-fragment that had a 29 bp insertion between exon 9 and 10. This sequence has high homology to the first part of the 53 bp human exon 9a. The 29 bp insertion induces a frame-shift and the introduction of a stop codon in exon 10. The protein product...

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-04-12

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

  19. Overexpression of insulin-like growth factor-II induces accelerated myoblast differentiation.

    Science.gov (United States)

    Stewart, C E; James, P L; Fant, M E; Rotwein, P

    1996-10-01

    Previous studies have shown that exogenous insulin-like growth factors (IGFs) can stimulate the terminal differentiation of skeletal myoblasts in culture and have established a correlation between the rate and the extent of IGF-II secretion by muscle cell lines and the rate of biochemical and morphological differentiation. To investigate the hypothesis that autocrine secretion of IGF-II plays a critical role in stimulating spontaneous myogenic differentiation in vitro, we have established C2 muscle cell lines that stably express a mouse IGF-II cDNA under control of the strong, constitutively active Moloney sarcoma virus promoter, enabling us to study directly the effects of IGF-II overproduction. Similar to observations with other muscle cell lines, IGF-II overexpressing myoblasts proliferated normally in growth medium containing 20% fetal serum, but they underwent enhanced differentiation compared with controls when incubated in low-serum differentiation medium. Accelerated differentiation of IGF-II overexpressing C2 cells was preceded by the rapid induction of myogenin mRNA and protein expression (within 1 h, compared with 24-48 h in controls) and was accompanied by an enhanced proportion of the retinoblastoma protein in an underphosphrylated and potentially active form, by a marked increase in activity of the muscle-specific enzyme, creatine phosphokinase, by extensive myotube formation by 48 h, and by elevated secretion of IGF binding protein-5 when compared with controls. These results confirm a role for IGF-II as an autocrine/paracrine differentiation factor for skeletal myoblasts, and they define a model cell system that will be useful in determining the biochemical mechanisms of IGF action in cellular differentiation.

  20. Functional KCa1.1 channels are crucial for regulating the proliferation, migration and differentiation of human primary skeletal myoblasts

    Science.gov (United States)

    Tajhya, Rajeev B; Hu, Xueyou; Tanner, Mark R; Huq, Redwan; Kongchan, Natee; Neilson, Joel R; Rodney, George G; Horrigan, Frank T; Timchenko, Lubov T; Beeton, Christine

    2016-01-01

    Myoblasts are mononucleated precursors of myofibers; they persist in mature skeletal muscles for growth and regeneration post injury. During myotonic dystrophy type 1 (DM1), a complex autosomal-dominant neuromuscular disease, the differentiation of skeletal myoblasts into functional myotubes is impaired, resulting in muscle wasting and weakness. The mechanisms leading to this altered differentiation are not fully understood. Here, we demonstrate that the calcium- and voltage-dependent potassium channel, KCa1.1 (BK, Slo1, KCNMA1), regulates myoblast proliferation, migration, and fusion. We also show a loss of plasma membrane expression of the pore-forming α subunit of KCa1.1 in DM1 myoblasts. Inhibiting the function of KCa1.1 in healthy myoblasts induced an increase in cytosolic calcium levels and altered nuclear factor kappa B (NFκB) levels without affecting cell survival. In these normal cells, KCa1.1 block resulted in enhanced proliferation and decreased matrix metalloproteinase secretion, migration, and myotube fusion, phenotypes all observed in DM1 myoblasts and associated with disease pathogenesis. In contrast, introducing functional KCa1.1 α-subunits into DM1 myoblasts normalized their proliferation and rescued expression of the late myogenic marker Mef2. Our results identify KCa1.1 channels as crucial regulators of skeletal myogenesis and suggest these channels as novel therapeutic targets in DM1. PMID:27763639

  1. Smad3 signaling is required for satellite cell function and myogenic differentiation of myoblasts

    Institute of Scientific and Technical Information of China (English)

    Xiaojia Ge; Ravi Kambadur; Craig McFarlane; Anuradha Vajjala; Sudarsanareddy Lokireddy; Zhi Hui Ng; Chek Kun Tan; Nguan Soon Tan; Walter Wahli; Mridula Sharma

    2011-01-01

    TGF-β and myostatin are the two most important regulators of muscle growth.Both growth factors have been shown to signal through a Smad3-dependent pathway.However to date,the role of Smad3 in muscle growth and differentiation is not investigated.Here,we demonstrate that Smad3-null mice have decreased muscle mass and pronounced skeletal muscle atrophy.Consistent with this,we also find increased protein ubiquitination and elevated levels of the ubiquitin E3 ligase MuRF1 in muscle tissue isolated from Smad3-null mice.Loss of Smad3 also led to defective satellite cell (SC) functionality.Smad3-null SCs showed reduced propensity for self-renewal,which may lead to a progressive loss of SC number.Indeed,decreased SC number was observed in skeletal muscle from Smad3- null mice showing signs of severe muscle wasting.Further in vitro analysis of primary myoblast cultures identified that Smad3-nuil myoblasts exhibit impaired proliferation,differentiation and fusion,resulting in the formation of atrophied myotubes.A search for the molecular mechanism revealed that loss of Smad3 results in increased myostatin expression in Smad3-null muscle and myoblasts.Given that myostatin is a negative regulator,we hypothesize that increased myostatin levels are responsible for the atrophic phenotype in Smad3-null mice.Consistent with this theory,inactivation of myostatin in Smad3-null mice rescues the muscle atrophy phenotype.

  2. Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts

    DEFF Research Database (Denmark)

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

    2015-01-01

    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...... 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...

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

    Directory of Open Access Journals (Sweden)

    Lomax Michael A

    2007-03-01

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

  4. Effect of fibroblast growth factor 9 on Runx2 gene promoter activity in MC3T3-E1 and C2C12 cells

    Institute of Scientific and Technical Information of China (English)

    YU Li-yun; PEI Yu; XIA Wei-bo; XING Xiao-ping; MENG Xun-wu; ZHOU Xue-ying

    2007-01-01

    Background Fibroblast growth factor 9 (FGF9), expressed in brain, kidney and developing skeletal tissues, can physiologically inhibit endochondral ossification; but little is known about how FGF9 affects osteoblasts and its detailed regulatory mechanism. Here we examined the effect of FGF9 on the activity of the murine Runt-related transcription factor2 (Runx2) gene promoter in preosteoblast MC3T3-E1 and premyoblast C2C12 cells.Methods Plasmids containing the Runx2 promoter region were transfected into MC3T3-E1 and C2C12 cells and stably transfected cell lines were established. The method of luciferase reporter gene activation was used to examine the effects of FGF9 on the promoter activity.Results FGF9 (10 ng/ml) increased Runx2 promoter activity in MC3T3-E1 cells. When MC3T3-E1 cells were treated with FGF9 plus the various inhibitors or activator of the intracellular signaling transducation pathways, including 10μmol/L U0126 (the inhibitor of mitogen-activated protein kinase kinase), 10 μmol/L SB203580 (the inhibitor of p38/mitogen activated protein kinase), or 1 μmol/L C6 ceramide (an activator of mitogen activated protein kinase), the luciferase expression did not change significantly compared with that of the cells treated with FGF9 only. However, when C2C12 cells were treated with 10 ng/ml FGF9, Runx2 gene promoter activity first decreased and then increased over a period of 1 to 5 days. Among the above inhibitors, only U0126 (10 μmol/L) completely blocked the effects of FGF9 on Runx2 gene promoter activity.Conclusions Our data showed that FGF9 can affect Runx2 gene promoter activity in MC3T3-E1 and C2C12 cells. The action of FGF9 appears to depend partly on the mitogen-activated protein kinase kinase/mitogen-activated protein kinase pathways in C2C12 cells.

  5. Critical Role Played by Cyclin D3 in the MyoD-Mediated Arrest of Cell Cycle during Myoblast Differentiation

    OpenAIRE

    1999-01-01

    During the terminal differentiation of skeletal myoblasts, the activities of myogenic factors regulate not only tissue-specific gene expressions but also the exit from the cell cycle. The induction of cell cycle inhibitors such as p21 and pRb has been shown to play a prominent role in the growth arrest of differentiating myoblasts. Here we report that, at the onset of differentiation, activation by MyoD of the Rb, p21, and cyclin D3 genes occurs in the absence of new protein synthesis and wit...

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

    Science.gov (United States)

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

    2013-06-01

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

  7. 红色发光二极管照射对C2C12细胞增殖的光生物调节作用%Photobiomodulation of red light emitting diodes in C2C12 cell proliferation

    Institute of Scientific and Technical Information of China (English)

    刘江; 陈小莹; 刘承宜; 王双喜; 郭红; 徐晓阳; 邓小元; 刘颂豪

    2006-01-01

    目的:采用小鼠成肌细胞C2C12作为模型,观察光生物调节作用对他汀类药物引起的肌病的作用.方法:实验于2004-09/2005-01在华南师范大学激光运动医学实验室完成.C2C12细胞用浓度分别为2.0×10-5,2.0×10-6,2.0×10-7,2.0×10-8mol/L的辛伐他汀培养,然后用强度分别为0,0.229,0.506,0.848,1.401,1.670 mW/cm2的红色发光二极管[波长(640±15)nm]照射2 d,15 min/d.用甲基噻唑基四唑比色法评价细胞增殖.结果:浓度为2.0×10-6,2.0×10-7,2.0×10-8 mol/L的辛伐他汀对C2C12的增殖没有影响,无光生物调节作用;浓度为2.0×10-5 mol/L的辛伐他汀抑制C2C12的增殖,发光二极管强度为0,0.229,0.506,0.848,1.401,1.670 mW/cm2时C2C12细胞增殖吸光度百分率分别降为(37.2±8.4)%,(58.4±24.9)%,(37.0±8.6)%,(63.0±8.8)%,(59.2±12.6)%,(28.9±20.3)%.强度为0.848 mW/cm2的红色发光二极管照射2 d,15 min/d可促进被抑制的C2C12增殖效应.结论:红色发光二极管可以促进被辛伐他汀抑制的C2C12细胞的增殖作用,对服用他汀类药物引起的肌病可能有光生物调节作用.

  8. Effects of Sunphenon and Polyphenon 60 on proteolytic pathways, inflammatory cytokines and myogenic markers in H22-treated C2C12 cells

    Indian Academy of Sciences (India)

    Allur Subramaniyan Sivakumar; Inho Hwang

    2015-03-01

    The effect of Sunphenon and Polyphenon 60 in oxidative stress response, myogenic regulatory factors, inflammatory cytokines, apoptotic and proteolytic pathways on H2O2-induced myotube atrophy was addressed. Cellular responses of H2O2-induced C2C12cells were examined, including mRNA expression of myogenic regulatory factors, such as MyoD and myogenin, inflammatory pathways, such as TNF- and NF-kB, as well as proteolytic enzymes, such as -calpain and m-calpain. The pre-treatment of Sunphenon (50 g/mL)/Polyphenon 60 (50 g/mL) on H2O2-treated C2C12 cells significantly down-regulated the mRNA expression of myogenin and MyoD when compared to those treated with H2O2-induced alone. Additionally, the mRNA expression of -calpain and m-calpain were significantly ( < 0.05) increased in H2O2-treated C2C12 cells, whereas pre-treatment with Sunphenon/Polyphenon significantly down-regulated the above genes, namely -calpain and m-calpain. Furthermore, the mRNA expression of TNF- and NF-kB were significantly increased in H2O2-treated C2C12 cells, while pre-treatment with Sunphenon (50 g/mL)/Polyphenon 60 (50 g/mL) significantly ( < 0.05) down-regulated it when compared to the untreated control group. Subsequent analysis of DNA degeneration and caspase activation revealed that Sunphenon (50 g/mL)/Polyphenon 60 (50 g/mL) inhibited activation of caspase-3 and showed an inhibitory effect on DNA degradation. From this result, we know that, in stress conditions, -calpain may be involved in the muscle atrophy through the suppression of myogenin and MyoD. Moreover, Sunphenon may regulate the skeletal muscle genes/promote skeletal muscle recovery by the up-regulation of myogenin and MyoD and suppression of -calpain and inflammatory pathways and may regulate the apoptosis pathways. Our findings suggest that dietary supplementation of Sunphenon might reduce inflammatory events in muscle-associated diseases, such as myotube atrophy.

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

    OpenAIRE

    Wang, Yanxin; Watford, Malcolm

    2006-01-01

    The cell-specific regulation of glutamine synthetase expression was studied in three cell lines. In C2C12 myotubes, glucocorticoids increased the abundance of both glutamine synthetase protein and mRNA. Culture in the absence of glutamine also resulted in very high glutamine synthetase protein abundance but mRNA levels were unchanged. Glucocorticoids also increased the abundance of glutamine synthetase mRNA in Hep G2 hepatoma cells but this was not reflected in changes in protein abundance. C...

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

    Science.gov (United States)

    Wang, Yanxin; Watford, Malcolm

    2007-04-01

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

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

    Science.gov (United States)

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

    2016-01-04

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

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

    Directory of Open Access Journals (Sweden)

    Tisdale Michael J

    2008-01-01

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

  13. Role of miR-181a-5p and endoplasmic reticulum stress in the regulation of myogenic differentiation.

    Science.gov (United States)

    Wei, Yingying; Tao, Xuelian; Xu, Huaming; Chen, Yan; Zhu, Li; Tang, Guoqing; Li, Mingzhou; Jiang, Anan; Shuai, Surong; Ma, Jideng; Jin, Long; Wen, Anxiang; Wang, Qin; Zhu, Guangxiang; Xie, Meng; Wu, Jiayun; He, Tao; Jiang, Yanzhi; Li, Xuewei

    2016-10-30

    Accumulating evidence has indicated that microRNAs (miRNAs) and endoplasmic reticulum (ER) stress play critical roles in myoblast differentiation. However, the regulation roles of miRNAs and ER stress in myogenic differentiation have not been fully revealed and need to be further studied. Here, we discovered that the expression levels of miR-181a-5p were strongly upregulated during C2C12 cell differentiation. miR-181a-5p overexpression promoted ER stress and differentiation of C2C12 cells, which was accompanied by increasing expression levels of marker genes related to ER stress-mediated apoptosis and myogenic differentiation. Opposite results were observed after inhibition of the miR-181a-5p expression. The gain- and loss-of-function experiments on C2C12 cells showed that miR-181a-5p affected the development of muscle fiber type, but had no significant influence on C2C12 cell proliferation. In the ER-stressed C2C12 cells induced by thapsigargin (Tg), the expression levels of both miR-181a-5p and marker genes related to ER stress and myogenesis were upregulated. In the ER-stressed C2C12 cells and porcine muscle fibroblast (PMF) cells pretreated with Tg, we found that miR-181a-5p targeted glucose-regulated protein, 78kDa/binding immunoglobulin protein (GRP78/BIP), and influenced cell apoptosis. In conclusion, these results indicate that miR-181a-5p and ER stress have positive synergistic effects on myogenic differentiation by increasing the expression levels of myogenic differentiation key genes and activating the ER stress-mediated apoptosis signaling pathway.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    In this study, stress in relation to slaughter was investigated in a model system by the use of (13)C, (1)H, and (31)P nuclear magnetic resonance (NMR) spectroscopy for elucidating changes in the metabolites in C2C12 myotubes exposed to H(2)O(2)-induced stress. Oxidative stress resulted in lower...... to lower levels of the unlabeled ((12)C) lactate were identified in the (1)H spectra after stress exposure. These data indicate an increase in de novo synthesis of alanine, concomitant with a release of lactate from the myotubes to the medium at oxidative stress conditions. The changes in the metabolite...... levels of several metabolites, mainly amino acids; however, higher levels of alanine were apparent in the (13)C spectra after incubation with [(13)C(1)]glucose. In the (13)C spectra [(13)C(3)]lactate tended to increase after exposure to increasing concentrations of H(2)O(2); conversely, a tendency...

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

    Directory of Open Access Journals (Sweden)

    Cui-Ping Zhang

    2014-01-01

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

  16. 转染脂联素cDNA对骨骼肌细胞株C2C12肌小管葡萄糖氧化和糖原合成的影响%Effects of transfection with adiponectin cDNA on glycogen synthesis and glucose oxidation in myotubes of skeletal muscle cell strain C2C12

    Institute of Scientific and Technical Information of China (English)

    张淼; 李芳萍; 杨川; 钱艳; 刘丹; 傅祖植

    2007-01-01

    背景:脂联素具有降低血脂、血糖,改善胰岛索敏感性的作用,但脂联素对骨骼肌糖代谢的影响尚无定论.目的:通过转染带小鼠脂联素基因的质粒,观察真核表达的脂联素对骨骼肌细胞株C2C12肌小管葡萄糖氧化和糖原合成的影响.设计:对照实验.单位:中山大学附属第二医院.材料:带小鼠脂联素cDNA的pcDNA3.0质粒,即pcDNA3.0-mad(由Dr.Da-Wei Gong惠赠,University of Maryland),C2C12细胞株(ATCC,CRL-1722),rabbit anti-mouse脂联素 IgG(ACRP303-A,Alpha Diagnostic International),SABC即用型免疫组化试剂盒(博士德),D-[U-14C]葡萄糖(放射比活度9.25~13.32 GBq/mmol,NEC),闪烁液POP,POPOP(Sigma),液体闪烁计数器(LS3801,Beckman,USA).方法:实验于2003-03/08在中山大学附属第二医院中心实验室完成.①pcDNA3.0-mad质粒抽提后采用XhoⅠ和XbaⅠ双酶切,及HindⅢ单酶切鉴定.②用阳离子脂质体介导分别将pcDNA3.0-mad和pcDNA3.0空载质粒转染至C2C12细胞,并通过G418(500 mg/L)筛选3周,收集G418抗性的转染后C2C12细胞,分别建立稳定转染pcDNA3.0-mad和pcDNA3.0的C2C12细胞株.③通过Western blot和免疫组化,鉴定C2C12细胞组、稳定转染pcDNA3.0和pcDNA3.0-mad的C2C12细胞组中有无脂联素蛋白表达.④C2C12肌细胞糖代谢实验分对照组、载体组和pcDNA3.0-mad组3组进行,每组又分别加入0,0.5,5,100 nmol/L胰岛素刺激4个浓度进行观察.在加入含D-[U-14C]葡萄糖的MEM培养液孵育一定时间后,通过液闪测定细胞株C2C12肌小管糖原合成和葡萄糖氧化情况.主要观察指标:稳定转染带小鼠脂联素基因的质粒后,骨骼肌细胞株C2C12肌小管葡萄糖氧化和糖原合成量的变化.结果:①质粒转化及酶切鉴定结果:pcDNA3.0-mad质粒抽提后采用XbaⅠ和XhoⅠ双酶切,及HindⅢ单酶切鉴定.得到的质粒酶切片段与预期相符,脂联素cDNA片段长度为781 bp,质粒片段长度为5 446 bp,脂联素cDNA被插

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

    Directory of Open Access Journals (Sweden)

    Hristina Obradović

    2016-01-01

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

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

    Science.gov (United States)

    Straadt, Ida K; Young, Jette F; Petersen, Bent O; Duus, Jens Ø; Gregersen, Niels; Bross, Peter; Oksbjerg, Niels; Theil, Peter K; Bertram, Hanne C

    2010-02-10

    In this study, stress in relation to slaughter was investigated in a model system by the use of (13)C, (1)H, and (31)P nuclear magnetic resonance (NMR) spectroscopy for elucidating changes in the metabolites in C2C12 myotubes exposed to H(2)O(2)-induced stress. Oxidative stress resulted in lower levels of several metabolites, mainly amino acids; however, higher levels of alanine were apparent in the (13)C spectra after incubation with [(13)C(1)]glucose. In the (13)C spectra [(13)C(3)]lactate tended to increase after exposure to increasing concentrations of H(2)O(2); conversely, a tendency to lower levels of the unlabeled ((12)C) lactate were identified in the (1)H spectra after stress exposure. These data indicate an increase in de novo synthesis of alanine, concomitant with a release of lactate from the myotubes to the medium at oxidative stress conditions. The changes in the metabolite levels could possibly be useful as markers for meat quality traits.

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

    Science.gov (United States)

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

    2012-12-01

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

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

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    Yan Shen

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

  1. Proinflammatory Macrophages Enhance the Regenerative Capacity of Human Myoblasts by Modifying Their Kinetics of Proliferation and Differentiation

    Science.gov (United States)

    Bencze, Maximilien; Negroni, Elisa; Vallese, Denis; Yacoub–Youssef, Houda; Chaouch, Soraya; Wolff, Annie; Aamiri, Ahmed; Di Santo, James P; Chazaud, Bénédicte; Butler-Browne, Gillian; Savino, Wilson; Mouly, Vincent; Riederer, Ingo

    2012-01-01

    Macrophages have been shown to be essential for muscle repair by delivering trophic cues to growing skeletal muscle precursors and young fibers. Here, we investigated whether human macrophages, either proinflammatory or anti-inflammatory, coinjected with human myoblasts into regenerating muscle of Rag2−/− γC−/− immunodeficient mice, could modify in vivo the kinetics of proliferation and differentiation of the transplanted human myogenic precursors. Our results clearly show that proinflammatory macrophages improve in vivo the participation of injected myoblasts to host muscle regeneration, extending the window of proliferation, increasing migration, and delaying differentiation. Interestingly, immunostaining of transplanted proinflammatory macrophages at different time points strongly suggests that these cells are able to switch to an anti-inflammatory phenotype in vivo, which then may stimulate differentiation during muscle regeneration. Conceptually, our data provide for the first time in vivo evidence strongly suggesting that proinflammatory macrophages play a supportive role in the regulation of myoblast behavior after transplantation into preinjured muscle, and could thus potentially optimize transplantation of myogenic progenitors in the context of cell therapy. PMID:23070116

  2. Proinflammatory macrophages enhance the regenerative capacity of human myoblasts by modifying their kinetics of proliferation and differentiation.

    Science.gov (United States)

    Bencze, Maximilien; Negroni, Elisa; Vallese, Denis; Yacoub-Youssef, Houda; Chaouch, Soraya; Wolff, Annie; Aamiri, Ahmed; Di Santo, James P; Chazaud, Bénédicte; Butler-Browne, Gillian; Savino, Wilson; Mouly, Vincent; Riederer, Ingo

    2012-11-01

    Macrophages have been shown to be essential for muscle repair by delivering trophic cues to growing skeletal muscle precursors and young fibers. Here, we investigated whether human macrophages, either proinflammatory or anti-inflammatory, coinjected with human myoblasts into regenerating muscle of Rag2(-/-) γC(-/-) immunodeficient mice, could modify in vivo the kinetics of proliferation and differentiation of the transplanted human myogenic precursors. Our results clearly show that proinflammatory macrophages improve in vivo the participation of injected myoblasts to host muscle regeneration, extending the window of proliferation, increasing migration, and delaying differentiation. Interestingly, immunostaining of transplanted proinflammatory macrophages at different time points strongly suggests that these cells are able to switch to an anti-inflammatory phenotype in vivo, which then may stimulate differentiation during muscle regeneration. Conceptually, our data provide for the first time in vivo evidence strongly suggesting that proinflammatory macrophages play a supportive role in the regulation of myoblast behavior after transplantation into preinjured muscle, and could thus potentially optimize transplantation of myogenic progenitors in the context of cell therapy.

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

    Science.gov (United States)

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

    2012-11-01

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

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

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    Lan eYe

    2012-09-01

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

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

    Science.gov (United States)

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

    2011-11-01

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

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

    Science.gov (United States)

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

    2014-02-01

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

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  8. PARP1 Differentially Interacts with Promoter region of DUX4 Gene in FSHD Myoblasts

    Science.gov (United States)

    Sharma, Vishakha; Pandey, Sachchida Nand; Khawaja, Hunain; Brown, Kristy J; Hathout, Yetrib; Chen, Yi-Wen

    2016-01-01

    Objective The goal of the study is to identity proteins, which interact with the promoter region of double homeobox protein 4 (DUX4) gene known to be causative for the autosomal dominant disorder Facioscapulohumeral Muscular Dystrophy (FSHD). Methods We performed a DNA pull down assay coupled with mass spectrometry analysis to identify proteins that interact with a DUX4 promoter probe in Rhabdomyosarcomca (RD) cells. We selected the top ranked protein poly (ADP-ribose) polymerase 1 (PARP1) from our mass spectrometry data for further ChIP-qPCR validation using patients' myoblasts. We then treated FSHD myoblasts with PARP1 inhibitors to investigate the role of PARP1 in the FSHD myoblasts. Results In our mass spectrometry analysis, PARP1 was found to be the top ranked protein interacting preferentially with the DUX4 promoter probe in RD cells. We further validated this interaction by immunoblotting in RD cells (2-fold enrichment compared to proteins pulled down by a control probe, pfisetin (0.5 mM), a polyphenol compound with PARP1 inhibitory property, for 24 h also suppressed the expression of DUX4 (44.8 fold, p<0.01) and ZSCAN4 (2.2 fold, p<0.05) in the FSHD myoblasts. We further showed that DNA methyltransferase 1 (DNMT1), a gene regulated by PARP1 was also enriched at the DUX4 promoter in RD cells through immunoblotting (2-fold, p<0.01) and immortalized FSHD myoblasts (42-fold, p<0.01) but not control myoblasts through ChIP qPCR. Conclusion Our results showed that PARP1 and DNMT1 interacted with DUX4 promoter and may be involved in modulating DUX4 expression in FSHD. PMID:27722032

  9. Macrophage colony-stimulating factor-induced macrophage differentiation promotes regrowth in atrophied skeletal muscles and C2C12 myotubes.

    Science.gov (United States)

    Dumont, Nicolas A; Frenette, Jérôme

    2013-02-01

    Skeletal muscle injury and regeneration are closely associated with an inflammatory reaction that is usually characterized by sequential recruitment of neutrophils and monocytes or macrophages. Selective macrophage depletion models have shown that macrophages are essential for complete regeneration of muscle fibers after freeze injuries, toxin injuries, ischemia-reperfusion, and hindlimb unloading and reloading. Although there is growing evidence that macrophages possess major myogenic capacities, it is not known whether the positive effects of macrophages can be optimized to stimulate muscle regrowth. We used in vivo and in vitro mouse models of atrophy to investigate the effects of stimulating macrophages with macrophage colony-stimulating factor (M-CSF) on muscle regrowth. When atrophied soleus muscles were injected intramuscularly with M-CSF, we observed a 1.6-fold increase in macrophage density and a faster recovery in muscle force (20%), combined with an increase in muscle fiber diameter (10%), after 7 days of reloading, compared with PBS-injected soleus muscles. Furthermore, coculture of atrophied myotubes with or without bone marrow-derived macrophages (BMDM) and/or M-CSF revealed that the combination of BMDMs and M-CSF was required to promote myotube growth (15%). More specifically, M-CSF promoted the anti-inflammatory macrophage phenotype, which in turn decreased protein degradation and MuRF-1 expression by 25% in growing myotubes. These results indicate that specific macrophage subsets can be stimulated to promote muscle cell regrowth after atrophy.

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

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    Shy Cian Khor

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

  11. 外源性分化抑制因子Id2在C2C12细胞中的表达%The expression of external Id2 protein gene containing green fluorescence in C2C12 cells

    Institute of Scientific and Technical Information of China (English)

    赖桂华; 余磊; 张黎声; 欧阳钧; 邱小忠

    2012-01-01

    目的:构建大鼠Id2基因真核荧光表达载体,并观察外源性Id2基因C2C12细胞中的表达.方法:RT-PCR扩增出Id2全长cDNA,T4 DNA连接酶将载体pGEM-T和Id2 cDNA进行连接,构建克隆载体,经限制性内切酶EcoR I酶切pGEM-Id2克隆载体和pEGFP-C2真核表达载体,构建出重组真核表达载体pEGFP-C2-Id2,经酶切分析、PCR鉴定及DNA测序证实cDNA片段大小和序列的正确性;通过电穿孔转染法将外源性Id2基因导入C2C12成肌细胞中.分别于转染4、8、12、24、36、72 h后通过荧光倒置显微镜下观察细胞整体情况,并计算转染效率.结果:经酶切分析和序列测定证实pEGFP-C2-Id2含大小正确的正向Id2 cDNA片段,获得高转染率和高表达外源性Id2基因的C2C12细胞,转染8h时,转染效率约为(10.5±2.8)%;转染12 h后,转染效率约为(20.9±3.1)%;转染24 h后,转染效率最高,约为(60.8±3.2)%.结论:成功构建了同时携带有G418筛选位点和Id2基因的真核表达载体;并获得高表达外源性Id2基因的C2C12细胞.%Objective:To construct the eukaryotic expression vector of rat Id2 and to observe the expression of 1(12 in CZC|2 cells for further study on skeletal muscle regeneration. Methods; RT-PCR method was used to amplify the entire Id2 cDNA. The pGEM-T and Id2 cDNA were ligated by T4 DNA ligase. The cloning vectors and the pEGFP-C2 (eukaryotic expression vector) were first cut by EcoR I and then ligated with Id2 by T4 DNA ligase again. The enzyme analysis and DNA sequencing were used to confirm the recombined vectors. The pEGFP-C2-Id2 vectors were transferred into C2C,2 cells by electric perforation. Fluorescence inverted microscopy was used to observe the global growth of the cells and to calculate the transfection efficiency 4,8,12,24,36 and 72 hours post-transfection. Results:The enzyme analysis and DNA sequencing analysis confirmed that the right Id2 gene was cloned. The Id2 transferred C2C12 cells with high expression and high

  12. Induction of group VIA phospholipase A2 activity during in vitro ischemia in C2C12 myotubes is associated with changes in the level of its splice variants

    DEFF Research Database (Denmark)

    Poulsen, K A; Petersen, Stine Helene Falsig; Kolko, M;

    2007-01-01

    The involvement of group VI Ca(2+)-independent PLA(2)s (iPLA(2)-VI) in in vitro ischemia [oxygen and glucose deprivation (OGD)] in mouse C2C12 myotubes was investigated. OGD induced a time-dependent (0-6 h) increase in bromoenol lactone (BEL)-sensitive iPLA(2) activity, which was suppressed...

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

    Directory of Open Access Journals (Sweden)

    Cherubino Paolo

    2005-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Shruti Haralalka

    Full Text Available 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

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

    Science.gov (United States)

    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

  16. Human myoblast genome therapy

    Institute of Scientific and Technical Information of China (English)

    Peter K Law; Leo A Bockeria; Choong-Chin Liew; Danlin M Law; Ping Lu; Eugene KW Sim; Khawja H Haider; Lei Ye; Xun Li; Margarita N Vakhromeeva; Ilia I Berishvili

    2006-01-01

    Human Myoblast Genome Therapy (HMGT) is a platform technology of cell transplantation, nuclear transfer, and tissue engineering. Unlike stem cells, myoblasts are differentiated, immature cells destined to become muscles. Myoblasts cultured from satellite cells of adult muscle biopsies survive, develop, and function to revitalize degenerative muscles upon transplantation. Injection injury activates regeneration of host myofibers that fuse with the engrafted myoblasts, sharing their nuclei in a common gene pool of the syncytium. Thus, through nuclear transfer and complementation, the normal human genome can be transferred into muscles of patients with genetic disorders to achieve phenotype repair or disease prevention. Myoblasts are safe and efficient gene transfer vehicles endogenous to muscles that constitute 50% of body weight. Results of over 280 HMGT procedures on Duchenne Muscular Dystrophy (DMD) subjects in the past 15 years demonstrated absolute safety. Myoblast-injected DMD muscles showed improved histology.Strength increase at 18 months post-operatively averaged 123%. In another application of HMGT on ischemic cardiomyopathy, the first human myoblast transfer into porcine myocardium revealed that it was safe and effective. Clinical trials on approximately 220 severe cardiomyopathy patients in 15 countries showed a <10% mortality. Most subjects received autologous cells implanted on the epicardial surface during coronory artery bypass graft, or injected on the endomyocardial surface percutaneously through guiding catheters. Significant increases in left ventricular ejection fraction, wall thickness, and wall motion have been reported, with reduction in perfusion defective areas, angina, and shortness of breath. As a new modality of treatment for disease in the skeletal muscle or myocardium, HMGT emerged as safe and effective. Large randomized multi-center trials are under way to confirm these preliminary results. The future of HMGT is bright and exciting

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

    Directory of Open Access Journals (Sweden)

    Stefania Rossi

    2010-01-01

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

  18. Celf1 regulates cell cycle and is partially responsible for defective myoblast differentiation in myotonic dystrophy RNA toxicity.

    Science.gov (United States)

    Peng, Xiaoping; Shen, Xiaopeng; Chen, Xuanying; Liang, Rui; Azares, Alon R; Liu, Yu

    2015-07-01

    Myotonic dystrophy is a neuromuscular disease of RNA toxicity. The disease gene DMPK harbors expanded CTG trinucleotide repeats on its 3'-UTR. The transcripts of this mutant DMPK led to misregulation of RNA-binding proteins including MBNL1 and Celf1. In myoblasts, CUG-expansion impaired terminal differentiation. In this study, we formally tested how the abundance of Celf1 regulates normal myocyte differentiation, and how Celf1 expression level mediates CUG-expansion RNA toxicity-triggered impairment of myocyte differentiation. As the results, overexpression of Celf1 largely recapitulated the defects of myocytes with CUG-expansion, by increasing myocyte cycling. Knockdown of endogenous Celf1 level led to precocious myotube formation, supporting a negative connection between Celf1 abundance and myocyte terminal differentiation. Finally, knockdown of Celf1 in myocyte with CUG-expansion led to partial rescue, by promoting cell cycle exit. Our results suggest that Celf1 plays a distinctive and negative role in terminal myocyte differentiation, which partially contribute to DM1 RNA toxicity. Targeting Celf1 may be a valid strategy in correcting DM1 muscle phenotypes, especially for congenital cases.

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

    Directory of Open Access Journals (Sweden)

    Chiara Sassoli

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

  20. Bone Marrow Mesenchymal Stromal Cells Stimulate Skeletal Myoblast Proliferation through the Paracrine Release of VEGF

    Science.gov (United States)

    Chellini, Flaminia; Mazzanti, Benedetta; Nistri, Silvia; Nosi, Daniele; Saccardi, Riccardo; Quercioli, Franco; Zecchi-Orlandini, Sandra; Formigli, Lucia

    2012-01-01

    Mesenchymal stromal cells (MSCs) are the leading cell candidates in the field of regenerative medicine. These cells have also been successfully used to improve skeletal muscle repair/regeneration; however, the mechanisms responsible for their beneficial effects remain to be clarified. On this basis, in the present study, we evaluated in a co-culture system, the ability of bone-marrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. We found that myoblast proliferation was greatly enhanced in the co-culture as judged by time lapse videomicroscopy, cyclin A expression and EdU incorporation. Moreover, myoblasts immunomagnetically separated from MSCs after co-culture expressed higher mRNA and protein levels of Notch-1, a key determinant of myoblast activation and proliferation, as compared with the single culture. Notch-1 intracellular domain and nuclear localization of Hes-1, a Notch-1 target gene, were also increased in the co-culture. Interestingly, the myoblastic response was mainly dependent on the paracrine release of vascular endothelial growth factor (VEGF) by MSCs. Indeed, the addition of MSC-derived conditioned medium (CM) to C2C12 cells yielded similar results as those observed in the co-culture and increased the phosphorylation and expression levels of VEGFR. The treatment with the selective pharmacological VEGFR inhibitor, KRN633, resulted in a marked attenuation of the receptor activation and concomitantly inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signaling. In conclusion, this study provides novel evidence for a role of MSCs in stimulating myoblast cell proliferation and suggests that the functional interaction between the two cell types may be exploited for the development of new and more efficient cell-based skeletal muscle repair strategies. PMID:22815682

  1. Inulin increases glucose transport in C2C12 myotubes and HepG2 cells via activation of AMP-activated protein kinase and phosphatidylinositol 3-kinase pathways.

    Science.gov (United States)

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

    2009-10-01

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

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

    Science.gov (United States)

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

    2014-01-15

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

  3. Nuclear translocation of the cytoskeleton-associated protein, smALP, upon induction of skeletal muscle differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Cambier, Linda [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France); Pomies, Pascal, E-mail: pascal.pomies@crbm.cnrs.fr [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France)

    2011-06-17

    Highlights: {yields} The cytoskeleton-associated protein, smALP, is expressed in differentiated skeletal muscle. {yields} smALP is translocated from the cytoplasm to the nucleus of C2C12 myoblasts upon induction of myogenesis. {yields} The differentiation-dependent nuclear translocation of smALP occurs in parallel with the nuclear accumulation of myogenin. {yields} The LIM domain of smALP is essential for the nuclear accumulation of the protein. {yields} smALP might act in the nucleus to control some critical aspect of the muscle differentiation process. -- Abstract: The skALP isoform has been shown to play a critical role in actin organization and anchorage within the Z-discs of skeletal muscles, but no data is available on the function of the smALP isoform in skeletal muscle cells. Here, we show that upon induction of differentiation a nuclear translocation of smALP from the cytoplasm to the nucleus of C2C12 myoblasts, concomitant to an up-regulation of the protein expression, occurs in parallel with the nuclear accumulation of myogenin. Moreover, we demonstrate that the LIM domain of smALP is essential for the nuclear translocation of the protein.

  4. PED/PEA-15 induces autophagy and mediates TGF-beta1 effect on muscle cell differentiation.

    Science.gov (United States)

    Iovino, S; Oriente, F; Botta, G; Cabaro, S; Iovane, V; Paciello, O; Viggiano, D; Perruolo, G; Formisano, P; Beguinot, F

    2012-07-01

    TGF-beta1 has been shown to induce autophagy in certain cells but whether and how this action is exerted in muscle and whether this activity relates to TGF-beta1 control of muscle cell differentiation remains unknown. Here, we show that expression of the autophagy-promoting protein phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) progressively declines during L6 and C2C12 skeletal muscle cell differentiation. PED/PEA-15 underwent rapid induction upon TGF-beta1 exposure of L6 and C2C12 myoblasts, accompanied by impaired differentiation into mature myotubes. TGF-beta1 also induced autophagy in the L6 and C2C12 cells through a PP2A/FoxO1-mediated mechanism. Both the TGF-beta1 effect on differentiation and that on autophagy were blocked by specific PED/PEA-15 ShRNAs. Myoblasts stably overexpressing PED/PEA-15 did not differentiate and showed markedly enhanced autophagy. In these same cells, the autophagy inhibitor 3-methyladenine rescued TGF-beta1 effect on both autophagy and myogenesis, indicating that PED/PEA-15 mediates TGF-beta1 effects in muscle. Muscles from transgenic mice overexpressing PED/PEA-15 featured a significant number of atrophic fibers, accompanied by increased light chain 3 (LC3)II to LC3I ratio and reduced PP2A/FoxO1 phosphorylation. Interestingly, these mice showed significantly impaired locomotor activity compared with their non-transgenic littermates. TGF-beta1 causes transcriptional upregulation of the autophagy-promoting gene PED/PEA-15, which in turn is capable to induce atrophic responses in skeletal muscle in vivo.

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

    Science.gov (United States)

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

    2013-11-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-06-22

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

  7. Methionine Regulates mTORC1 via the T1R1/T1R3-PLCβ-Ca2+-ERK1/2 Signal Transduction Process in C2C12 Cells

    Directory of Open Access Journals (Sweden)

    Yuanfei Zhou

    2016-10-01

    Full Text Available The mammalian target of rapamycin complex 1 (mTORC1 integrates amino acid (AA availability to support protein synthesis and cell growth. Taste receptor type 1 member (T1R is a G protein-coupled receptor that functions as a direct sensor of extracellular AA availability to regulate mTORC1 through Ca2+ stimulation and extracellular signal–regulated kinases 1 and 2 (ERK1/2 activation. However, the roles of specific AAs in T1R1/T1R3-regulated mTORC1 are poorly defined. In this study, T1R1 and T1R3 subunits were expressed in C2C12 myotubes, and l-AA sensing was accomplished by T1R1/T1R3 to activate mTORC1. In response to l-AAs, such as serine (Ser, arginine (Arg, threonine (Thr, alanine (Ala, methionine (Met, glutamine (Gln, and glycine (Gly, Met induced mTORC1 activation and promoted protein synthesis. Met also regulated mTORC1 via T1R1/T1R3-PLCβ-Ca2+-ERK1/2 signal transduction. Results revealed a new role for Met-regulated mTORC1 via an AA receptor. Further studies should be performed to determine the role of T1R1/T1R3 in mediating extracellular AA to regulate mTOR signaling and to reveal its mechanism.

  8. Methionine Regulates mTORC1 via the T1R1/T1R3-PLCβ-Ca2+-ERK1/2 Signal Transduction Process in C2C12 Cells

    Science.gov (United States)

    Zhou, Yuanfei; Ren, Jiao; Song, Tongxing; Peng, Jian; Wei, Hongkui

    2016-01-01

    The mammalian target of rapamycin complex 1 (mTORC1) integrates amino acid (AA) availability to support protein synthesis and cell growth. Taste receptor type 1 member (T1R) is a G protein-coupled receptor that functions as a direct sensor of extracellular AA availability to regulate mTORC1 through Ca2+ stimulation and extracellular signal–regulated kinases 1 and 2 (ERK1/2) activation. However, the roles of specific AAs in T1R1/T1R3-regulated mTORC1 are poorly defined. In this study, T1R1 and T1R3 subunits were expressed in C2C12 myotubes, and l-AA sensing was accomplished by T1R1/T1R3 to activate mTORC1. In response to l-AAs, such as serine (Ser), arginine (Arg), threonine (Thr), alanine (Ala), methionine (Met), glutamine (Gln), and glycine (Gly), Met induced mTORC1 activation and promoted protein synthesis. Met also regulated mTORC1 via T1R1/T1R3-PLCβ-Ca2+-ERK1/2 signal transduction. Results revealed a new role for Met-regulated mTORC1 via an AA receptor. Further studies should be performed to determine the role of T1R1/T1R3 in mediating extracellular AA to regulate mTOR signaling and to reveal its mechanism. PMID:27727170

  9. Peptide separations by on-line MudPIT compared to isoelectric focusing in an off-gel format: application to a membrane-enriched fraction from C2C12 mouse skeletal muscle cells.

    Science.gov (United States)

    Elschenbroich, Sarah; Ignatchenko, Vladimir; Sharma, Parveen; Schmitt-Ulms, Gerold; Gramolini, Anthony O; Kislinger, Thomas

    2009-10-01

    High-resolution peptide separation is pivotal for successful shotgun proteomics. The need for capable techniques propels invention and improvement of ever more sophisticated approaches. Recently, Agilent Technologies has introduced the OFFGEL fractionator, which conducts peptide separation by isoelectric focusing in an off-gel setup. This platform has been shown to accomplish high resolution of peptides for diverse sample types, yielding valuable advantages over comparable separation techniques. In this study, we deliver the first comparison of the newly emerging OFFGEL approach to the well-established on-line MudPIT platform. Samples from a membrane-enriched fraction isolated from murine C2C12 cells were subjected to replicate analysis by OFFGEL (12 fractions, pH 3-10) followed by RP-LC-MS/MS or 12-step on-line MudPIT. OFFGEL analyses yielded 1398 proteins (identified by 10,269 peptides), while 1428 proteins (11,078 peptides) were detected with the MudPIT approach. Thus, our data shows that both platforms produce highly comparable results in terms of protein/peptide identifications and reproducibility for the sample type analyzed. We achieve more accurate peptide focusing after OFFGEL fractionation with 88% of all peptides binned to a single fraction, as compared to 61% of peptides detected in only one step in MudPIT analyses. Our study suggests that both platforms are equally capable of high quality peptide separation of a sample with medium complexity, rendering them comparably valuable for comprehensive proteomic analyses.

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

    Science.gov (United States)

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

    2013-03-19

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

  11. 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.

    Science.gov (United States)

    Miyata, Kohei; Miyata, Tomoko; Nakabayashi, Kazuhiko; Okamura, Kohji; Naito, Masashi; Kawai, Tomoko; Takada, Shuji; Kato, Kiyoko; Miyamoto, Shingo; Hata, Kenichiro; Asahara, Hiroshi

    2015-01-15

    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.

  12. Platelet-rich plasma, especially when combined with a TGF-β inhibitor promotes proliferation, viability and myogenic differentiation of myoblasts in vitro.

    Directory of Open Access Journals (Sweden)

    Robi Kelc

    Full Text Available 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.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

    In vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro. PMID:28134270

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

    Science.gov (United States)

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

    2011-07-10

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

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

    Science.gov (United States)

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

    2014-01-01

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

  17. Mesenchymal stromal cell secreted sphingosine 1-phosphate (S1P exerts a stimulatory effect on skeletal myoblast proliferation.

    Directory of Open Access Journals (Sweden)

    Chiara Sassoli

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

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

    DEFF Research Database (Denmark)

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

    fusion, cell membrane and cytoskeleton component reorganization due to the activity of ubiquitous proteolytic enzymes known as calpains has been reported. Whether there is a link between stretch- or load induced signals, the MAPK pathway and calpain expression and activation is not known. Using......, with each individual myotube containing fewer nuclei. Mechanical stimulation increases not only the expression of m-calpain but also the overall activity of calpain in the cells through the MAPK signaling cascade. Our findings underline that the mechanical modulation of MAPK signaling cascade enhances...... the expression and activity of m-calpain, which play a pivotal role during myoblast fusion, strengthening the idea of its implication during the initial events of muscle development....

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

    Directory of Open Access Journals (Sweden)

    Pornthida Poosala

    2016-01-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2016-11-23

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

  1. The pesticide methoxychlor decreases myotube formation in cell culture by slowing myoblast proliferation.

    Science.gov (United States)

    Steffens, Bradley W; Batia, Lyn M; Baarson, Chad J; Choi, Chang-Kun Charles; Grow, Wade A

    2007-08-01

    We studied the effect of the estrogenic pesticide methoxychlor (MXC) on skeletal muscle development using C2C12 cell culture. Myoblast cultures were exposed to various concentrations of MXC at various times during the process of myoblast fusion into myotubes. We observed that MXC exposure decreased myotube formation. In addition, we observed myoblasts with cytoplasmic vacuoles in cultures exposed to MXC. Because cytoplasmic vacuoles can be characteristic of cell death, apoptosis assays and trypan blue exclusion assays were performed. We found no difference in the frequency of apoptosis or in the frequency of cell death for cultures exposed to MXC and untreated cultures. Collectively, these results indicate that MXC exposure decreases myotube formation without causing cell death. In contrast, when cell proliferation was assessed, untreated cultures had a myoblast proliferation rate 50% greater than cultures exposed to MXC. We conclude that MXC decreases myotube formation at least in part by slowing myoblast proliferation. Furthermore, we suggest that direct exposure to MXC could affect skeletal muscle development in animals or humans, in addition to the defects in reproductive development that have previously been reported.

  2. The regulation of myoblast plasticity and its mechanism

    Institute of Scientific and Technical Information of China (English)

    Peng ZHANG; Xiao-ping CHEN

    2012-01-01

    The development of skeletal muscle is a highly regulated,multi-step process in which pluripotent mesodermal cells give rise to myoblasts that subsequently withdraw from the cell cycle and differentiate into myotubes as well as myofibers.The plasticity of myoblasts plays a critical role in maintaining skeletal muscle structure and function by myoblast activation,migration,adhesion,membrane reorganization,nuclear fusion,finally forming myotubes/myofibers.Our studies demonstrate that the local hypoxic microenvironment,a great diversity of regulatory factors such as IL-6 superfamily factors (IL-6,LIF,CNTF) and TGF-β1 could regulate the myoblast plasticity.The aim of this paper is to review the previous studies focused on the regulation of myoblast plasticity and its mechanism in our laboratory.Knowledge about the microenvironment or factors involved in regulating the myoblast plasticity will help develop the prevention and cure measures of skeletal muscle diseases.

  3. Heterogeneous nuclear ribonucleoprotein M associates with mTORC2 and regulates muscle differentiation

    Science.gov (United States)

    Chen, Wei-Yen; Lin, Chia-Lung; Chuang, Jen-Hua; Chiu, Fu-Yu; Sun, Yun-Ya; Liang, Mei-Chih; Lin, Yenshou

    2017-01-01

    Mammalian target of rapamycin (mTOR) plays a range of crucial roles in cell survival, growth, proliferation, metabolism, and morphology. However, mTOR forms two distinct complexes, mTOR complex 1 and mTOR complex 2 (mTORC1 and mTORC2), via association with a series of different components; this allows the complexes to execute their wide range of functions. This study explores further the composition of the mTORC2 complex. Utilizing Rictor knock-out cells, immunoprecipitation and mass spectrometry, a novel Rictor associated protein, heterogeneous nuclear ribonucleoprotein M (hnRNP M), was identified. The association between hnRNP M and Rictor was verified using recombinant and endogenous protein and the binding site was found to be within aa 1~532 of hnRNP M. The presence of hnRNP M significantly affects phosphorylation of SGK1 S422, but not of Akt S473, PKCα S657 and PKCζ T560. Furthermore, hnRNP M also plays a critical role in muscle differentiation because knock-down of either hnRNP M or Rictor in C2C12 myoblasts reduced differentiation. This decrease is able to be rescued by overexpression SGK S422D in hnRNP M knockdown C2C12 myoblasts. Taken together, we have identified a novel Rictor/mTOR binding molecule, hnRNP M, that allows mTORC2 signaling to phosphorylate SGK1 thus regulating muscle differentiation. PMID:28106162

  4. Heterogeneous nuclear ribonucleoprotein M associates with mTORC2 and regulates muscle differentiation.

    Science.gov (United States)

    Chen, Wei-Yen; Lin, Chia-Lung; Chuang, Jen-Hua; Chiu, Fu-Yu; Sun, Yun-Ya; Liang, Mei-Chih; Lin, Yenshou

    2017-01-20

    Mammalian target of rapamycin (mTOR) plays a range of crucial roles in cell survival, growth, proliferation, metabolism, and morphology. However, mTOR forms two distinct complexes, mTOR complex 1 and mTOR complex 2 (mTORC1 and mTORC2), via association with a series of different components; this allows the complexes to execute their wide range of functions. This study explores further the composition of the mTORC2 complex. Utilizing Rictor knock-out cells, immunoprecipitation and mass spectrometry, a novel Rictor associated protein, heterogeneous nuclear ribonucleoprotein M (hnRNP M), was identified. The association between hnRNP M and Rictor was verified using recombinant and endogenous protein and the binding site was found to be within aa 1~532 of hnRNP M. The presence of hnRNP M significantly affects phosphorylation of SGK1 S422, but not of Akt S473, PKCα S657 and PKCζ T560. Furthermore, hnRNP M also plays a critical role in muscle differentiation because knock-down of either hnRNP M or Rictor in C2C12 myoblasts reduced differentiation. This decrease is able to be rescued by overexpression SGK S422D in hnRNP M knockdown C2C12 myoblasts. Taken together, we have identified a novel Rictor/mTOR binding molecule, hnRNP M, that allows mTORC2 signaling to phosphorylate SGK1 thus regulating muscle differentiation.

  5. Sp3 controls fibroblast growth factor receptor 4 gene activity during myogenic differentiation.

    Science.gov (United States)

    Cavanaugh, Eric; DiMario, Joseph X

    2017-03-27

    Fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling is a critical component in the regulation of myoblast proliferation and differentiation. The transient FGFR4 gene expression during the transition from proliferating myoblasts to differentiated myotubes indicates that FGFR4 regulates this critical phase of myogenesis. The Specificity Protein (SP) family of transcription factors controls FGFR family member gene activity. We sought to determine if members of the Sp family regulate mouse FGFR4 gene activity during myogenic differentiation. RT-PCR and western blot analysis of FGFR4 mRNA and protein revealed transient expression over 72h, with peak expression between 24 and 36h after addition of differentiation medium to C2C12 myogenic cultures. Sp3 also displayed a transient expression pattern with peak expression occurring after 6h of differentiation. We cloned a 1527bp fragment of the mouse FGFR4 promoter into a luciferase reporter. This FGFR4 promoter contains eight putative Sp binding sites and directed luciferase gene activity comparable to native FGFR4 expression. Overexpression of Sp1 and Sp3 showed that Sp1 repressed FGFR4 gene activity, and Sp3 activated FGFR4 gene activity during myogenic differentiation. Mutational analyses of multiple Sp binding sites within the FGFR4 promoter revealed that three of these sites were transcriptionally active. Electromobility shift assays and chromatin immunoprecipitation of the area containing the activator sites showed that Sp3 bound to this promoter location.

  6. Non-invasive monitoring of osteogenic differentiation on microtissue arrays under physiological conditions using scanning electrochemical microscopy

    NARCIS (Netherlands)

    Sridhar, Adithya; Berg, van den Albert; Le Gac, Séverine

    2014-01-01

    In this paper, we present a non-invasive assay using scanning electrochemical microscopy (SECM) for detecting osteogenic differentiation at physiological conditions (pH 7.5) on arrays of C2C12 microtissues. Upon exposure to bone morphogenic protein 2 (BMP-2), C2C12 microtissues differentiate and exp

  7. Ankrd2/ARPP is a novel Akt2 specific substrate and regulates myogenic differentiation upon cellular exposure to H2O2

    Science.gov (United States)

    Cenni, Vittoria; Bavelloni, Alberto; Beretti, Francesca; Tagliavini, Francesca; Manzoli, Lucia; Lattanzi, Giovanna; Maraldi, Nadir M.; Cocco, Lucio; Marmiroli, Sandra

    2011-01-01

    Activation of Akt-mediated signaling pathways is crucial for survival, differentiation, and regeneration of muscle cells. A proteomic-based search for novel substrates of Akt was therefore undertaken in C2C12 murine muscle cells exploiting protein characterization databases in combination with an anti–phospho-Akt substrate antibody. A Scansite database search predicted Ankrd2 (Ankyrin repeat domain protein 2, also known as ARPP) as a novel substrate of Akt. In vitro and in vivo studies confirmed that Akt phosphorylates Ankrd2 at Ser-99. Moreover, by kinase assay with recombinant Akt1 and Akt2, as well as by single-isoform silencing, we demonstrated that Ankrd2 is a specific substrate of Akt2. Ankrd2 is typically found in skeletal muscle cells, where it mediates the transcriptional response to stress conditions. In an attempt to investigate the physiological implications of Ankrd2 phosphorylation by Akt2, we found that oxidative stress induced by H2O2 triggers this phosphorylation. Moreover, the forced expression of a phosphorylation-defective mutant form of Ankrd2 in C2C12 myoblasts promoted a faster differentiation program, implicating Akt-dependent phosphorylation at Ser-99 in the negative regulation of myogenesis in response to stress conditions. PMID:21737686

  8. ROCK-2 is associated with focal adhesion maturation during myoblast migration.

    Science.gov (United States)

    Goetsch, K P; Snyman, C; Myburgh, K H; Niesler, C U

    2014-07-01

    Satellite cell migration is critical for skeletal muscle growth and regeneration. Controlled cell migration is dependent on the formation of mature focal adhesions between the cell and the underlying extracellular matrix (ECM). These cell-ECM interactions trigger the activation of signalling events such as the Rho/ROCK pathway. We have previously identified a specific role for ROCK-2 during myoblast migration. In this study we report that ROCK inhibition with Y-27632 increases C2C12 myoblast velocity, but at the expense of directional migration. In response to Y-27632 an increased number of smaller focal adhesions were distributed across adhesion sites that in turn were clearly larger than sites in untreated cells, suggesting a reduction in focal adhesion maturation. We also confirm ROCK-2 localisation to the focal adhesion sites in migrating myoblasts and demonstrate a change in the distribution of these ROCK-2 containing adhesions in response to Y-27632. Taken together, our observations provide further proof that ROCK-2 regulates directional myoblast migration through focal adhesion formation and maturation.

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

    Science.gov (United States)

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

    2016-02-23

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

  10. Promoting osteoblast differentiation by the flavanes from Huangshan Maofeng tea is linked to a reduction of oxidative stress.

    Science.gov (United States)

    Zeng, Xiaobin; Tian, Jun; Cai, Kangyong; Wu, Xin; Wang, Yang; Zheng, Yayuan; Su, Yanjie; Cui, Liao

    2014-02-15

    Epidemiological evidence has shown an association between tea consumption and the prevention of bone loss in the elderly. Previous studies indicated that green tea exerted osteoprotective effect in vivo. This study aims to investigate the constituents in Huangshan Maofeng tea and systemically evaluate their antioxidative and osteogenic effects in vitro. Five flavanes, isolated from Huangshan Maofeng tea, showed effects on proliferation of osteoblastic cells and ameliorated H2O2-induced C2C12 mouse myoblast cell apoptosis at 3.125-50 μg/ml. (-)-Epicatechin observably increased alkaline phosphatase (ALP) activity and hydroxyproline content. (-)-Epiafzelechin at 25 μg/ml significantly increased the area of mineralized bone nodules. The activities of flavanes in promoting osteblastic proliferation and differentiation are positively correlated with activities in protecting against apoptosis in C2C12 cells. It indicates that anti-osteoporosis effect of these flavanes may be linked to their antioxidative activity. The observed effects of these flavanes suggest that these flavanes may have beneficial effects on bone health.

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

    Science.gov (United States)

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

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

    Science.gov (United States)

    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.

  13. In-depth analysis of the secretome identifies three major independent secretory pathways in differentiating human myoblasts

    DEFF Research Database (Denmark)

    Le Bihan, Marie-Catherine; Bigot, Anne; Jensen, Søren Skov;

    2012-01-01

    and genomic strategies, we show that human muscle cells release not only soluble secreted proteins through conventional secretory mechanisms but also complex protein and nucleic acid cargos via membrane microvesicle shedding. The soluble secretome of muscle cells contains 253 conventionally secreted signaling...... features and larger, morphologically distinct, microvesicles. While they share some common features, their distinct protein and RNA cargos suggest independent functions in myogenesis. We further demonstrate that both types of microvesicles can dock and fuse with adjacent muscle cells but also deliver...... functional protein cargo. Thus, the intercellular signaling networks invoked during muscle differentiation and regeneration may employ conventional soluble signaling molecules acting in concert with muscle derived microvesicles delivering their cargos directly into target cells....

  14. Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice

    Science.gov (United States)

    Hayashi, Shinichiro; Manabe, Ichiro; Suzuki, Yumi; Relaix, Frédéric; Oishi, Yumiko

    2016-01-01

    Krüppel-like factor 5 (Klf5) is a zinc-finger transcription factor that controls various biological processes, including cell proliferation and differentiation. We show that Klf5 is also an essential mediator of skeletal muscle regeneration and myogenic differentiation. During muscle regeneration after injury (cardiotoxin injection), Klf5 was induced in the nuclei of differentiating myoblasts and newly formed myofibers expressing myogenin in vivo. Satellite cell-specific Klf5 deletion severely impaired muscle regeneration, and myotube formation was suppressed in Klf5-deleted cultured C2C12 myoblasts and satellite cells. Klf5 knockdown suppressed induction of muscle differentiation-related genes, including myogenin. Klf5 ChIP-seq revealed that Klf5 binding overlaps that of MyoD and Mef2, and Klf5 physically associates with both MyoD and Mef2. In addition, MyoD recruitment was greatly reduced in the absence of Klf5. These results indicate that Klf5 is an essential regulator of skeletal muscle differentiation, acting in concert with myogenic transcription factors such as MyoD and Mef2. DOI: http://dx.doi.org/10.7554/eLife.17462.001 PMID:27743478

  15. NOV/CCN3 impairs muscle cell commitment and differentiation.

    Science.gov (United States)

    Calhabeu, Frederico; Lafont, Jérome; Le Dreau, Gwenvael; Laurent, Maryvonne; Kazazian, Chantal; Schaeffer, Laurent; Martinerie, Cécile; Dubois, Catherine

    2006-06-10

    NOV (nephroblastoma overexpressed) is a member of a family of proteins which encodes secreted matrix-associated proteins. NOV is expressed during development in dermomyotome and limb buds, but its functions are still poorly defined. In order to understand the role of NOV in myogenic differentiation, C2C12 cells overexpressing NOV (C2-NOV) were generated. These cells failed to engage into myogenic differentiation, whereas they retained the ability to differentiate into osteoblasts. In differentiating conditions, C2-NOV cells remained proliferative, failed to express differentiation markers and lost their ability to form myotubes. Inhibition of differentiation by NOV was also observed with human primary muscle cells. Further examination of C2-NOV cells revealed a strong downregulation of the myogenic determination genes MyoD and Myf5 and of IGF-II expression. MyoD forced expression in C2-NOV was sufficient to restore differentiation and IGF-II induction whereas 10(-6) M insulin treatment had no effects. NOV therefore acts upstream of MyoD and does not affect IGF-II induction and signaling. HES1, a target of Notch, previously proposed to mediate NOV action, was not implicated in the inhibition of differentiation. We propose that NOV is a specific cell fate regulator in the myogenic lineage, acting negatively on key myogenic genes thus controlling the transition from progenitor cells to myoblasts.

  16. Integrated functions of Pax3 and Pax7 in the regulation of proliferation, cell size and myogenic differentiation.

    Directory of Open Access Journals (Sweden)

    Charlotte A Collins

    Full Text Available Pax3 and Pax7 are paired-box transcription factors with roles in developmental and adult regenerative myogenesis. Pax3 and Pax7 are expressed by postnatal satellite cells or their progeny but are down regulated during myogenic differentiation. We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size. Conversely, expression of dominant-negative constructs leads to slowing of cell division, a dramatic increase in cell size and altered morphology. Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation. However, expression of Pax3 or Pax7 dominant-negative constructs inhibits expression of Myf5, MyoD and myogenin, and prevents differentiation from proceeding. In fibroblasts, expression of Pax3 or Pax7, or dominant-negative inhibition of these factors, reproduce the effects on cell size, morphology and proliferation seen in myoblasts. Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed.

  17. A Cistanches Herba Fraction/β-Sitosterol Causes a Redox-Sensitive Induction of Mitochondrial Uncoupling and Activation of Adenosine Monophosphate-Dependent Protein Kinase/Peroxisome Proliferator-Activated Receptor γ Coactivator-1 in C2C12 Myotubes: A Possible Mechanism Underlying the Weight Reduction Effect

    Directory of Open Access Journals (Sweden)

    Hoi Shan Wong

    2015-01-01

    Full Text Available Previous studies have demonstrated that HCF1, a semipurified fraction of Cistanches Herba, causes weight reduction in normal diet- and high fat diet-fed mice. The weight reduction was associated with the induction of mitochondrial uncoupling and changes in metabolic enzyme activities in mouse skeletal muscle. To further investigate the biochemical mechanism underlying the HCF1-induced weight reduction, the effect of HCF1 and its active component, β-sitosterol (BSS, on C2C12 myotubes was examined. Incubation with HCF1/BSS caused a transient increase in mitochondrial membrane potential (MMP, possibly by fluidizing the mitochondrial inner membrane. The increase in MMP was paralleled to an increase in mitochondrial reactive oxygen species (ROS production. Mitochondrial ROS, in turn, triggered a redox-sensitive induction of mitochondrial uncoupling by uncoupling protein 3 (UCP3. Biochemical analysis indicated that HCF1 was capable of activating an adenosine monophosphate-dependent protein kinase/peroxisome proliferator-activated receptor γ coactivator-1 pathway and thereby increased the expression of cytochrome c oxidase and UCP3. Animal studies using mitochondrial recoupler also confirmed the role of mitochondrial uncoupling in the HCF1-induced weight reduction. In conclusion, a HCF1/BSS causes the redox-sensitive induction of mitochondrial uncoupling and activation of AMPK/PGC-1 in C2C12 myotubes, with resultant reductions in body weight and adiposity by increased energy consumption.

  18. Electroactive polyurethane/siloxane derived from castor oil as a versatile cardiac patch, part I: Synthesis, characterization, and myoblast proliferation and differentiation.

    Science.gov (United States)

    Baheiraei, Nafiseh; Gharibi, Reza; Yeganeh, Hamid; Miragoli, Michele; Salvarani, Nicolò; Di Pasquale, Elisa; Condorelli, Gianluigi

    2015-11-05

    Tissue-engineered cardiac patch aims at regenerating an infarcted heart by improving cardiac function and providing mechanical support to the diseased myocardium. In order to take advantages of electroactivity, a new synthetic method was developed for the introduction of an electroactive oligoaniline into the backbone of prepared patches. For this purpose, a series of electroactive polyurethane/siloxane films containing aniline tetramer (AT) was prepared through sol-gel reaction of trimethoxysilane functional intermediate polyurethane prepolymers made from castor oil and poly(ethylene glycol). Physicochemical, mechanical, and electrical conductivity of samples were evaluated and the recorded results were correlated to their structural characteristics. The optimized films were proved to be biodegradable and have tensile properties suitable for cardiac patch application. The embedded AT moieties in the backbone of the prepared samples preserved their electroactivity with the electrical conductivity in the range of 10(-4) S/cm. The prepared films were compatible with proliferation of C2C12 and had potential for enhancing myotube formation even without external electrical stimulation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2015.

  19. Myoblast fusion in Drosophila

    Energy Technology Data Exchange (ETDEWEB)

    Haralalka, Shruti [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Abmayr, Susan M., E-mail: sma@stowers.org [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, MO 66160 (United States)

    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.

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

    Science.gov (United States)

    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.

  1. Combining a micro/nano-hierarchical scaffold with cell-printing of myoblasts induces cell alignment and differentiation favorable to skeletal muscle tissue regeneration.

    Science.gov (United States)

    Yeo, Miji; Lee, Hyeongjin; Kim, Geun Hyung

    2016-09-16

    Biomedical scaffolds must be used in tissue engineering to provide physical stability and topological/biochemical properties that directly affect tissue regeneration. In this study, a new cell-laden scaffold was developed that supplies micro/nano-topological cues and promotes efficient release of cells. The hierarchical structure consisted of poly(ε-caprolactone) macrosized struts for sustaining a three-dimensional structural shape, aligned nanofibers obtained with optimized electrospinning, and cell-printed myoblasts. Importantly, the printed myoblasts were fully safe and were efficiently released from the cell-laden struts to neighboring nanofiber networks. The incorporation of micro/nanofibers in the hierarchical scaffold significantly affected myoblast proliferation, alignment, and even facilitated the formation of myotubes. We observed that myosin heavy chain expression and the expression levels of various myogenic genes (MyoD, myogenin, and troponin T) were significantly affected by the fiber alignment achieved in our hierarchical cell-laden structure. We believe that the combination of cell-printing and a hierarchical scaffold that encourages fiber alignment is a highly promising technique for skeletal muscle tissue engineering.

  2. Non-invasive Bioluminescence Imaging of Myoblast-Mediated Hypoxia-Inducible Factor-1 Alpha Gene Transfer

    Science.gov (United States)

    Gheysens, Olivier; Chen, Ian Y.; Rodriguez-Porcel, Martin; Chan, Carmel; Rasooly, Julia; Vaerenberg, Caroline; Paulmurugan, Ramasamy; Willmann, Juergen K.; Deroose, Christophe; Wu, Joseph; Gambhir, Sanjiv S.

    2011-01-01

    Purpose We tested a novel imaging strategy, in which both the survival of transplanted myoblasts and their therapeutic transgene expression, a recombinant hypoxia-inducible factor-1α (HIF-1α-VP2), can be monitored using firefly luciferase (fluc) and Renilla luciferase (hrl) bioluminescence reporter genes, respectively. Procedures The plasmid pUbi-hrl-pUbi-HIF-1α-VP2, which expresses both hrl and HIF-1α-VP2 using two ubiquitin promoters, was characterized in vitro. C2c12 myoblasts stably expressing fluc and transiently transfected with pUbi-hrl-pUbi-HIF-1α-VP2 were injected into the mouse hindlimb. Both hrl and fluc expression were monitored using bioluminescence imaging (BLI). Results Strong correlations existed between the expression of hRL and each of HIF-1α-VP2, VEGF, and PlGF (r2>0.83, r2>0.82, and r2>0.97, respectively). In vivo, both transplanted cells and HIF-1α-VP2 transgene expression were successfully imaged using BLI. Conclusions An objective evaluation of myoblast-mediated gene transfer in living mice can be performed by monitoring both the survival and the transgene expression of transplanted myoblasts using the techniques developed herein. PMID:21267661

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

    Science.gov (United States)

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

    2016-12-01

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

  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.

    Science.gov (United States)

    Chaudhuri, Biswadeep; Bhadra, Debabrata; Moroni, Lorenzo; Pramanik, Krishna

    2015-02-18

    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.

  5. Engineering skeletal myoblasts: roles of three-dimensional culture and electrical stimulation.

    Science.gov (United States)

    Pedrotty, Dawn M; Koh, Jennifer; Davis, Bryce H; Taylor, Doris A; Wolf, Patrick; Niklason, Laura E

    2005-04-01

    Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty in attempts to regenerate cardiac muscle tissue by injection of cells into damaged myocardium. In some studies, muscle tissue within myoblast implant sites may be morphologically similar to cardiac muscle. We hypothesized that identifiable aspects of the cardiac milieu may contribute to growth and development of implanted myoblasts in vivo. To test this hypothesis, we designed a novel in vitro system to mimic some aspects of the electrical and biochemical environment of native myocardium. This system enabled us to separate the three-dimensional (3-D) electrical and biochemical signals that may be involved in myoblast proliferation and plasticity. Myoblasts were grown on 3-D polyglycolic acid mesh scaffolds under control conditions, in the presence of cardiac-like electrical current fluxes, or in the presence of culture medium that had been conditioned by mature cardiomyocytes. Cardiac-like electrical current fluxes caused increased myoblast number in 3-D culture, as determined by DNA assay. The increase in cell number was due to increased cellular proliferation and not differences in apoptosis, as determined by proliferating cell nuclear antigen and TdT-mediated dUTP nick-end labeling. Cardiomyocyte-conditioned medium also significantly increased myoblast proliferation. Expression of transcription factors governing differentiation along skeletal or cardiac lineages was evaluated by immunoblotting. Although these assays are qualitative, no changes in differentiation state along skeletal or cardiac lineages were observed in response to electrical current fluxes. Furthermore, from these experiments, conditioned medium did not appear to alter the differentiation state of skeletal myoblasts. Hence, cardiac milieu appears to stimulate proliferation but does not affect differentiation of skeletal myoblasts.

  6. PLLA/ZnO nanocomposites: Dynamic surfaces to harness cell differentiation.

    Science.gov (United States)

    Trujillo, Sara; Lizundia, Erlantz; Vilas, José Luis; Salmeron-Sanchez, Manuel

    2016-08-01

    This work investigates the effect of the sequential availability of ZnO nanoparticles, (nanorods of ∼40nm) loaded within a degradable poly(lactic acid) (PLLA) matrix, in cell differentiation. The system constitutes a dynamic surface, in which nanoparticles are exposed as the polymer matrix degrades. ZnO nanoparticles were loaded into PLLA and the system was measured at different time points to characterise the time evolution of the physicochemical properties, including wettability and thermal properties. The micro and nanostructure were also investigated using AFM, SEM and TEM images. Cellular experiments with C2C12 myoblasts show that cell differentiation was significantly enhanced on ZnO nanoparticles-loaded PLLA, as the polymer degrades and the availability of nanoparticles become more apparent, whereas the release of zinc within the culture medium was negligible. Our results suggest PLLA/ZnO nanocomposites can be used as a dynamic system where nanoparticles are exposed during degradation, activating the material surface and driving cell differentiation.

  7. Clone-derived human AF-amniotic fluid stem cells are capable of skeletal myogenic differentiation in vitro and in vivo.

    Science.gov (United States)

    Ma, Xiaorong; Zhang, Shengli; Zhou, Junmei; Chen, Baisong; Shang, Yafeng; Gao, Tongbing; Wang, Xue; Xie, Hua; Chen, Fang

    2012-08-01

    Stem cell-based therapy may be the most promising method to cure skeletal muscle degenerative diseases such as Duchenne muscular dystrophy (DMD) and trauma in the future. Human amniotic fluid is enriched with early-stage stem cells from developing fetuses and these cells have cardiomyogenic potential both in vitro and in vivo. In the present study, we investigated the characteristics of human amniotic fluid-derived AF-type stem (HAF-AFS) cells by flow cytometry, immunofluorescence staining, reverse-transcription polymerase chain reaction, and osteogenic and adipogenic differentiation analysis. After confirming the stemness of HAF-AFS cells, we tested whether HAF-AFS cells could differentiate into skeletal myogenic cells in vitro and incorporate into regenerating skeletal muscle in vivo. By temporary exposure to the DNA demethylation agent 5-aza-2'-deoxycytidine (5-Aza dC) or co-cultured with C2C12 myoblasts, HAF-AFS cells differentiated into skeletal myogenic cells, expressing skeletal myogenic cell-specific markers such as Desmin, Troponin I (Tn I) and α-Actinin. Four weeks after transplantation into cardiotoxin-injured and X-ray-irradiated tibialis anterior (TA) muscles of NOD/SCID mice, HAF-AFS cells survived, differentiated into myogenic precursor cells and fused with host myofibres. The findings that HAF-AFS cells differentiate into myogenic cells in vitro and incorporate in skeletal muscle regeneration in vivo hold the promise of HAF-AFS cell-based therapy for skeletal muscle degenerative diseases.

  8. A conserved role for calpains during myoblast fusion.

    Science.gov (United States)

    Buffolo, Marcio; Batista Possidonio, Ana Claudia; Mermelstein, Claudia; Araujo, Helena

    2015-07-01

    Myoblast fusion is a key step during skeletal muscle differentiation as it enables the formation of contractile fibers. Calpains have been implicated in some aspects of myogenesis in mammals, but whether they exert a conserved function during myoblast fusion has not been investigated. Here, we studied Calpain function in two models of myogenesis: in vitro analysis of chick myogenic cultures and in vivo analysis of Drosophila melanogaster muscle development. First we showed that Calpain A is important for fly muscle function. In addition, Calpain A knockdown reduced lateral body wall muscle length and width, as well as the number of nuclei in dorsal oblique muscles, consistent with fewer cells fusing to form fibers. Treatment of chick cultures with a selective Calpain inhibitor led to the formation of thinner myotubes containing a reduced number of nuclei, consistent with decreased myoblast fusion. Dynamic changes in IκBα labeling and transfection with a dominant-negative IκBα suggest a role for the NFκB pathway during chick myogenesis and a possible role of Calpains in attenuating NFκB signals that restrict myoblast fusion. Our data suggest that different model organisms may be used to study the role of Calpains in regular myogenesis and Calpain-related muscular degenerative disorders.

  9. Id2从核迁移到细胞质后通过调节凋亡诱导因子表达促进骨骼肌细胞分化%Id2 translocation from nucleus to cytoplasm accelerating differentiation of skeletal muscle cells by regulating the expression of apoptosis inducing factor

    Institute of Scientific and Technical Information of China (English)

    胡晓芳; 赖桂华; 王乐禹; 欧阳钧; 余磊; 邱小忠

    2011-01-01

    目的 探讨Id2在骨骼肌再生中的作用机制.方法 用绿色荧光蛋白(GFP)-Id2-C2表达载体转染C2C12成肌细胞,对转染组和非转染组进行H2O2处理和2%马血清处理,用RT-PCR法观察两组细胞Id2基因表达的差异;Western blotting观察两组细胞的成肌分化相关蛋白的表达情况;免疫荧光显微镜观察正常组、纤维损伤组以及去神经支配组大鼠的骨骼肌中Id2和凋亡诱导因子(AIF)蛋白的表达情况.结果 与非转染组相比,Id2转染组细胞成肌分化明显增强.免疫荧光染色法显示,50μmol/L H2O2能增加核Id2蛋白的表达.在氧化应激条件下,Id2能抑制成肌调节因子(MyoD)而活化肌浆蛋白(myogenin).2%马血清能引起大多数Id2从细胞核迁移到细胞质,从而抑制活性氧(ROS)诱导的线粒体AIF表达.免疫荧光分析显示,去神经支配组大鼠的骨骼肌中细胞内的Id2和AIF蛋白表达增多.结论 Id2从细胞核迁移到细胞质后能促进骨骼肌细胞分化,其作用与AIF表达水平相关.%Objective To explore the functional role of Id2 in skeletal muscle regeneration. Methods Id2 expression vectors were transferred into C2C12 cells. The transferred and un-transferred C2C12 skeletal muscle cells were exposed to 50μmol/L H2O2 and 2% horse serum for 12 hours without fetal bovine serum( FBS ). Expression of Id2 gene in transferred and untransferred C2C12 cells was observed by RT-PCR. Expression of various myogenesis related proteins in the transferred and untransferred C2C12 cells were observed by Western blotting. Expression of Id2 and AIF proteins in the normal, fiber-damaged and denervated skeletal muscles were observed by immunofluorescence. Results Compared with un-transferred cells, the Id2 transferred cells exhibited higher differentiation. Immunofluorescence staining revealed that 50μmol/L H2O2 treatment increased the expression of nucleic Id2. Under the oxidative stress, Id2 repressed both MyoD repressors and myogenin

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

    Science.gov (United States)

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

    2015-02-01

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

  11. A gene-trap strategy identifies quiescence-induced genes in synchronized myoblasts

    Indian Academy of Sciences (India)

    Ramkumar Sambasivan; Grace K Pavlath; Jyotsna Dhawan

    2008-03-01

    Cellular quiescence is characterized not only by reduced mitotic and metabolic activity but also by altered gene expression. Growing evidence suggests that quiescence is not merely a basal state but is regulated by active mechanisms. To understand the molecular programme that governs reversible cell cycle exit, we focused on quiescence-related gene expression in a culture model of myogenic cell arrest and activation. Here we report the identification of quiescence-induced genes using a gene-trap strategy. Using a retroviral vector, we generated a library of gene traps in C2C12 myoblasts that were screened for arrest-induced insertions by live cell sorting (FACS-gal). Several independent genetrap lines revealed arrest-dependent induction of gal activity, confirming the efficacy of the FACS screen. The locus of integration was identified in 15 lines. In three lines, insertion occurred in genes previously implicated in the control of quiescence, i.e. EMSY – a BRCA2-interacting protein, p8/com1– a p300HAT-binding protein and MLL5 – a SET domain protein. Our results demonstrate that expression of chromatin modulatory genes is induced in G0, providing support to the notion that this reversibly arrested state is actively regulated.

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

    Science.gov (United States)

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

    2013-02-01

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

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

    Science.gov (United States)

    Bhuyan, Mohammod Kabir; Rodriguez-Devora, 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-09-10

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

  15. Myoblast transplantation can repair heart damage

    Institute of Scientific and Technical Information of China (English)

    Jonathan Dinsmore; Nabil Dib

    2006-01-01

    Myocardial regeneration is an exciting new frontier for the treatment of heart disease. Many approaches are currently being tested. The use of autologous skeletal myoblasts has been the earliest, with over 10 years of research having been conducted.Current progress in the area of skeletal myoblasts for cardiac regeneration is presented. Reviewed is work from both pre-clinical and clinical studies. Work in this area continues to progress and definitive studies to assess efficacy of myoblasts for heart failure either have been initiated or will be initiated shortly. One result that is clear is that myoblasts can survive and form myotubes and myofibers in the area of myocardial infarction. In the early clinical trials, arrhythmia was a concern. However, further studies have shown that the risk was assumed prematurely based on limited human studies. Myoblasts, therefore, provide a highly promising treatment for heart disease. (J Geriatr Cardiol 2006;3 :168-70.)

  16. Overexpression of miRNA-133a on the in vitro proliferation and differentiation of L6 myoblasts%miRNA-133a过表达对体外L6成肌细胞增殖分化作用机制的研究

    Institute of Scientific and Technical Information of China (English)

    李波; 弓贺炜; 李文斌; 李永平; 冯勇; 贾英伟; 田江华; 李刚; 梁炳生

    2014-01-01

    Objective To construct recombinant lentiviral vector of micro RNA-133a and observe the proliferation,differentiation and expression of transcription factor MEF2A of L6 myoblasts transfected with the vector system.Methods Recombinant lentiviral vector containing micro RNA-133a gene was constructed and transfected into L6 myoblasts.The expression of micro RNA-133a gene was detected by real-time PCR (Taqman probe).The effect of micro RNA-133a overexpression on L6 myoblast proliferation was quantified using cell counting kit (CCK-8).Its effect on cell differentiation was detected by inverted fluorescence microscope.Western blot assay was used to detect the expression level of transcription factor MEF2A in these cells.Results The successful construction of micro RNA-133a recombinant lentiviral vector was confirmed by plasmid enzyme digestion and DNA sequencing.Compared with the control group,relative expression of micro RNA-133a gene in L6 myoblasts was significantly increased (P < 0.01) 24h after the vector transfection.L6 cell proliferation was increased significantly (P < 0.01),while its differentiation was effectively inhibited.The expression level of MEF2A was significantly reduced (P < 0.01).Conclusion Micro RNA-133a recombinant lentiviral vector can successfully transfect L6 myoblasts causing the cells to overexpress micro RNA-133a.This overexpression promotes L6 myoblast proliferation and inhibits its differentiation in an in vitro cell culture system.%目的 构建微小RNA-133a重组慢病毒载体,观察其转染后对L6成肌细胞增殖、分化及转录因子MEF2A的影响.方法 构建表达含微小RNA-133a基因的重组慢病毒载体,转染L6成肌细胞,以实时定量PCR(Taqman探针法)对微小RNA-133a基因表达水平进行检测;细胞计数试剂盒(CCK-8)试验评价微小RNA-133a过表达后对L6成肌细胞增殖的影响;倒置荧光显微镜观察L6成肌细胞增殖、分化的影响;Western blot法检测转录因子MEF2A

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-01-15

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

  18. Characterization of human myoblast cultures for tissue engineering.

    Science.gov (United States)

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

    2008-01-01

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

  19. Heterogeneity of myotubes generated by the MyoD and E12 basic helix-loop-helix transcription factors in otherwise non-differentiation growth conditions.

    Science.gov (United States)

    Grubišić, Vladimir; Gottipati, Manoj K; Stout, Randy F; Grammer, J Robert; Parpura, Vladimir

    2014-02-01

    We used a synthetic biology approach to produce myotubes from mammalian C2C12 myoblasts in non-differentiation growth conditions using the expression of basic helix-loop-helix transcription factors, MyoD and E12, in various combinations and configurations. Our approach not only recapitulated the basics of muscle development and physiology, as the obtained myotubes showed qualities similar to those seen in striated muscle fibers in vivo, but also allowed for the synthesis of populations of myotubes which assumed distinct morphology, myofibrillar development and Ca(2+) dynamics. This fashioned class of biomaterials is suitable for the building blocks of soft actuators in micro-scale biomimetic robotics. This production line strategy can be embraced in reparative medicine as synthetic human myotubes with predetermined morphological/functional properties could be obtained using this very approach. This methodology can be adopted beyond striated muscle for the engineering of other tissue components/cells whose differentiation is governed by the principles of basic helix-loop-helix transcription factors, as in the case, for example, of neural or immune cell types.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-06-15

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

  1. MAPK signaling pathways and HDAC3 activity are disrupted during emerin-null myogenic progenitor differentiation.

    Science.gov (United States)

    Collins, Carol M; Ellis, Joseph; Holaska, James M

    2017-02-10

    Mutations in the gene encoding emerin cause Emery-Dreifuss muscular dystrophy (EDMD). Emerin is an integral inner nuclear membrane protein and a component of the nuclear lamina. EDMD is characterized by skeletal muscle wasting, cardiac conduction defects and tendon contractures. The failure to regenerate skeletal muscle is predicted to contribute to the skeletal muscle pathology of EDMD. We hypothesize muscle regeneration defects are caused by impaired muscle stem cell differentiation. Myogenic progenitors derived from emerin-null mice were used to confirm their impaired differentiation and analyze selected myogenic molecular pathways. Emerin-null progenitors were delayed in their cell cycle exit, had decreased myosin heavy chain (MyHC) expression and formed fewer myotubes. Emerin binds to and activates histone deacetylase 3 (HDAC3). Here we show theophylline, an HDAC3-specific activator, improved myotube formation in emerin-null cells. Addition of the HDAC3-specific inhibitor RGFP966 blocked myotube formation and MyHC expression in wildtype and emerin-null myogenic progenitors, but did not affect cell cycle exit. Downregulation of emerin was previously shown to affect the p38 and ERK MAPK pathways in C2C12 myoblast differentiation. Using a pure population of myogenic progenitors completely lacking emerin expression we show these pathways are also disrupted. ERK inhibition improved MyHC expression in emerin-null cells, but failed to rescue myotube formation or cell cycle exit. p38 MAPK inhibition prevented differentiation in both wildtype and emerin-null progenitors. These results show each of these molecular pathways specifically regulate particular stages of myogenic differentiation in an emerin-dependent manner. Thus, pharmacological targeting of multiple pathways acting at specific differentiation stages may be a better therapeutic approach in the future to rescue muscle regeneration in vivo.

  2. Smurf1 plays a role in EGF inhibition of BMP2-induced osteogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hye-Lim; Park, Hyun-Jung; Kwon, Arang [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Baek, Kyunghwa [Department of Pharmacology, College of Dentistry and Research Institute of Oral Science, Gangneung-Wonju National University, Gangneung 210-702, Gangwondo (Korea, Republic of); Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Baek, Jeong-Hwa, E-mail: baekjh@snu.ac.kr [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of)

    2014-05-01

    It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression, which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation. - Highlights: • EGF increases the expression level of Smurf1 in mesenchymal precursor cells. • EGF reduces the protein levels and transcriptional activity of Runx2 and Smad1. • EGF suppresses BMP2-induced osteogenic differentiation, which is rescued by Smurf1 knockdown.

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

    Directory of Open Access Journals (Sweden)

    Oliver Couture

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Piestun, Yogev; Yahav, Shlomo; Halevy, Orna

    2015-10-01

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

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

    Science.gov (United States)

    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)...

  7. Diversity in the utilization of glucose and lactate in synthetic mammalian myotubes generated by engineered configurations of MyoD and E12 in otherwise non-differentiation growth conditions.

    Science.gov (United States)

    Grubišić, Vladimir; Parpura, Vladimir

    2015-03-01

    We previously used the expression of various combinations and configurations of MyoD and E12, two basic helix-loop-helix transcription factors (TF), to produce populations of myotubes assuming distinct morphology, myofibrillar development and Ca2+ dynamics, from mammalian C2C12 myoblasts in non-differentiation growth conditions. Here, we assessed the synthetically generated myotubes in terms of energetics, otherwise necessary to sustain their mechanical output as bio-actuators. We found that the myotubes exhibit changed expression of key regulators for the uptake and utilization of two major cellular fuels, glucose and lactate. Furthermore, while lactate transport was uniformly slowed in all the populations of myotubes, glucose uptake and utilization were modified by particular TF configuration. Our approach allows the production of a class of biomaterials with predetermined energetics that could be applied in biorobotics, where fuel of choice could be used, and also in reparative medicine where, for example, particular population of myotubes could be additionally employed as glucose sinks to mitigate effects of secondary metabolic syndrome.

  8. Modified methods for culturing myoblasts of rats: Combination of multi-enzymatic digestion and double purification

    Institute of Scientific and Technical Information of China (English)

    Li Zhang; Wei Wang; Ming Fan; Xiaoping Chen; Shuhong Liu; Liang Sun

    2007-01-01

    BACKGROUND: With developments of tissue engineering and genetic engineering, we aim to culture myoblasts, which are characterized by high purity, high quality and high production, for wide application in neural regeneration researches.OBJECTIVE: To modify traditional dissociation method in order to obtain myoblasts, which are characterized by high purity, high quality and high production, and explore the biological properties under in vitro culture.DESIGN: Observational study.SETTING: Basic Institute of Academy of Military Medical Sciences of Chinese PLA.MATERIALS: Four neonatal Wistar rats of 5 days old, both genders and mean body mass of 10 g were selected in this study. The main reagents and devices were detailed as follows: DMEM medium (Gibco Company), fetus bovine serum (FBS, Hycolne Company), collagenase Ⅱ (Sigma Company), trypsin (Sigma Company), dispase Ⅱ (Sigma Company), desmin antibody (Fuzhou Maixin Company), antibody Ⅱ and ABC kit (Wuhan Boster Biotechnology Company), desk centrifuge (KUBATO, Japan), and inverted phase contrast microscope (LEICA DMIRB, Germany).METHODS: The experiment was carried out in the Basic Institute of Academy of Military Medical Sciences of Chinese PLA from June to October 2006. Neonatal rats were sacrificed under sterile condition to obtain skeletal muscles of limbs, which were washed with cold PBS (containing benzylpenicillin and estreptomicina), and muscular tissue was sheared into pieces. Then, those muscular pieces were added with mixed digestive enzyme (containing 2 g/L collagenase Ⅱ + 5 g/L dispase Ⅱ + 0.28 g/L CaCl2) as twice volume as pieces, dealt with mechanical pipetting for 5 minutes and cultured in CO2 incubator for 10 minutes.The operation was done for three times and the muscular pieces were digested for 45 minutes in total.Moreover, cells were suspended again in order to obtain myoblasts from skeletal muscle of neonatal rats. In addition, myoblasts were purified with differential attachment technique

  9. An integrated strategy for analyzing the unique developmental programs of different myoblast subtypes.

    Directory of Open Access Journals (Sweden)

    2006-02-01

    Full Text Available An important but largely unmet challenge in understanding the mechanisms that govern the formation of specific organs is to decipher the complex and dynamic genetic programs exhibited by the diversity of cell types within the tissue of interest. Here, we use an integrated genetic, genomic, and computational strategy to comprehensively determine the molecular identities of distinct myoblast subpopulations within the Drosophila embryonic mesoderm at the time that cell fates are initially specified. A compendium of gene expression profiles was generated for primary mesodermal cells purified by flow cytometry from appropriately staged wild-type embryos and from 12 genotypes in which myogenesis was selectively and predictably perturbed. A statistical meta-analysis of these pooled datasets--based on expected trends in gene expression and on the relative contribution of each genotype to the detection of known muscle genes--provisionally assigned hundreds of differentially expressed genes to particular myoblast subtypes. Whole embryo in situ hybridizations were then used to validate the majority of these predictions, thereby enabling true-positive detection rates to be estimated for the microarray data. This combined analysis reveals that myoblasts exhibit much greater gene expression heterogeneity and overall complexity than was previously appreciated. Moreover, it implicates the involvement of large numbers of uncharacterized, differentially expressed genes in myogenic specification and subsequent morphogenesis. These findings also underscore a requirement for considerable regulatory specificity for generating diverse myoblast identities. Finally, to illustrate how the developmental functions of newly identified myoblast genes can be efficiently surveyed, a rapid RNA interference assay that can be scored in living embryos was developed and applied to selected genes. This integrated strategy for examining embryonic gene expression and function provides

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

    Science.gov (United States)

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

    2016-01-15

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

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

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    D. T. Yates

    2012-01-01

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

  12. Developmental programming in response to intrauterine growth restriction impairs myoblast function and skeletal muscle metabolism.

    Science.gov (United States)

    Yates, D T; Macko, A R; Nearing, M; Chen, X; Rhoads, R P; Limesand, S W

    2012-01-01

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

  13. Myoblast fusion: Experimental systems and cellular mechanisms.

    Science.gov (United States)

    Schejter, Eyal D

    2016-12-01

    Fusion of myoblasts gives rise to the large, multi-nucleated muscle fibers that power and support organism motion and form. The mechanisms underlying this prominent form of cell-cell fusion have been investigated by a variety of experimental approaches, in several model systems. The purpose of this review is to describe and discuss recent progress in the field, as well as point out issues currently unresolved and worthy of further investigation. Following a description of several new experimental settings employed in the study of myoblast fusion, a series of topics relevant to the current understanding of the process are presented. These pertain to elements of three major cellular machineries- cell-adhesion, the actin-based cytoskeleton and membrane-associated elements- all of which play key roles in mediating myoblast fusion. Among the issues raised are the diversity of functions ascribed to different adhesion proteins (e.g. external cell apposition and internal recruitment of cytoskeleton regulators); functional significance of fusion-associated actin structures; and discussion of alternative mechanisms employing single or multiple fusion pore formation as the basis for muscle cell fusion.

  14. Bupivacaine can enhance lysosomal activity in mouse muscle myoblasts%布比卡因增强小鼠成肌细胞溶酶体的活性

    Institute of Scientific and Technical Information of China (English)

    熊静薇; 毛雨; 李荣荣; 丁正年

    2015-01-01

    Objective To investigate the effects of bupivacaine on lysosomal abundance and activity in mouse muscle myoblasts.Methods Mouse myoblasts C2C12 was randomly divided into control group (without any treatment) and bupivacaine group (treated with bupivacaine 600 μ mol/L for 6 h).After then,the changes of lysosomal pH was assessed by LysoSensor pH indicator.The content of lysosomes was detected by LysoTracker probe.The expression of lysosomal-associated membrane protein-1 (LAMP-1) and Cathepsin B was detected by Western blot analysis.The activity of lysosomal proteolytic enzymes Cathepsin B was determined by MagicRed assay kit.Results Bupivacaine did not affect lysosomal pH.However,compared with the controls,lysosomal abundance was significantly increased 15.15% following bupivacaine treatment(P<0.01).Moreover,protein expression levels of LAMP-1 and Cathepsin B were significantly upregulated 36.41% and 35.29% respetctively by bupivacaine (P<0.01).Furthermore,the activity of Cathepsin B was significantly increased 23.74% by bupivacaine(P<0.01).Conclusions Bupivacaine increased lysosomal content and enhance lysosomal activity in mouse muscle myoblasts.%目的 探讨局部麻醉药布比卡因对小鼠成肌细胞溶酶体的影响. 方法 将体外培养的小鼠成肌细胞C2C12分为2组.对照组:不加任何药物;布比卡因组:以600μmol/L布比卡因刺激细胞6h.实验结束后,用LysoSensor探针评价溶酶体腔pH,用LysoTrackor探针检测溶酶体含量,用蛋白免疫印迹法检测溶酶体相关膜蛋白-1(LAMP-1)和溶酶体蛋白水解酶Cathepsin B的表达水平,并以MagicRed染色法测定Cathepsin B的活性.结果 布比卡因对溶酶体腔pH没有影响.但是,与对照组相比,布比卡因组溶酶体含量增加15.15% (P<0.01),LAMP-1与Cathepsin B表达量分别增加36.41%、35.29% (P<0.01),Cathepsin B活性增加23.74%(P<0.01).结论 布比卡因能增加小鼠成肌细胞溶酶体含量,增强溶酶体活性.

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

    Directory of Open Access Journals (Sweden)

    Sebastian Gehmert

    2014-01-01

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

  16. LIM homeobox transcription factor Lhx2 inhibits skeletal muscle differentiation in part via transcriptional activation of Msx1 and Msx2.

    Science.gov (United States)

    Kodaka, Yusaku; Tanaka, Kiyoko; Kitajima, Kenji; Tanegashima, Kosuke; Matsuda, Ryoichi; Hara, Takahiko

    2015-02-15

    LIM homeobox transcription factor Lhx2 is known to be an important regulator of neuronal development, homeostasis of hair follicle stem cells, and self-renewal of hematopoietic stem cells; however, its function in skeletal muscle development is poorly understood. In this study, we found that overexpression of Lhx2 completely inhibits the myotube-forming capacity of C2C12 cells and primary myoblasts. The muscle dedifferentiation factors Msx1 and Msx2 were strongly induced by the Lhx2 overexpression. Short interfering RNA-mediated knockdown of Lhx2 in the developing limb buds of mouse embryos resulted in a reduction in Msx1 and Msx2 mRNA levels, suggesting that they are downstream target genes of Lhx2. We found two Lhx2 consensus-binding sites in the -2097 to -1189 genomic region of Msx1 and two additional sites in the -536 to +73 genomic region of Msx2. These sequences were shown by luciferase reporter assay to be essential for Lhx2-mediated transcriptional activation. Moreover, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that Lhx2 is present in chromatin DNA complexes bound to the enhancer regions of the Msx1 and Msx2 genes. These data demonstrate that Msx1 and Msx2 are direct transcriptional targets of Lhx2. In addition, overexpression of Lhx2 significantly enhanced the mRNA levels of bone morphogenetic protein 4 and transforming growth factor beta family genes. We propose that Lhx2 is involved in the early stage of skeletal muscle development by inducing multiple differentiation inhibitory factors.

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

    Science.gov (United States)

    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

  18. Compatibility of hyaluronic acid hydrogel and skeletal muscle myoblasts

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-04-15

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

  19. Reversal of Myoblast Aging by Tocotrienol Rich Fraction Posttreatment

    Directory of Open Access Journals (Sweden)

    Jing Jye Lim

    2013-01-01

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

  20. Experiment list: DRX013455 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available q of Pol II-Ser5ph, C2C12 myotube sample_name=DRS013213 || sample comment=The sample was fixed with 0.5% formaldehyde for 5 min. Diff...erentiation from myoblast state was done by transferring to Dulbecco's modified Eag

  1. Drosophila Kette coordinates myoblast junction dissolution and the ratio of Scar-to-WASp during myoblast fusion

    Science.gov (United States)

    Hamp, Julia; Löwer, Andreas; Dottermusch-Heidel, Christine; Beck, Lothar; Moussian, Bernard; Flötenmeyer, Matthias

    2016-01-01

    ABSTRACT The fusion of founder cells and fusion-competent myoblasts (FCMs) is crucial for muscle formation in Drosophila. Characteristic events of myoblast fusion include the recognition and adhesion of myoblasts, and the formation of branched F-actin by the Arp2/3 complex at the site of cell–cell contact. At the ultrastructural level, these events are reflected by the appearance of finger-like protrusions and electron-dense plaques that appear prior to fusion. Severe defects in myoblast fusion are caused by the loss of Kette (a homolog of Nap1 and Hem-2, also known as NCKAP1 and NCKAP1L, respectively), a member of the regulatory complex formed by Scar or WAVE proteins (represented by the single protein, Scar, in flies). kette mutants form finger-like protrusions, but the electron-dense plaques are extended. Here, we show that the electron-dense plaques in wild-type and kette mutant myoblasts resemble other electron-dense structures that are known to function as cellular junctions. Furthermore, analysis of double mutants and attempts to rescue the kette mutant phenotype with N-cadherin, wasp and genes of members of the regulatory Scar complex revealed that Kette has two functions during myoblast fusion. First, Kette controls the dissolution of electron-dense plaques. Second, Kette controls the ratio of the Arp2/3 activators Scar and WASp in FCMs. PMID:27521427

  2. Tracing myoblast fusion in Drosophila embryos by fluorescent actin probes.

    Science.gov (United States)

    Haralalka, Shruti; Abmayr, Susan M

    2015-01-01

    Myoblast fusion in the Drosophila embryo is a highly elaborate process that is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs). It occurs through an asymmetric event in which actin foci assemble in the FCMs at points of cell-cell contact and direct the formation of membrane protrusions that drive fusion. Herein, we describe the approach that we have used to image in living embryos the highly dynamic actin foci and actin-rich projections that precede myoblast fusion. We discuss resources currently available for imaging actin and myogenesis, and our experience with these resources if available. This technical report is not intended to be comprehensive on providing instruction on standard microscopy practices or software utilization. However, we discuss microscope parameters that we have used in data collection, and our experience with image processing tools in data analysis.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-10-18

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

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

    Full Text Available InP.Myo.10.AllAg.Myoblasts mm9 Input control Muscle Myoblasts SRX029148,SRX150190,S...RX497480,SRX497481,SRX497484,SRX497485,SRX766227 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Myo.10.AllAg.Myoblasts.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.20.AllAg.Myoblasts mm9 TFs and others Muscle Myoblasts SRX030143,SRX497478,...4,SRX029143,SRX227233,SRX227231,SRX227229,SRX984580 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Myo.20.AllAg.Myoblasts.bed ...

  15. Regulation of expression of osteoblast transcription factor SATB2 by BMP2 in osteoblastic differentiation of MSCs%MSCs成骨分化中BMP2对成骨转录因子SATB2表达的调控作用

    Institute of Scientific and Technical Information of China (English)

    左炽健; 剌婷; 张宁; 戴尅戎; 张晓玲

    2011-01-01

    目的 探索间充质干细胞(MSCs)成骨分化中骨形态发生蛋白2(BMP2)对成骨转录因子SATB2表达的调控作用.方法 体外培养小鼠间充质细胞系C2C12,腺病毒介导的BMP2 (Adv-BMP2)诱导其向成骨细胞分化,建立并验证C2C12细胞成骨分化细胞模型.Real-Time PCR和Western blotting分别检测C2C12细胞成骨分化过程中经不同浓度Adv-BMP2处理不同时间时SATB2 mRNA和SATB2蛋白表达;以经相应浓度Adv-β-Gal处理细胞作对照.结果 经150 pfu/cell Adv-BMP2处理C2C12细胞5d后,成骨细胞标志基因Ⅰ型胶原,骨唾液酸蛋白和骨钙素表达以及碱性磷酸酶活性均显著增加,MSCs成骨分化模型构建成功.150 pfu/cell Adv-BMP2诱导C2C12细胞成骨分化过程中,SATB2 mRNA和SATB蛋白表达随分化进程而增加;Adv-BMP2浓度为0~225 pfu/cell时,SATB2表达随Adv-BMP2浓度升高而增加.结论BMP2可调控SATB2的表达,从而影响MSCs成骨分化.%Objective To investigate the role of bone morphogenetic protein 2 (BMP2) in the regulation of expression of osteoblast transcription factor SATB2 in osteoblastic differentiation of mesenchymal stem cells ( MSCs). Methods Mesenchymal cell line C2C12 was stimulated by adenovirus-derived BMP2 (Adv-BMP2) for osteoblastic differentiation, and model of osteoblastic differentiation of C2C12 cells was established and verified. Real-Time PCR and Western blotting were employed to detect the expression of SATB2 mRNA and SATB2 protein respectively in osteoblastic differentiation treated by Adv-BMP2 for different time and by Adv-BMP2 of different concentrations. C2CI2 cells treated with Adv-β-Gal were served as controls. Results After treatment by ISO pfu/cell Adv-BMP2 for 5 d, the expression of collagen type I , bone sialoprotein and osteocalcin and the activity of alkaline phosphatase in C2C12 cells significantly increased, and the model of osteoblastic differentiation of C2C12 cells was established. The expression of SATB2 mRNA and

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    -energy phosphate compounds adenosine triphosphate and phosphocreatine with increasing severity of stress were identified. At anoxic conditions, an increase in (13)C-labeled lactate and appearance of glycerol-3-phosphate were observed. Accumulation of lactate and glycerol-3-phosphate is in agreement with a shift...... to anaerobic metabolism due to inhibition of the aerobic pathway in the mitochondria. Conversely, lower levels of unlabeled ((12)C) lactate were apparent at increasing severity of stress, which indicate that lactate is released from the myotubes to the medium. In conclusion, the metabolites identified...

  17. DUX4c is up-regulated in FSHD. It induces the MYF5 protein and human myoblast proliferation.

    Directory of Open Access Journals (Sweden)

    Eugénie Ansseau

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is a dominant disease linked to contractions of the D4Z4 repeat array in 4q35. We have previously identified a double homeobox gene (DUX4 within each D4Z4 unit that encodes a transcription factor expressed in FSHD but not control myoblasts. DUX4 and its target genes contribute to the global dysregulation of gene expression observed in FSHD. We have now characterized the homologous DUX4c gene mapped 42 kb centromeric of the D4Z4 repeat array. It encodes a 47-kDa protein with a double homeodomain identical to DUX4 but divergent in the carboxyl-terminal region. DUX4c was detected in primary myoblast extracts by Western blot with a specific antiserum, and was induced upon differentiation. The protein was increased about 2-fold in FSHD versus control myotubes but reached 2-10-fold induction in FSHD muscle biopsies. We have shown by Western blot and by a DNA-binding assay that DUX4c over-expression induced the MYF5 myogenic regulator and its DNA-binding activity. DUX4c might stabilize the MYF5 protein as we detected their interaction by co-immunoprecipitation. In keeping with the known role of Myf5 in myoblast accumulation during mouse muscle regeneration DUX4c over-expression activated proliferation of human primary myoblasts and inhibited their differentiation. Altogether, these results suggested that DUX4c could be involved in muscle regeneration and that changes in its expression could contribute to the FSHD pathology.

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

    Science.gov (United States)

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

    1997-01-01

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

  19. The Endocytic Recycling Protein EHD2 Interacts with Myoferlin to Regulate Myoblast Fusion*

    Science.gov (United States)

    Doherty, Katherine R.; Demonbreun, Alexis R.; Wallace, Gregory Q.; Cave, Andrew; Posey, Avery D.; Heretis, Konstantina; Pytel, Peter; McNally, Elizabeth M.

    2008-01-01

    Skeletal muscle is a multinucleated syncytium that develops and is maintained by the fusion of myoblasts to the syncytium. Myoblast fusion involves the regulated coalescence of two apposed membranes. Myoferlin is a membrane-anchored, multiple C2 domain-containing protein that is highly expressed in fusing myoblasts and required for efficient myoblast fusion to myotubes. We found that myoferlin binds directly to the eps15 homology domain protein, EHD2. Members of the EHD family have been previously implicated in endocytosis as well as endocytic recycling, a process where membrane proteins internalized by endocytosis are returned to the plasma membrane. EHD2 binds directly to the second C2 domain of myoferlin, and EHD2 is reduced in myoferlin null myoblasts. In contrast to normal myoblasts, myoferlin null myoblasts accumulate labeled transferrin and have delayed recycling. Introduction of dominant negative EHD2 into myoblasts leads to the sequestration of myoferlin and inhibition of myoblast fusion. The interaction of myoferlin with EHD2 identifies molecular overlap between the endocytic recycling pathway and the machinery that regulates myoblast membrane fusion. PMID:18502764

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

    Directory of Open Access Journals (Sweden)

    Takashi Matsumoto

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

  1. Mechanical stimulation increases proliferation, differentiation and protein expression in culture

    DEFF Research Database (Denmark)

    Grossi, Alberto; Yadav, Kavita; Lawson, Moira Ann

    2007-01-01

    Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. Myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Stimulation due to stretch- or load...

  2. Analysis of Mitochondrial Network Morphology in Cultured Myoblasts from Patients with Mitochondrial Disorders.

    Science.gov (United States)

    Sládková, J; Spáčilová, J; Čapek, M; Tesařová, M; Hansíková, H; Honzík, T; Martínek, J; Zámečník, J; Kostková, O; Zeman, J

    2015-01-01

    Mitochondrial morphology was studied in cultivated myoblasts obtained from patients with mitochondrial disorders, including CPEO, MELAS and TMEM70 deficiency. Mitochondrial networks and ultrastructure were visualized by fluorescence microscopy and transmission electron microscopy, respectively. A heterogeneous picture of abnormally sized and shaped mitochondria with fragmentation, shortening, and aberrant cristae, lower density of mitochondria and an increased number of "megamitochondria" were found in patient myoblasts. Morphometric Fiji analyses revealed different mitochondrial network properties in myoblasts from patients and controls. The small number of cultivated myoblasts required for semiautomatic morphometric image analysis makes this tool useful for estimating mitochondrial disturbances in patients with mitochondrial disorders.

  3. Inhibition of methylation decreases osteoblast differentiation via a non-DNA-dependent methylation mechanism.

    Science.gov (United States)

    Vaes, Bart L T; Lute, Carolien; van der Woning, Sebastian P; Piek, Ester; Vermeer, Jenny; Blom, Henk J; Mathers, John C; Müller, Michael; de Groot, Lisette C P G M; Steegenga, Wilma T

    2010-02-01

    S-adenosylmethionine (SAM)-dependent methylation of biological molecules including DNA and proteins is rapidly being uncovered as a critical mechanism for regulation of cellular processes. We investigated the effects of reduced SAM-dependent methylation on osteoblast differentiation by using periodate oxidized adenosine (ADOX), an inhibitor of SAM-dependent methyltransferases. The capacity of this agent to modulate osteoblast differentiation was analyzed under non-osteogenic control conditions and during growth factor-induced differentiation and compared with the effect of inhibition of DNA methylation by 5-Aza-2'-deoxycytidine (5-Aza-CdR). Without applying specific osteogenic triggers, both ADOX and 5-Aza-CdR induced mRNA expression of the osteoblast markers Alp, Osx, and Ocn in murine C2C12 cells. Under osteogenic conditions, ADOX inhibited differentiation of both human mesenchymal stem cells and C2C12 cells. Gene expression analysis of early (Msx2, Dlx5, Runx2) and late (Alp, Osx, Ocn) osteoblast markers during bone morphogenetic protein 2-induced C2C12 osteoblast differentiation revealed that ADOX only reduced expression of the late phase Runx2 target genes. By using a Runx2-responsive luciferase reporter (6xOSE), we showed that ADOX reduced the activity of Runx2, while 5-Aza-CdR had no effect. Taken together, our data suggest that decreased SAM-dependent methyltransferase activity leads to impaired osteoblast differentiation via non-DNA-dependent methylation mechanisms and that methylation is a regulator of Runx2-controlled gene expression.

  4. Muscle Specific Fragile X Related Protein 1 Isoforms are Sequestered in the Nucleus of Undifferentiated Myoblast

    Directory of Open Access Journals (Sweden)

    Khandjian Edouard W

    2000-12-01

    Full Text Available Abstract Background The family of Fragile X Mental Retardation Proteins is composed of three members: Fragile Mental Retardation 1, Fragile X Related 1 and X Related 2 proteins. These proteins are associated with mRNPs within translating ribosomes and have the capacity to shuttle between the nucleus and the cytoplasm. Great attention has been given to FMRP due to its implication in human hereditary mental retardation while FXR1P and FXR2P have only recently been studied. Results Using antibodies directed against several epitopes of FXR1P, we have detected protein isoforms generated by small peptides pocket inserts. Four isoforms of MW 70, 74, 78, 80 kDa are widely distributed in mouse organs, while in striated muscles these isoforms are replaced by proteins of 82 and 84 kDa containing an extra pocket of 27 aa. Expression of these muscle isoforms is an early event during in vitro differentiation of myoblasts into myotubes and correlates with the activation of muscle-specific genes. However, while FXR1P82,84 are associated with cytoplasmic mRNPs in myotubes, they are sequestered in the nuclei of undifferentiated myoblasts. These observations suggest that, in addition to a cytoplasmic function yet to be defined, FXR1P82,84 may play a nuclear role in pre-mRNA metabolism. Conclusions The pattern of subcellular partitioning of FXR1P isoforms during myogenesis is unique among the family of the FXR proteins. The model system described here should be considered as a powerful tool for ongoing attempts to unravel structure-function relationships of the different FMR family members since the potential role(s of FXR1P as a compensatory factor in Fragile X syndrome is still elusive.

  5. Polyunsaturated fatty acids incorporation into cardiolipin in H9c2 cardiac myoblast.

    Science.gov (United States)

    Ting, Hsiu-Chi; Chao, Yu-Jen; Hsu, Yuan-Hao Howard

    2015-07-01

    Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), known as ω-3 polyunsaturated fatty acid (PUFA), are common nutrients in daily food intake and have been shown to prevent cardiovascular disease and improve cardiac functions. Cardiolipin is a mitochondrial phospholipid necessary for maintaining physiological function of mitochondria. Several studies have indicated that the cardiolipin acyl chain compositions affect the function of cardiolipin and mitochondria. Here, we investigated the structural changes of cardiolipin after DHA and EPA supplementation and compared them to arachidonic acid (AA) treatment. H9c2 cardiac myoblast was used as a cell model, and cardiolipin species was monitored and identified via LC-MS and MS/MS. Our results showed distinct mass envelopes of cardiolipin with the same carbon number but different double bonds in mass spectrum. There were 116 cardiolipin species with 36 distinct mass in 6 mass envelopes identified by MS/MS. Three days of PUFA treatment resulted in decreases of low-molecular-weight cardiolipin and increases of high-molecular-weight cardiolipin, suggesting the incorporation of exogenous DHA, EPA and AA into mitochondrial cardiolipin. PUFA incorporation was further verified by MS/MS analysis. More importantly, we found that DHA supplementation elevated the percent content of less unsaturated cardiolipin species and highly unsaturated cardiolipin species, containing ω-3 fatty acyl chains, indicating a ω-3 fatty acid incorporation mechanism with peroxidation protection. Our results indicate that PUFA supplementation differentially perturbed the fatty acyl chain compositions in the mitochondrial cardiolipin in the H9c2 cardiac myoblast, suggesting that mitochondrial membrane and the function of mitochondria are susceptible to exogenous lipid species.

  6. Absence of muscle regeneration after implantation of a collagen matrix seeded with myoblasts

    NARCIS (Netherlands)

    van Wachem, PB; Brouwer, LA; van Luyn, MJA

    1999-01-01

    Collagens are widely used as biomaterials for e.g. soft tissue reconstruction. The present study was aimed at reconstruction of abdominal wall muscle using processed dermal sheep collagen (DSC) and myoblast seeding. Myoblasts were harvested from foetal quadriceps muscle of an inbred rat strain, cult

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

    Science.gov (United States)

    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.

  8. PAX3-FOXO1 is essential for tumour initiation and maintenance but not recurrence in a human myoblast model of rhabdomyosarcoma.

    Science.gov (United States)

    Pandey, Puspa R; Chatterjee, Bishwanath; Olanich, Mary E; Khan, Javed; Miettinen, Markku M; Hewitt, Stephen M; Barr, Frederic G

    2017-01-31

    The PAX3-FOXO1 fusion gene is generated by a 2;13 chromosomal translocation and is a characteristic feature of an aggressive subset of rhabdomyosarcoma (RMS). To dissect the mechanism of oncogene action during RMS tumourigenesis and progression, doxycycline-inducible PAX3-FOXO1 and constitutive MYCN expression constructs were introduced into immortalised human myoblasts. Though myoblasts expressing PAX3-FOXO1 or MYCN alone were not transformed in focus formation assays, combined PAX3-FOXO1 and MYCN expression resulted in transformation. Following intramuscular injection into immunodeficient mice, myoblasts expressing PAX3-FOXO1 and MYCN formed rapidly growing RMS tumours whereas myoblasts expressing only PAX3-FOXO1 formed tumours after a longer latency period. Doxycycline withdrawal in myoblasts expressing inducible PAX3-FOXO1 and constitutive MYCN following tumour formation in vivo or focus formation in vitro resulted in tumour regression or smaller foci associated with myogenic differentiation and cell death. Following regression, most tumours recurred in the absence of doxycycline. Analysis of recurrent tumours revealed a subset without PAX3-FOXO1 expression, and cell lines derived from these recurrent tumours demonstrated transformation in the absence of doxycycline. The doxycycline-independent oncogenicity in these recurrent tumour-derived lines persisted even after PAX3-FOXO1 was inactivated by a CRISPR-Cas9 editing strategy. Whereas cell lines derived from primary tumours were dependent on PAX3-FOXO1 and differentiated following doxycycline withdrawal, recurrent tumour-derived cells without PAX3-FOXO1 expression did not differentiate under these conditions. These findings indicate that PAX3-FOXO1 collaborates with MYCN during early RMS tumourigenesis to dysregulate proliferation and inhibit myogenic differentiation and cell death. Although most cells in the primary tumours are dependent on PAX3-FOXO1, recurrent tumours can develop by a PAX3-FOXO1

  9. Experiment list: SRX062122 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available le|Tissue Diagnosis=NOS 102757855,83.0,18.6,1196 GSM721306: Sonicated input MB source_name=Growing C2C12 myo...blasts || cell line=C2C12 || cell type=myoblasts || chromatin preparation method=sonication || chip antibody

  10. Heat shock pretreatment enhances porcine myoblasts survival after autotransplantation in intact skeletal muscle

    Institute of Scientific and Technical Information of China (English)

    YANG Sheng; Thomas LAUMONIER; Jacques MENETREY

    2007-01-01

    Myoblast transplantation (MT) is a cell-based gene therapy treatment, representing a potential treatment for Duchenne muscular dystrophy (DMD), cardiac failure and muscle trauma. The rapid and massive death of transplanted cells after MT is considered as a major hurdle which limits the efficacy of MT treatment. Heat shock proteins (HSPs) are overexpressed when cells undergo various insults. HSPs have been described to protect cells in vivo and in vitro against diverse insults. The aim of our study is to investigate whether HSP overexpression could increase myoblast survival after autotransplantation in pig intact skeletal muscle. HSP expression was induced by warming the cells at 42℃ for 1 h. HSP70 expression was quantified by Western blot and flow cytometry 24 h after the treatment. To investigate the myogenic characteristics of myoblasts, desmin and CD56 were analysed by Western blot and flow cytometry; and the fusion index was measured. We also quantified cell survival after autologous transplantation in pig intact skeletal muscle and followed cell integration. Results showed that heat shock treatment of myoblasts induced a significative overexpression of the HSP70 (P<0.01) without loss of their myogenic characteristics as assessed by FACS and fusion index. In vivo (n=7), the myoblast survival rate was not significantly different at 24 h between heat shock treated and nontreated cells (67.69%±8.35% versus 58.79%±8.35%, P>0.05). However, the myoblast survival rate in the heat shocked cells increased by twofold at 48 h (53.32%±8.22% versus 28.27%±6.32%, P<0.01)and more than threefold at 120 h (26.33%±5.54% versus 8.79%±2.51%, P<0.01). Histological analysis showed the presence of non-heat shocked and heat shocked donor myoblasts fused with host myoblasts. These results suggested that heat shock pretreatment increased the HSP70 expression in porcine myoblasts, and improved the survival rate after autologous transplantation. Therefore, heat shock

  11. Heat shock pretreatment enhances porcine myoblasts survival after autotransplantation in intact skeletal muscle

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Myoblast transplantation (MT) is a cell-based gene therapy treatment, representing a potential treat-ment for Duchenne muscular dystrophy (DMD), cardiac failure and muscle trauma. The rapid and mas-sive death of transplanted cells after MT is considered as a major hurdle which limits the efficacy of MT treatment. Heat shock proteins (HSPs) are overexpressed when cells undergo various insults. HSPs have been described to protect cells in vivo and in vitro against diverse insults. The aim of our study is to investigate whether HSP overexpression could increase myoblast survival after autotransplantation in pig intact skeletal muscle. HSP expression was induced by warming the cells at 42℃ for 1 h. HSP70 expression was quantified by Western blot and flow cytometry 24 h after the treatment. To investigate the myogenic characteristics of myoblasts, desmin and CD56 were analysed by Western blot and flow cytometry; and the fusion index was measured. We also quantified cell survival after autologous transplantation in pig intact skeletal muscle and followed cell integration. Results showed that heat shock treatment of myoblasts induced a significative overexpression of the HSP70 (P < 0.01) without loss of their myogenic characteristics as assessed by FACS and fusion index. In vivo (n=7), the myoblast survival rate was not significantly different at 24 h between heat shock treated and non- treated cells (67.69% ± 8.35% versus 58.79% ± 8.35%, P > 0.05). However, the myoblast survival rate in the heat shocked cells increased by twofold at 48 h (53.32% ± 8.22% versus 28.27% ± 6.32%, P < 0.01) and more than threefold at 120 h (26.33% ± 5.54% versus 8.79% ± 2.51%, P < 0.01). Histological analy-sis showed the presence of non-heat shocked and heat shocked donor myoblasts fused with host myoblasts. These results suggested that heat shock pretreatment increased the HSP70 expression in porcine myoblasts, and improved the survival rate after autologous transplantation

  12. Reductive stress impairs myoblasts mitochondrial function and triggers mitochondrial hormesis.

    Science.gov (United States)

    Singh, François; Charles, Anne-Laure; Schlagowski, Anna-Isabel; Bouitbir, Jamal; Bonifacio, Annalisa; Piquard, François; Krähenbühl, Stephan; Geny, Bernard; Zoll, Joffrey

    2015-07-01

    Even though oxidative stress damage from excessive production of ROS is a well known phenomenon, the impact of reductive stress remains poorly understood. This study tested the hypothesis that cellular reductive stress could lead to mitochondrial malfunction, triggering a mitochondrial hormesis (mitohormesis) phenomenon able to protect mitochondria from the deleterious effects of statins. We performed several in vitro experiments on L6 myoblasts and studied the effects of N-acetylcysteine (NAC) at different exposure times. Direct NAC exposure (1mM) led to reductive stress, impairing mitochondrial function by decreasing maximal mitochondrial respiration and increasing H₂O₂production. After 24h of incubation, the reactive oxygen species (ROS) production was increased. The resulting mitochondrial oxidation activated mitochondrial biogenesis pathways at the mRNA level. After one week of exposure, mitochondria were well-adapted as shown by the decrease of cellular ROS, the increase of mitochondrial content, as well as of the antioxidant capacities. Atorvastatin (ATO) exposure (100μM) for 24h increased ROS levels, reduced the percentage of live cells, and increased the total percentage of apoptotic cells. NAC exposure during 3days failed to protect cells from the deleterious effects of statins. On the other hand, NAC pretreatment during one week triggered mitochondrial hormesis and reduced the deleterious effect of statins. These results contribute to a better understanding of the redox-dependant pathways linked to mitochondria, showing that reductive stress could trigger mitochondrial hormesis phenomenon.

  13. Culture Conditions Affect Expression of DUX4 in FSHD Myoblasts

    Directory of Open Access Journals (Sweden)

    Sachchida Nand Pandey

    2015-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Antti Siltanen

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-21

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

  16. Spatial and functional restriction of regulatory molecules during mammalian myoblast fusion

    Energy Technology Data Exchange (ETDEWEB)

    Pavlath, Grace K., E-mail: gpavlat@emory.edu [Department of Pharmacology, Emory University, 1510 Clifton Rd., Room 5027, Atlanta, GA 30322 (United States)

    2010-11-01

    Myoblast fusion is a highly regulated process that is key for forming skeletal muscle during development and regeneration in mammals. Much remains to be understood about the molecular regulation of myoblast fusion. Some molecules that influence mammalian muscle fusion display specific cellular localization during myogenesis. Such molecules can be localized to the contact region between two fusing cells either in both cells or only in one of the cells. How distinct localization of molecules contributes to fusion is not clear. Further complexity exists as other molecules are functionally restricted to myoblasts at later stages of myogenesis to regulate their fusion with multinucleated myotubes. This review examines these three categories of molecules and discusses how spatial and functional restriction may contribute to the formation of a multinucleated cell. Understanding how and why molecules become restricted in location or function is likely to provide further insights into the mechanisms regulating mammalian muscle fusion.

  17. Myoblast transplantation for heart repair: A review of the state of the field

    Institute of Scientific and Technical Information of China (English)

    Howard J. Leonhardt; Michael Brown

    2006-01-01

    Over 200 humans have been treated with myoblast transplantation for heart muscle repair since June 2000. Bioheart sponsored percutaneous delivery studies began in May 2001 in Europe. Approximately one third of the patients have exhibited substantial improvement in left ventricular ejection fraction (LVEF) of over 30% and two heart failure class improvements. Over 80% of the patients have exhibited one heart failure class improvement with moderate improvement of LVEF. Clinical trials seem to demonstrate a marked reduction in emergency hospitalizations in myoblast treated patients. Many years of careful studies have lead to randomized controlled studies that are enrolling patients now at numerous centers worldwide. A firm conclusion on the safety and efficacy of myoblast transplantation cannot be determined until these randomized studies are completed. Final results from randomized controlled studies should be available soon. (J Geriatr Cardiol 2006;3:165-7.)

  18. The development of adult abdominal muscles in Drosophila: myoblasts express twist and are associated with nerves.

    Science.gov (United States)

    Currie, D A; Bate, M

    1991-09-01

    During metamorphosis, the adult muscles of the Drosophila abdomen develop from pools of myoblasts that are present in the larva. The adult myoblasts express twist in the third larval instar and the early pupa and are closely associated with nerves. Growing adult nerves and the twist-expressing cells migrate out across the developing abdominal epidermis, and as twist expression declines, the myoblasts begin to synthesize beta 3 tubulin. There follows a process involving cell fusion and segregation into cell groups to form multinucleate muscle precursors. These bipolar precursors migrate at both ends to find their correct attachment points. beta 3 tubulin expression continues at least until 51 h APF by which time the adult muscle pattern has been established.

  19. Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders

    Directory of Open Access Journals (Sweden)

    Mamchaoui Kamel

    2011-11-01

    Full Text Available Abstract Background Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies. Methods Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders. Results The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both in vitro and in vivo after transplantation into regenerating muscle of immunodeficient mice. Conclusions Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess in vivo the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.

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

    OpenAIRE

    Yang, Chuanbin; Li, Xuechen; Rong, Jianhui

    2014-01-01

    Background The Chinese medicine formulation ISF-1 (also known as Bu-Yang-Huan-Wu-Tang) for post-stroke rehabilitation could increase the expression of growth-regulating protein follistatin by approximately 4-fold. This study aims to identify the active compounds of ISF-1 for the induction of follistatin expression. Methods Active compounds in ISF-1 responsible for induction of follistatin were identified by a bioactivity-guided fractionation procedure involving liquid-liquid extraction, HPLC ...

  1. Conjugated linoleic acid (CLA) stimulates mitochondrial biogenesis signaling by the upregulation of PPARγ coactivator 1α (PGC-1α) in C2C12 cells.

    Science.gov (United States)

    Kim, Yoo; Park, Yeonhwa

    2015-04-01

    Along with its effect on body fat reduction, dietary conjugated linoleic acid (CLA) has been reported to improve physical activity and endurance capacity in mice. It has been suggested these effects may in part be due to physiological changes in skeletal muscle, however, the mode of action is not completely understood. Thus, the purpose of this study was to determine the relevant mechanisms of CLA isomers for mitochondrial biogenesis, one of the most important adaptive responses in skeletal muscle. Both cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12) CLA isomers increased the expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), however, only the t10,c12 isomer, but not c9,t11, increased phosphorylation of AMP-activated protein kinase (AMPK) compared to the control. Among downstream biomarkers of PGC-1α, the CLA mixed isomer enhanced the expression of peroxisome proliferator-activated receptor-δ (PPARδ). Both c9,t11 and t10,c12 CLA isomers increased expression of nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (Tfam), while the c9,t11 increased expression of cytochrome c (Cyt C) and t10,c12 CLA increased expression of voltage-dependent anion channel (VDAC), respectively. Both CLA isomers significantly increased mitochondrial DNA copy number compared to that of control. These findings suggest that the individual CLA isomers potentiate mitochondrial biogenesis via PGC-1α-NRF-1-Tfam signaling cascade, although downstream regulation may be isomer dependent.

  2. The immune system modulator a1-acid glycoprotein inhibits insulin and IGF1 induced protein synthesis in C2C12 myotubes

    Science.gov (United States)

    Alpha-1 acid glycoprotein (AGP) has previously been demonstrated by our laboratory to be negatively correlated with growth rate in newborn piglets. However, a mechanism of action for AGP in growth has not been identified. Previous research has demonstrated that AGP can modify adipose tissue metabo...

  3. The physical interaction of myoblasts with the microenvironment during remodeling of the cytoarchitecture.

    Directory of Open Access Journals (Sweden)

    Daniel J Modulevsky

    Full Text Available Integrins, focal adhesions, the cytoskeleton and the extracellular matrix, form a structural continuum between the external and internal environment of the cell and mediate the pathways associated with cellular mechanosensitivity and mechanotransduction. This continuum is important for the onset of muscle tissue generation, as muscle precursor cells (myoblasts require a mechanical stimulus to initiate myogenesis. The ability to sense a mechanical cue requires an intact cytoskeleton and strong physical contact and adhesion to the microenvironment. Importantly, myoblasts also undergo reorientation, alignment and large scale remodeling of the cytoskeleton when they experience mechanical stretch and compression in muscle tissue. It remains unclear if such dramatic changes in cell architecture also inhibit physical contact and adhesion with the tissue microenvironment that are clearly important to myoblast physiology. In this study, we employed interference reflection microscopy to examine changes in the close physical contact of myoblasts with a substrate during induced remodeling of the cytoarchitecture (de-stabilization of the actin and microtubule cytoskeleton and inhibition of acto-myosin contractility. Our results demonstrate that while each remodeling pathway caused distinct effects on myoblast morphology and sub-cellular structure, we only observed a ~13% decrease in close physical contact with the substrate, regardless of the pathway inhibited. However, this decrease did not correlate well with changes in cell adhesion strength. On the other hand, there was a close correlation between cell adhesion and β1-integrin expression and the presence of cell-secreted fibronectin, but not with the presence of intact focal adhesions. In this study, we have shown that myoblasts are able to maintain a large degree of physical contact and adhesion to the microenvironment, even during shot periods (<60 min of large scale remodeling and physiological

  4. Hedgehog can drive terminal differentiation of amniote slow skeletal muscle

    Directory of Open Access Journals (Sweden)

    Bildsoe Heidi

    2004-07-01

    Full Text Available Abstract Background Secreted Hedgehog (Hh signalling molecules have profound influences on many developing and regenerating tissues. Yet in most vertebrate tissues it is unclear which Hh-responses are the direct result of Hh action on a particular cell type because Hhs frequently elicit secondary signals. In developing skeletal muscle, Hhs promote slow myogenesis in zebrafish and are involved in specification of medial muscle cells in amniote somites. However, the extent to which non-myogenic cells, myoblasts or differentiating myocytes are direct or indirect targets of Hh signalling is not known. Results We show that Sonic hedgehog (Shh can act directly on cultured C2 myoblasts, driving Gli1 expression, myogenin up-regulation and terminal differentiation, even in the presence of growth factors that normally prevent differentiation. Distinct myoblasts respond differently to Shh: in some slow myosin expression is increased, whereas in others Shh simply enhances terminal differentiation. Exposure of chick wing bud cells to Shh in culture increases numbers of both muscle and non-muscle cells, yet simultaneously enhances differentiation of myoblasts. The small proportion of differentiated muscle cells expressing definitive slow myosin can be doubled by Shh. Shh over-expression in chick limb bud reduces muscle mass at early developmental stages while inducing ectopic slow muscle fibre formation. Abundant later-differentiating fibres, however, do not express extra slow myosin. Conversely, Hh loss of function in the limb bud, caused by implanting hybridoma cells expressing a functionally blocking anti-Hh antibody, reduces early slow muscle formation and differentiation, but does not prevent later slow myogenesis. Analysis of Hh knockout mice indicates that Shh promotes early somitic slow myogenesis. Conclusions Taken together, the data show that Hh can have direct pro-differentiative effects on myoblasts and that early-developing muscle requires Hh for

  5. Autologous myoblast transplantation for oculopharyngeal muscular dystrophy: a phase I/IIa clinical study.

    Science.gov (United States)

    Périé, Sophie; Trollet, Capucine; Mouly, Vincent; Vanneaux, Valérie; Mamchaoui, Kamel; Bouazza, Belaïd; Marolleau, Jean Pierre; Laforêt, Pascal; Chapon, Françoise; Eymard, Bruno; Butler-Browne, Gillian; Larghero, Jérome; St Guily, Jean Lacau

    2014-01-01

    Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomal dominant genetic disease mainly characterized by ptosis and dysphagia. We conducted a phase I/IIa clinical study (ClinicalTrials.gov NCT00773227) using autologous myoblast transplantation following myotomy in adult OPMD patients. This study included 12 patients with clinical diagnosis of OPMD, indication for cricopharyngeal myotomy, and confirmed genetic diagnosis. The feasibility and safety end points of both autologous myoblast transplantation and the surgical procedure were assessed by videoendoscopy in addition to physical examinations. Potential therapeutic benefit was also assessed through videoendoscopy and videofluoroscopy of swallowing, quality of life score, dysphagia grade, and a drink test. Patients were injected with a median of 178 million myoblasts following myotomy. Short and long-term (2 years) safety and tolerability were observed in all the patients, with no adverse effects. There was an improvement in the quality of life score for all 12 patients, and no functional degradation in swallowing was observed for 10 patients. A cell dose-dependant improvement in swallowing was even observed in this study. This trial supports the hypothesis that a local injection of autologous myoblasts in the pharyngeal muscles is a safe and efficient procedure for OPMD patients.

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

    Science.gov (United States)

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

    2015-04-01

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

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

    Science.gov (United States)

    Dhanyasi, Nagaraju; Segal, Dagan; Shimoni, Eyal; Shinder, Vera; Shilo, Ben-Zion; VijayRaghavan, K; Schejter, Eyal D

    2015-10-12

    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.

  8. Histone H3 Methyltransferase Suv39h1 Prevents Myogenic Terminal Differentiation by Repressing MEF2 Activity in Muscle Cells

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    Wei Jin

    2016-11-01

    Full Text Available The myogenic regulatory factors (MRFs and myocyte enhancer factor 2 (MEF2 transcription factors have been extensively studied as key transcription factors that regulate myogenic gene expression. However, few reports on the molecular mechanism that modulates chromatin remodeling during skeletal muscle differentiation are available. We reported here that the expression of the H3-K9 methyltransferase Suv39h1 was decreased during myoblast differentiation. Ectopic expression of Suv39h1 could inhibit myoblast differentiation, increasing H3-K9 methylation levels, whereas knockdown of Suv39h1 stimulated myoblast differentiation. Furthermore, Suv39h1 interacted with MEF2C directly and inhibited MEF2 transcription activity in a dose-dependent manner. Together, our studies revealed a molecular mechanism wherein Suv39h1 modulated myogenic gene expression and activation during skeletal muscle differentiation.

  9. Basal Lamina Mimetic Nanofibrous Peptide Networks for Skeletal Myogenesis

    Science.gov (United States)

    Yasa, I. Ceren; Gunduz, Nuray; Kilinc, Murat; Guler, Mustafa O.; Tekinay, Ayse B.

    2015-11-01

    Extracellular matrix (ECM) is crucial for the coordination and regulation of cell adhesion, recruitment, differentiation and death. Therefore, equilibrium between cell-cell and cell-matrix interactions and matrix-associated signals are important for the normal functioning of cells, as well as for regeneration. In this work, we describe importance of adhesive signals for myoblast cells’ growth and differentiation by generating a novel ECM mimetic peptide nanofiber scaffold system. We show that not only structure but also composition of bioactive signals are important for cell adhesion, growth and differentiation by mimicking the compositional and structural properties of native skeletal muscle basal lamina. We conjugated laminin-derived integrin binding peptide sequence, “IKVAV”, and fibronectin-derived well known adhesive sequence, “RGD”, into peptide nanostructures to provide adhesive and myogenic cues on a nanofibrous morphology. The myogenic and adhesive signals exhibited a synergistic effect on model myoblasts, C2C12 cells. Our results showed that self-assembled peptide nanofibers presenting laminin derived epitopes support adhesion, growth and proliferation of the cells and significantly promote the expression of skeletal muscle-specific marker genes. The functional peptide nanofibers used in this study present a biocompatible and biodegradable microenvironment, which is capable of supporting the growth and differentiation of C2C12 myoblasts into myotubes.

  10. A surgical robot with a heart-surface-motion synchronization mechanism for myoblast cell sheet transplantation.

    Science.gov (United States)

    Xu, Kangyi; Nakamura, Ryoichi

    2013-01-01

    Myoblast cell sheets are employed in the clinical treatment of heart disorders. We propose a surgical robot system with two endoscopic cameras, characterized by a double remote center of motion (RCM) mechanism, to realize heart-surface-motion synchronization movement for myoblast cell sheet transplantation on a beating heart surface. A robot system with the double RCM mechanism was developed for which the linear and rotation motions are totally isolated, and an experiment was conducted to evaluate the tracking accuracy of the robot system when tracking a randomly moving target. The tracking data were updated with a Polaris system at 30 Hz. The experiment results showed linear and rotation tracking errors of 4.93 ± 5.92 mm and 2.54 ± 5.44°, respectively.

  11. Cargo delivery to adhering myoblast cells from liposome-containing poly(dopamine) composite coatings

    DEFF Research Database (Denmark)

    Madsen, Martin Elias Lynge; Mian Teo, Boon; Laursen, Marie Bækgaard;

    2013-01-01

    Designing surfaces to deliver therapeutic compounds to adhering cells is of paramount importance for both implantable devices and tissue engineering. We report the assembly of composite films consisting of liposomes as drug deposits in a poly(dopamine) matrix. We monitor the film assembly using...... a quartz crystal microbalance with dissipation. We assess the response of adhering myoblast cells to these films containing fluorescent lipids in terms of uptake efficiency and cell mean fluorescence using flow cytometry. The viability of adhering myoblast cells, when the hydrophobic cytotoxic compound...... thiocoraline is entrapped in the lipid membrane, is assessed for different films. Coatings with one or two liposome deposition steps are considered. Further, the effect of the polymer separation layers between the liposome layers is determined. We found that it is possible to use the different nano...

  12. Milk-derived ribonuclease 5 preparations induce myogenic differentiation in vitro and muscle growth in vivo.

    Science.gov (United States)

    Knight, Matthew I; Tester, Angus M; McDonagh, Matthew B; Brown, Andrew; Cottrell, Jeremy; Wang, Jianghui; Hobman, Peter; Cocks, Benjamin G

    2014-12-01

    Ribonuclease 5, also known as angiogenin, is a stable and abundant ribonuclease in milk whey protein, which is able to regulate several cellular functions, including capillary formation, neuron survival, and epithelial cell growth. Ribonuclease 5 is important for protein synthesis directly stimulating rRNA synthesis in the nucleolus. Here, we show that biologically active RNase5 can be purified from bovine milk. Furthermore, we show that milk-derived RNase5 directly stimulates muscle cell differentiation in vitro, inducing C2C12 cell differentiation and myogenesis. When supplemented into the diet of healthy adult mice, milk-derived RNase5 preparations promoted muscle weight gain and grip strength. Collectively, these data indicate that milk-derived RNase5 preparations exhibit a novel role in skeletal muscle cell function.

  13. The intracellular domain of Dumbfounded affects myoblast fusion efficiency and interacts with Rolling pebbles and Loner.

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    Sarada Bulchand

    Full Text Available 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.

  14. The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation.

    Science.gov (United States)

    Deng, Su; Bothe, Ingo; Baylies, Mary K

    2015-08-01

    The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, is essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease.

  15. The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation.

    Directory of Open Access Journals (Sweden)

    Su Deng

    2015-08-01

    Full Text Available The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia, which nucleates and facilitates the elongation of actin filaments, is essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease.

  16. Maged1, a new regulator of skeletal myogenic differentiation and muscle regeneration

    Directory of Open Access Journals (Sweden)

    Achouri Younes

    2010-07-01

    Full Text Available Abstract Background In normal adult skeletal muscle, cell turnover is very slow. However, after an acute lesion or in chronic pathological conditions, such as primary myopathies, muscle stem cells, called satellite cells, are induced to proliferate, then withdraw definitively from the cell cycle and fuse to reconstitute functional myofibers. Results We show that Maged1 is expressed at very low levels in normal adult muscle but is strongly induced after injury, during the early phase of myoblast differentiation. By comparing in vitro differentiation of myoblasts derived from wild-type or Maged1 knockout mice, we observed that Maged1 deficiency results in reduced levels of p21CIP1/WAF1, defective cell cycle exit and impaired myotube maturation. In vivo, this defect results in delayed regeneration of injured muscle. Conclusions These data demonstrate for the first time that Maged1 is an important factor required for proper skeletal myoblast differentiation and muscle healing.

  17. PKC-Mediated ZYG1 Phosphorylation Induces Fusion of Myoblasts as well as of Dictyostelium Cells

    Directory of Open Access Journals (Sweden)

    Aiko Amagai

    2012-01-01

    Full Text Available We have previously demonstrated that a novel protein ZYG1 induces sexual cell fusion (zygote formation of Dictyostelium cells. In the process of cell fusion, involvements of signal transduction pathways via Ca2+ and PKC (protein kinase C have been suggested because zygote formation is greatly enhanced by PKC activators. In fact, there are several deduced sites phosphorylated by PKC in ZYG1 protein. Thereupon, we designed the present work to examine whether or not ZYG1 is actually phosphorylated by PKC and localized at the regions of cell-cell contacts where cell fusion occurs. These were ascertained, suggesting that ZYG1 might be the target protein for PKC. A humanized version of zyg1 cDNA (mzyg1 was introduced into myoblasts to know if ZYG1 is also effective in cell fusion of myoblasts. Quite interestingly, enforced expression of ZYG1 in myoblasts was found to induce markedly their cell fusion, thus strongly suggesting the existence of a common signaling pathway for cell fusion beyond the difference of species.

  18. Annexin A1 induces skeletal muscle cell migration acting through formyl peptide receptors.

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    Valentina Bizzarro

    Full Text Available Annexin A1 (ANXA1, lipocortin-1 is a glucocorticoid-regulated 37-kDa protein, so called since its main property is to bind (i.e. to annex to cellular membranes in a Ca(2+-dependent manner. Although ANXA1 has predominantly been studied in the context of immune responses and cancer, the protein can affect a larger variety of biological phenomena, including cell proliferation and migration. Our previous results show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. In this work, we have evaluated the hypothesis that ANXA1 is able to exert effects on myoblast cell migration acting through formyl peptide receptors (FPRs following changes in its subcellular localization as in other cell types and tissues. The analysis of the subcellular localization of ANXA1 in C2C12 myoblasts during myogenic differentiation showed an interesting increase of extracellular ANXA1 starting from the initial phases of skeletal muscle cell differentiation. The investigation of intracellular Ca(2+ perturbation following exogenous administration of the ANXA1 N-terminal derived peptide Ac2-26 established the engagement of the FPRs which expression in C2C12 cells was assessed by qualitative PCR. Wound healing assay experiments showed that Ac2-26 peptide is able to increase migration of C2C12 skeletal muscle cells and to induce cell surface translocation and secretion of ANXA1. Our results suggest a role for ANXA1 as a highly versatile component in the signaling chains triggered by the proper calcium perturbation that takes place during active migration and differentiation or membrane repair since the protein is strongly redistributed onto the plasma membranes after an rapid increase of intracellular levels of Ca(2+. These properties indicate that ANXA1 may be involved in a novel repair mechanism for skeletal muscle and may have therapeutic implications with

  19. FHL1 reduces dystrophy in transgenic mice overexpressing FSHD muscular dystrophy region gene 1 (FRG1.

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    Sandra J Feeney

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.

  20. Hypergravity Stimulation Enhances PC12 Neuron-Like Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Giada Graziana Genchi

    2015-01-01

    Full Text Available Altered gravity is a strong physical cue able to elicit different cellular responses, representing a largely uninvestigated opportunity for tissue engineering/regenerative medicine applications. Our recent studies have shown that both proliferation and differentiation of C2C12 skeletal muscle cells can be enhanced by hypergravity treatment; given these results, PC12 neuron-like cells were chosen to test the hypothesis that hypergravity stimulation might also affect the behavior of neuronal cells, in particular promoting an enhanced differentiated phenotype. PC12 cells were thus cultured under differentiating conditions for either 12 h or 72 h before being stimulated with different values of hypergravity (50 g and 150 g. Effects of hypergravity were evaluated at transcriptional level 1 h and 48 h after the stimulation, and at protein level 48 h from hypergravity exposure, to assess its influence on neurite development over increasing differentiation times. PC12 differentiation resulted strongly affected by the hypergravity treatments; in particular, neurite length was significantly enhanced after exposure to high acceleration values. The achieved results suggest that hypergravity might induce a faster and higher neuronal differentiation and encourage further investigations on the potential of hypergravity in the preparation of cellular constructs for regenerative medicine and tissue engineering purposes.

  1. G-protein coupled receptor 56 promotes myoblast fusion through serum response factor- and nuclear factor of activated T-cell-mediated signalling but is not essential for muscle development in vivo.

    Science.gov (United States)

    Wu, Melissa P; Doyle, Jamie R; Barry, Brenda; Beauvais, Ariane; Rozkalne, Anete; Piao, Xianhua; Lawlor, Michael W; Kopin, Alan S; Walsh, Christopher A; Gussoni, Emanuela

    2013-12-01

    Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the gene for G-protein coupled receptor 56 (GPR56) was transiently up-regulated during the early fusion of human myoblasts. Human mutations in the gene for GPR56 cause the disease bilateral frontoparietal polymicrogyria; however, the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR56 in skeletal muscle. GPR56(-/-) myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, a loss of GPR56 expression in muscle cells results in decreases or delays in the expression of myogenic differentiation 1, myogenin and nuclear factor of activated T-cell (NFAT)c2. Our data suggest that these abnormalities result from decreased GPR56-mediated serum response element and NFAT signalling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase compared to wild-type. In agreement with these findings, clinical data from 13 bilateral frontoparietal polymicrogyria patients revealed mild serum creatine kinase increase in only two patients. In summary, targeted disruption of GPR56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR56 knockout mice and human patients suggests that other factors may compensate for the lack of this G-protein coupled receptor during muscle development and that the motor delay observed in these patients is likely not a result of primary muscle abnormalities.

  2. Constructing retroviral vector carrying green fluorescent protein (GFP) and investigating the expression of GFP in primary rat myoblast

    Institute of Scientific and Technical Information of China (English)

    Shuling Rong; Yongxin Lu; Yuhua Liao; Xiaolin Wang; Xiaoqing Li; Jiahua Zhang; Yanli He

    2006-01-01

    Objective: To construct green fluorescent protein (GFP) retroviral vector (pLgXSN), and to investigate the expression of GFP in primary rat myoblast. Methods: GFP cDNA was subcloned into the plasmid pLgXSN, and the recombinant vector was transfected into packaging cell PT67. G418 was used to select positive colony. Myoblasts were infected by a high-titer viral supernatant. The recombinant retroviral plasmid vector was identified by restriction endonuclease analysis and DNA sequence analysis. Confocal microscopy and flow cytometry were used to detect the expression of GFP. Results: The GFP cDNA sequence was identical to that of GenBank. Recombinant retroviral plasmid vector pLgGFPSN was constructed successfully. The titer of the packaged recombinant retrovirus was 1 × 106 cfu/ml. Bright green fluorescence of the transfected cells was observed under confocal microscope 48 h after transfection. The transfection rate was 33%. The effective expression of GFP in myoblast infected by recombinant retrovirus lasted for 6 weeks. Conclusion: GFP gene could be effectively and stably expressed in myoblast, which suggests that GFP could act as a marker for studies on myoblast.

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

    Science.gov (United States)

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

    2011-02-01

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

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

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    Pearson Stephen

    2008-07-01

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

  5. Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Erica M. [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Niu, MengMeng; Bergholz, Johann [Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China); Jim Xiao, Zhi-Xiong, E-mail: jxiao@bu.edu [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China)

    2015-05-29

    The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation. - Highlights: • Nutlin-3 inhibits myoblast proliferation and prevents differentiation into myotubes. • Nutlin-3 increases MDM2 expression and down-regulates Rb protein levels. • This study has implication in nutlin-3 treatment of rhabdomyosarcomas.

  6. Expression of myosin heavy-chain mRNA in cultured myoblasts induced by centrifugal force.

    Science.gov (United States)

    Kurokawa, Katsuhide; Sakiyama, Koji; Abe, Shinichi; Hiroki, Emi; Naito, Kaoru; Nakajima, Kazunori; Takeda, Tomotaka; Inoue, Takashi; Ide, Yoshinobu; Ishigami, Keiichi

    2008-11-01

    Ballistic muscle training leads to hypertrophy of fast type fibers and training for endurance induces that of slow type fibers. Numerous studies have been conducted on electrical, extending and magnetic stimulation of cells, but the effect of centrifugal force on cells remains to be investigated. In this study, we investigated the effect of stimulating cultured myoblasts with centrifugal force at different speeds on cell proliferation and myosin heavy-chain (MyHC) mRNA expression in muscle fiber. Stimulation of myoblasts was carried out at 2 different speeds for 20 min using the Himac CT6D, a desk centrifuge, and cells were observed at 1, 3 and 5 days later. Number of cells 1 and 5 days after centrifugal stimulation was significantly larger in the 62.5 x g and 4,170 x g stimulation groups than in the control group. Expression of MyHC-2b mRNA 1 day after centrifugal stimulation was significantly higher in the 2 stimulation groups than in the control group. Almost no expression of MyHC-2a was observed in any group at 1 and 3 days after centrifugal stimulation. However, 5 days after stimulation, MyHC-2a was strongly expressed in the 2 stimulation groups in comparison to the control group. Three days after centrifugal stimulation, expression of MyHC-1 was significantly higher in the 2 stimulation groups than in the control group. The results of this study clarified the effect of different centrifugal stimulation speeds on muscle fiber characteristics, and suggest that centrifugal stimulation of myoblasts enhances cell proliferation.

  7. Gene therapy for rat renal anemia with implantation of erythropoietin-transgenic myoblasts

    Institute of Scientific and Technical Information of China (English)

    刘永学; 魏汉东; 吴祖泽; 贺福初

    1999-01-01

    To investigate whether an erythropoietin (EPO) gene-based therapy could serve as an alternative to the repeated injection of rhEPO in treatment to renal anemia, the genetically modified myoblasts of rats, named Myo/ EPO, were implanted through intramuscular injection to model rats with renal anemia. The hemoglobin (Hb) and hematocrit (HCT) of the rats increased from (92. 5±3.0) g/L and 0.29 ±0.04 to the peak values of (103.8 ±5.0) g/L and 0. 32 ±0. 04 respectively 14 d after implantation, and sustained the pre-implantation level for 90 d. Otherwise, the control rats implanted with Myo/X, which carried the parent retroviral vector, gradually became severe in anemia. The PCR detection for hEPO cDNA in the rat muscle adjacent to injection sites indicated that the Myo/EPO cells survived for a long period in the muscle of rats. The results primarily demonstrate that myoblast gene transfer of EPO is effective for the treatment of rat renal anemia.

  8. Action of lovastatin, simvastatin, and pravastatin on sterol synthesis and their antiproliferative effect in cultured myoblasts from human striated muscle

    NARCIS (Netherlands)

    Vliet, A.K. van; Nègre-Arrariou, P.; Thiel, G.C.F. van; Bolhuis, P.A.; Cohen, L.H.

    1996-01-01

    Lovastatin, simvastatin, and pravastatin are fairly strong inhibitors of sterol synthesis in human myoblasts in culture. Lovastatin and simvastatin have IC50 values of 19 ± 6 nM and 4.0 ± 2.3 nM, respectively. Pravastatin is a weaker inhibitor of sterol synthesis (IC50 value of 110 ± 38 nM). Through

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

    DEFF Research Database (Denmark)

    Vasyutina, Elena; Martarelli, Benedetta; Brakebusch, Cord;

    2009-01-01

    Rac1 and Cdc42 are small G-proteins that regulate actin dynamics and affect plasma membrane protrusion and vesicle traffic. We used conditional mutagenesis in mice to demonstrate that Rac1 and Cdc42 are essential for myoblast fusion in vivo and in vitro. The deficit in fusion of Rac1 or Cdc42...

  10. Molecular Basis for the Regulation of Transcriptional Coactivator p300 in Myogenic Differentiation.

    Science.gov (United States)

    Chen, Jihong; Wang, Yingjian; Hamed, Munerah; Lacroix, Natascha; Li, Qiao

    2015-09-10

    Skeletal myogenesis is a highly ordered process which specifically depends on the function of transcriptional coactivator p300. Previous studies have established that Akt/protein kinase B (PKB), a positive regulator of p300 in proliferating cells, is also important for proper skeletal muscle development. Nevertheless, it is not clear as to how the p300 is regulated by myogenic signaling events given that both p300 and Akt are involved in many cellular processes. Our studies revealed that the levels of p300 protein are temporally maintained in ligand-enhanced skeletal myocyte development. Interestingly, this maintenance of p300 protein is observed at the stage of myoblast differentiation, which coincides with an increase in Akt phosphorylation. Moreover, regulation of p300 during myoblast differentiation appears to be mediated by Akt signaling. Blunting of p300 impairs myogenic expression and myoblast differentiation. Thus, our data suggests a particular role for Akt in myoblast differentiation through interaction with p300. Our studies also establish the potential of exploiting p300 regulation and Akt activation to decipher the complex signaling cascades involved in skeletal muscle development.

  11. Functional analysis of the Myostatin gene promoter in sheep

    Institute of Scientific and Technical Information of China (English)

    DU; Rong; AN; XiaoRong; CHEN; YongFu; QIN; Jian

    2007-01-01

    Compared with the understanding for the functional mechanism of the myostatin gene, little is known about the regulatory mechanism of the myostatin gene transcription and expression. To better understand the function of the myostatin gene promoter (MSTNpro) in the transcriptional regulation of the myostatin gene and to further investigate the transcriptional regulation mechanism of the myostatin gene, the promoter region of the myostatin gene in sheep has been cloned in our recent study (AY918121). In this study, the wild (W) type MSTNProW-EGFP vectors and E-box (E) (CANNTG) mutant (M) type MSTNProE(3+5+7)M-EGFP vectors were constructed and the transcriptional regulation activities were compared by detecting the fluorescent strength of EGFP (enhanced green fluorescent protein) in C2C12 myoblasts (or myotubes) and sheep fibroblasts transfected with the vectors. Results showed that the 0.3―1.2 kb sheep myostatin promoter could activate the transcription and expression of EGFP gene in C2C12 myoblasts to different extent and the 1.2 kb promoter was the strongest. However, fluorescence was not observed in the sheep fibroblasts transfected with the 1.2 kb sheep myostatin promoter. These results suggested that the specific nature of the myostatin gene expression in skeletal muscle was attributed to the specific nature of the myostatin promoter activity. The increasing growth density of C2C12 myoblasts inhibited the transcriptional regulation activity of the wild type sheep myostatin promoter by a mechanism of feedback. The transcriptional regulation activity of the 1.2 kb wild type sheep myostatin promoter increased significantly after C2C12 myoblasts were differentiated, while the activity of 1.2 kb E(3+5+7)-mutant type myostatin promoter had no obvious change. This result suggested that MyoD may be responsible for the difference of the myostatin gene transcription and expression between growing and differentiating conditions by binding to E-box of the myostatin

  12. Mast cell tryptase stimulates myoblast proliferation; a mechanism relying on protease-activated receptor-2 and cyclooxygenase-2

    Directory of Open Access Journals (Sweden)

    Côté Claude H

    2011-10-01

    Full Text Available Abstract Background Mast cells contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase which activates protease-activated receptor-2 (PAR-2. The possibility that a tryptase/PAR-2 signaling pathway exists in skeletal muscle cell has never been investigated. The aim of this study was to evaluate whether tryptase can stimulate myoblast proliferation and determine the downstream cascade. Methods Proliferation of L6 rat skeletal myoblasts stimulated with PAR-2 agonists (tryptase, trypsin and SLIGKV was assessed. The specificity of the tryptase effect was evaluated with a specific inhibitor, APC-366. Western blot analyses were used to evaluate the expression and functionality of PAR-2 receptor and to assess the expression of COX-2. COX-2 activity was evaluated with a commercial activity assay kit and by measurement of PGF2α production. Proliferation assays were also performed in presence of different prostaglandins (PGs. Results Tryptase increased L6 myoblast proliferation by 35% above control group and this effect was completely inhibited by APC-366. We confirmed the expression of PAR-2 receptor in vivo in skeletal muscle cells and in satellite cells and in vitro in L6 cells, where PAR-2 was found to be functional. Trypsin and SLIGKV increased L6 cells proliferation by 76% and 26% above control, respectively. COX-2 activity was increased following stimulation with PAR-2 agonist but its expression remained unchanged. Inhibition of COX-2 activity by NS-398 abolished the stimulation of cell proliferation induced by tryptase and trypsin. Finally, 15-deoxy-Δ-12,14-prostaglandin J2 (15Δ-PGJ2, a product of COX-2-derived prostaglandin D2, stimulated myoblast proliferation, but not PGE2 and PGF2α. Conclusions Taken together, our data show that tryptase can stimulate myoblast proliferation and this effect is part of a signaling cascade dependent on PAR-2 activation and on the downstream

  13. YB-1 gene expression is kept constant during myocyte differentiation through replacement of different transcription factors and then falls gradually under the control of neural activity.

    Science.gov (United States)

    Kobayashi, Shunsuke; Tanaka, Toru; Moue, Masamitsu; Ohashi, Sachiyo; Nishikawa, Taishi

    2015-11-01

    We have previously reported that translation of acetylcholine receptor α-subunit (AChR α) mRNA in skeletal muscle cells is regulated by Y-box binding protein 1 (YB-1) in response to neural activity, and that in the postnatal mouse developmental changes in the amount of YB-1 mRNA are similar to those of AChR α mRNA, which is known to be regulated by myogenic transcription factors. Here, we examined transcriptional regulation of the YB-1 gene in mouse skeletal muscle and differentiating C2C12 myocytes. Although neither YB-1 nor AChR α was detected at either the mRNA or protein level in adult hind limb muscle, YB-1 expression was transiently activated in response to denervation of the sciatic nerve and completely paralleled that of AChR α, suggesting that these genes are regulated by the same transcription factors. However, during differentiation of C2C12 cells to myotubes, the level of YB-1 remained constant even though the level of AChR α increased markedly. Reporter gene, gel mobility shift and ChIP assays revealed that in the initial stage of myocyte differentiation, transcription of the YB-1 gene was regulated by E2F1 and Sp1, and was then gradually replaced under the control of both MyoD and myogenin through an E-box sequence in the proximal region of the YB-1 gene promoter. These results suggest that transcription factors for the YB-1 gene are exchanged during skeletal muscle cell differentiation, perhaps playing a role in translational control of mRNAs by YB-1 in both myotube formation and the response of skeletal muscle tissues to neural stimulation.

  14. Effect of oxygen tension on bioenergetics and proteostasis in young and old myoblast precursor cells

    Directory of Open Access Journals (Sweden)

    M. Konigsberg

    2013-01-01

    Full Text Available In the majority of studies using primary cultures of myoblasts, the cells are maintained at ambient oxygen tension (21% O2, despite the fact that physiological O2 at the tissue level in vivo is much lower (~1–5% O2. We hypothesized that the cellular response in presence of high oxygen concentration might be particularly important in studies comparing energetic function or oxidative stress in cells isolated from young versus old animals. To test this, we asked whether oxygen tension plays a role in mitochondrial bioenergetics (oxygen consumption, glycolysis and fatty acid oxidation or oxidative damage to proteins (protein disulfides, carbonyls and aggregates in myoblast precursor cells (MPCs isolated from young (3–4 m and old (29–30 m C57BL/6 mice. MPCs were grown under physiological (3% or ambient (21% O2 for two weeks prior to exposure to an acute oxidative insult (H2O2. Our results show significantly higher basal mitochondrial respiration in young versus old MPCs, an increase in basal respiration in young MPCs maintained at 3% O2 compared to cells maintained at 21% O2, and a shift toward glycolytic metabolism in old MPCs grown at 21% O2. H2O2 treatment significantly reduced respiration in old MPCs grown at 3% O2 but did not further repress respiration at 21% O2 in old MPCs. Oxidative damage to protein was higher in cells maintained at 21% O2 and increased in response to H2O2 in old MPCs. These data underscore the importance of understanding the effect of ambient oxygen tension in cell culture studies, in particular studies measuring oxidative damage and mitochondrial function.

  15. Methylglyoxal impairs GLUT4 trafficking and leads to increased glucose uptake in L6 myoblasts.

    Science.gov (United States)

    Engelbrecht, B; Mattern, Y; Scheibler, S; Tschoepe, D; Gawlowski, T; Stratmann, B

    2014-02-01

    Methylglyoxal (MG) is a highly reactive dicarbonyl compound derived mainly from glucose degradation pathways, but also from protein and fatty acid metabolism. MG modifies structure and function of different biomolecules and thus plays an important role in the pathogenesis of diabetic complications. Hyperglycemia-associated accumulation of MG might be associated with generation of oxidative stress and subsequently insulin resistance. Therefore, the effects of MG on insulin signaling and on translocation of glucose transporter 4 (GLUT4) were investigated in the rat skeletal muscle cell line L6-GLUT4myc stably expressing myc-tagged GLUT4. Twenty four-hour MG treatment resulted in elevated GLUT4 presentation on the surface of L6 myoblasts and in an increased uptake of glucose even without insulin stimulation. Exogenously added MG neither effected IRS-1 expression nor IRS-1 phosphorylation. A decreased expression of Akt1 but not Akt2 and concomitantly increased apoptosis were detected following MG treatment. To exclude that oxidative stress caused by MG treatment leads to increased GLUT4 translocation, effects of pretreatment with 2 antioxidants were investigated. The antioxidant and MG scavenger NAC prevented the MG-induced GLUT4 translocation. In contrast, tiron, a well-known antioxidant that does not exert MG-scavenger function, had no impact on MG-induced GLUT4 translocation supporting the hypothesis of a direct effect of MG on GLUT4 trafficking. In conclusion, prolonged treatment with MG augments GLUT4 level on the surface of L6 myoblasts, at least in part through a higher translocation of GLUT4 from the intracellular compartment as well as a reduction of GLUT4 internalization, resulting in increased glucose uptake.

  16. Enhancement of energy production by black ginger extract containing polymethoxy flavonoids in myocytes through improving glucose, lactic acid and lipid metabolism.

    Science.gov (United States)

    Toda, Kazuya; Takeda, Shogo; Hitoe, Shoketsu; Nakamura, Seikou; Matsuda, Hisashi; Shimoda, Hiroshi

    2016-04-01

    Enhancement of muscular energy production is thought to improve locomotive functions and prevent metabolic syndromes including diabetes and lipidemia. Black ginger (Kaempferia parviflora) has been cultivated for traditional medicine in Thailand. Recent studies have shown that black ginger extract (KPE) activated brown adipocytes and lipolysis in white adipose tissue, which may cure obesity-related dysfunction of lipid metabolism. However, the effect of KPE on glucose and lipid utilization in muscle cells has not been examined yet. Hence, we evaluated the effect of KPE and its constituents on energy metabolism in pre-differentiated (p) and differentiated (d) C2C12 myoblasts. KPE (0.1-10 μg/ml) was added to pC2C12 cells in the differentiation process for a week or used to treat dC2C12 cells for 24 h. After culturing, parameters of glucose and lipid metabolism and mitochondrial biogenesis were assessed. In terms of the results, KPE enhanced the uptake of 2-deoxyglucose and lactic acid as well as the mRNA expression of glucose transporter (GLUT) 4 and monocarboxylate transporter (MCT) 1 in both types of cells. The expression of peroxisome proliferator-activated receptor γ coactivator (PGC)-1α was enhanced in pC2C12 cells. In addition, KPE enhanced the production of ATP and mitochondrial biogenesis. Polymethoxy flavonoids in KPE including 5-hydroxy-7-methoxyflavone, 5-hydroxy-3,7,4'-trimethoxyflavone and 5,7-dimethoxyflavone enhanced the expression of GLUT4 and PGC-1α. Moreover, KPE and 5,7-dimethoxyflavone enhanced the phosphorylation of 5'AMP-activated protein kinase (AMPK). In conclusion, KPE and its polymethoxy flavonoids were found to enhance energy metabolism in myocytes. KPE may improve the dysfunction of muscle metabolism that leads to metabolic syndrome and locomotive dysfunction.

  17. Effects of transplanted myoblasts transfected with human growth hormone gene on improvement of ventricular function of rats

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Background Cell transplantation for myocardial repair is limited by early cell death.Gene therapy with human growth hormone(hGH)has been shown to promote angiogensis and attenuate apoptosis in the experimental animal.This study was conducted to explore the effects of myoblast-based hGH gene therapy on heart function restoration and angiogenesis after myocardial infarction,and to compare the differences between myoblast-based hGH gene therapy and myoblast therapy.Methods Myoblasts were isolated from several SD rats,cultured,purified,and transfected with plasmid pLghGHSN and pLgGFPSN.Radioimmunoassay(RIA)was used to detect the expression of hGH in these myoblasts.SD rats underwent the ligation of the left anterior descending coronary artery so as to establish a heart ischemia model.Thirty surviving rats that underwent ligation were randomly divided into 3 equal groups 2 weeks after left coronary artery occlusion:pLghGHSN group received myoblast infected with hGH gene transplantation;pLgGFPSN group received myoblast infected with GFP gene transplantation;control group:received cultured medium only.Four weeks after the injection the surviving rat underwent evaluation of cardiac function by echocardiography.The rats were killed and ventricular samples were undergone immunohistochemistry with hematoxylin-eosin and factorⅧ.Cryosection was analyzed by fluorescence microscopy to examine the expression of green fluorescent protein.Reverse transcriptase-polymerase chain reaction(RT-PCR)was used to examine the mRNA expression of vascular endothelial growth factor(VEGF),bax and Bcl-2.hGH expression in myocardium was examined by Western blot.Results Myoblast can be successfully isolated,cultured and transfected.The expression of hGH in transfected myoblast was demonstrated with RIA.Four weeks after therapy,the cardiac function was improved significantly in pLghGHSN group and pLgGFPSN group.Fractional shortening(FS)and ejection fraction(EF)in pLghGHSN group were elevated

  18. Systematic analysis of cis-elements in unstable mRNAs demonstrates that CUGBP1 is a key regulator of mRNA decay in muscle cells.

    Directory of Open Access Journals (Sweden)

    Jerome E Lee

    Full Text Available BACKGROUND: Dramatic changes in gene expression occur in response to extracellular stimuli and during differentiation. Although transcriptional effects are important, alterations in mRNA decay also play a major role in achieving rapid and massive changes in mRNA abundance. Moreover, just as transcription factor activity varies between different cell types, the factors influencing mRNA decay are also cell-type specific. PRINCIPAL FINDINGS: We have established the rates of decay for over 7000 transcripts expressed in mouse C2C12 myoblasts. We found that GU-rich (GRE and AU-rich (ARE elements are over-represented in the 3'UTRs of short-lived mRNAs and that these mRNAs tend to encode factors involved in cell cycle and transcription regulation. Stabilizing elements were also identified. By comparing mRNA decay rates in C2C12 cells with those previously measured for pluripotent and differentiating embryonic stem (ES cells, we identified several groups of transcripts that exhibit cell-type specific decay rates. Further, whereas in C2C12 cells the impact of GREs on mRNA decay appears to be greater than that of AREs, AREs are more significant in ES cells, supporting the idea that cis elements make a cell-specific contribution to mRNA stability. GREs are recognized by CUGBP1, an RNA-binding protein and instability factor whose function is affected in several neuromuscular diseases. We therefore utilized RNA immunoprecipitation followed by microarray (RIP-Chip to identify CUGBP1-associated transcripts. These mRNAs also showed dramatic enrichment of GREs in their 3'UTRs and encode proteins linked with cell cycle, and intracellular transport. Interestingly several CUGBP1 substrate mRNAs, including those encoding the myogenic transcription factors Myod1 and Myog, are also bound by the stabilizing factor HuR in C2C12 cells. Finally, we show that several CUGBP1-associated mRNAs containing 3'UTR GREs, including Myod1, are stabilized in cells depleted of CUGBP1

  19. Cultured senescent myoblasts derived from human vastus lateralis exhibit normal mitochondrial ATP synthesis capacities with correlating concomitant ROS production while whole cell ATP production is decreased.

    Science.gov (United States)

    Minet, Ariane D; Gaster, Michael

    2012-06-01

    The free radical theory of aging says that increased oxidative stress and mitochondrial dysfunction are associated with old age. In the present study we have investigated the effects of cellular senescence on muscle energetic by comparing mitochondrial content and function in cultured muscle satellite cells at early and late passage numbers. We show that cultured muscle satellite cells undergoing senescence express a reduced mitochondrial mass, decreased whole cell ATP level, normal to increased mitochondrial ATP production under ATP utilization, increased mitochondrial membrane potential and increased superoxide/mitochondrial mass and hydrogen peroxide/mitochondrial mass ratios. Moreover, the increased ROS production correlates with the corresponding mitochondrial ATP production. Thus, myotubes differentiated from human myoblasts undergoing senescence have a reduced mitochondrial content, but the existent mitochondria express normal to increased functional capabilities. The present data suggest that the origin of aging lies outside the mitochondria and that a malfunction in the cell might be preceding and initiating the increase of mitochondrial ATP synthesis and concomitant ROS production in the single mitochondrion in response to decreased mitochondrial mass and reduced extra-mitochondrial energy supply. This then can lead to the increased damage of DNA, lipids and proteins of the mitochondria as postulated by the free radical theory of aging.

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

    Directory of Open Access Journals (Sweden)

    Haohan Wang

    2016-03-01

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

  1. Mast cell tryptase stimulates myoblast proliferation; a mechanism relying on protease-activated receptor-2 and cyclooxygenase-2

    OpenAIRE

    Côté Claude H; Tremblay Marie-Hélène; Duchesne Elise

    2011-01-01

    Abstract Background Mast cells contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase which activates protease-activated receptor-2 (PAR-2). The possibility that a tryptase/PAR-2 signaling pathway exists in skeletal muscle cell has never been investigated. The aim of this study was to evaluate whether tryptase can stimulate myoblast proliferation and determine the downstream cascade. Methods Proliferation of L6 rat skeletal m...

  2. Experiment list: SRX344977 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available gged ING1 || cell type=Growing C1C12 myoblasts || chromatin preparation method=So...ource_name=Input_ING1_flag_myoblasts || cell line=C2C12 || genotype/variation=ectopically expressing Flag-ta

  3. Experiment list: SRX344978 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available urce_name=Input_ING1_flag_myoblasts || cell line=C2C12 || genotype/variation=ectopically expressing Flag-tagged ING1 || cell type=Gro...wing C1C12 myoblasts || chromatin preparation method=Son

  4. Cultured myoblasts from patients affected by myotonic dystrophy type 2 exhibit senescence-related features: ultrastructural evidence

    Directory of Open Access Journals (Sweden)

    M. Malatesta

    2011-09-01

    Full Text Available Myotonic dystrophy type 2 (DM2 is an autosomal dominant disorder caused by the expansion of the tetranucleotidic repeat (CCTGn in the first intron of the Zinc Finger Protein-9 gene. In DM2 tissues, the expanded mutant transcripts accumulate in nuclear focal aggregates where splicing factors are sequestered, thus impairing the whole mRNA processing. Interestingly, the ultrastructural alterations in the splicing machinery observed in the myonuclei of DM2 skeletal muscles are reminiscent of the nuclear changes occurring in age-related muscle atrophy. Here, we investigated structural and functional features of satellite cell-derived myoblasts from biceps brachii, in the attempt to investigate cell senescence indices in DM2 patients by ultrastructural cytochemistry. We observed that in satellite cell-derived DM2 myoblasts, cell-senescence alterations and impairment of the pre-mRNA maturation pathways occur earlier than in myoblasts from healthy patients. This suggests that also in vivo the regeneration capability of satellite cells could be reduced in dystrophic muscles.

  5. Age-associated repression of type 1 inositol 1, 4, 5-triphosphate receptor impairs muscle regeneration

    Science.gov (United States)

    Lee, Bora; Lee, Seung-Min; Bahn, Young Jae; Lee, Kwang-Pyo; Kang, Moonkyung; Kim, Yeon-Soo; Woo, Sun-Hee; Lim, Jae-Young; Kim, Eunhee; Kwon, Ki-Sun

    2016-01-01

    Skeletal muscle mass and power decrease with age, leading to impairment of mobility and metabolism in the elderly. Ca2+ signaling is crucial for myoblast differentiation as well as muscle contraction through activation of transcription factors and Ca2+-dependent kinases and phosphatases. Ca2+ channels, such as dihydropyridine receptor (DHPR), two-pore channel (TPC) and inositol 1,4,5-triphosphate receptor (ITPR), function to maintain Ca2+ homeostasis in myoblasts. Here, we observed a significant decrease in expression of type 1 IP3 receptor (ITPR1), but not types 2 and 3, in aged mice skeletal muscle and isolated myoblasts, compared with those of young mice. ITPR1 knockdown using shRNA-expressing viruses in C2C12 myoblasts and tibialis anterior muscle of mice inhibited myotube formation and muscle regeneration after injury, respectively, a typical phenotype of aged muscle. This aging phenotype was associated with repression of muscle-specific genes and activation of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. ERK inhibition by U0126 not only induced recovery of myotube formation in old myoblasts but also facilitated muscle regeneration after injury in aged muscle. The conserved decline in ITPR1 expression in aged human skeletal muscle suggests utility as a potential therapeutic target for sarcopenia, which can be treated using ERK inhibition strategies. PMID:27658230

  6. Irisin Enhances Osteoblast Differentiation In Vitro

    Directory of Open Access Journals (Sweden)

    Graziana Colaianni

    2014-01-01

    Full Text Available It has been recently demonstrated that exercise activity increases the expression of the myokine Irisin in skeletal muscle, which is able to drive the transition of white to brown adipocytes, likely following a phenomenon of transdifferentiation. This new evidence supports the idea that muscle can be considered an endocrine organ, given its ability to target adipose tissue by promoting energy expenditure. In accordance with these new findings, we hypothesized that Irisin is directly involved in bone metabolism, demonstrating its ability to increase the differentiation of bone marrow stromal cells into mature osteoblasts. Firstly, we confirmed that myoblasts from mice subjected to 3 weeks of free wheel running increased Irisin expression compared to nonexercised state. The conditioned media (CM collected from myoblasts of exercised mice induced osteoblast differentiation in vitro to a greater extent than those of mice housed in resting conditions. Furthermore, the differentiated osteoblasts increased alkaline phosphatase and collagen I expression by an Irisin-dependent mechanism. Our results show, for the first time, that Irisin directly targets osteoblasts, enhancing their differentiation. This finding advances notable perspectives in future studies which could satisfy the ongoing research of exercise-mimetic therapies with anabolic action on the skeleton.

  7. Involvement of M-cadherin in terminal differentiation of skeletal muscle cells.

    Science.gov (United States)

    Zeschnigk, M; Kozian, D; Kuch, C; Schmoll, M; Starzinski-Powitz, A

    1995-09-01

    Cadherins are a gene family encoding calcium-dependent cell adhesion proteins which are thought to act in the establishment and maintenance of tissue organization. M-cadherin, one member of the family, has been found in myogenic cells of somitic origin during embryogenesis and in the adult. These findings have suggested that M-cadherin is involved in the regulation of morphogenesis of skeletal muscle cells. Therefore, we investigated the function of M-cadherin in the fusion of myoblasts into myotubes (terminal differentiation) in cell culture. Furthermore, we tested whether M-cadherin might influence (a) the expression of troponin T, a typical marker of biochemical differentiation of skeletal muscle cells, and (b) withdrawal of myoblasts from the cell cycle (called terminal commitment). The studies were performed by using antagonistic peptides which correspond to sequences of the putative M-cadherin binding domain. Analogous peptides of N-cadherin have previously been shown to interfere functionally with the N-cadherin-mediated cell adhesion. In the presence of antagonistic M-cadherin peptides, the fusion of myoblasts into myotubes was inhibited. Analysis of troponin T revealed that it was downregulated at the protein level although its mRNA was still detectable. In addition, withdrawal from the cell cycle typical for terminal commitment of muscle cells was not complete in fusion-blocked myogenic cells. Finally, expression of M-cadherin antisense RNA reducing the expression of the endogenous M-cadherin protein interfered with the fusion process of myoblasts. Our data imply that M-cadherin-mediated myoblast interaction plays an important role in terminal differentiation of skeletal muscle cells.

  8. Switch from ER-mitochondrial to SR-mitochondrial calcium coupling during muscle differentiation.

    Science.gov (United States)

    Yi, Muqing; Weaver, David; Eisner, Verónica; Várnai, Péter; Hunyady, László; Ma, Jianjie; Csordás, György; Hajnóczky, György

    2012-11-01

    Emerging evidence indicates that mitochondria are locally coupled to endoplasmic reticulum (ER) Ca2+ release in myoblasts and to sarcoplasmic reticulum (SR) Ca2+ release in differentiated muscle fibers in order to regulate cytoplasmic calcium dynamics and match metabolism with cell activity. However, the mechanism of the developmental transition from ER to SR coupling remains unclear. We have studied mitochondrial sensing of IP3 receptor (IP3R)- and ryanodine receptor (RyR)-mediated Ca2+ signals in H9c2 myoblasts and differentiating myotubes, as well as the attendant changes in mitochondrial morphology. Mitochondria in myoblasts were largely elongated, luminally connected and relatively few in number, whereas the myotubes were densely packed with globular mitochondria that displayed limited luminal continuity. Vasopressin, an IP3-linked agonist, evoked a large cytoplasmic Ca2+ ([Ca2+]c) increase in myoblasts, whereas it elicited a smaller response in myotubes. Conversely, RyR-mediated Ca2+ release induced by caffeine, was not observed in myoblasts, but triggered a large [Ca2+]c signal in myotubes. Both the IP3R and the RyR-mediated [Ca2+]c rise was closely associated with a mitochondrial matrix Ca2+ ([Ca2+]m) signal. Every myotube that showed a [Ca2+]c spike also displayed a [Ca2+]m response. Addition of IP3 to permeabilized myoblasts and caffeine to permeabilized myotubes also resulted in a rapid [Ca2+]m rise, indicating that Ca2+ was delivered via local coupling of the ER/SR and mitochondria. Thus, as RyRs are expressed during muscle differentiation, the local connection between RyR and mitochondrial Ca2+ uptake sites also appears. When RyR1 was exogenously introduced to myoblasts by overexpression, the [Ca2+]m signal appeared together with the [Ca2+]c signal, however the mitochondrial morphology remained unchanged. Thus, RyR expression alone is sufficient to induce the steps essential for their alignment with mitochondrial Ca2+ uptake sites, whereas the

  9. The methyl-CpG-binding protein CIBZ suppresses myogenic differentiation by directly inhibiting myogenin expression

    Institute of Scientific and Technical Information of China (English)

    Yu Oikawa; Reiko Omori; Tomonori Nishii; Yasumasa Ishida; Masashi Kawaichi; Eishou Matsuda

    2011-01-01

    Postnatal growth and regeneration of skeletal muscle are carried out mainly by satellite cells,which,upon stimulation,begin to express myogenin (Myog),the critical determinant of myogenic differentiation.DNA methylation status has been associated with the expression of Myog,but the causative mechanism remains almost unknown.Here,we report that the level of CIBZ,a methyI-CpG-binding protein,decreases upon myogenic differentiation of satellitederived C2C12 cells,and during skeletal muscle regeneration in mice.We present data showing that the loss of CIBZ promotes myogenic differentiation,whereas exogenous expression of CIBZ impairs it,in cultured cells.CIBZ binds to a Myog promoter-proximal region and inhibits Myog transcription in a methylation-dependent manner.These data suggest that the suppression of myogenic differentiation by CIBZ is dependent,at least in part,on the regulation of Myog.Our data show that the methylation status of this proximal Myog promoter inversely correlates with Myog transcription in cells and tissues,and during postnatal growth of skeletal muscle.Notably,induction of Myog transcription by CIBZ suppression is independent of the demethylation of CpG sites in the Myog promoter.These observations provide the first reported molecular mechanism illustrating how Myog transcription is coordinately regulated by a methyI-CpG-binding protein and the methylation status of the proximal Myog promoter.

  10. Expression of Lecithin: Cholesterol Acyltransferaseand/or apoA-I Mediated by Recombinant Adeno-as-sociated Virus in Myogenic Cell

    Institute of Scientific and Technical Information of China (English)

    王立峰; 范乐明; 陈丙莺; 刘宝瑞; 王若宁; 魏恩会

    2002-01-01

    Objective Lecithia: cholesterol acyltrmsfer ase (LCAT) is the major enzyme producing most plasma cholesterol esters( CE )and a key partiipant in the process of reverse cholesterol traansfer ( RCT). The aim of the study was to co-express LCAT and its nature activator apoA- I medi ated by recombinant adeno-associated virus vectors in the skeletal muscle cells, and open a new avenue of gene therapy touard the primary or secondary LCAT deficiency. Methods 293T cells were cotrans fected with pDG and rAAVAIL/rAAVL plasmid to produce infectious rAAV, and non-iouic iodixanol gradients centri f ngation followed by heparin affinity chromatography was per formed f or separation . pu rification and concentration of rAAV. The particle numbers of rAAV were assayed by dot-blot, then these vectors transduced C2C12 myoblasts. ELISA and Western Blot asasayed for human apoA- I and 3H-cholesterol labeled radiochemical methods for LCAT activity. Genomic DNA was extracted from transduced C2C12 and analyzed fo the presence of vector sequence by PCR amplifiations. Results The particle mumbers of rAAV were 7× 1014/L (rAAAIL) and 1 × 1014/L (rAAVL). The expres sion of human apoA- I cDNA and/or human LCAT cDNA in transduced C2C12 cells lasted for 3 0 d, even after myoblasts were differentiated into myotubes. PCR products for transgene indiated the long-term persistence of transduced vector sequences. Conclusion The result indicated that the meth ods used for production and purification of rAAV is an effiient and rAAV vector mediate the expres sion and secretion of LCAT and apoA- I gene in C2C12 myoblasts successfully. It suggested that the use of rAAV vectors mediating the high efficiency, long-term expression of human LCAT cDNA and/ or apoA- I cDNA in skeletal muscle in vivo might be a safe and fessible strategy to the gene therapy of LCAT deficiency.

  11. Efeito do laser de baixa potência sobre células musculares C2C12 submetidas à lesão por veneno da serpente Bothrops jararacussu.

    OpenAIRE

    Silva, Camila Aparecida Alves da

    2012-01-01

    O veneno das serpentes do gênero Bothrops, induz uma intensa reação inflamatória local podendo evoluir para necrose tecidual. A soroterapia apresenta eficácia em neutralizar os efeitos sistêmicos, porém sua ação não se estende as manifestações locais. O laser de baixa potência (LBP) é usado em situações de lesão muscular, pois apresenta efeitos biológicos, tais como analgésicos, antiinflamatórios e cicatrizantes. O objetivo deste trabalho foi analisar o efeito do LBP em células musculares C2C...

  12. Efeito do laser de baixa potência sobre células musculares c2c12 submetidas à lesão por veneno da serpente Bothrops jararacussu.

    OpenAIRE

    Silva, Camila Aparecida Alves da

    2012-01-01

    O veneno das serpentes do gênero Bothrops, induz uma intensa reação inflamatória local podendo evoluir para necrose tecidual. A soroterapia apresenta eficácia em neutralizar os efeitos sistêmicos, porém sua ação não se estende as manifestações locais. O laser de baixa potência (LBP) é usado em situações de lesão muscular, pois apresenta efeitos biológicos, tais como analgésicos, antiinflamatórios e cicatrizantes. O objetivo deste trabalho foi analisar o efeito do LBP em células musculares C2C...

  13. efeito do laser de baixa potência sobre células musculares c2c12 submetidas à lesão por miotoxinas BTHTX - I e BTHTX - II isoladas do veneno da serpente bothrops jararacussu

    OpenAIRE

    Santos, Adriano Silvio dos

    2015-01-01

    O veneno das serpentes do gênero Bothrops induz uma reação inflamatória local intensa, caracterizada por dor, formação de edema, migração leucocitária, podendo ser acompanhada por necrose tecidual. A utilização do soro antibotrópico desempenha a função de neutralizar a maior quantidade possível do veneno circulante, minimizando assim seus efeitos sistêmicos, porém sua ação não se estende às manifestações locais, sendo assim necessário o uso de outro recurso terapêutico para o controle dessa m...

  14. Stimulative Effects of Low Intensity He-Ne Laser Irradiation on the Proliferative Potential and Cell-Cycle Progression of Myoblasts in Culture

    Directory of Open Access Journals (Sweden)

    Cui-Ping Zhang

    2014-01-01

    Full Text Available Low intensity laser irradiation (LILI was found to promote the regeneration of skeletal muscle in vivo but the cellular mechanisms are not fully understood. Myoblasts, normally quiescent and inactivated in adult skeletal muscle, are a type of myogenic progenitor cells and considered as the major candidates responsible for muscle regeneration. The aim of the present study was to study the effect of LILI on the growth potential and cell-cycle progression of the cultured myoblasts. Primary myoblasts isolated from rat hind legs were cultured in nutrient-deficient medium for 36 hours and then irradiated by helium-neon laser at a certain energy density. Immunohistochemical and flow cytometric analysis revealed that laser irradiation could increase the expression of cellular proliferation marker and the amount of cell subpopulations in the proliferative phase as compared with the nonirradiated control group. Meanwhile, the expressions of cell-cycle regulatory proteins in the laser-treated myoblasts were markedly upregulated as compared to the unirradiated cells, indicating that LILI could promote the reentry of quiescent myoblasts into the cell division cycle. These results suggest that LILI at certain fluences could promote their proliferation, thus contributing to the skeletal muscle regeneration following trauma and myopathic diseases.

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

    Science.gov (United States)

    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.

  16. Differential regulation of the histone chaperone HIRA during muscle cell differentiation by a phosphorylation switch.

    Science.gov (United States)

    Yang, Jae-Hyun; Song, Tae-Yang; Jo, Chanhee; Park, Jinyoung; Lee, Han-Young; Song, Ilang; Hong, Suji; Jung, Kwan Young; Kim, Jaehoon; Han, Jeung-Whan; Youn, Hong-Duk; Cho, Eun-Jung

    2016-08-12

    Replication-independent incorporation of variant histone H3.3 has a profound impact on chromatin function and numerous cellular processes, including the differentiation of muscle cells. The histone chaperone HIRA and H3.3 have essential roles in MyoD regulation during myoblast differentiation. However, the precise mechanism that determines the onset of H3.3 deposition in response to differentiation signals is unclear. Here we show that HIRA is phosphorylated by Akt kinase, an important signaling modulator in muscle cells. By generating a phosphospecific antibody, we found that a significant amount of HIRA was phosphorylated in myoblasts. The phosphorylation level of HIRA and the occupancy of phosphorylated protein on muscle genes gradually decreased during cellular differentiation. Remarkably, the forced expression of the phosphomimic form of HIRA resulted in reduced H3.3 deposition and suppressed the activation of muscle genes in myotubes. Our data show that HIRA phosphorylation limits the expression of myogenic genes, while the dephosphorylation of HIRA is required for proficient H3.3 deposition and gene activation, demonstrating that the phosphorylation switch is exploited to modulate HIRA/H3.3-mediated muscle gene regulation during myogenesis.

  17. Mild heat stress enhances differentiation and proliferation of Japanese quail myoblasts and enhances slow muscle fiber characteristics.

    Science.gov (United States)

    Choi, Y M; Chen, P R; Shin, S; Zhang, J; Hwang, S; Lee, K

    2016-08-01

    The objective of this study was to investigate the effect of mild heat stress on muscle fiber hyperplastic and hypertrophic growth in quail primary myogenic cells to better understand the mechanisms leading to increased skeletal muscle development in avian embryos incubated at a higher temperature. Compared to control cultures maintained at 37°C, incubation at 39°C enhanced myotube length (P fusion index (56.7 vs. 46.2%, P heat stress compared to the control cells. On the other hand, mild heat stress enhanced protein levels of slow myosin heavy chain isoform (P heat stress plays a significant role in myogenic mechanisms related to muscle mass and development.

  18. Effect of myeloperoxidase and anoxia/reoxygenation on mitochondrial respiratory function of cultured primary equine skeletal myoblasts.

    Science.gov (United States)

    Ceusters, Justine D; Mouithys-Mickalad, Ange A; Franck, Thierry J; Derochette, Sandrine; Vanderplasschen, Alain; Deby-Dupont, Ginette P; Serteyn, Didier A

    2013-09-01

    Horses are particularly sensitive to excessive inflammatory reaction where myeloperoxidase, a marker of inflammation, may contribute to mitochondrial dysfunctions. This study investigated the interaction between myeloperoxidase and cultured primary equine skeletal myoblasts, particularly its effect on mitochondrial respiration combined or not with anoxia followed by reoxygenation (AR). We showed that active myeloperoxidase entered into the cells, interacted with mitochondria and decreased routine and maximal respirations. When combined with AR, myeloperoxidase caused a further decrease of these respiratory parameters while the leak increased. Our results indicate that myeloperoxidase amplifies the mitochondrial damages initiated by AR phenomenon and alters the mitochondrial function.

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

    Science.gov (United States)

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

    2015-08-01

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

  20. Stem Cell Differentiation Toward the Myogenic Lineage for Muscle Tissue Regeneration: A Focus on Muscular Dystrophy.

    Science.gov (United States)

    Ostrovidov, Serge; Shi, Xuetao; Sadeghian, Ramin Banan; Salehi, Sahar; Fujie, Toshinori; Bae, Hojae; Ramalingam, Murugan; Khademhosseini, Ali

    2015-12-01

    Skeletal muscle tissue engineering is one of the important ways for regenerating functionally defective muscles. Among the myopathies, the Duchenne muscular dystrophy (DMD) is a progressive disease due to mutations of the dystrophin gene leading to progressive myofiber degeneration with severe symptoms. Although current therapies in muscular dystrophy are still very challenging, important progress has been made in materials science and in cellular technologies with the use of stem cells. It is therefore useful to review these advances and the results obtained in a clinical point of view. This article focuses on the differentiation of stem cells into myoblasts, and their application in muscular dystrophy. After an overview of the different stem cells that can be induced to differentiate into the myogenic lineage, we introduce scaffolding materials used for muscular tissue engineering. We then described some widely used methods to differentiate different types of stem cell into myoblasts. We highlight recent insights obtained in therapies for muscular dystrophy. Finally, we conclude with a discussion on stem cell technology. We discussed in parallel the benefits brought by the evolution of the materials and by the expansion of cell sources which can differentiate into myoblasts. We also discussed on future challenges for clinical applications and how to accelerate the translation from the research to the clinic in the frame of DMD.

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

    Energy Technology Data Exchange (ETDEWEB)

    Nordgren, Kendra K.S., E-mail: knordgre@d.umn.edu; Wallace, Kendall B., E-mail: kwallace@d.umn.edu

    2014-01-01

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

  2. Rbfox2-coordinated alternative splicing of Mef2d and Rock2 controls myoblast fusion during myogenesis.

    Science.gov (United States)

    Singh, Ravi K; Xia, Zheng; Bland, Christopher S; Kalsotra, Auinash; Scavuzzo, Marissa A; Curk, Tomaz; Ule, Jernej; Li, Wei; Cooper, Thomas A

    2014-08-21

    Alternative splicing plays important regulatory roles during periods of physiological change. During development, a large number of genes coordinately express protein isoform transitions regulated by alternative splicing; however, the mechanisms that coordinate splicing and the functional integration of the resultant tissue-specific protein isoforms are typically unknown. Here we show that the conserved Rbfox2 RNA binding protein regulates 30% of the splicing transitions observed during myogenesis and is required for the specific step of myoblast fusion. Integration of Rbfox2-dependent splicing outcomes from RNA-seq with Rbfox2 iCLIP data identified Mef2d and Rock2 as Rbfox2 splicing targets. Restored activities of Mef2d and Rock2 rescued myoblast fusion in Rbfox2-depleted cultures, demonstrating functional cooperation of protein isoforms generated by coordinated alterative splicing. The results demonstrate that coordinated alternative splicing by a single RNA binding protein modulates transcription (Mef2d) and cell signaling (Rock2) programs to drive tissue-specific functions (cell fusion) to promote a developmental transition.

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

    Science.gov (United States)

    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.

  4. Globular adiponectin induces differentiation and fusion of skeletal muscle cells

    Institute of Scientific and Technical Information of China (English)

    Tania Fiaschi; Domenico Cirelli; Giuseppina Comito; Stefania Gelmini; Giampietro Ramponi; Maria Serio; Paola Chiarugi

    2009-01-01

    The growing interest in skeletal muscle regeneration is associated with the opening of new therapeutic strategies for muscle injury after trauma, as well as several muscular degenerative pathologies, including dystrophies, muscu-lar atrophy, and cachexia. Studies focused on the ability of extracellular factors to promote myogenesis are therefore highly promising. We now report that an adipocyte-derived factor, globular adiponectin (gAd), is able to induce mus-cle gene expression and cell differentiation, gAd, besides its well-known ability to regulate several metabolic func-tions in muscle, including glucose uptake and consumption and fatty acid catabolism, is able to block cell cycle entry of myoblasts, to induce the expression of specific skeletal muscle markers such as myosin heavy chain or eaveolin-3, as well as to provoke cell fusion into multinucleated syneytia and, finally, muscle fibre formation, gAd exerts its pro-differentiative activity through redox-dependent activation of p38, Akt and 5'-AMP-activated protein kinase path-ways. Interestingly, differentiating myoblasts are autocrine for adiponectiu, and the mimicking of pro-inflammatory settings or exposure to oxidative stress strongly increases the production of the hormone from differentiating cells. These data suggest a novel function of adiponectin, directly coordinating the myogenic differentiation program and serving an autocrine function during skeletal myogenesis.

  5. Zinc promotes proliferation and activation of myogenic cells via the PI3K/Akt and ERK signaling cascade

    Energy Technology Data Exchange (ETDEWEB)

    Ohashi, Kazuya, E-mail: asuno10k@yahoo.co.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Wada, Eiji, E-mail: gacchu1@yahoo.co.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Zammit, Peter S., E-mail: peter.zammit@kcl.ac.uk [Randall Division of Cell and Molecular Biophysics, King' s College London, London SE1 1UL (United Kingdom); Shiozuka, Masataka, E-mail: cmuscle@mail.ecc.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Matsuda, Ryoichi, E-mail: cmatsuda@mail.ecc.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan)

    2015-05-01

    Skeletal muscle stem cells named muscle satellite cells are normally quiescent but are activated in response to various stimuli, such as injury and overload. Activated satellite cells enter the cell cycle and proliferate to produce a large number of myogenic progenitor cells, and these cells then differentiate and fuse to form myofibers. Zinc is one of the essential elements in the human body, and has multiple roles, including cell growth and DNA synthesis. However, the role of zinc in myogenic cells is not well understood, and is the focus of this study. We first examined the effects of zinc on differentiation of murine C2C12 myoblasts and found that zinc promoted proliferation, with an increased number of cells incorporating EdU, but inhibited differentiation with reduced myogenin expression and myotube formation. Furthermore, we used the C2C12 reserve cell model of myogenic quiescence to investigate the role of zinc on activation of myogenic cells. The number of reserve cells incorporating BrdU was increased by zinc in a dose dependent manner, with the number dramatically further increased using a combination of zinc and insulin. Akt and extracellular signal-regulated kinase (ERK) are downstream of insulin signaling, and both were phosphorylated after zinc treatment. The zinc/insulin combination-induced activation involved the phosphoinositide 3-kinase (PI3K)/Akt and ERK cascade. We conclude that zinc promotes activation and proliferation of myogenic cells, and this activation requires phosphorylation of PI3K/Akt and ERK as part of the signaling cascade. - Highlights: • Zinc has roles for promoting proliferation and inhibition differentiation of C2C12. • Zinc promotes activation of reserve cells. • Insulin and zinc synergize activation of reserve cells. • PI3K/Akt and ERK cascade affect zinc/insulin-mediated activation of reserve cells.

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

    Directory of Open Access Journals (Sweden)

    In Young Choi

    2016-06-01

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

  7. Distribution, phosphorylation, and activities of Hsp25 in heat-stressed H9c2 myoblasts : a functional link to cytoprotection

    NARCIS (Netherlands)

    Bryantsev, AL; Loktionova, SA; Ilyinskaya, OP; Tararak, EM; Kampinga, HH; Kabakov, AE

    2002-01-01

    The behavior of the endogenous heat shock protein 25 (Hsp25) in heat-stressed rat H9c2 myoblasts was studied. After mild or severe heating, this protein became less extractable with Triton X-100 and displayed characteristic immunofluorescence patterns, namely (1) granules in the nucleus, and (2) ass

  8. Dock mediates Scar- and WASp-dependent actin polymerization through interaction with cell adhesion molecules in founder cells and fusion-competent myoblasts.

    Science.gov (United States)

    Kaipa, Balasankara Reddy; Shao, Huanjie; Schäfer, Gritt; Trinkewitz, Tatjana; Groth, Verena; Liu, Jianqi; Beck, Lothar; Bogdan, Sven; Abmayr, Susan M; Önel, Susanne-Filiz

    2013-01-01

    The formation of the larval body wall musculature of Drosophila depends on the asymmetric fusion of two myoblast types, founder cells (FCs) and fusion-competent myoblasts (FCMs). Recent studies have established an essential function of Arp2/3-based actin polymerization during myoblast fusion, formation of a dense actin focus at the site of fusion in FCMs, and a thin sheath of actin in FCs and/or growing muscles. The formation of these actin structures depends on recognition and adhesion of myoblasts that is mediated by cell surface receptors of the immunoglobulin superfamily. However, the connection of the cell surface receptors with Arp2/3-based actin polymerization is poorly understood. To date only the SH2-SH3 adaptor protein Crk has been suggested to link cell adhesion with Arp2/3-based actin polymerization in FCMs. Here, we propose that the SH2-SH3 adaptor protein Dock, like Crk, links cell adhesion with actin polymerization. We show that Dock is expressed in FCs and FCMs and colocalizes with the cell adhesion proteins Sns and Duf at cell-cell contact points. Biochemical data in this study indicate that different domains of Dock are involved in binding the cell adhesion molecules Duf, Rst, Sns and Hbs. We emphasize the importance of these interactions by quantifying the enhanced myoblast fusion defects in duf dock, sns dock and hbs dock double mutants. Additionally, we show that Dock interacts biochemically and genetically with Drosophila Scar, Vrp1 and WASp. Based on these data, we propose that Dock links cell adhesion in FCs and FCMs with either Scar- or Vrp1-WASp-dependent Arp2/3 activation.

  9. Growth factor array fabrication using a color ink jet printer.

    Science.gov (United States)

    Watanabe, Kohei; Miyazaki, Takeshi; Matsuda, Ryoichi

    2003-04-01

    We have developed a novel method for growth factor analysis using a commercial color ink jet printer to fabricate substrata patterned with growth factors. We prepared substrata with insulin printed in a simple pattern or containing multiple areas of varying quantities of printed insulin. When we cultured the mouse myoblast cell line, C2C12, on the insulin-patterned substrata, the cells were grown in the same pattern with the insulin-printed pattern. Cell culture with the latter substrata demonstrated that quantity control of insulin deposition by a color ink jet printer is possible. For further applications, we developed substrata with insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF) spotted in 16 different areas in varying combinations and concentrations (growth factor array). With this growth factor array, C2C12 cells were cultured, and the onset of muscle cell differentiation was monitored for the expression of the myogenic regulator myogenin. The ratio of cells expressing myogenin varied with the doses of IGF-I and bFGF in the sections, demonstrating a feasibility of growth factor array fabrication by a color ink jet printer. Since a printer manipulates several colors, this method can be easily applied to multivariate analyses of growth factors and attachment factors affecting cell growth and differentiation. This method may provide a powerful tool for cell biology and tissue engineering, especially for stem cell research in investigating unknown conditions for differentiation.

  10. The differentiation and morphogenesis of craniofacial muscles.

    Science.gov (United States)

    Noden, Drew M; Francis-West, Philippa

    2006-05-01

    Unraveling the complex tissue interactions necessary to generate the structural and functional diversity present among craniofacial muscles is challenging. These muscles initiate their development within a mesenchymal population bounded by the brain, pharyngeal endoderm, surface ectoderm, and neural crest cells. This set of spatial relations, and in particular the segmental properties of these adjacent tissues, are unique to the head. Additionally, the lack of early epithelialization in head mesoderm necessitates strategies for generating discrete myogenic foci that may differ from those operating in the trunk. Molecular data indeed indicate dissimilar methods of regulation, yet transplantation studies suggest that some head and trunk myogenic populations are interchangeable. The first goal of this review is to present key features of these diversities, identifying and comparing tissue and molecular interactions regulating myogenesis in the head and trunk. Our second focus is on the diverse morphogenetic movements exhibited by craniofacial muscles. Precursors of tongue muscles partly mimic migrations of appendicular myoblasts, whereas myoblasts destined to form extraocular muscles condense within paraxial mesoderm, then as large cohorts they cross the mesoderm:neural crest interface en route to periocular regions. Branchial muscle precursors exhibit yet another strategy, establishing contacts with neural crest populations before branchial arch formation and maintaining these relations through subsequent stages of morphogenesis. With many of the prerequisite stepping-stones in our knowledge of craniofacial myogenesis now in place, discovering the cellular and molecular interactions necessary to initiate and sustain the differentiation and morphogenesis of these neglected craniofacial muscles is now an attainable goal.

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

    Science.gov (United States)

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

    2016-03-15

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

  12. Automated high-content assay for compounds selectively toxic to Trypanosoma cruzi in a myoblastic cell line.

    Directory of Open Access Journals (Sweden)

    Julio Alonso-Padilla

    2015-01-01

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

  13. 成骨细胞分化、骨形成与修复中转录因子Osx和Satb2的调控作用%Osx and Satb2 regulate osteoblast differentiation, bone formation and repair

    Institute of Scientific and Technical Information of China (English)

    侯秋科; 黄永铨; 李昀骏; 陈东风

    2016-01-01

    背景:成骨细胞主要来自于骨髓细胞向骨基质中的间充质细胞,一些转录因子或局部因素可促进骨髓基质细胞调节分化为成骨细胞。  目的:明确C2C12细胞以及Osx与Satb2在骨质疏松修复过程中的作用。  方法:取野生型SD大鼠20只,分为正常组10只,假手术组和骨质疏松模型组(模型组)各5只,同时取Osx-KO大鼠10只。模型组和Osx-KO大鼠切除双侧卵巢构建野生型大鼠与Osx-KO大鼠进行骨质疏松模型;假手术组找出双侧卵巢但不切除。检测各组大鼠术后体质量的变化及股骨骨密度含量。体外培养C2C12细胞,并设计了siRNA-Satb2、siRNA-Osx,通过细胞实验、基因沉默、western blot法,观察成骨细胞分化的相关Osx与Satb2的表达及对骨质疏松的影响。  结果与结论:①体质量:造模12周后,模型组、Osx-KO组大鼠较正常组和假手术组大鼠显著增加(P OBJECTIVE: To study the role of Osx and Satb2 in C2C12cels in the repair process of osteoporosis. METHODS: Twenty wild-type Sprague-Dawley rats were assigned into normal control group (n=10), sham group (n=5) and osteoporosis group (model group,n=5). Another 10 Osx-KO rats were enroled in the study. Osteoporosis models were established by removal of both ovaries in the model group and Osx-KO group. In the sham group, bilateral ovaries were exposed but not removed. Changes in body mass and femoral bone density were detected in the four groups post operation. C2C12 cels were culturedin vitro, and siRNA-Satb2 and siRNA-Osx were designed. Expressions of Osx and Satb2 and their effects on osteoporosis were observed using cel experiments, gene silencing and western blot assay. RESULTS AND CONCLUSION:After 12 weeks, the body mass in the model and Osx-KO groups was significantly increased compared with the normal control and sham groups (P< 0.01); the bone density in the model and Osx-KO group was significantly decreased

  14. Robust fabrication of electrospun-like polymer mats to direct cell behaviour.

    Science.gov (United States)

    Ballester-Beltrán, José; Lebourg, Myriam; Capella, Hector; Diaz Lantada, Andres; Salmerón-Sánchez, Manuel

    2014-09-01

    Currently, cell culture systems that include nanoscale topography are widely used in order to provide cells additional cues closer to the in vivo environment, seeking to mimic the natural extracellular matrix. Electrospinning is one of the most common techniques to produce nanofiber mats. However, since many sensitive parameters play an important role in the process, a lack of reproducibility is a major drawback. Here we present a simple and robust methodology to prepare reproducible electrospun-like samples. It consists of a polydimethylsiloxane mold reproducing the fiber pattern to solvent-cast a polymer solution and obtain the final sample. To validate this methodology, poly(L-lactic) acid (PLLA) samples were obtained and, after characterisation, bioactivity and ability to direct cell response were assessed. C2C12 myoblasts developed focal adhesions on the electrospun-like fibers and, when cultured under myogenic differentiation conditions, similar differentiation levels to electrospun PLLA fibers were obtained.

  15. Metformin Treatment Prevents Sedentariness Related Damages in Mice

    Directory of Open Access Journals (Sweden)

    Pamela Senesi

    2016-01-01

    Full Text Available Metformin (METF, historical antihyperglycemic drug, is a likely candidate for lifespan extension, treatment and prevention of sedentariness damages, insulin resistance, and obesity. Skeletal muscle is a highly adaptable tissue, capable of hypertrophy response to resistance training and of regeneration after damage. Aims of this work were to investigate METF ability to prevent sedentariness damage and to enhance skeletal muscle function. Sedentary 12-week-old C57BL/6 mice were treated with METF (250 mg/kg per day, in drinking water for 60 days. METF role on skeletal muscle differentiation was studied in vitro using murine C2C12 myoblasts. Muscular performance evaluation revealed that METF enhanced mice physical performance (Estimated VO2max. Biochemical analyses of hepatic and muscular tissues indicated that in liver METF increased AMPK and CAMKII signaling. In contrast, METF inactivated ERKs, the principal kinases involved in hepatic stress. In skeletal muscle, METF activated AKT, key kinase in skeletal muscle mass maintenance. In in vitro studies, METF did not modify the C2C12 proliferation capacity, while it positively influenced the differentiation process and myotube maturation. In conclusion, our novel results suggest that METF has a positive action not only on the promotion of healthy aging but also on the prevention of sedentariness damages.

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

    Energy Technology Data Exchange (ETDEWEB)

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany); Renkawitz-Pohl, Renate, E-mail: renkawit@biologie.uni-marburg.de [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany)

    2013-02-15

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

  17. DNA replication timing is maintained genome-wide in primary human myoblasts independent of D4Z4 contraction in FSH muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Benjamin D Pope

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is linked to contraction of an array of tandem 3.3-kb repeats (D4Z4 at 4q35.2 from 11-100 copies to 1-10 copies. The extent to which D4Z4 contraction at 4q35.2 affects overall 4q35.2 chromatin organization remains unclear. Because DNA replication timing is highly predictive of long-range chromatin interactions, we generated genome-wide replication-timing profiles for FSHD and control myogenic precursor cells. We compared non-immortalized myoblasts from four FSHD patients and three control individuals to each other and to a variety of other human cell types. This study also represents the first genome-wide comparison of replication timing profiles in non-immortalized human cell cultures. Myoblasts from both control and FSHD individuals all shared a myoblast-specific replication profile. In contrast, male and female individuals were readily distinguished by monoallelic differences in replication timing at DXZ4 and other regions across the X chromosome affected by X inactivation. We conclude that replication timing is a robust cell-type specific feature that is unaffected by FSHD-related D4Z4 contraction.

  18. Endocytic Recycling Proteins EHD1 and EHD2 Interact with Fer-1-like-5 (Fer1L5) and Mediate Myoblast Fusion*

    Science.gov (United States)

    Posey, Avery D.; Pytel, Peter; Gardikiotes, Konstantina; Demonbreun, Alexis R.; Rainey, Mark; George, Manju; Band, Hamid; McNally, Elizabeth M.

    2011-01-01

    The mammalian ferlins are calcium-sensing, C2 domain-containing proteins involved in vesicle trafficking. Myoferlin and dysferlin regulate myoblast fusion and muscle membrane resealing, respectively. Correspondingly, myoferlin is most highly expressed in singly nucleated myoblasts, whereas dysferlin expression is increased in mature, multinucleated myotubes. Myoferlin also mediates endocytic recycling and participates in trafficking the insulin-like growth factor receptor. We have now characterized a novel member of the ferlin family, Fer1L5, because of its high homology to dysferlin and myoferlin. We found that Fer1L5 protein is expressed in small myotubes that contain only two to four nuclei. We also found that Fer1L5 protein binds directly to the endocytic recycling proteins EHD1 and EHD2 and that the second C2 domain in Fer1L5 mediates this interaction. Reduction of EHD1 and/or EHD2 inhibits myoblast fusion, and EHD2 is required for normal translocation of Fer1L5 to the plasma membrane. The characterization of Fer1L5 and its interaction with EHD1 and EHD2 underscores the complex requirement of ferlin proteins and mediators of endocytic recycling for membrane trafficking events during myotube formation. PMID:21177873

  19. Endocytic recycling proteins EHD1 and EHD2 interact with fer-1-like-5 (Fer1L5) and mediate myoblast fusion.

    Science.gov (United States)

    Posey, Avery D; Pytel, Peter; Gardikiotes, Konstantina; Demonbreun, Alexis R; Rainey, Mark; George, Manju; Band, Hamid; McNally, Elizabeth M

    2011-03-01

    The mammalian ferlins are calcium-sensing, C2 domain-containing proteins involved in vesicle trafficking. Myoferlin and dysferlin regulate myoblast fusion and muscle membrane resealing, respectively. Correspondingly, myoferlin is most highly expressed in singly nucleated myoblasts, whereas dysferlin expression is increased in mature, multinucleated myotubes. Myoferlin also mediates endocytic recycling and participates in trafficking the insulin-like growth factor receptor. We have now characterized a novel member of the ferlin family, Fer1L5, because of its high homology to dysferlin and myoferlin. We found that Fer1L5 protein is expressed in small myotubes that contain only two to four nuclei. We also found that Fer1L5 protein binds directly to the endocytic recycling proteins EHD1 and EHD2 and that the second C2 domain in Fer1L5 mediates this interaction. Reduction of EHD1 and/or EHD2 inhibits myoblast fusion, and EHD2 is required for normal translocation of Fer1L5 to the plasma membrane. The characterization of Fer1L5 and its interaction with EHD1 and EHD2 underscores the complex requirement of ferlin proteins and mediators of endocytic recycling for membrane trafficking events during myotube formation.

  20. The CELF1 RNA-Binding Protein Regulates Decay of Signal Recognition Particle mRNAs and Limits Secretion in Mouse Myoblasts

    Science.gov (United States)

    Russo, Joseph; Lee, Jerome E.; López, Carolina M.; Anderson, John; Nguyen, Thuy-mi P.; Heck, Adam M.; Wilusz, Jeffrey

    2017-01-01

    We previously identified several mRNAs encoding components of the secretory pathway, including signal recognition particle (SRP) subunit mRNAs, among transcripts associated with the RNA-binding protein CELF1. Through immunoprecipitation of RNAs crosslinked to CELF1 in myoblasts and in vitro binding assays using recombinant CELF1, we now provide evidence that CELF1 directly binds the mRNAs encoding each of the subunits of the SRP. Furthermore, we determined the half-lives of the Srp transcripts in control and CELF1 knockdown myoblasts. Our results indicate CELF1 is a destabilizer of at least five of the six Srp transcripts and that the relative abundance of the SRP proteins is out of balance when CELF1 is depleted. CELF1 knockdown myoblasts exhibit altered secretion of a luciferase reporter protein and are impaired in their ability to migrate and close a wound, consistent with a defect in the secreted extracellular matrix. Importantly, similar defects in wound healing are observed when SRP subunit imbalance is induced by over-expression of SRP68. Our studies support the existence of an RNA regulon containing Srp mRNAs that is controlled by CELF1. One implication is that altered function of CELF1 in myotonic dystrophy may contribute to changes in the extracellular matrix of affected muscle through defects in secretion. PMID:28129347

  1. Microrna-221 and microrna-222 modulate differentiation and maturation of skeletal muscle cells.

    Directory of Open Access Journals (Sweden)

    Beatrice Cardinali

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are a class of small non-coding RNAs that have recently emerged as important regulators of gene expression. They negatively regulate gene expression post-transcriptionally by translational repression and target mRNA degradation. miRNAs have been shown to play crucial roles in muscle development and in regulation of muscle cell proliferation and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: By comparing miRNA expression profiling of proliferating myoblasts versus differentiated myotubes, a number of modulated miRNAs, not previously implicated in regulation of myogenic differentiation, were identified. Among these, miR-221 and miR-222 were strongly down-regulated upon differentiation of both primary and established myogenic cells. Conversely, miR-221 and miR-222 expression was restored in post-mitotic, terminally differentiated myotubes subjected to Src tyrosine kinase activation. By the use of specific inhibitors we provide evidence that expression of miR-221 and miR-222 is under the control of the Ras-MAPK pathway. Both in myoblasts and in myotubes, levels of the cell cycle inhibitor p27 inversely correlated with miR-221 and miR-222 expression, and indeed we show that p27 mRNA is a direct target of these miRNAs in myogenic cells. Ectopic expression of miR-221 and miR-222 in myoblasts undergoing differentiation induced a delay in withdrawal from the cell cycle and in myogenin expression, followed by inhibition of sarcomeric protein accumulation. When miR-221 and miR-222 were expressed in myotubes undergoing maturation, a profound alteration of myofibrillar organization was observed. CONCLUSIONS/SIGNIFICANCE: miR-221 and miR-222 have been found to be modulated during myogenesis and to play a role both in the progression from myoblasts to myocytes and in the achievement of the fully differentiated phenotype. Identification of miRNAs modulating muscle gene expression is crucial for the understanding of the circuits

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-02-15

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

  3. EFFECTS OF MECHANICAL STRETCH WITH VARIANT FREQUENCIES ON ALIGNMENT AND DIFFERENTIATION OF MULTILAYER MYOTUBES CULTURED IN VITRO%不同频率周期性应力加载对体外多层肌管极性及分化的影响

    Institute of Scientific and Technical Information of China (English)

    黄维一; 刘幸卉; 陈荣; 冯利强; 廖华; 余磊; 曾慧君

    2012-01-01

    .05). Conclusion Low frequency (0.25 Hz) and suitable time (7 days) periodic mechanical stretch is beneficial to the differentiation of the multilayer myotubes cultured in the groove casts of Sylgard 184, but as the stretch time goes on the aging of myotubes will be accelerated.%目的 探讨不同频率的周期性应力加载对体外培养多层肌管极性与分化的影响,筛选优化的肌组织体外应力加载培养条件. 方法 体外培养C2C12成肌细胞于Sylgard 184铸型凹槽诱导分化形成多层肌管组织,采用自制体外细胞拉伸仪,对培养并分化的肌管进行间歇性应力加载:加载幅度10%,加载频率分别为0(A组)、0.25(B组)、0.50(C组)、1.00 Hz (D组),加载时间3次/d,每次1h.连续加载5、7、10 d时,观察各组肌管形态;RT-PCR和实时荧光定量PCR(real-time fluorescent quantitative PCR,QRT-PCR)分析检测成肌相关基因成肌分化抗原(myogenic differentiation antigen,MyoD)、肌细胞生成素(Myogenin)、结蛋白(Desmin)、肌球重链蛋白(myosin heavy chain,MyHC) mRNA的表达差异. 结果 倒置相差显微镜观察示,应力加载促进各组肌管的极性融合及数量增加,其中B组加载培养7d时,多层肌管排列紧密,极性显著.加载条件能促进成肌相关基因mRNA的表达:组内随加载时间延长,各组MyoD的mRNA表达逐渐下降,7、10d与5d比较差异均有统计学意义(P< 0.05); Myogenin、Desmin、MyHC的mRNA表达呈先升高后降低趋势,以7d表达量最高;B组除7d与10d Desmin的mRNA表达比较差异无统计学意义(P>0.05)外,其余各时间点比较差异均有统计学意义(P<0.05).同时间点随加载频率增加,MyoD、Myogenin、Desmin、MyHC的mRNA表达呈先升高后降低趋势,以B组表达量最高;除5dB、C组Desmin和10dA、B组MyHC的mRNA表达比较差异无统计学意义(P>0.05)外,其余各组与B组各相关基因mRNA表达比较,差异均有统计学意义(P<0.05).结论 低频(0.25 Hz)、适时(7 d)的周期性应

  4. 骨骼肌成肌干细胞体外三维与平面培养的比较研究%Comparative study of spatial and monolayer cultures of myoblasts in vitro

    Institute of Scientific and Technical Information of China (English)

    艾鹤英; 秦建强; 余磊; 廖华

    2010-01-01

    目的 比较三维与平面培养C2C12细胞形态及功能差异.方法 Ⅰ型胶原和MatrigelMatrix修饰Sylgard 184铸槽和普通培养皿,分别接种C2C12细胞悬液,细胞增殖至80%融合时换含2%马血清的DMEM/F12诱导分化获取成熟的肌管;倒置显微镜观察肌管形态,RT-PCR和免疫荧光检测.结果 Sylgard 184铸槽表面的C2C12细胞诱导分化5d出现多核、极性肌管,17 d时肌管增粗成熟、极性平行,融合形成肌组织类似物,厚0.15 mm,有自主收缩性;扫描电镜见肌管之间排列紧密、重叠,具有三维性;而平面培养肌管小而排列紊乱.RT-PCR表明三维培养的MyoD、Myogenin mRNA表达更为显著;Myogenin、Desmin、F-actin、MHC和nAChR蛋白检测显示,极性肌管内荧光蛋白的表达更密集.结论 三维培养更有利于成肌细胞的体外极性分化和肌管间细胞连接的形成,分化肌管的存活时间较长,有利于成肌分化因子和收缩蛋白的表达,是骨骼肌的发生发育、应力加载和骨骼肌肌病良好的体外研究模型.

  5. Cell entry of lymphocytic choriomeningitis virus is restricted in myotubes.

    Science.gov (United States)

    Iwasaki, Masaharu; Urata, Shuzo; Cho, Yoshitake; Ngo, Nhi; de la Torre, Juan C

    2014-06-01

    In mice persistently infected since birth with the prototypic arenavirus lymphocytic choriomeningitis viurs, viral antigen and RNA are readily detected in most organs and cell types but remarkably absent in skeletal muscle. Here we report that mouse C2C12 myoblasts that are readily infected by LCMV, become highly refractory to LCMV infection upon their differentiation into myotubes. Myotube's resistance to LCMV was not due to an intracellular restriction of virus replication but rather an impaired cell entry mediated by the LCMV surface glycoprotein. Our findings provide an explanation for the observation that in LCMV carrier mice myotubes, which are constantly exposed to blood-containing virus, remain free of viral antigen and RNA despite myotubes express high levels of the LCMV receptor alpha dystroglycan and do not pose an intracellular blockade to LCMV multiplication.

  6. Skeletal myoblasts for heart regeneration and repair: state of the art and perspectives on the mechanisms for functional cardiac benefits.

    Science.gov (United States)

    Formigli, L; Zecchi-Orlandini, S; Meacci, E; Bani, D

    2010-01-01

    Until recently, skeletal myoblasts (SkMBs) have been the most widely used cells in basic research and clinical trials of cell based therapy for cardiac repair and regeneration. Although SkMB engraftment into the post-infarcted heart has been consistently found to improve cardiac contractile function, the underlying therapeutic mechanisms remain still a matter of controversy and debate. This is basically because SkMBs do not attain a cardiac-like phenotype once homed into the diseased heart nor they form a contractile tissue functionally coupled with the surrounding viable myocardium. This issue of concern has generated the idea that the cardiotropic action of SkMBs may depend on the release of paracrine factors. However, the paracrine hypothesis still remains ill-defined, particularly concerning the identification of the whole spectrum of cell-derived soluble factors and details on their cardiac effects. In this context, the possibility to genetically engineering SkMBs to potentate their paracrine attitudes appears particularly attractive and is actually raising great expectation. Aim of the present review is not to cover all the aspects of cell-based therapy with SkMBs, as this has been the object of previous exhaustive reviews in this field. Rather, we focused on novel aspects underlying the interactions between SkMBs and the host cardiac tissues which may be relevant for directing the future basic and applied research on SkMB transplantation for post ischemic cardiac dysfunction.

  7. PI(4,5)P2 regulates myoblast fusion through Arp2/3 regulator localization at the fusion site.

    Science.gov (United States)

    Bothe, Ingo; Deng, Su; Baylies, Mary

    2014-06-01

    Cell-cell fusion is a regulated process that requires merging of the opposing membranes and underlying cytoskeletons. However, the integration between membrane and cytoskeleton signaling during fusion is not known. Using Drosophila, we demonstrate that the membrane phosphoinositide PI(4,5)P2 is a crucial regulator of F-actin dynamics during myoblast fusion. PI(4,5)P2 is locally enriched and colocalizes spatially and temporally with the F-actin focus that defines the fusion site. PI(4,5)P2 enrichment depends on receptor engagement but is upstream or parallel to actin remodeling. Regulators of actin branching via Arp2/3 colocalize with PI(4,5)P2 in vivo and bind PI(4,5)P2 in vitro. Manipulation of PI(4,5)P2 availability leads to impaired fusion, with a reduction in the F-actin focus size and altered focus morphology. Mechanistically, the changes in the actin focus are due to a failure in the enrichment of actin regulators at the fusion site. Moreover, improper localization of these regulators hinders expansion of the fusion interface. Thus, PI(4,5)P2 enrichment at the fusion site encodes spatial and temporal information that regulates fusion progression through the localization of activators of actin polymerization.

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

    Directory of Open Access Journals (Sweden)

    Ieva Antanavičiūtė

    2014-01-01

    Full Text Available 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.

  9. Hyperthermia differently affects connexin43 expression and gap junction permeability in skeletal myoblasts and HeLa cells.

    Science.gov (United States)

    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.

  10. Cell type-specific and common characteristics of exosomes derived from mouse cell lines: Yield, physicochemical properties, and pharmacokinetics.

    Science.gov (United States)

    Charoenviriyakul, Chonlada; Takahashi, Yuki; Morishita, Masaki; Matsumoto, Akihiro; Nishikawa, Makiya; Takakura, Yoshinobu

    2017-01-01

    Exosomes are small membrane vesicles secreted from cells and are expected to be used as drug delivery systems. Important characteristics of exosomes, such as yield, physicochemical properties, and pharmacokinetics, may be different among different cell types. However, there is limited information about the effect of cell type on these characteristics. In the present study, we evaluated these characteristics of exosomes derived from five different types of mouse cell lines: B16BL6 murine melanoma cells, C2C12 murine myoblast cells, NIH3T3 murine fibroblasts cells, MAEC murine aortic endothelial cells, and RAW264.7 murine macrophage-like cells. Exosomes were collected using a differential ultracentrifugation method. The exosomes collected from all the cell types were negatively charged globular vesicles with a diameter of approximately 100nm. C2C12 and RAW264.7 cells produced more exosomes than the other types of cells. The exosomes were labeled with a fusion protein of Gaussia luciferase and lactadherin to evaluate their pharmacokinetics. After intravenous injection into mice, all the exosomes rapidly disappeared from the systemic circulation and mainly distributed to the liver. In conclusion, the exosome yield was significantly different among the cell types, and all the exosomes evaluated in this study showed comparable physicochemical and pharmacokinetic properties.

  11. Orientation of Cells Cultured in Vortex Flow with Swinging Plate in Vitro

    Directory of Open Access Journals (Sweden)

    Shigehiro Hashimoto

    2011-06-01

    Full Text Available An effect of flow on cell culture has been studied in vitro. A silicone disk was placed in the center of culture dish of 52 mm internal diameter to make a doughnut-shaped canal. The dish was placed on a tilted plate, which rotates to make a vortex flow around the silicone disk with a swing motion. Variations were made on the diameter (20 mm, 30 mm, and 40 mm of the silicone disk and the rotational speed (2.1 rad/sec, 5.2 rad/sec of the swinging plate, which tilts with 0.1 rad from the horizontal plane. Five kinds of cells were cultured in the vortex flow of Dulbecco’s Modified Eagle’s Medium for seven days: C2C12 (mouse myoblast, L6 (rat skeletal muscle cell, A7r5 (rat aortic smooth muscle cell, CS-2P2-C75 (primary normal porcine aortic endothelial cell, and L929 (mouse fibroblast. The experiments show the following results. The orientation of cells depends on flow and on kinds of cells. A7r5 and CS-2P2-C75 line along the streamline of the flow. C2C12 and L6 adhere along the direction of the flow in the first stage, and tilt to the perpendicular direction to the flow differentiating to myotubes with fusion in the second stage.

  12. A Novel Synaptobrevin/VAMP Homologous Protein (VAMP5) Is Increased during In Vitro Myogenesis and Present in the Plasma Membrane

    Science.gov (United States)

    Zeng, Qi; Subramaniam, V. Nathan; Wong, Siew Heng; Tang, Bor Luen; Parton, Robert G.; Rea, Shane; James, David E.; Hong, Wanjin

    1998-01-01

    cDNA clones encoding a novel protein (VAMP5) homologous to synaptobrevins/VAMPs are detected during database searches. The predicted 102–amino acid VAMP5 harbors a 23-residue hydrophobic region near the carboxyl terminus and exhibits an overall amino acid identity of 33% with synaptobrevin/VAMP1 and 2 and cellubrevin. Northern blot analysis reveals that the mRNA for VAMP5 is preferentially expressed in the skeletal muscle and heart, whereas significantly lower levels are detected in several other tissues but not in the brain. During in vitro differentiation (myogenesis) of C2C12 myoblasts into myotubes, the mRNA level for VAMP5 is increased ∼8- to 10-fold. Immunoblot analysis using antibodies specific for VAMP5 shows that the protein levels are also elevated ∼6-fold during in vitro myogenesis of C2C12 cells. Indirect immunofluorescence microscopy and immunoelectron microscopy reveal that VAMP5 is associated with the plasma membrane as well as intracellular perinuclear and peripheral vesicular structures of myotubes. Epitope-tagged versions of VAMP5 are similarly targeted to the plasma membrane. PMID:9725904

  13. Nrf2 Protects Against TWEAK-mediated Skeletal Muscle Wasting

    Science.gov (United States)

    Al-Sawaf, Othman; Fragoulis, Athanassios; Rosen, Christian; Kan, Yuet Wai; Sönmez, Tolga Taha; Pufe, Thomas; Wruck, Christoph Jan

    2014-01-01

    Skeletal muscle (SM) regeneration after injury is impaired by excessive inflammation. Particularly, the inflammatory cytokine tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a potent inducer of skeletal muscle wasting and fibrosis. In this study we investigated the role of Nrf2, a major regulator of oxidative stress defence, in SM ischemia/reperfusion (I/R) injury and TWEAK induced atrophy. We explored the time-dependent expression of TWEAK after I/R in SM of Nrf2-wildtype (WT) and knockout (KO) mice. Nrf2-KO mice expressed significant higher levels of TWEAK as compared to WT mice. Consequently, Nrf2-KO mice present an insufficient regeneration as compared to Nrf2-WT mice. Moreover, TWEAK stimulation activates Nrf2 in the mouse myoblast cell line C2C12. This Nrf2 activation inhibits TWEAK induced atrophy in C2C12 differentiated myotubes. In summary, we show that Nrf2 protects SM from TWEAK-induced cell death in vitro and that Nrf2-deficient mice therefore have poorer muscle regeneration.

  14. The parafibromin tumor suppressor protein interacts with actin-binding proteins actinin-2 and actinin-3

    Directory of Open Access Journals (Sweden)

    Marx Stephen J

    2008-08-01

    Full Text Available Abstract Background Germline and somatic inactivating mutations in the HRPT2 gene occur in the inherited hyperparathyroidism-jaw tumor syndrome, in some cases of parathyroid cancer and in some cases of familial hyperparathyroidism. HRPT2 encodes parafibromin. To identify parafibromin interacting proteins we used the yeast two-hybrid system for screening a heart cDNA library with parafibromin as the bait. Results Fourteen parafibromin interaction positive preys representing 10 independent clones encoding actinin-2 were isolated. Parafibromin interacted with muscle alpha-actinins (actinin-2 and actinin-3, but not with non-muscle alpha-actinins (actinin-1 and actinin-4. The parafibromin-actinin interaction was verified by yeast two-hybrid, GST pull-down, and co-immunoprecipitation. Yeast two-hybrid analysis revealed that the N-terminal region of parafibromin interacted with actinins. In actin sedimentation assays parafibromin did not dissociate skeletal muscle actinins from actin filaments, but interestingly, parafibromin could also bundle/cross-link actin filaments. Parafibromin was predominantly nuclear in undifferentiated proliferating myoblasts (C2C12 cells, but in differentiated C2C12 myotubes parafibromin co-localized with actinins in the cytoplasmic compartment. Conclusion These data support a possible contribution of parafibromin outside the nucleus through its interaction with actinins and actin bundling/cross-linking. These data also suggest that actinins (and actin participate in sequestering parafibromin in the cytoplasmic compartment.

  15. Patterned three-dimensional encapsulation of embryonic stem cells using dielectrophoresis and stereolithography.

    Science.gov (United States)

    Bajaj, Piyush; Marchwiany, Daniel; Duarte, Carlos; Bashir, Rashid

    2013-03-01

    Controlling the assembly of cells in three dimensions is very important for engineering functional tissues, drug screening, probing cell-cell/cell-matrix interactions, and studying the emergent behavior of cellular systems. Although the current methods of cell encapsulation in hydrogels can distribute them in three dimensions, these methods typically lack spatial control of multi-cellular organization and do not allow for the possibility of cell-cell contacts as seen for the native tissue. Here, we report the integration of dielectrophoresis (DEP) with stereolithography (SL) apparatus for the spatial patterning of cells on custom made gold micro-electrodes. Afterwards, they are encapsulated in poly (ethylene glycol) diacrylate (PEGDA) hydrogels of different stiffnesses. This technique can mimic the in vivo microscale tissue architecture, where the cells have a high degree of three dimensional (3D) spatial control. As a proof of concept, we show the patterning and encapsulation of mouse embryonic stem cells (mESCs) and C2C12 skeletal muscle myoblasts. mESCs show high viability in both the DEP (91.79% ± 1.4%) and the no DEP (94.27% ± 0.5%) hydrogel samples. Furthermore, we also show the patterning of mouse embryoid bodies (mEBs) and C2C12 spheroids in the hydrogels, and verify their viability. This robust and flexible in vitro platform can enable various applications in stem cell differentiation and tissue engineering by mimicking elements of the native 3D in vivo cellular micro-environment.

  16. Insulin-like growth factor binding protein-5 modulates muscle differentiation through an insulin-like growth factor-dependent mechanism.

    Science.gov (United States)

    James, P L; Stewart, C E; Rotwein, P

    1996-05-01

    The insulin-like growth factor binding proteins (IGFBPs) are a family of six secreted proteins which bind to and modulate the actions of insulin-like growth factors-I and -II (IGF-I and -II). IGFBP-5 is more conserved than other IGFBPs characterized to date, and is expressed in adult rodent muscle and in the developing myotome. We have shown previously that C2 myoblasts secrete IGFBP-5 as their sole IGFBP. Here we use these cells to study the function of IGFBP-5 during myogenesis, a process stimulated by IGFs. We stably transfected C2 cells with IGFBP-5 cDNAs under control of a constitutively active promoter. Compared with vector-transfected control cells, C2 myoblasts expressing the IGFBP-5 transgene in the sense orientation exhibit increased IGFBP-5 levels in the extracellular matrix during proliferation, and subsequently fail to differentiate normally, as assessed by both morphological and biochemical criteria. Compared to controls, IGFBP-5 sense myoblasts show enhanced survival in low serum medium, remaining viable for at least four weeks in culture. By contrast, myoblasts expressing the IGFBP-5 antisense transcript differentiate prematurely and more extensively than control cells. The inhibition of myogenic differentiation by high level expression of IGFBP-5 could be overcome by exogenous IGFs, with des (1-3) IGF-I, an analogue with decreased affinity for IGFBP-5 but normal affinity for the IGF-I receptor, showing the highest potency. These results are consistent with a model in which IGFBP-5 blocks IGF-stimulated myogenesis, and indicate that sequestration of IGFs in the extracellular matrix could be a possible mechanism of action. Our observations also suggest that IGFBP-5 normally inhibits muscle differentiation, and imply a role for IGFBP-5 in regulating IGF action during myogenic development in vivo.

  17. M19 modulates skeletal muscle differentiation and insulin secretion in pancreatic β-cells through modulation of respiratory chain activity.

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    Linda Cambier

    Full Text Available Mitochondrial dysfunction due to nuclear or mitochondrial DNA alterations contributes to multiple diseases such as metabolic myopathies, neurodegenerative disorders, diabetes and cancer. Nevertheless, to date, only half of the estimated 1,500 mitochondrial proteins has been identified, and the function of most of these proteins remains to be determined. Here, we characterize the function of M19, a novel mitochondrial nucleoid protein, in muscle and pancreatic β-cells. We have identified a 13-long amino acid sequence located at the N-terminus of M19 that targets the protein to mitochondria. Furthermore, using RNA interference and over-expression strategies, we demonstrate that M19 modulates mitochondrial oxygen consumption and ATP production, and could therefore regulate the respiratory chain activity. In an effort to determine whether M19 could play a role in the regulation of various cell activities, we show that this nucleoid protein, probably through its modulation of mitochondrial ATP production, acts on late muscle differentiation in myogenic C2C12 cells, and plays a permissive role on insulin secretion under basal glucose conditions in INS-1 pancreatic β-cells. Our results are therefore establishing a functional link between a mitochondrial nucleoid protein and the modulation of respiratory chain activities leading to the regulation of major cellular processes such as myogenesis and insulin secretion.

  18. Experiment list: SRX142521 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available of C3H mice after crush injury || cell sex=F || antibody=Input || antibody description=Control signal which ...ncing || cell=C2C12 || cell organism=mouse || cell description=Myoblast cell line derived from thigh muscle

  19. Experiment list: SRX143620 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available of C3H mice after crush injury || cell sex=F || antibody=Input || antibody description=Control signal which ...ncing || cell=C2C12 || cell organism=mouse || cell description=Myoblast cell line derived from thigh muscle

  20. Experiment list: SRX143622 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available of C3H mice after crush injury || cell sex=F || antibody=Input || antibody description=Control signal which ...ncing || cell=C2C12 || cell organism=mouse || cell description=Myoblast cell line derived from thigh muscle

  1. Experiment list: SRX142511 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available of C3H mice after crush injury || cell sex=F || antibody=Input || antibody description=Control signal which ...ncing || cell=C2C12 || cell organism=mouse || cell description=Myoblast cell line derived from thigh muscle

  2. Experiment list: SRX143623 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available f C3H mice after crush injury || cell sex=F || antibody=H3ac_(06-599) || antibody antibodydescription=rabbit...cing || cell=C2C12 || cell organism=mouse || cell description=Myoblast cell line derived from thigh muscle o

  3. Experiment list: SRX142523 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available of C3H mice after crush injury || cell sex=F || antibody=Input || antibody description=Control signal which ...ncing || cell=C2C12 || cell organism=mouse || cell description=Myoblast cell line derived from thigh muscle

  4. Prolonged Culture of Aligned Skeletal Myotubes on Micromolded Gelatin Hydrogels

    Science.gov (United States)

    Bettadapur, Archana; Suh, Gio C.; Geisse, Nicholas A.; Wang, Evelyn R.; Hua, Clara; Huber, Holly A.; Viscio, Alyssa A.; Kim, Joon Young; Strickland, Julie B.; McCain, Megan L.

    2016-06-01

    In vitro models of skeletal muscle are critically needed to elucidate disease mechanisms, identify therapeutic targets, and test drugs pre-clinically. However, culturing skeletal muscle has been challenging due to myotube delamination from synthetic culture substrates approximately one week after initiating differentiation from myoblasts. In this study, we successfully maintained aligned skeletal myotubes differentiated from C2C12 mouse skeletal myoblasts for three weeks by utilizing micromolded (μmolded) gelatin hydrogels as culture substrates, which we thoroughly characterized using atomic force microscopy (AFM). Compared to polydimethylsiloxane (PDMS) microcontact printed (μprinted) with fibronectin (FN), cell adhesion on gelatin hydrogel constructs was significantly higher one week and three weeks after initiating differentiation. Delamination from FN-μprinted PDMS precluded robust detection of myotubes. Compared to a softer blend of PDMS μprinted with FN, myogenic index, myotube width, and myotube length on μmolded gelatin hydrogels was similar one week after initiating differentiation. However, three weeks after initiating differentiation, these parameters were significantly higher on μmolded gelatin hydrogels compared to FN-μprinted soft PDMS constructs. Similar results were observed on isotropic versions of each substrate, suggesting that these findings are independent of substrate patterning. Our platform enables novel studies into skeletal muscle development and disease and chronic drug testing in vitro.

  5. Brown fat determination and development from muscle precursor cells by novel action of bone morphogenetic protein 6.

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    Ankur Sharma

    Full Text Available Brown adipose tissue (BAT plays a pivotal role in promoting energy expenditure by the virtue of uncoupling protein-1 (UCP-1 that differentiates BAT from its energy storing white adipose tissue (WAT counterpart. The clinical implication of "classical" BAT (originates from Myf5 positive myoblastic lineage or the "beige" fat (originates through trans-differentiation of WAT activation in improving metabolic parameters is now becoming apparent. However, the inducers and endogenous molecular determinants that govern the lineage commitment and differentiation of classical BAT remain obscure. We report here that in the absence of any forced gene expression, stimulation with bone morphogenetic protein 6 (BMP6 induces brown fat differentiation from skeletal muscle precursor cells of murine and human origins. Through a comprehensive transcriptional profiling approach, we have discovered that two days of BMP6 stimulation in C2C12 myoblast cells is sufficient to induce genes characteristic of brown preadipocytes. This developmental switch is modulated in part by newly identified regulators, Optineurin (Optn and Cyclooxygenase-2 (Cox2. Furthermore, pathway analyses using the Causal Reasoning Engine (CRE identified additional potential causal drivers of this BMP6 induced commitment switch. Subsequent analyses to decipher key pathway that facilitates terminal differentiation of these BMP6 primed cells identified a key role for Insulin Like Growth Factor-1 Receptor (IGF-1R. Collectively these data highlight a therapeutically innovative role for BMP6 by providing a means to enhance the amount of myogenic lineage derived brown fat.

  6. Experiment list: SRX062123 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available le|Tissue Diagnosis=NOS 86008497,80.9,21.1,1101 GSM721307: Sonicated input MT source_name=C2C12 myotubes dif...ferentiated for 96h || cell line=C2C12 || cell type=myotubes || chromatin preparation method=sonication || c

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

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    Volkan Gurel

    2015-11-01

    Full Text Available 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.

  8. Efficient non-viral reprogramming of myoblasts to stemness with a single small molecule to generate cardiac progenitor cells.

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    Zeeshan Pasha

    Full Text Available UNLABELLED: The current protocols for generation of induced pluripotent stem (iPS cells involve genome integrating viral vectors which may induce tumorgenesis. The aim of this study was to develop and optimize a non-viral method without genetic manipulation for reprogramming of skeletal myoblasts (SMs using small molecules. METHODS AND RESULTS: SMs from young male Oct3/4-GFP(+ transgenic mouse were treated with DNA methyltransferase (DNMT inhibitor, RG108. Two weeks later, GFP(+ colonies of SM derived iPS cells (SiPS expressing GFP and with morphological similarity of mouse embryonic stem (ESCs were formed and propagated in vitro. SiPS were positive for alkaline phosphatase activity, expressed SSEA1, displayed ES cell specific pluripotency markers and formed teratoma in nude mice. Optimization of culture conditions for embryoid body (EBs formation yielded spontaneously contracting EBs having morphological, molecular, and ultra-structural similarities with cardiomyocytes and expressed early and late cardiac markers. miR profiling showed abrogation of let-7 family and upregulation of ESCs specific miR-290-295 cluster thus indicating that SiPS were similar to ESCs in miR profile. Four weeks after transplantation into the immunocompetent mice model of acute myocardial infarction (n = 12 per group, extensive myogenesis was observed in SiPS transplanted hearts as compared to DMEM controls (n = 6 per group. A significant reduction in fibrosis and improvement in global heart function in the hearts transplanted with SiPS derived cardiac progenitor cells were observed. CONCLUSIONS: Reprogramming of SMs by DNMT inhibitor is a simple, reproducible and efficient technique more likely to generate transgene integration-free iPS cells. Cardiac progenitors derived from iPS cells propagated extensively in the infarcted myocardium without tumorgenesis and improved cardiac function.

  9. RNA/MBNL1-containing foci in myoblast nuclei from patients affected by myotonic dystrophy type 2: an immunocytochemical study

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    F. Perdoni

    2009-09-01

    Full Text Available Myotonic dystrophy type 2 (DM2 is a dominantly inherited autosomal disease with multi-systemic clinical features and it is caused by expansion of a CCTG tetranucleotide repeat in the first intron of the zinc finger protein 9 (ZNF9 gene in 3q21.The expanded-CCUG-containing transcripts are retained in the cell nucleus and accumulate in the form of focal aggregates which specifically sequester the muscleblind-like 1 (MBNL1 protein, a RNA binding factor involved in the regulation of alternative splicing. The structural organization and composition of the foci are still incompletely known. In this study, the nuclear foci occurring in cultured myoblasts from DM2 patients were characterised at fluorescence and transmission electron microscopy by using a panel of antibodies recognizing transcription and processing factors of pre-mRNAs. MBNL1 proved to co-locate in the nuclear foci with snRNPs and hnRNPs, whereas no co-location was observed with RNA polymerase II, the non-RNP splicing factor SC35, the cleavage factor CStF and the PML protein. At electron microscopy the MBNL1-containing nuclear foci appeared as roundish domains showing a rather homogeneous structure and proved to contain snRNPs and hnRNPs. The sequestration of splicing factors involved in early phases of pre-mRNA processing supports the hypothesis of a general alteration in the maturation of several mRNAs, which could lead to the multiple pathological dysfunctions observed in dystrophic patients.

  10. Effects of Ghrelin on Triglyceride Accumulation and Glucose Uptake in Primary Cultured Rat Myoblasts under Palmitic Acid-Induced High Fat Conditions

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    Lingling Han

    2015-01-01

    Full Text Available This study aimed to study the effects of acylated ghrelin on glucose and triglyceride metabolism in rat myoblasts under palmitic acid- (PA- induced high fat conditions. Rat myoblasts were treated with 0, 10−11, 10−9, or 10−7 M acylated ghrelin and 0.3 mM PA for 12 h. Triglyceride accumulation was determined by Oil-Red-O staining and the glycerol phosphate dehydrogenase-peroxidase enzymatic method, and glucose uptake was determined by isotope tracer. The glucose transporter 4 (GLUT4, AMP-activated protein kinase (AMPK, acetyl-CoA carboxylase (ACC, and uncoupling protein 3 (UCP3 were assessed by RT-PCR and western blot. Compared to 0.3 mM PA, ghrelin at 10−9 and 10−7 M reduced triglyceride content (5.855 ± 0.352 versus 5.030 ± 0.129 and 4.158 ± 0.254 mM, P<0.05 and prevented PA-induced reduction of glucose uptake (1.717 ± 0.264 versus 2.233 ± 0.333 and 2.333 ± 0.273 10−2 pmol/g/min, P<0.05. The relative protein expression of p-AMPKα/AMPKα, UCP3, and p-ACC under 0.3 mM PA was significantly reduced compared to controls (all P<0.05, but those in the 10−9 and 10−7 M ghrelin groups were significantly protected from 0.3 mM PA (all P<0.05. In conclusion, acylated ghrelin reduced PA-induced triglyceride accumulation and prevented the PA-induced decrease in glucose uptake in rat myoblasts. These effects may involve fatty acid oxidation.

  11. A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA.

    KAUST Repository

    Cesana, Marcella

    2011-10-01

    Recently, a new regulatory circuitry has been identified in which RNAs can crosstalk with each other by competing for shared microRNAs. Such competing endogenous RNAs (ceRNAs) regulate the distribution of miRNA molecules on their targets and thereby impose an additional level of post-transcriptional regulation. Here we identify a muscle-specific long noncoding RNA, linc-MD1, which governs the time of muscle differentiation by acting as a ceRNA in mouse and human myoblasts. Downregulation or overexpression of linc-MD1 correlate with retardation or anticipation of the muscle differentiation program, respectively. We show that linc-MD1 "sponges" miR-133 and miR-133 [corrected] to regulate the expression of MAML1 and MEF2C, transcription factors that activate muscle-specific gene expression. Finally, we demonstrate that linc-MD1 exerts the same control over differentiation timing in human myoblasts, and that its levels are strongly reduced in Duchenne muscle cells. We conclude that the ceRNA network plays an important role in muscle differentiation.

  12. Cardiac Shock Wave Therapy Attenuates H9c2 Myoblast Apoptosis by Activating the AKT Signal Pathway

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    Weiwei Yu

    2014-04-01

    Full Text Available Background: Previous studies have demonstrated that Cardiac Shock Wave Therapy (CSWT improves myocardial perfusion and cardiac function in a porcine model of chronic myocardial ischemia and also ameliorates myocardial ischemia in patients with severe coronary artery disease (CAD. Apoptosis plays a key role in ischemic myocardial pathogenesis. However, it remains unclear whether CSWT is beneficial for ischemia/hypoxia (I/H-induced myocardial cell apoptosis and by which mechanism CSWT could improve heart function. We put forward the hypothesis that CSWT might protect heart function during ischemia/hypoxia by decreasing apoptosis. Methods: We generated ischemia/hypoxia (I/H-induced apoptosis in the H9c2 myoblast cell line to examine the CSWT function and possible mechanisms. H9c2 cells were treated under hypoxic serum-starved conditions for 24 h and then treated with or without CSWT (500 shots, 0.06, 0.09, 0.12mJ/mm2. The apoptotic cell rate was determined by flow cytometry assay, cell viability was examined by the MTT assay, nuclear fragmentation was detected by Hoechst 33342 staining, and the mitochondrial-mediated intrinsic pathway of apoptosis was assessed by the expression of Bax and Bcl-2 protein and Caspase3 activation. Results: First, apoptosis could be induced by ischemia/hypoxia in H9c2 cells. Second, CSWT attenuates the cell death and decreases the H9c2 cell apoptosis rate induced by ischemia and hypoxia. Third, CSWT suppresses the expression of apoptosis molecules that regulate the intrinsic pathway of apoptosis in H9c2 cells. Fourth, CSWT increases the phosphorylation of AKT, which indicates the activation of the PI3K-AKT pathway. Conclusions: These results indicate that CSWT exerts a protective effect against I/H-induced cell death, potentially by preventing the activation of components of the mitochondrial-dependent intrinsic apoptotic pathway. We also demonstrate that the PI3K-Akt pathway may be involved in the CSWT effects on

  13. ATOH8, a regulator of skeletal myogenesis in the hypaxial myotome of the trunk.

    Science.gov (United States)

    Balakrishnan-Renuka, Ajeesh; Morosan-Puopolo, Gabriela; Yusuf, Faisal; Abduelmula, Aisha; Chen, Jingchen; Zoidl, Georg; Philippi, Susanne; Dai, Fangping; Brand-Saberi, Beate

    2014-03-01

    The embryonic muscles of the axial skeleton and limbs take their origin from the dermomyotomes of the somites. During embryonic myogenesis, muscle precursors delaminate from the dermomyotome giving rise to the hypaxial and epaxial myotome. Mutant studies for myogenic regulatory factors have shown that the development of the hypaxial myotome differs from the formation of the epaxial myotome and that the development of the hypaxial myotome depends on the latter within the trunk region. The transcriptional networks that regulate the transition of proliferative dermomyotomal cells into the predominantly post-mitotic hypaxial myotome, as well as the eventual patterning of the myotome, are not fully understood. Similar transitions occurring during the development of the neural system have been shown to be controlled by the Atonal family of helix-loop-helix transcription factors. Here, we demonstrate that ATOH8, a member of the Atonal family, is expressed in a subset of embryonic muscle cells in the dermomyotome and myotome. Using the RNAi approach, we show that loss of ATOH8 in the lateral somites at the trunk level results in a blockage of differentiation and thus causes cells to be maintained in a predetermined state. Furthermore, we show that ATOH8 is also expressed in cultured C2C12 mouse myoblasts and becomes dramatically downregulated during their differentiation. We propose that ATOH8 plays a role during the transition of myoblasts from the proliferative phase to the differentiation phase and in the regulation of myogenesis in the hypaxial myotome of the trunk.

  14. BPAG1a and b associate with EB1 and EB3 and modulate vesicular transport, Golgi apparatus structure, and cell migration in C2.7 myoblasts.

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    Kseniia Poliakova

    Full Text Available BPAG1a and BPAG1b (BPAG1a/b constitute two major isoforms encoded by the dystonin (Dst gene and show homology with MACF1a and MACF1b. These proteins are members of the plakin family, giant multi-modular proteins able to connect the intermediate filament, microtubule and microfilament cytoskeletal networks with each other and to distinct cell membrane sites. They also serve as scaffolds for signaling proteins that modulate cytoskeletal dynamics. To gain better insights into the functions of BPAG1a/b, we further characterized their C-terminal region important for their interaction with microtubules and assessed the role of these isoforms in the cytoskeletal organization of C2.7 myoblast cells. Our results show that alternative splicing does not only occur at the 5' end of Dst and Macf1 pre-mRNAs, as previously reported, but also at their 3' end, resulting in expression of additional four mRNA variants of BPAG1 and MACF1. These isoform-specific C-tails were able to bundle microtubules and bound to both EB1 and EB3, two microtubule plus end proteins. In the C2.7 cell line, knockdown of BPAG1a/b had no major effect on the organization of the microtubule and microfilament networks, but negatively affected endocytosis and maintenance of the Golgi apparatus structure, which became dispersed. Finally, knockdown of BPAG1a/b caused a specific decrease in the directness of cell migration, but did not impair initial cell adhesion. These data provide novel insights into the complexity of alternative splicing of Dst pre-mRNAs and into the role of BPAG1a/b in vesicular transport, Golgi apparatus structure as well as in migration in C2.7 myoblasts.

  15. Overexpression of ryanodine receptor type 1 enhances mitochondrial fragmentation and Ca2+-induced ATP production in cardiac H9c2 myoblasts.

    Science.gov (United States)

    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; Sheu, Shey-Shing

    2013-12-01

    Ca(+) influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca(2+) influx is mainly mediated by the mitochondrial Ca(2+) uniporter (MCU). Growing evidence also indicates that mitochondrial Ca(2+) 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 Ca(2+) uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+)-induced ATP production in cardiac H9c2 myoblasts.

  16. Differential characters

    CERN Document Server

    Bär, Christian

    2014-01-01

    Providing a systematic introduction to differential characters as introduced by Cheeger and Simons, this text describes important concepts such as fiber integration, higher dimensional holonomy, transgression, and the product structure in a geometric manner. Differential characters form a model of what is nowadays called differential cohomology, which is the mathematical structure behind the higher gauge theories in physics.  

  17. MicroRNA-206 is highly expressed in newly formed muscle fibers: implications regarding potential for muscle regeneration and maturation in muscular dystrophy.

    Science.gov (United States)

    Yuasa, Katsutoshi; Hagiwara, Yasuko; Ando, Masanori; Nakamura, Akinori; Takeda, Shin'ichi; Hijikata, Takao

    2008-01-01

    miR-1, miR-133a, and miR-206 are muscle-specific microRNAs expressed in skeletal muscles and have been shown to contribute to muscle development. To gain insight into the pathophysiological roles of these three microRNAs in dystrophin-deficient muscular dystrophy, their expression in the tibialis anterior (TA) muscles of mdx mice and CXMD(J) dogs were evaluated by semiquantitative RT-PCR and in situ hybridization. Their temporal and spatial expression patterns were also analyzed in C2C12 cells during muscle differentiation and in cardiotoxin (CTX)-injured TA muscles to examine how muscle degeneration and regeneration affect their expression. In dystrophic TA muscles of mdx mice, miR-206 expression was significantly elevated as compared to that in control TA muscles of age-matched B10 mice, whereas there were no differences in miR-1 or miR-133a expression between B10 and mdx TA muscles. On in situ hybridization analysis, intense signals for miR-206 probes were localized in newly formed myotubes with centralized nuclei, or regenerating muscle fibers, but not in intact pre-degenerated fibers or numerous small mononucleated cells, possibly proliferating myoblasts and inflammatory infiltrates. Similar increased expression of miR-206 was also found in C2C12 differentiation and CTX-induced regeneration, in which differentiated myotubes or regenerating fibers showed abundant expression of miR-206. However, CXMD(J) TA muscles contained smaller amounts of miR-206, miR-1, and miR-133a than controls. They exhibited more severe and more progressive degenerative alterations than mdx TA muscles. Taken together, these observations indicated that newly formed myotubes showed markedly increased expression of miR-206, which might reflect active regeneration and efficient maturation of skeletal muscle fibers.

  18. Isolation and maintenance-free culture of contractile myotubes from Manduca sexta embryos.

    Directory of Open Access Journals (Sweden)

    Amanda L Baryshyan

    Full Text Available Skeletal muscle tissue engineering has the potential to treat tissue loss and degenerative diseases. However, these systems are also applicable for a variety of devices where actuation is needed, such as microelectromechanical systems (MEMS and robotics. Most current efforts to generate muscle bioactuators are focused on using mammalian cells, which require exacting conditions for survival and function. In contrast, invertebrate cells are more environmentally robust, metabolically adaptable and relatively autonomous. Our hypothesis is that the use of invertebrate muscle cells will obviate many of the limitations encountered when mammalian cells are used for bioactuation. We focus on the tobacco hornworm, Manduca sexta, due to its easy availability, large size and well-characterized muscle contractile properties. Using isolated embryonic cells, we have developed culture conditions to grow and characterize contractile M. sexta muscles. The insect hormone 20-hydroxyecdysone was used to induce differentiation in the system, resulting in cells that stained positive for myosin, contract spontaneously for the duration of the culture, and do not require media changes over periods of more than a month. These cells proliferate under normal conditions, but the application of juvenile hormone induced further proliferation and inhibited differentiation. Cellular metabolism under normal and low glucose conditions was compared for C2C12 mouse and M. sexta myoblast cells. While differentiated C2C12 cells consumed glucose and produced lactate over one week as expected, M. sexta muscle did not consume significant glucose, and lactate production exceeded mammalian muscle production on a per cell basis. Contractile properties were evaluated using index of movement analysis, which demonstrated the potential of these cells to perform mechanical work. The ability of cultured M. sexta muscle to continuously function at ambient conditions without medium replenishment

  19. Measurement of Periodical Contraction of Cultured Muscle Tube with Laser

    Directory of Open Access Journals (Sweden)

    Jun Takase

    2009-06-01

    Full Text Available Periodical contraction of a cultured muscle tube has been measured with laser in vitro. C2C12 (mouse myoblast cell line was cultured with High-glucose Dulbecco's Modified Eagle's Medium on a dish to make muscle tubes. Differentiation from myoblasts to myotubes was induced with an additional horse serum. Repetitive contraction of the tube was generated by electric pulses lower than sixty volts of amplitude with one milli-second of width through the electrodes of platinum, and observed with a phase-contrast microscope. A laser beam of 632.8 nm wavelength was restricted to 0.096 mm diameter, and applied to the muscle tubes on the bottom of the culture dish. Fluctuating intensity of the transmitted laser beam through the periodically contracting muscle tubes was measured, and its spectrum was analyzed. The analyzed data show that the repetitive contraction is synchronized with stimulation of the periodical electric pulses between 0.2 s and 2 s.

  20. The vitamin C transporter SVCT2 is down-regulated during postnatal development of slow skeletal muscles.

    Science.gov (United States)

    Sandoval, Daniel; Ojeda, Jorge; Low, Marcela; Nualart, Francisco; Marcellini, Sylvain; Osses, Nelson; Henríquez, Juan Pablo

    2013-06-01

    Vitamin C plays key roles in cell homeostasis, acting as a potent antioxidant as well as a positive modulator of cell differentiation. In skeletal muscle, the vitamin C/sodium co-transporter SVCT2 is preferentially expressed in oxidative slow fibers. Besides, SVCT2 is up-regulated upon the early fusion of primary myoblasts. However, our knowledge of the postnatal expression profile of SVCT2 remains scarce. Here we have analyzed the expression of SVCT2 during postnatal development of the chicken slow anterior and fast posterior latissimus dorsi muscles, ranging from day 7 to adulthood. SVCT2 expression is consistently higher in the slow than in the fast muscle at all stages. After hatching, SVCT2 expression is significantly down-regulated in the anterior latissimus dorsi, which nevertheless maintains a robust slow phenotype. Taking advantage of the C2C12 cell line to recapitulate myogenesis, we confirmed that SVCT2 is expressed in a biphasic fashion, reaching maximal levels upon early myoblasts fusion and decreasing during myotube growth. Together, these findings suggest that the dynamic expression levels of SVCT2 could be relevant for different features of skeletal muscle physiology, such as muscle cell formation, growth and activity.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-05-01

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

  2. Differential microRNA Expression in Fast- and Slow-Twitch Skeletal Muscle of Piaractus mesopotamicus during Growth.

    Directory of Open Access Journals (Sweden)

    Bruno Oliveira da Silva Duran

    Full Text Available Pacu (Piaractus mesopotamicus is a Brazilian fish with a high economic value in pisciculture due to its rusticity and fast growth. Postnatal growth of skeletal muscle in fish occurs by hyperplasia and/or hypertrophy, processes that are dependent on the proliferation and differentiation of myoblasts. A class of small noncoding RNAs, known as microRNAs (miRNAs, represses the expression of target mRNAs, and many studies have demonstrated that miR-1, miR-133, miR-206 and miR-499 regulate different processes in skeletal muscle through the mRNA silencing of hdac4 (histone deacetylase 4, srf (serum response factor, pax7 (paired box 7 and sox6 ((sex determining region Y-box 6, respectively. The aim of our work was to evaluate the expression of these miRNAs and their putative target mRNAs in fast- and slow-twitch skeletal muscle of pacu during growth. We used pacus in three different development stages: larval (aged 30 days, juvenile (aged 90 days and 150 days and adult (aged 2 years. To complement our study, we also performed a pacu myoblast cell culture, which allowed us to investigate miRNA expression in the progression from myoblast proliferation to differentiation. Our results revealed an inverse correlation between the expression of the miRNAs and their target mRNAs, and there was evidence that miR-1 and miR-206 may regulate the differentiation of myoblasts, whereas miR-133 may regulate the proliferation of these cells. miR-499 was highly expressed in slow-twitch muscle, which suggests its involvement in the specification of the slow phenotype in muscle fibers. The expression of these miRNAs exhibited variations between different development stages and between distinct muscle twitch phenotypes. This work provides the first identification of miRNA expression profiles in pacu skeletal muscle and suggests an important role of these molecules in muscle growth and in the maintenance of the muscle phenotype.

  3. Differential microRNA Expression in Fast- and Slow-Twitch Skeletal Muscle of Piaractus mesopotamicus during Growth.

    Science.gov (United States)

    Duran, Bruno Oliveira da Silva; Fernandez, Geysson Javier; Mareco, Edson Assunção; Moraes, Leonardo Nazario; Salomão, Rondinelle Artur Simões; Gutierrez de Paula, Tassiana; Santos, Vander Bruno; Carvalho, Robson Francisco; Dal-Pai-Silva, Maeli; Dal-Pai-Silvca, Maeli

    2015-01-01

    Pacu (Piaractus mesopotamicus) is a Brazilian fish with a high economic value in pisciculture due to its rusticity and fast growth. Postnatal growth of skeletal muscle in fish occurs by hyperplasia and/or hypertrophy, processes that are dependent on the proliferation and differentiation of myoblasts. A class of small noncoding RNAs, known as microRNAs (miRNAs), represses the expression of target mRNAs, and many studies have demonstrated that miR-1, miR-133, miR-206 and miR-499 regulate different processes in skeletal muscle through the mRNA silencing of hdac4 (histone deacetylase 4), srf (serum response factor), pax7 (paired box 7) and sox6 ((sex determining region Y)-box 6), respectively. The aim of our work was to evaluate the expression of these miRNAs and their putative target mRNAs in fast- and slow-twitch skeletal muscle of pacu during growth. We used pacus in three different development stages: larval (aged 30 days), juvenile (aged 90 days and 150 days) and adult (aged 2 years). To complement our study, we also performed a pacu myoblast cell culture, which allowed us to investigate miRNA expression in the progression from myoblast proliferation to differentiation. Our results revealed an inverse correlation between the expression of the miRNAs and their target mRNAs, and there was evidence that miR-1 and miR-206 may regulate the differentiation of myoblasts, whereas miR-133 may regulate the proliferation of these cells. miR-499 was highly expressed in slow-twitch muscle, which suggests its involvement in the specification of the slow phenotype in muscle fibers. The expression of these miRNAs exhibited variations between different development stages and between distinct muscle twitch phenotypes. This work provides the first identification of miRNA expression profiles in pacu skeletal muscle and suggests an important role of these molecules in muscle growth and in the maintenance of the muscle phenotype.

  4. Differential manifolds

    CERN Document Server

    Kosinski, Antoni A

    2007-01-01

    The concepts of differential topology form the center of many mathematical disciplines such as differential geometry and Lie group theory. Differential Manifolds presents to advanced undergraduates and graduate students the systematic study of the topological structure of smooth manifolds. Author Antoni A. Kosinski, Professor Emeritus of Mathematics at Rutgers University, offers an accessible approach to both the h-cobordism theorem and the classification of differential structures on spheres.""How useful it is,"" noted the Bulletin of the American Mathematical Society, ""to have a single, sho

  5. Skeletal myoblast based delivery of angiogenic growth factors:a comparison between angiopoietin-1 and VEGF gene delivery for therapeutic angiogenesis in the heart

    Institute of Scientific and Technical Information of China (English)

    Lei Ye; Husnain Kh Haider; Shujia Jiang; Rusan Tan; In-Chin Song; Ruowen Ge; Peter K Law; Eugene KW Sim

    2006-01-01

    Objectives This study investigated the efficacy of human skeletal myoblasts (SkM) mediated either human vascular endothelial growth factor-165 (hVEGF165) or angiopoietin-1 (Ang-1) on vascular development and myocardial regional perfusion. Methods A porcine heart model of chronic infarction was created in 28 female swine by coronary artery ligation. The animals were randomized into:(1) group-1, DMEM injected (n=6), (2) group-2, Ad-null transduced SkM transplanted (n=6), (3) group-3, Ad-hVEGF165 transduced SkM transplanted (n=8), and (4) group-4, Ad-Ang-1 transduced SkM (n=8). Three weeks later, 5 ml DMEM containing 3× 108 SkM carrying exogenous genes were intramyocardially injected into 20 sites in left ventricle in groups-2, -3 and -4. Animals in group-1 were injected 5 ml DMEM without cells. Animals were kept on 5 mg/kg cyclosporine per day for 6 weeks. Regional blood flow was measured using fluorescent microspheres. The heart was explanted at 2, 6 and 12 weeks after transplantation for histological studies. Results Histological examination showed survival of lac-z expressing myoblasts in host tissue. Capillary density based on Von Willebrand factor-Ⅷ (vWF-Ⅷ) at low power field (× 100) was 57.13+11.85 in group-3 at 6 weeks and declined to 32.1±5.21 at 12 weeks, while it was 39.9±10.26 at 6 weeks and increased to 45.14±6.54 at 12 weeks in group-4. The mature blood vessel index was highest in group4 at 6 and 12 weeks after transplantation. The regional blood flow in the center and peri-infarct area was significantly increased in animals of groups-3 and -4. Conclusions SkM carrying either hVEGF165 or Ang- 1 induced neovascularization with increased blood flow. Ang- 1 overexpression resulted in mature and stable blood vessel formation and may be a more potent arteriogenic inducer for neovascularization.(J Geriatr Cardiol 2006;3:152-60.)

  6. Differential meadows

    NARCIS (Netherlands)

    J.A. Bergstra; A. Ponse

    2008-01-01

    A meadow is a zero totalised field (0^{-1}=0), and a cancellation meadow is a meadow without proper zero divisors. In this paper we consider differential meadows, i.e., meadows equipped with differentiation operators. We give an equational axiomatization of these operators and thus obtain a finite b

  7. New insights into the trophic and cytoprotective effects of creatine in in vitro and in vivo models of cell maturation.

    Science.gov (United States)

    Sestili, Piero; Ambrogini, Patrizia; Barbieri, Elena; Sartini, Stefano; Fimognari, Carmela; Calcabrini, Cinzia; Diaz, Anna Rita; Guescini, Michele; Polidori, Emanuela; Luchetti, Francesca; Canonico, Barbara; Lattanzi, Davide; Cuppini, Riccardo; Papa, Stefano; Stocchi, Vilberto

    2016-08-01

    A growing body of scientific reports indicates that the role of creatine (Cr) in cellular biochemistry and physiology goes beyond its contribution to cell energy. Indeed Cr has been shown to exert multiple effects promoting a wide range of physiological responses in vitro as well as in vivo. Included in these, Cr promotes in vitro neuron and muscle cell differentiation, viability and survival under normal or adverse conditions; anabolic, protective and pro-differentiative effects have also been observed in vivo. For example Cr has been shown to accelerate in vitro differentiation of cultured C2C12 myoblasts into myotubes, where it also induces a slight but significant hypertrophic effect as compared to unsupplemented cultures; Cr also prevents the anti-differentiation effects caused by oxidative stress in the same cells. In trained adults, Cr increases the mRNA expression of relevant myogemic factors, protein synthesis, muscle strength and size, in cooperation with physical exercise. As to neurons and central nervous system, Cr favors the electrophysiological maturation of chick neuroblasts in vitro and protects them from oxidative stress-caused killing; similarly, Cr promotes the survival and differentiation of GABA-ergic neurons in fetal spinal cord cultures in vitro; in vivo, maternal Cr supplementation promotes the morpho-functional development of hippocampal neurons in rat offsprings. This article, which presents also some new experimental data, focuses on the trophic, pro-survival and pro-differentiation effects of Cr and examines the ensuing preventive and therapeutic potential in pathological muscle and brain conditions.

  8. Differential equations

    CERN Document Server

    Barbu, Viorel

    2016-01-01

    This textbook is a comprehensive treatment of ordinary differential equations, concisely presenting basic and essential results in a rigorous manner. Including various examples from physics, mechanics, natural sciences, engineering and automatic theory, Differential Equations is a bridge between the abstract theory of differential equations and applied systems theory. Particular attention is given to the existence and uniqueness of the Cauchy problem, linear differential systems, stability theory and applications to first-order partial differential equations. Upper undergraduate students and researchers in applied mathematics and systems theory with a background in advanced calculus will find this book particularly useful. Supplementary topics are covered in an appendix enabling the book to be completely self-contained.

  9. Comparative expression profiling identifies differential roles for Myogenin and p38α MAPK signaling in myogenesis

    Institute of Scientific and Technical Information of China (English)

    Qi-Cai Liu; Marjorie Brand; Carol Perez-Iratxeta; F. Jeffrey Dilworth; Xiao-Hui Zha; Hervé Faralli; Hang Yin; Caroline Louis-Jeune; Eusebio Perdiguero; Erinija Pranckeviciene; Pura Mu(n)oz-Cànoves; Michael A. Rudnicki

    2012-01-01

    Skeletal muscle differentiation is mediated by a complex gene expression program requiring both the muscle-specific transcription factor Myogenin (Myog) and p38α MAPK (p38α) signaling.However,the relative contribution of Myog and p38α to the formation of mature myotubes remains unknown.Here,we have uncoupled the activity of Myog from that of p38α to gain insight into the individual roles of these proteins in myoganesis.Comparative expression profiling confirmed that Myog activates the expression of genes involved in muscle function.Furthermore,we found that in the absence of p38α signaling,Myog expression leads to the down-regulation of genes involved in cell cycle progression.Consistent with this,the expression of Myog is sufficient to induce cell cycle exit.Interestingly,p38α-defective,Myog-expressing myoblasts fail to form multinucleated myotubes,suggesting an important role for p38α in cell fusion.Through the analysis of p38α up-regulated genes,the tetraspanin CD53 was identified as a candidate fusion protein,a role confirmed both ex vivo in primary myoblasts,and in vivo during myofiber regeneration in mice.Thus,our study has revealed an unexpected role for Myog in mediating cell cycle exit and has identified an essential role for p38α in cell fusion through the up-regulation of CD53.

  10. Fine-Tuning of the Actin Cytoskeleton and Cell Adhesion During Drosophila Development by the Unconventional Guanine Nucleotide Exchange Factors Myoblast City and Sponge.

    Science.gov (United States)

    Biersmith, Bridget; Wang, Zong-Heng; Geisbrecht, Erika R

    2015-06-01

    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.

  11. Norepinephrine-induced apoptotic and hypertrophic responses in H9c2 cardiac myoblasts are characterized by different repertoire of reactive oxygen species generation

    Directory of Open Access Journals (Sweden)

    Anita Thakur

    2015-08-01

    Full Text Available 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.

  12. Differential games

    CERN Document Server

    Friedman, Avner

    2006-01-01

    This volume lays the mathematical foundations for the theory of differential games, developing a rigorous mathematical framework with existence theorems. It begins with a precise definition of a differential game and advances to considerations of games of fixed duration, games of pursuit and evasion, the computation of saddle points, games of survival, and games with restricted phase coordinates. Final chapters cover selected topics (including capturability and games with delayed information) and N-person games.Geared toward graduate students, Differential Games will be of particular interest

  13. Differential topology

    CERN Document Server

    Mukherjee, Amiya

    2015-01-01

    This book presents a systematic and comprehensive account of the theory of differentiable manifolds and provides the necessary background for the use of fundamental differential topology tools. The text includes, in particular, the earlier works of Stephen Smale, for which he was awarded the Fields Medal. Explicitly, the topics covered are Thom transversality, Morse theory, theory of handle presentation, h-cobordism theorem, and the generalised Poincaré conjecture. The material is the outcome of lectures and seminars on various aspects of differentiable manifolds and differential topology given over the years at the Indian Statistical Institute in Calcutta, and at other universities throughout India. The book will appeal to graduate students and researchers interested in these topics. An elementary knowledge of linear algebra, general topology, multivariate calculus, analysis, and algebraic topology is recommended.

  14. Distinct amino acid-sensing mTOR pathways regulate skeletal myogenesis.

    Science.gov (United States)

    Yoon, Mee-Sup; Chen, Jie

    2013-12-01

    Signaling through the mammalian target of rapamycin (mTOR) in response to amino acid availability controls many cellular and developmental processes. mTOR is a master regulator of myogenic differentiation, but the pathways mediating amino acid signals in this process are not known. Here we examine the Rag GTPases and the class III phosphoinositide 3-kinase (PI3K) Vps34, two mediators of amino acid signals upstream of mTOR complex 1 (mTORC1) in cell growth regulation, for their potential involvement in myogenesis. We find that, although both Rag and Vps34 mediate amino acid activation of mTORC1 in C2C12 myoblasts, they have opposing functions in myogenic differentiation. Knockdown of RagA/B enhances, whereas overexpression of active RagB/C mutants impairs, differentiation, and this inhibitory function of Rag is mediated by mTORC1 suppression of the IRS1-PI3K-Akt pathway. On the other hand, Vps34 is required for myogenic differentiation. Amino acids activate a Vps34-phospholipase D1 (PLD1) pathway that controls the production of insulin-like growth factor II, an autocrine inducer of differentiation, through the Igf2 muscle enhancer. The product of PLD, phosphatidic acid, activates the enhancer in a rapamycin-sensitive but mTOR kinase-independent manner. Our results uncover amino acid-sensing mechanisms controlling the homeostasis of myogenesis and underline the versatility and context dependence of mTOR signaling.

  15. Differential Krull dimension in differential polynomial extensions

    OpenAIRE

    Smirnov, Ilya

    2011-01-01

    We investigate the differential Krull dimension of differential polynomials over a differential ring. We prove a differential analogue of Jaffard's Special Chain Theorem and show that differential polynomial extensions of certain classes of differential rings have no anomaly of differential Krull dimension.

  16. Alpha B-crystallin induction in skeletal muscle cells under redox imbalance is mediated by a JNK-dependent regulatory mechanism.

    Science.gov (United States)

    Fittipaldi, Simona; Mercatelli, Neri; Dimauro, Ivan; Jackson, Malcolm J; Paronetto, Maria Paola; Caporossi, Daniela

    2015-09-01

    The small heat shock protein α-B-crystallin (CRYAB) is critically involved in stress-related cellular processes such as differentiation, apoptosis, and redox homeostasis. The up-regulation of CRYAB plays a key role in the cytoprotective and antioxidant response, but the molecular pathway driving its expression in muscle cells during oxidative stress still remains unknown. Here we show that noncytotoxic exposure to sodium meta-arsenite (NaAsO2) inducing redox imbalance is able to increase the CRYAB content of C2C12 myoblasts in a transcription-dependent manner. Our in silico analysis revealed a genomic region upstream of the Cryab promoter containing two putative antioxidant-responsive elements motifs and one AP-1-like binding site. The redox-sensitive transcription factors Nrf2 and the AP-1 component c-Jun were found to be up-regulated in NaAsO2-treated cells, and we demonstrated a specific NaAsO2-mediated increase of c-Jun and Nrf2 binding activity to the genomic region identified, supporting their putative involvement in CRYAB regulation following a shift in redox balance. These changes also correlated with a specific phosphorylation of JNK and p38 MAPK kinases, the well-known molecular mediators of signaling pathways leading to the activation of these transcription factors. Pretreatment of C2C12 cells with the JNK inhibitor SP600125 induced a decrease in c-Jun and Nrf2 content and was able to counteract the NaAsO2-mediated increase in CRYAB expression. Thus these data show a direct role of JNK in CRYAB regulation under redox imbalance and also point to a previously unrecognized link between c-Jun and Nrf2 transcription factors and redox-induced CRYAB expression in muscle cells.

  17. Porous polycaprolactone scaffold for cardiac tissue engineering fabricated by selective laser sintering.

    Science.gov (United States)

    Yeong, W Y; Sudarmadji, N; Yu, H Y; Chua, C K; Leong, K F; Venkatraman, S S; Boey, Y C F; Tan, L P

    2010-06-01

    An advanced manufacturing technique, selective laser sintering (SLS), was utilized to fabricate a porous polycaprolactone (PCL) scaffold designed with an automated algorithm in a parametric library system named the "computer-aided system for tissue scaffolds" (CASTS). Tensile stiffness of the sintered PCL strut was in the range of 0.43+/-0.15MPa when a laser power of 3W and scanning speed of 150 in s(-1) was used. A series of compressive mechanical characterizations was performed on the parametric scaffold design and an empirical formula was presented to predict the compressive stiffness of the scaffold as a function of total porosity. In this work, the porosity of the scaffold was selected to be 85%, with micropores (40-100mum) throughout the scaffold. The compressive stiffness of the scaffold was 345kPa. The feasibility of using the scaffold for cardiac tissue engineering was investigated by culturing C2C12 myoblast cells in vitro for 21days. Fluorescence images showed cells were located throughout the scaffold. High density of cells at 1.2x10(6)cellsml(-1) was recorded after 4days of culture. Fusion and differentiation of C2C12 were observed as early as 6days in vitro and was confirmed with myosin heavy chain immunostaining after 11days of cell culture. A steady population of cells was then maintained throughout 21days of culturing. This work demonstrated the feasibility of tailoring the mechanical property of the scaffold for soft tissue engineering using CASTS and SLS. The macroarchitecture of the scaffold can be modified efficiently to fabricate scaffolds with different macropore sizes or changing the elemental cell design in CASTS. Further process and design optimization could be carried out in the future to fabricate scaffolds that match the tensile strength of native myocardium, which is of the order of tens of kPa.

  18. Statin-Induced Myopathy Is Associated with Mitochondrial Complex III Inhibition.

    Science.gov (United States)

    Schirris, Tom J J; Renkema, G Herma; Ritschel, Tina; Voermans, Nicol C; Bilos, Albert; van Engelen, Baziel G M; Brandt, Ulrich; Koopman, Werner J H; Beyrath, Julien D; Rodenburg, Richard J; Willems, Peter H G M; Smeitink, Jan A M; Russel, Frans G M

    2015-09-01

    Cholesterol-lowering statins effectively reduce the risk of major cardiovascular events. Myopathy is the most important adverse effect, but its underlying mechanism remains enigmatic. In C2C12 myoblasts, several statin lactones reduced respiratory capacity and appeared to be strong inhibitors of mitochondrial complex III (CIII) activity, up to 84% inhibition. The lactones were in general three times more potent inducers of cytotoxicity than their corresponding acid forms. The Qo binding site of CIII was identified as off-target of the statin lactones. These findings could be confirmed in muscle tissue of patients suffering from statin-induced myopathies, in which CIII enzyme activity was reduced by 18%. Respiratory inhibition in C2C12 myoblasts could be attenuated by convergent electron flow into CIII, restoring respiration up to 89% of control. In conclusion, CIII inhibition was identified as a potential off-target mechanism associated with statin-induced myopathies.

  19. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp; Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

  20. The Adhesion Modulating Properties of Tenascin-W

    Directory of Open Access Journals (Sweden)

    Florence Brellier, Enrico Martina, Matthias Chiquet, Jacqueline Ferralli, Michael van der Heyden, Gertraud Orend, Johannes C. Schittny, Ruth Chiquet-Ehrismann, Richard P. Tucker

    2012-01-01

    Full Text Available Tenascins are extracellular matrix glycoproteins associated with cell motility, proliferation and differentiation. Tenascin-C inhibits cell spreading by binding to fibronectin; tenascin-R and tenascin-X also have anti-adhesive properties in vitro. Here we have studied the adhesion modulating properties of the most recently characterized tenascin, tenascin-W. C2C12 cells, a murine myoblast cell line, will form broad lamellipodia with stress fibers and focal adhesion complexes after culture on fibronectin. In contrast, C2C12 cells cultured on tenascin-W fail to spread and form stress fibers or focal adhesion complexes, and instead acquire a multipolar shape with short, actin-tipped pseudopodia. The same stellate morphology is observed when C2C12 cells are cultured on a mixture of fibronectin and tenascin-W, or on fibronectin in the presence of soluble tenascin-W. Tenascin-W combined with fibronectin also inhibits the spreading of mouse embryo fibroblasts when compared with cells cultured on fibronectin alone. The similarity between the adhesion modulating effects of tenascin-W and tenascin-C in vitro led us to study the possibility of tenascin-W compensating for tenascin-C in tenascin-C knockout mice, especially during epidermal wound healing. Dermal fibroblasts harvested from a tenascin-C knockout mouse express tenascin-W, but dermal fibroblasts taken from a wild type mouse do not. However, there is no upregulation of tenascin-W in the dermis of tenascin-C knockout mice, or in the granulation tissue of skin wounds in tenascin-C knockout animals. Similarly, tenascin-X is not upregulated in early wound granulation tissue in the tenascin-C knockout mice. Thus, tenascin-W is able to inhibit cell spreading in vitro and it is upregulated in dermal fibroblasts taken from the tenascin-C knockout mouse, but neither it nor tenascin-X are likely to compensate for missing tenascin-C during wound healing.

  1. Differential geometry

    CERN Document Server

    Graustein, William C

    2006-01-01

    This first course in differential geometry presents the fundamentals of the metric differential geometry of curves and surfaces in a Euclidean space of three dimensions. Written by an outstanding teacher and mathematician, it explains the material in the most effective way, using vector notation and technique. It also provides an introduction to the study of Riemannian geometry.Suitable for advanced undergraduates and graduate students, the text presupposes a knowledge of calculus. The first nine chapters focus on the theory, treating the basic properties of curves and surfaces, the mapping of

  2. Constraint Differentiation

    DEFF Research Database (Denmark)

    Mödersheim, Sebastian Alexander; Basin, David; Viganò, Luca

    2010-01-01

    , under the assumption that the original constraint-based approach has these properties. Practically, as a concrete case study, we have integrated this technique into OFMC, a state-of-the-art model-checker for security protocol analysis, and demonstrated its effectiveness by extensive experimentation. Our......We introduce constraint differentiation, a powerful technique for reducing search when model-checking security protocols using constraint-based methods. Constraint differentiation works by eliminating certain kinds of redundancies that arise in the search space when using constraints to represent...

  3. Differential geometry

    CERN Document Server

    Guggenheimer, Heinrich W

    1977-01-01

    This is a text of local differential geometry considered as an application of advanced calculus and linear algebra. The discussion is designed for advanced undergraduate or beginning graduate study, and presumes of readers only a fair knowledge of matrix algebra and of advanced calculus of functions of several real variables. The author, who is a Professor of Mathematics at the Polytechnic Institute of New York, begins with a discussion of plane geometry and then treats the local theory of Lie groups and transformation groups, solid differential geometry, and Riemannian geometry, leading to a

  4. Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1

    Directory of Open Access Journals (Sweden)

    Qiushi Chen

    2015-10-01

    Full Text Available 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.

  5. Cytotoxicity of an ebulin l-anti-human CD105 immunotoxin on mouse fibroblasts (L929) and rat myoblasts (L6E9) cells expressing human CD105.

    Science.gov (United States)

    Benítez, Jorge; Ferreras, J Miguel; Muñoz, Raquel; Arias, Yolanda; Iglesias, Rosario; Córdoba-Díaz, Manuel; del Villar, Rosario; Girbés, Tomás

    2005-01-01

    Tumour growth is characterised by the formation of a fine vessel network or neovasculature which nourishes tumour cells. Two kinds of novel anti-angiogenic therapies are based on the prevention of vessels growth and on the destruction of those vessels already formed. We report here on the design and construction of a novel immunotoxin formed with the non-toxic type II ribosome-inactivating protein ebulin l and the mouse anti-human CD105 monoclonal antibody 44G4. The 44G4-ebulin immunotoxin was formed by covalent linking of both proteins with N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and was purified by chromatography on Superdex 200 HiLoad. The analysis of the anti-ribosomal effects in a cell-free translation system indicated that conjugation does not affect the activity of ebulin l. The immunotoxin displays cytotoxicity with nanomolar IC50 values on human CD105+ cells like the mouse fibroblasts L929 cells transfected with the short form of human CD105 and the rat myoblasts L6E9 transfected with the long form of human CD105. In contrast, cells lacking human CD105 were 2-2.5 logs less sensitive to the immunotoxin. Free ebulin displays IC50 values in the range 10(-6) M. Since CD105 is being considered as a potential target for the anti-vascular therapy of tumours, the present immunotoxin could be a promising tool for the anticancer therapy, especially due to the very low in vivo toxicity of ebulin l as compared ricin and other toxins used for immunotoxins.

  6. Transfection of rat myoblasts with leuflvirus carrying autocrine motility factor gene%携带自分泌运动因子基因的慢病毒载体转染大鼠成肌细胞

    Institute of Scientific and Technical Information of China (English)

    李任; 金岚; 田怡; 牙祖蒙

    2009-01-01

    目的 探索高效、安全的自分泌运动因子(autocrine motility factor,AMF)基因转染方法 ,为携带AMF基因的成肌细胞移植提供实验依据. 方法 取SD大鼠胸肌,用组织块培养法原代培养成肌细胞,纯化、鉴定、扩增成肌细胞;构建携带AMF及增强型绿色荧光蛋白(enhancedgreen fluorescent protein,EGFP)基因的猫免疫缺陷病毒(feline immuneddieiency vires,FIV)慢病毒载体;后者转染至成肌细胞;用荧光显微镜、激光共聚焦显微镜检测EGFP以确定转染的阳性率;应用免疫组化方法 检测AMF的表达. 结果 经过2周的原代培养及纯化,可获得纯度为98%的成肌细胞,在转染复数(multiplieity ofinfection,MOI)为100时,可获得90.4%(P<0.01)的转染阳性率,而转染后的AMF基因能正常表达. 结论 组织块培养法适合成肌细胞的原代培养;FIV载体能以高转染率将AMF基因转至大鼠成肌细胞,并获得高效的表达.该方法 为一种较理想的AMF基因转染模式.%Objective To explore a safe and high efficiency way of gene transfection of autocrine motility factor(AMF) in order to provide experimental basis for transplantation of myoblasts carrying AMF gone. Methods Sprague Dawley rat myoblasts were cultured, purified, proliferated and immunohisto-chemically verified. Then, the myoblasts were transfected w