MicroRNA-27a promotes myoblast proliferation by targeting myostatin

Energy Technology Data Exchange (ETDEWEB)

Huang, Zhiqing; Chen, Xiaoling; Yu, Bing; He, Jun [Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Yaan, Sichuan 625014 (China); Chen, Daiwen, E-mail: dwchen@sicau.edu.cn [Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Yaan, Sichuan 625014 (China)

2012-06-29

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

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

International Nuclear Information System (INIS)

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

2013-01-01

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 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

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.

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

Science.gov (United States)

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

2012-01-01

Sarcopenia is defined as an age-related decrease in skeletal muscle mass and function while adjacent satellite cells are unable to compensate for this loss. However, myoblast cultures can be established even in the presence of sarcopenia. It is yet unknown whether satellite cells from failing muscle in older age are equally affected, as human satellite cells have been assessed using myoblast mixed cultures and not by using myoblast clonal cultures. We questioned to what extent myoblast mixed cultures reflect the in vivo characteristics of single satellite cells from adult skeletal muscle. We established a myoblast mixed culture and three myoblast clonal cultures out of the same muscle biopsy and cultured these cells for 100 days. Replicative capacity and oxidative stress resistance were compared. We found marked heterogeneity between the myoblast clonal cultures that all had a significantly lower replicative capacity when compared to the mixed culture. Replicative capacity of the clonal cultures was inversely related to the β-galactosidase activity after exposure to oxidative stress. Addition of L-carnosine enhanced the remaining replicative capacity in all cultures with a concomitant marginal decrease in β-galactosidase activity. It is concluded that myoblast mixed cultures in vitro do not reflect the marked heterogeneity between single isolated satellite cells. The consequences of the heterogeneity on muscle performance remain to be established. Copyright © 2011 S. Karger AG, Basel.

NEU3 sialidase strictly modulates GM3 levels in skeletal myoblasts C2C12 thus favoring their differentiation and protecting them from apoptosis.

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Anastasia, Luigi; Papini, Nadia; Colazzo, Francesca; Palazzolo, Giacomo; Tringali, Cristina; Dileo, Loredana; Piccoli, Marco; Conforti, Erika; Sitzia, Clementina; Monti, Eugenio; Sampaolesi, Maurilio; Tettamanti, Guido; Venerando, Bruno

2008-12-26

Membrane-bound sialidase NEU3, often referred to as the "ganglioside sialidase," has a critical regulatory function on the sialoglycosphingolipid pattern of the cell membrane, with an anti-apoptotic function, especially in cancer cells. Although other sialidases have been shown to be involved in skeletal muscle differentiation, the role of NEU3 had yet to be disclosed. Herein we report that NEU3 plays a key role in skeletal muscle differentiation by strictly modulating the ganglioside content of adjacent cells, with special regard to GM3. Induced down-regulation of NEU3 in murine C2C12 myoblasts, even when partial, totally inhibits their capability to differentiate by increasing the GM3 level above a critical point, which causes epidermal growth factor receptor inhibition (and ultimately its down-regulation) and an higher responsiveness of myoblasts to the apoptotic stimuli.

Isolation and Characterization of Human Myoblast Culture In Vitro for Technologies of Cell and Gene Therapy of Skeletal Muscle Pathologies.

Science.gov (United States)

Tabakov, V Yu; Zinov'eva, O E; Voskresenskaya, O N; Skoblov, M Yu

2018-03-01

We analyzed cultures of 5 independent myoblast lines from human skeletal muscles. It was shown that the content of desmin-positive cells in cultures at early passages exceeds 90%. Typical morphofunctional signs of myogenic differentiation disturbances were identified and their dynamics was studied. Signs of alternative adipogenic and chondrogenic differentiation of cells were revealed. Based on these data, limitations for the use of myoblast cultures of certain passages for biomedical research and cell therapy were evaluated.

TIPS to manipulate myogenesis: retention of myoblast differentiation capacity using microsphere culture

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N Parmar

2015-07-01

Full Text Available Cell therapy is an emerging option for regenerating skeletal muscle. Improved delivery methods for anchorage-dependent myoblasts are likely to improve integration and function of transplanted muscle cells. Highly porous microspheres, produced using thermally induced phase separation (TIPS, have features ideally suited for minimally invasive cell delivery. The purpose of this study was to investigate, for the first time, the use of TIPS microspheres as highly porous microcarriers for manipulation of human skeletal muscle myoblasts (HSMM under defined culture conditions. HSMM cells readily attached to the surface of poly (DL-lactide-co-glycolide (PLGA TIPS microcarriers, where they were induced to continue proliferating or to be driven towards differentiation whilst under static-dynamic culture conditions for 7 days. Switching from proliferation medium to differentiation medium for 7 days, resulted in increased protein expression of skeletal muscle cell contractile apparatus components, MyoD and skeletal muscle myosin heavy chain, compared with cells cultured on conventional culture plasticware for the same duration (p < 0.001. Growth of myoblasts on the surface of the microcarriers and their migration following simulated delivery, caused no change to the proliferative capacity of cells over 7 days. Results from this study demonstrate that TIPS microspheres provide an ideal vehicle for the expansion and delivery of myoblasts for therapeutic applications. Transplantation of myoblasts anchored to a substrate, rather than in suspension, will reduce the amount of ex vivo manipulation required during preparation of the product and allows cells to be delivered in a more natural state. This will improve the ability to control cell dosage and increase the likelihood of efficacy.

Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

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Gogos, J A; Thompson, R; Lowry, W; Sloane, B F; Weintraub, H; Horwitz, M

1996-08-01

To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors, containing a promoterless marker gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning. The vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.

Effect of ionizing radiation on human skeletal muscle precursor cells

International Nuclear Information System (INIS)

Jurdana, Mihaela; Cemazar, Maja; Pegan, Katarina; Mars, Tomaz

2013-01-01

Long term effects of different doses of ionizing radiation on human skeletal muscle myoblast proliferation, cytokine signalling and stress response capacity were studied in primary cell cultures. Human skeletal muscle myoblasts obtained from muscle biopsies were cultured and irradiated with a Darpac 2000 X-ray unit at doses of 4, 6 and 8 Gy. Acute effects of radiation were studied by interleukin – 6 (IL-6) release and stress response detected by the heat shock protein (HSP) level, while long term effects were followed by proliferation capacity and cell death. Compared with non-irradiated control and cells treated with inhibitor of cell proliferation Ara C, myoblast proliferation decreased 72 h post-irradiation, this effect was more pronounced with increasing doses. Post-irradiation myoblast survival determined by measurement of released LDH enzyme activity revealed increased activity after exposure to irradiation. The acute response of myoblasts to lower doses of irradiation (4 and 6 Gy) was decreased secretion of constitutive IL-6. Higher doses of irradiation triggered a stress response in myoblasts, determined by increased levels of stress markers (HSPs 27 and 70). Our results show that myoblasts are sensitive to irradiation in terms of their proliferation capacity and capacity to secret IL-6. Since myoblast proliferation and differentiation are a key stage in muscle regeneration, this effect of irradiation needs to be taken in account, particularly in certain clinical conditions

Perturbations of NAD+ salvage systems impact mitochondrial function and energy homeostasis in mouse myoblasts and intact skeletal muscle

DEFF Research Database (Denmark)

Andersen, Marianne Agerholm; Dall, Morten; Jensen, Benjamin Anderschou Holbech

2018-01-01

Nicotinamide adenine dinucleotide (NAD+) can be synthesized by nicotinamide phosphoribosyltransferase (NAMPT). We aimed to determine the role of NAMPT for maintaining NAD+ levels, mitochondrial function, and metabolic homeostasis in skeletal muscle cells. We generated stable Nampt knockdown (sh......Nampt KD) C2C12 cells using a shRNA lentiviral approach. Moreover, we applied gene electrotransfer to express cre recombinase in tibialis anterior muscle of floxed Nampt mice. In shNampt KD C2C12 myoblasts, Nampt and NAD+ levels were reduced by 70% and 50%, respectively, and maximal respiratory capacity...... was reduced by 25%. Moreover, anaerobic glycolytic flux increased by 55% and 2-deoxyglucose uptake increased by 25% in shNampt KD cells. Treatment with the NAD+ precursor nicotinamide riboside restored NAD+ levels in shNampt cells and increased maximal respiratory capacity by 18% and 32% in control and sh...

Short-hairpin Mediated Myostatin Knockdown Resulted in Altered Expression of Myogenic Regulatory Factors with Enhanced Myoblast Proliferation in Fetal Myoblast Cells of Goats.

Science.gov (United States)

Kumar, Rohit; Singh, Satyendra Pal; Mitra, Abhijit

2018-01-02

Myostatin (MSTN) is a well-known negative regulator of skeletal muscle development. Reduced expression due to natural mutations in the coding region and knockout as well as knockdown of MSTN results in an increase in the muscle mass. In the present study, we demonstrated as high as 60 and 52% downregulation (p < 0.01) of MSTN mRNA and protein in the primary fetal myoblast cells of goats using synthetic shRNAs (n = 3), without any interferon response. We, for the first time, evaluated the effect of MSTN knockdown on the expression of MRFs (namely, MyoD, Myf5), follistatin (FST), and IGFs (IGF-1 & IGF-2) in goat myoblast cells. MSTN knockdown caused an upregulation (p < 0.05) of MyoD and downregulation (p < 0.01) of MYf5 and FST expression. Moreover, we report up to ∼four fold (p < 0.001) enhanced proliferation in myoblasts after four days of culture. The anti-MSTN shRNA demonstrated in the present study could be used for the production of transgenic goats to increase the muscle mass.

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

NARCIS (Netherlands)

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

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2014-09-26

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

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

International Nuclear Information System (INIS)

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

2014-01-01

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

Sphingosine kinase/sphingosine 1-phosphate axis: a new player for insulin-like growth factor-1-induced myoblast differentiation

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Bernacchioni Caterina

2012-07-01

Full Text Available Abstract Background Insulin-like growth factor-1 (IGF-1 is the most important physiological regulator of skeletal muscle progenitor cells, which are responsible for adult skeletal muscle regeneration. The ability of IGF-1 to affect multiple aspects of skeletal muscle cell biology such as proliferation, differentiation, survival and motility is well recognized, although the molecular mechanisms implicated in its complex biological action are not fully defined. Since sphingosine 1-phosphate (S1P has recently emerged as a key player in skeletal muscle regeneration, we investigated the possible involvement of the sphingosine kinase (SK/S1P receptor axis on the biological effects of IGF-1 in murine myoblasts. Methods RNA interference, chemical inhibition and immunofluorescence approaches were used to assess the role of the SK/S1P axis on the myogenic and mitogenic effects of IGF-1 in C2C12 myoblasts. Results We show that IGF-1 increases SK activity in mouse myoblasts. The effect of the growth factor does not involve transcriptional regulation of SK1 or SK2, since the protein content of both isoforms is not affected; rather, IGF-1 enhances the fraction of the active form of SK. Moreover, transactivation of the S1P2 receptor induced by IGF-1 via SK activation appears to be involved in the myogenic effect of the growth factor. Indeed, the pro-differentiating effect of IGF-1 in myoblasts is impaired when SK activity is pharmacologically inhibited, or SK1 or SK2 are specifically silenced, or the S1P2 receptor is downregulated. Furthermore, in this study we show that IGF-1 transactivates S1P1/S1P3 receptors via SK activation and that this molecular event negatively regulates the mitogenic effect elicited by the growth factor, since the specific silencing of S1P1 or S1P3 receptors increases cell proliferation induced by IGF-1. Conclusions We demonstrate a dual role of the SK/S1P axis in response to myoblast challenge with IGF-1, that likely is important to

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

International Nuclear Information System (INIS)

Shafey, Dina; Cote, Patrice D.; Kothary, Rashmi

2005-01-01

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

Selenium regulates gene expression of selenoprotein W in chicken skeletal muscle system.

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Ruan, Hongfeng; Zhang, Ziwei; Wu, Qiong; Yao, Haidong; Li, Jinlong; Li, Shu; Xu, Shiwen

2012-01-01

Selenoprotein W (SelW) is abundantly expressed in skeletal muscles of mammals and necessary for the metabolism of skeletal muscles. However, its expression pattern in skeletal muscle system of birds is still uncovered. Herein, to investigate the distribution of SelW mRNA in chicken skeletal muscle system and its response to different selenium (Se) status, 1-day-old chickens were exposed to various concentrations of Se as sodium selenite in the feed for 35 days. In addition, myoblasts were treated with different concentrations of Se in the medium for 72 h. Then the levels of SelW mRNA in skeletal muscles (wing muscle, pectoral muscle, thigh muscle) and myoblasts were determined on days 1, 15, 25, and 35 and at 0, 24, 48, and 72 h, respectively. The results showed that SelW was detected in all these muscle components and it increased both along with the growth of organism and the differentiation process of myoblasts. The thigh muscle is more responsive to Se intake than the other two skeletal muscle tissues while the optimal Se supplementation for SelW mRNA expression in chicken myoblasts was 10(-7) M. In summary, Se plays important roles in the development of chicken skeletal muscles. To effect optimal SelW gene expression, Se must be provided in the diet and the media in adequate amounts and neither at excessive nor deficient levels.

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.

The critical role of myostatin in differentiation of sheep myoblasts

International Nuclear Information System (INIS)

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

2012-01-01

Highlights: ► Identification of the effective and specific shRNA to knockdown MSTN. ► Overexpression of MSTN reversibly suppressed myogenic differentiation. ► shRNA knockdown of endogenous MSTN promoted ovine myoblast differentiation. ► MSTN inhibits myogenic differentiation through down-regulation of MyoD and Myogenin and up-regulation of Smad3. ► 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.

Group I Paks Promote Skeletal Myoblast Differentiation In Vivo and In Vitro

DEFF Research Database (Denmark)

Joseph, Giselle A; Lu, Min; Radu, Maria

2017-01-01

fusion in Drosophila We report that both Pak1 and Pak2 are activated during mammalian myoblast differentiation. One pathway of activation is initiated by N-cadherin ligation and involves the cadherin coreceptor Cdo with its downstream effector, Cdc42. Individual genetic deletion of Pak1 and Pak2 in mice....... Furthermore, primary myoblasts lacking Pak1 and Pak2 display delayed expression of myogenic differentiation markers and myotube formation. These results identify Pak1 and Pak2 as redundant regulators of myoblast differentiation in vitro and in vivo and as components of the promyogenic Ncad/Cdo/Cdc42 signaling...

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

Combined use of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and platelet rich plasma (PRP) stimulates proliferation and differentiation of myoblasts in vitro: new therapeutic perspectives for skeletal muscle repair/regeneration.

Science.gov (United States)

Sassoli, Chiara; Vallone, Larissa; Tani, Alessia; Chellini, Flaminia; Nosi, Daniele; Zecchi-Orlandini, Sandra

2018-02-05

Satellite cell-mediated skeletal muscle repair/regeneration is compromised in cases of extended damage. Bone marrow mesenchymal stromal cells (BM-MSCs) hold promise for muscle healing but some criticisms hamper their clinical application, including the need to avoid animal serum contamination for expansion and the scarce survival after transplant. In this context, platelet-rich plasma (PRP) could offer advantages. Here, we compare the effects of PRP or standard culture media on C2C12 myoblast, satellite cell and BM-MSC viability, survival, proliferation and myogenic differentiation and evaluate PRP/BM-MSC combination effects in promoting myogenic differentiation. PRP induced an increase of mitochondrial activity and Ki67 expression comparable or even greater than that elicited by standard media and promoted AKT signaling activation in myoblasts and BM-MSCs and Notch-1 pathway activation in BM-MSCs. It stimulated MyoD, myogenin, α-sarcomeric actin and MMP-2 expression in myoblasts and satellite cell activation. Notably, PRP/BM-MSC combination was more effective than PRP alone. We found that BM-MSCs influenced myoblast responses through a paracrine activation of AKT signaling, contributing to shed light on BM-MSC action mechanisms. Our results suggest that PRP represents a good serum substitute for BM-MSC manipulation in vitro and could be beneficial towards transplanted cells in vivo. Moreover, it might influence muscle resident progenitors' fate, thus favoring the endogenous repair/regeneration mechanisms. Finally, within the limitations of an in vitro experimentation, this study provides an experimental background for considering the PRP/BM-MSC combination as a potential therapeutic tool for skeletal muscle damage, combining the beneficial effects of BM-MSCs and PRP on muscle tissue, while potentiating BM-MSC functionality.

Skeletal muscle tissue engineering: methods to form skeletal myotubes and their applications.

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

2014-10-01

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

Heterogeneity among muscle precursor cells in adult skeletal muscles with differing regenerative capacities.

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Pavlath, G K; Thaloor, D; Rando, T A; Cheong, M; English, A W; Zheng, B

1998-08-01

Skeletal muscle has a remarkable capacity to regenerate after injury, although studies of muscle regeneration have heretofore been limited almost exclusively to limb musculature. Muscle precursor cells in skeletal muscle are responsible for the repair of damaged muscle. Heterogeneity exists in the growth and differentiation properties of muscle precursor cell (myoblast) populations throughout limb development but whether the muscle precursor cells differ among adult skeletal muscles is unknown. Such heterogeneity among myoblasts in the adult may give rise to skeletal muscles with different regenerative capacities. Here we compare the regenerative response of a masticatory muscle, the masseter, to that of limb muscles. After exogenous trauma (freeze or crush injuries), masseter muscle regenerated much less effectively than limb muscle. In limb muscle, normal architecture was restored 12 days after injury, whereas in masseter muscle, minimal regeneration occurred during the same time period. Indeed, at late time points, masseter muscles exhibited increased fibrous connective tissue in the region of damage, evidence of ineffective muscle regeneration. Similarly, in response to endogenous muscle injury due to a muscular dystrophy, widespread evidence of impaired regeneration was present in masseter muscle but not in limb muscle. To explore the cellular basis of these different regenerative capacities, we analyzed the myoblast populations of limb and masseter muscles both in vivo and in vitro. From in vivo analyses, the number of myoblasts in regenerating muscle was less in masseter compared with limb muscle. Assessment of population growth in vitro indicated that masseter myoblasts grow more slowly than limb myoblasts under identical conditions. We conclude that the impaired regeneration in masseter muscles is due to differences in the intrinsic myoblast populations compared to limb muscles.

MURC, a muscle-restricted coiled-coil protein, is involved in the regulation of skeletal myogenesis.

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Tagawa, Masashi; Ueyama, Tomomi; Ogata, Takehiro; Takehara, Naofumi; Nakajima, Norio; Isodono, Koji; Asada, Satoshi; Takahashi, Tomosaburo; Matsubara, Hiroaki; Oh, Hidemasa

2008-08-01

Skeletal myogenesis is a multistep process by which multinucleated mature muscle fibers are formed from undifferentiated, mononucleated myoblasts. However, the molecular mechanisms of skeletal myogenesis have not been fully elucidated. Here, we identified muscle-restricted coiled-coil (MURC) protein as a positive regulator of myogenesis. In skeletal muscle, MURC was localized to the cytoplasm with accumulation in the Z-disc of the sarcomere. In C2C12 myoblasts, MURC expression occurred coincidentally with myogenin expression and preceded sarcomeric myosin expression during differentiation into myotubes. RNA interference (RNAi)-mediated knockdown of MURC impaired differentiation in C2C12 myoblasts, which was accompanied by impaired myogenin expression and ERK activation. Overexpression of MURC in C2C12 myoblasts resulted in the promotion of differentiation with enhanced myogenin expression and ERK activation during differentiation. During injury-induced muscle regeneration, MURC expression increased, and a higher abundance of MURC was observed in immature myofibers compared with mature myofibers. In addition, ERK was activated in regenerating tissue, and ERK activation was detected in MURC-expressing immature myofibers. These findings suggest that MURC is involved in the skeletal myogenesis that results from modulation of myogenin expression and ERK activation. MURC may play pivotal roles in the molecular mechanisms of skeletal myogenic differentiation.

Dynamics of the Skeletal Muscle Secretome during Myoblast Differentiation

DEFF Research Database (Denmark)

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

2010-01-01

During recent years, increased efforts have focused on elucidating the secretory function of skeletal muscle. Through secreted molecules, skeletal muscle affects local muscle biology in an auto/paracrine manner as well as having systemic effects on other tissues. Here we used a quantitative...... proteomics platform to investigate the factors secreted during the differentiation of murine C2C12 skeletal muscle cells. Using triple encoding stable isotope labeling by amino acids in cell culture, we compared the secretomes at three different time points of muscle differentiation and followed the dynamics...... of the skeletal muscle as a prominent secretory organ. In addition to previously reported molecules, we identified many secreted proteins that have not previously been shown to be released from skeletal muscle cells nor shown to be differentially released during the process of myogenesis. We found 188...

β‐Taxilin participates in differentiation of C2C12 myoblasts into myotubes

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

2016-07-15

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

Macrophages improve survival, proliferation and migration of engrafted myogenic precursor cells into MDX skeletal muscle.

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Pierre-François Lesault

Full Text Available Transplantation of muscle precursor cells is of therapeutic interest for focal skeletal muscular diseases. However, major limitations of cell transplantation are the poor survival, expansion and migration of the injected cells. The massive and early death of transplanted myoblasts is not fully understood although several mechanisms have been suggested. Various attempts have been made to improve their survival or migration. Taking into account that muscle regeneration is associated with the presence of macrophages, which are helpful in repairing the muscle by both cleansing the debris and deliver trophic cues to myoblasts in a sequential way, we attempted in the present work to improve myoblast transplantation by coinjecting macrophages. The present data showed that in the 5 days following the transplantation, macrophages efficiently improved: i myoblast survival by limiting their massive death, ii myoblast expansion within the tissue and iii myoblast migration in the dystrophic muscle. This was confirmed by in vitro analyses showing that macrophages stimulated myoblast adhesion and migration. As a result, myoblast contribution to regenerating host myofibres was increased by macrophages one month after transplantation. Altogether, these data demonstrate that macrophages are beneficial during the early steps of myoblast transplantation into skeletal muscle, showing that coinjecting these stromal cells may be used as a helper to improve the efficiency of parenchymal cell engraftment.

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

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

2016-01-01

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

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

International Nuclear Information System (INIS)

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

2015-01-01

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

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

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

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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

Function of skeletal muscle tissue formed after myoblast transplantation into irradiated mouse muscles.

Science.gov (United States)

Wernig, A; Zweyer, M; Irintchev, A

2000-01-15

1. Pretreatment of muscles with ionising radiation enhances tissue formation by transplanted myoblasts but little is known about the effects on muscle function. We implanted myoblasts from an expanded, male-donor-derived, culture (i28) into X-ray irradiated (16 Gy) or irradiated and damaged soleus muscles of female syngeneic mice (Balb/c). Three to 6 months later the isometric contractile properties of the muscles were studied in vitro, and donor nuclei were visualised in muscle sections with a Y chromosome-specific DNA probe. 2. Irradiated sham-injected muscles had smaller masses than untreated solei and produced less twitch and tetanic force (all by about 18 %). Injection of 106 myoblasts abolished these deficiencies and innervation appeared normal. 3. Cryodamage of irradiated solei produced muscle remnants with few (1-50) or no fibres. Additional myoblast implantation led to formation of large muscles (25 % above normal) containing numerous small-diameter fibres. Upon direct electrical stimulation, these muscles produced considerable twitch (53 % of normal) and tetanic forces (35 % of normal) but innervation was insufficient as indicated by weak nerve-evoked contractions and elevated ACh sensitivity. 4. In control experiments on irradiated muscles, reinnervation was found to be less complete after botulinum toxin paralysis than after nerve crush indicating that proliferative arrest of irradiated Schwann cells may account for the observed innervation deficits. 5. Irradiation appears to be an effective pretreatment for improving myoblast transplantation. The injected cells can even produce organised contractile tissue replacing whole muscle. However, impaired nerve regeneration limits the functional performance of the new muscle.

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

Science.gov (United States)

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

2017-03-01

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

Expression of androgen receptor target genes in skeletal muscle

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Kesha Rana

2014-10-01

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

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

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Wen Luo

2015-11-01

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

Growth of Limb Muscle is Dependent on Skeletal-Derived Indian Hedgehog

Science.gov (United States)

Bren-Mattison, Yvette; Hausburg, Melissa; Olwin, Bradley B.

2011-01-01

During embryogenesis, muscle and bone develop in close temporal and spatial proximity. We show that Indian Hedgehog, a bone-derived signaling molecule, participates in growth of skeletal muscle. In Ihh−/− embryos, skeletal muscle development appears abnormal at embryonic day 14.5 and at later ages through embryonic day 20.5, dramatic losses of hindlimb muscle occur. To further examine the role of Ihh in myogenesis, we manipulated Ihh expression in the developing chick hindlimb. Reduction of Ihh in chicken embryo hindlimbs reduced skeletal muscle mass similar to that seen in Ihh−/− mouse embryos. The reduction in muscle mass appears to be a direct effect of Ihh since ectopic expression of Ihh by RCAS retroviral infection of chicken embryo hindlimbs restores muscle mass. These effects are independent of bone length, and occur when Shh is not expressed, suggesting Ihh acts directly on fetal myoblasts to regulate secondary myogenesis. Loss of muscle mass in Ihh null mouse embryos is accompanied by a dramatic increase in myoblast apoptosis accompanied by a loss of p21 protein. Our data suggest that Ihh promotes fetal myoblast survival during their differentiation into secondary myofibers by maintaining p21 protein levels. PMID:21683695

Spatial and functional restriction of regulatory molecules during mammalian myoblast fusion

International Nuclear Information System (INIS)

Pavlath, Grace K.

2010-01-01

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.

Identification of Histone Deacetylase 2 as a Functional Gene for Skeletal Muscle Development in Chickens

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Md. Shahjahan

2016-04-01

Full Text Available A previous genome-wide association study (GWAS exposed histone deacetylase 2 (HDAC2 as a possible candidate gene for breast muscle weight in chickens. The present research has examined the possible role of HDAC2 in skeletal muscle development in chickens. Gene expression was measured by quantitative polymerase chain reaction in breast and thigh muscles during both embryonic (four ages and post-hatch (five ages development and in cultures of primary myoblasts during both proliferation and differentiation. The expression of HDAC2 increased significantly across embryonic days (ED in breast (ED 14, 16, 18, and 21 and thigh (ED 14 and 18, and ED 14 and 21 muscles suggesting that it possibly plays a role in myoblast hyperplasia in both breast and thigh muscles. Transcript abundance of HDAC2 identified significantly higher in fast growing muscle than slow growing in chickens at d 90 of age. Expression of HDAC2 during myoblast proliferation in vitro declined between 24 h and 48 h when expression of the marker gene paired box 7 (PAX7 increased and cell numbers increased throughout 72 h of culture. During induced differentiation of myoblasts to myotubes, the abundance of HDAC2 and the marker gene myogenic differentiation 1 (MYOD1, both increased significantly. Taken together, it is suggested that HDAC2 is most likely involved in a suppressive fashion in myoblast proliferation and may play a positive role in myoblast differentiation. The present results confirm the suggestion that HDAC2 is a functional gene for pre-hatch and post-hatch (fast growing muscle development of chicken skeletal muscle.

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

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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

mTOR is necessary for proper satellite cell activity and skeletal muscle regeneration

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Zhang, Pengpeng [Key Laboratory of Swine Genetics and Breeding of Agricultural Ministry & Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Liang, Xinrong; Shan, Tizhong [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Jiang, Qinyang [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); College of Animal Science and Technology, Guangxi University, Nanning 530004 (China); Deng, Changyan [Key Laboratory of Swine Genetics and Breeding of Agricultural Ministry & Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Zheng, Rong, E-mail: zhengrong@mail.hzau.edu.cn [Key Laboratory of Swine Genetics and Breeding of Agricultural Ministry & Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Kuang, Shihuan, E-mail: skuang@purdue.edu [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States)

2015-07-17

The serine/threonine kinase mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive deletion of Mtor gene results in embryonic lethality, the function of mTOR in muscle stem cells (satellite cells) and skeletal muscle regeneration remains to be determined. In this study, we established a satellite cell specific Mtor conditional knockout (cKO) mouse model by crossing Pax7{sup CreER} and Mtor{sup flox/flox} mice. Skeletal muscle regeneration after injury was severely compromised in the absence of Mtor, indicated by increased number of necrotic myofibers infiltrated by Evans blue dye, and reduced number and size of regenerated myofibers in the Mtor cKO mice compared to wild type (WT) littermates. To dissect the cellular mechanism, we analyzed satellite cell-derived primary myoblasts grown on single myofibers or adhered to culture plates. The Mtor cKO myoblasts exhibited defective proliferation and differentiation kinetics when compared to myoblasts derived from WT littermates. At the mRNA and protein levels, the Mtor cKO myoblasts expressed lower levels of key myogenic determinant genes Pax7, Myf5, Myod, Myog than did the WT myoblasts. These results suggest that mTOR is essential for satellite cell function and skeletal muscle regeneration through controlling the expression of myogenic genes. - Highlights: • Pax7{sup CreER} was used to delete Mtor gene in satellite cells. • Satellite cell specific deletion of Mtor impairs muscle regeneration. • mTOR is necessary for satellite cell proliferation and differentiation. • Deletion of Mtor leads to reduced expression of key myogenic genes.

mTOR is necessary for proper satellite cell activity and skeletal muscle regeneration

International Nuclear Information System (INIS)

Zhang, Pengpeng; Liang, Xinrong; Shan, Tizhong; Jiang, Qinyang; Deng, Changyan; Zheng, Rong; Kuang, Shihuan

2015-01-01

The serine/threonine kinase mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive deletion of Mtor gene results in embryonic lethality, the function of mTOR in muscle stem cells (satellite cells) and skeletal muscle regeneration remains to be determined. In this study, we established a satellite cell specific Mtor conditional knockout (cKO) mouse model by crossing Pax7 CreER and Mtor flox/flox mice. Skeletal muscle regeneration after injury was severely compromised in the absence of Mtor, indicated by increased number of necrotic myofibers infiltrated by Evans blue dye, and reduced number and size of regenerated myofibers in the Mtor cKO mice compared to wild type (WT) littermates. To dissect the cellular mechanism, we analyzed satellite cell-derived primary myoblasts grown on single myofibers or adhered to culture plates. The Mtor cKO myoblasts exhibited defective proliferation and differentiation kinetics when compared to myoblasts derived from WT littermates. At the mRNA and protein levels, the Mtor cKO myoblasts expressed lower levels of key myogenic determinant genes Pax7, Myf5, Myod, Myog than did the WT myoblasts. These results suggest that mTOR is essential for satellite cell function and skeletal muscle regeneration through controlling the expression of myogenic genes. - Highlights: • Pax7 CreER was used to delete Mtor gene in satellite cells. • Satellite cell specific deletion of Mtor impairs muscle regeneration. • mTOR is necessary for satellite cell proliferation and differentiation. • Deletion of Mtor leads to reduced expression of key myogenic genes

Let-7b Regulates Myoblast Proliferation by Inhibiting IGF2BP3 Expression in Dwarf and Normal Chicken

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Lin, Shumao; Luo, Wen; Ye, Yaqiong; Bekele, Endashaw J.; Nie, Qinghua; Li, Yugu; Zhang, Xiquan

2017-01-01

The sex-linked dwarf chicken is caused by the mutation of growth hormone receptor (GHR) gene and characterized by shorter shanks, lower body weight, smaller muscle fiber diameter and fewer muscle fiber number. However, the precise regulatory pathways that lead to the inhibition of skeletal muscle growth in dwarf chickens still remain unclear. Here we found a let-7b mediated pathway might play important role in the regulation of dwarf chicken skeletal muscle growth. Let-7b has higher expression in the skeletal muscle of dwarf chicken than in normal chicken, and the expression of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which is a translational activator of IGF2, showed opposite expression trend to let-7b. In vitro cellular assays validated that let-7b directly inhibits IGF2BP3 expression through binding to its 3′UTR region, and the protein level but not mRNA level of IGF2 would be reduced in let-7b overexpressed chicken myoblast. Let-7b can inhibit cell proliferation and induce cell cycle arrest in chicken myoblast through let-7b-IGF2BP3-IGF2 signaling pathway. Additionally, let-7b can also regulate skeletal muscle growth through let-7b-GHR-GHR downstream genes pathway, but this pathway is non-existent in dwarf chicken because of the deletion mutation of GHR 3′UTR. Notably, as the loss binding site of GHR for let-7b, let-7b has enhanced its binding and inhibition on IGF2BP3 in dwarf myoblast, suggesting that the miRNA can balance its inhibiting effect through dynamic regulate its binding to target genes. Collectively, these results not only indicate that let-7b can inhibit skeletal muscle growth through let-7b-IGF2BP3-IGF2 signaling pathway, but also show that let-7b regulates myoblast proliferation by inhibiting IGF2BP3 expression in dwarf and normal chickens. PMID:28736533

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

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

2016-02-01

Improving both systolic and diastolic function may be the most important factor in treating heart failure. In this study, we hypothesized that cell-sheet transplantation could improve these function in the damaged heart. We generated a dilated cardiomyopathy model in beagles by continuous ventricle pacing at 240 beats per minute. After 4 weeks, the beagles underwent skeletal myoblast cell sheet transplantation (SMCST) or a sham operation, and rapid ventricle pacing continued for an additional 4 weeks. Six of the e8 beagles treated by SMCST were still alive 4 weeks after the procedure. We evaluated SMCST's cardiotherapeutic effects by comparing beagles treated by SMCST with beagles that underwent a sham operation (control, n = 5). Diastolic function, as well as systolic function improved significantly in the SMCST group as compared with the sham group (control vs SMCST group, median [interquartile range]: E/E', 16 [0.9] vs 11 [1.0]; P dilated cardiomyopathy heart.

Impact of Hepatocyte Growth Factor on Skeletal Myoblast Transplantation Late after Myocardial Infarction

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Stacy B. O'blenes

2013-01-01

Full Text Available In clinical studies, skeletal myoblast (SKMB transplantation late after myocardial infarction (MI has minimal impact on left ventricular (LV function. This may be related to our previous observation that the extent of SKMB engraftment is minimal in chronic MI when compared to acute MI, which correlates with decreased hepatocyte growth factor (HGF expression, an important regulator of SKMB function. Here, we investigated delivery of exogenous HGF as a strategy for augmenting SKMB engraftment late after MI. Rats underwent SKMB transplantation 4 weeks after coronary ligation. HGF or vehicle control was delivered intravenously during the subsequent 2 weeks. LV function was assessed by MRI before and 2 weeks after SKMB transplantation. We evaluated HGF delivery, SKMB engraftment, and expression of genes associated with post-MI remodeling. Serum HGF was 6.2 ± 2.4 ng/mL after 2 weeks of HGF infusion (n = 7, but undetectable in controls (n = 7. LV end-diastolic volume and ejection fraction did not improve with HGF treatment (321 ± 27 mm 3 , 42% ± 2% vs. 285 ± 33 mm 3 , 43% ± 2%, HGF vs. control. MIs were larger in HGF-treated animals (50 ± 7 vs. 30 ± 6 mm 3 , P = 0.046, but the volume of engrafted SKMBs or percentage of MIs occupied by SKMBs did not increase with HGF (1.7 ± 0.3 mm 3 , 4.7% ± 1.9% vs. 1.4 ± 0.4 mm 3 , 5.3% ± 1.6%, HGF vs. control. Expression of genes associated with post-infarction remodeling was not altered by HGF. Delivery of exogenous HGF failed to augment SKMB engraftment and functional recovery in chronic MI. Expression of genes associated with LV remodeling was not altered by HGF. Alternative strategies to enhance engraftment of SKMB must be explored to optimize the clinical efficacy of SKMB transplantation.

A Simplified Method for Tissue Engineering Skeletal Muscle Organoids in Vitro

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Shansky, Janet; DelTatto, Michael; Chromiak, Joseph; Vandenburgh, Herman

1996-01-01

Tissue-engineered three dimensional skeletal muscle organ-like structures have been formed in vitro from primary myoblasts by several different techniques. This report describes a simplified method for generating large numbers of muscle organoids from either primary embryonic avian or neonatal rodent myoblasts, which avoids the requirements for stretching and other mechanical stimulation.

Liposome-based vascular endothelial growth factor-165 transfection with skeletal myoblast for treatment of ischaemic limb disease.

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Ye, Lei; Haider, Husnain Kh; Esa, Wahidah Bte; Su, Liping; Law, Peter K; Zhang, Wei; Lim, Yeanteng; Poh, Kian Keong; Sim, Eugene K W

2010-01-01

The study aims to use cholesterol (Chol) + DOTAP liposome (CD liposome) based human vascular endothelial growth factor-165 (VEGF(165)) gene transfer into skeletal myoblasts (SkMs) for treatment of acute hind limb ischaemia in a rabbit model. The feasibility and efficacy of CD liposome mediated gene transfer with rabbit SkMs were characterized using plasmid carrying enhanced green fluorescent protein (pEGFP) and assessed by flow cytometry. After optimization, SkMs were transfected with CD lipoplexes carrying plasmid-VEGF(165) (CD-pVEGF(165)) and transplanted into rabbit ischaemic limb. Animals were randomized to receive intramuscular injection of Medium199 (M199; group 1), non-transfected SkM (group 2) or CD-pVEGF(165) transfected SkM (group 3). Flow cytometry revealed that up to 16% rabbit SkMs were successfully transfected with pEGFP. Based on the optimized transfection condition, transfected rabbit SkM expressed VEGF(165) up to day 18 with peak at day 2. SkMs were observed in all cell-transplanted groups, as visualized with 6-diamidino-2-phenylindole and bromodeoxyuridine. Angiographic blood vessel score revealed increased collateral vessel development in group 3 (39.7 +/- 2.0) compared with group 2 (21.6 +/- 1.1%, P limb and may serve as a safe and new therapeutic modality for the repair of acute ischaemic limb disease.

Effects of heat stimulation and l-ascorbic acid 2-phosphate supplementation on myogenic differentiation of artificial skeletal muscle tissue constructs.

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Ikeda, Kazushi; Ito, Akira; Sato, Masanori; Kanno, Shota; Kawabe, Yoshinori; Kamihira, Masamichi

2017-05-01

Although skeletal muscle tissue engineering has been extensively studied, the physical forces produced by tissue-engineered skeletal muscles remain to be improved for potential clinical utility. In this study, we examined the effects of mild heat stimulation and supplementation of a l-ascorbic acid derivative, l-ascorbic acid 2-phosphate (AscP), on myoblast differentiation and physical force generation of tissue-engineered skeletal muscles. Compared with control cultures at 37°C, mouse C2C12 myoblast cells cultured at 39°C enhanced myotube diameter (skeletal muscle hypertrophy), whereas mild heat stimulation did not promote myotube formation (differentiation rate). Conversely, AscP supplementation resulted in an increased differentiation rate but did not induce skeletal muscle hypertrophy. Following combined treatment with mild heat stimulation and AscP supplementation, both skeletal muscle hypertrophy and differentiation rate were enhanced. Moreover, the active tension produced by the tissue-engineered skeletal muscles was improved following combined treatment. These findings indicate that tissue culture using mild heat stimulation and AscP supplementation is a promising approach to enhance the function of tissue-engineered skeletal muscles. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Shikonin increases glucose uptake in skeletal muscle cells and improves plasma glucose levels in diabetic Goto-Kakizaki rats.

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Anette I Öberg

Full Text Available BACKGROUND: There is considerable interest in identifying compounds that can improve glucose homeostasis. Skeletal muscle, due to its large mass, is the principal organ for glucose disposal in the body and we have investigated here if shikonin, a naphthoquinone derived from the Chinese plant Lithospermum erythrorhizon, increases glucose uptake in skeletal muscle cells. METHODOLOGY/PRINCIPAL FINDINGS: Shikonin increases glucose uptake in L6 skeletal muscle myotubes, but does not phosphorylate Akt, indicating that in skeletal muscle cells its effect is medaited via a pathway distinct from that used for insulin-stimulated uptake. Furthermore we find no evidence for the involvement of AMP-activated protein kinase in shikonin induced glucose uptake. Shikonin increases the intracellular levels of calcium in these cells and this increase is necessary for shikonin-mediated glucose uptake. Furthermore, we found that shikonin stimulated the translocation of GLUT4 from intracellular vesicles to the cell surface in L6 myoblasts. The beneficial effect of shikonin on glucose uptake was investigated in vivo by measuring plasma glucose levels and insulin sensitivity in spontaneously diabetic Goto-Kakizaki rats. Treatment with shikonin (10 mg/kg intraperitoneally once daily for 4 days significantly decreased plasma glucose levels. In an insulin sensitivity test (s.c. injection of 0.5 U/kg insulin, plasma glucose levels were significantly lower in the shikonin-treated rats. In conclusion, shikonin increases glucose uptake in muscle cells via an insulin-independent pathway dependent on calcium. CONCLUSIONS/SIGNIFICANCE: Shikonin increases glucose uptake in skeletal muscle cells via an insulin-independent pathway dependent on calcium. The beneficial effects of shikonin on glucose metabolism, both in vitro and in vivo, show that the compound possesses properties that make it of considerable interest for developing novel treatment of type 2 diabetes.

Comparison of arrhythmogenicity and proinflammatory activity induced by intramyocardial or epicardial myoblast sheet delivery in a rat model of ischemic heart failure.

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Tommi Pätilä

Full Text Available Although cell therapy of the failing heart by intramyocardial injections of myoblasts to results in regenerative benefit, it has also been associated with undesired and prospectively fatal arrhythmias. We hypothesized that intramyocardial injections of myoblasts could enhance inflammatory reactivity and facilitate electrical cardiac abnormalities that can be reduced by epicardial myoblast sheet delivery. In a rat model of ischemic heart failure, myoblast therapy either by intramyocardial injections or epicardial cell sheets was given 2 weeks after occlusion of the coronary artery. Ventricular premature contractions (VPCs were assessed, using an implanted three-lead electrocardiograph at 1, 7, and 14 days after therapy, and 16-point epicardial electropotential mapping (EEPM was used to evaluate ventricular arrhythmogenicity under isoproterenol stress. Cardiac functioning was assessed by echocardiography. Both transplantation groups showed therapeutic benefit over sham therapy. However, VPCs were more frequent in the Injection group on day 1 and day 14 after therapy than in animals receiving epicardial or sham therapy (p < 0.05 and p < 0.01, respectively. EEPM under isoproterenol stress showed macroreentry at the infarct border area, leading to ventricular tachycardias in the Injection group, but not in the myoblast sheet- or sham-treated groups (p = 0.045. Both transplantation types modified the myocardial cytokine expression profile. In animals receiving epicardial myoblast therapy, selective reductions in the expressions of interferon gamma, interleukin (IL-1β and IL12 were observed, accompanied by reduced infiltration of inflammatory CD11b- and CD68-positive leukocytes, compared with animals receiving myoblasts as intramyocardial injections. Intramyocardial myoblast delivery was associated with enhanced inflammatory and immunomodulatory reactivity and increased frequency of VPCs. In comparison to intramyocardial injection, the epicardial

Insulin acutely improves mitochondrial function of rat and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation.

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Nisr, Raid B; Affourtit, Charles

2014-02-01

Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells. © 2013.

Insulin acutely improves mitochondrial function of rat and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation☆

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Nisr, Raid B.; Affourtit, Charles

2014-01-01

Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells. PMID:24212054

Lipopolysaccharide inhibits myogenic differentiation of C2C12 myoblasts through the Toll-like receptor 4-nuclear factor-κB signaling pathway and myoblast-derived tumor necrosis factor-α.

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Yuko Ono

Full Text Available Circulating lipopolysaccharide (LPS concentrations are often elevated in patients with sepsis or with various endogenous diseases that are associated with metabolic endotoxemia. Involuntary loss of skeletal muscle, termed muscle wasting, is commonly observed in these conditions, suggesting that circulating LPS might play an essential role in its development. Although impairment of muscle regeneration is an important determinant of skeletal muscle wasting, it is unclear whether LPS affects this process and, if so, by what mechanism. Here, we used the C2C12 myoblast cell line to investigate the effects of LPS on myogenesis.C2C12 myoblasts were grown to 80% confluence and induced to differentiate in the absence or presence of LPS (0.1 or 1 μg/mL; TAK-242 (1 μM, a specific inhibitor of Toll-like receptor 4 (TLR4 signaling; and a tumor necrosis factor (TNF-α neutralizing antibody (5 μg/mL. Expression of a skeletal muscle differentiation marker (myosin heavy chain II, two essential myogenic regulatory factors (myogenin and MyoD, and a muscle negative regulatory factor (myostatin was analyzed by western blotting. Nuclear factor-κB (NF-κB DNA-binding activity was measured using an enzyme-linked immunosorbent assay.LPS dose-dependently and significantly decreased the formation of multinucleated myotubes and the expression of myosin heavy chain II, myogenin, and MyoD, and increased NF-κB DNA-binding activity and myostatin expression. The inhibitory effect of LPS on myogenic differentiation was reversible, suggesting that it was not caused by nonspecific toxicity. Both TAK-242 and anti-TNF-α reduced the LPS-induced increase in NF-κB DNA-binding activity, downregulation of myogenic regulatory factors, and upregulation of myostatin, thereby partially rescuing the impairment of myogenesis.Our data suggest that LPS inhibits myogenic differentiation via a TLR4-NF-κB-dependent pathway and an autocrine/paracrine TNF-α-induced pathway. These pathways

Enhancement of contractile force generation of artificial skeletal muscle tissues by mild and transient heat treatment.

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Sato, Masanori; Ikeda, Kazushi; Kanno, Shota; Ito, Akira; Kawabe, Yoshinori; Kamihira, Masamichi

2014-01-01

Artificial skeletal muscle tissues composed of cells are expected to be used for applications of regenerative medicine and drug screening. Generally, however, the physical forces generated by tissue-engineered skeletal muscle are lower than those of skeletal muscle tissues found in the body. Local hyperthermia is used for many diseases including muscle injuries. It was recently reported that mild heat treatment improved skeletal muscle functions. In this study, we investigated the effects of mild heat treatment on the tissue-engineered skeletal muscle tissues in vitro. We used magnetite cationic liposomes to label C2C12 myoblast cells magnetically, and constructed densely packed artificial skeletal muscle tissues by using magnetic force. Cell culture at 39°C promoted the differentiation of myoblast cells into myotubes. Moreover, the mild and transient heat treatment improved the contractile properties of artificial skeletal muscle tissue constructs. These findings indicate that the culture method using heat treatment is a useful approach to enhance functions of artificial skeletal muscle tissue.

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

DEFF Research Database (Denmark)

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

2011-01-01

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

Enhanced glucose metabolism in cultured human skeletal muscle after Roux-en-Y gastric bypass surgery.

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Nascimento, Emmani B M; Riedl, Isabelle; Jiang, Lake Qunfeng; Kulkarni, Sameer S; Näslund, Erik; Krook, Anna

2015-01-01

Roux-en-Y gastric bypass (RYGB) surgery rapidly increases whole body insulin sensitivity, with changes in several organs including skeletal muscle. Objectives were to determine whether improvements in insulin action in skeletal muscle may occur directly at the level of the myocyte or secondarily from changes in systemic factors associated with weight loss. Myotubes were derived before and after RYGB surgery. The setting was Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden. Eight patients (body mass index (BMI) 41.8 kg/m(2); age 41 yr) underwent RYGB surgery. Before and 6 months after RYGB surgery, skeletal muscle biopsies were collected from vastus lateralis muscle. Satellite cells derived from skeletal muscle biopsies were propagated in vitro as myoblasts and differentiated into myotubes. Expression of myogenic markers is increased in myoblasts derived from biopsies taken 6 months after bypass surgery, compared with their respective presurgery condition. Furthermore, glycogen synthesis, tyrosine phosphorylation of insulin receptor (IRS)-1-Tyr612 and Interleukin (IL)-8 secretion were increased, while fatty acid oxidation and circulating IL8 levels remain unaltered. Myotubes derived from muscle biopsies obtained after RYGB surgery displayed increased insulin-stimulated phosphorylation of protein kinase B (PKB)-Thr308 and proline-rich Akt substrate of 40 kDa (PRAS40)-Thr246. RYGB surgery is accompanied by enhanced glucose metabolism and insulin signaling, altered IL8 secretion and changes in mRNA levels and myogenic markers in cultured skeletal muscle cells. Thus, RYGB surgery involves intrinsic reprogramming of skeletal muscle to increase peripheral insulin sensitivity and glucose metabolism. Copyright © 2015 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.

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

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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.

Possible role of TIEG1 as a feedback regulator of myostatin and TGF-β in myoblasts

International Nuclear Information System (INIS)

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

2010-01-01

Myostatin and TGF-β negatively regulate skeletal muscle development and growth. Both factors signal through the Smad2/3 pathway. However, the regulatory mechanism of myostatin and TGF-β signaling remains unclear. TGF-β inducible early gene (TIEG) 1 is highly expressed in skeletal muscle and has been implicated in the modulation of TGF-β signaling. These findings prompted us to investigate the effect of TIEG1 on myostatin and TGF-β signaling using C2C12 myoblasts. Myostatin and TGF-β 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-β enhanced TIEG1 expression at an early stage. Overexpression of TIEG1 prevented the transcriptional activation of Smad by myostatin and TGF-β 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-β. These results demonstrate that TIEG1 is one possible feedback regulator of myostatin and TGF-β that prevents excess action in myoblasts.

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

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

2017-01-01

Scaffolds are materials used for delivery of cells for regeneration of tissues. They support three-dimensional organization and improve cell survival. For the repair of small skeletal muscles, injections of small volumes of cells are attractive, and injectable scaffolds for delivery of cells offer...... a minimally invasive technique. In this study, we examined in vitro the cell instructive effects of three types of injectable scaffolds, fibrin, alginate, and poly(lactic-co-glycolic acid)-based microparticles on primary human myoblasts. The myoblast morphology and progression in the myogenic program differed......, depending on the type of scaffold material. In alginate gel, the cells obtained a round morphology, they ceased to proliferate, and entered quiescence. In the fibrin gels, differentiation was promoted, and myotubes were observed within a few days in culture, while poly(lactic-co-glycolic acid...

Peripheral endocannabinoids regulate skeletal muscle development and maintenance

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Dongjiao Zhao

2010-12-01

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

Uniaxial cyclic strain enhances adipose-derived stem cell fusion with skeletal myocytes

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Andersen, Jens Isak; Juhl, Morten; Nielsen, Thøger; Emmersen, Jeppe; Fink, Trine; Zachar, Vladimir; Pennisi, Cristian Pablo, E-mail: cpennisi@hst.aau.dk

2014-07-25

Highlights: • Uniaxial cyclic tensile strain (CTS) applied to ASCs alone or in coculture with myogenic precursors. • CTS promoted the formation of a highly ordered array of parallel ASCs. • Without biochemical supplements, CTS did not support advanced myogenic differentiation of ASCs. • Mechanical stimulation of cocultures boosted fusion of ASCs with skeletal myoblasts. - Abstract: Although adult muscle tissue possesses an exceptional capacity for regeneration, in the case of large defects, the restoration to original state is not possible. A well-known source for the de novo regeneration is the adipose-derived stem cells (ASCs), which can be readily isolated and have been shown to have a broad differentiation and regenerative potential. In this work, we employed uniaxial cyclic tensile strain (CTS), to mechanically stimulate human ASCs to participate in the formation skeletal myotubes in an in vitro model of myogenesis. The application of CTS for 48 h resulted in the formation of a highly ordered array of parallel ASCs, but failed to support skeletal muscle terminal differentiation. When the same stimulation paradigm was applied to cocultures with mouse skeletal muscle myoblasts, the percentage of ASCs contributing to the formation of myotubes significantly exceeded the levels reported in the literature hitherto. In perspective, the mechanical strain may be used to increase the efficiency of incorporation of ASCs in the skeletal muscles, which could be found useful in diverse traumatic or pathologic scenarios.

Human skeletal muscle drug transporters determine local exposure and toxicity of statins.

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Knauer, Michael J; Urquhart, Bradley L; Meyer zu Schwabedissen, Henriette E; Schwarz, Ute I; Lemke, Christopher J; Leake, Brenda F; Kim, Richard B; Tirona, Rommel G

2010-02-05

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, are important drugs used in the treatment and prevention of cardiovascular disease. Although statins are well tolerated, many patients develop myopathy manifesting as muscle aches and pain. Rhabdomyolysis is a rare but severe toxicity of statins. Interindividual differences in the activities of hepatic membrane drug transporters and metabolic enzymes are known to influence statin plasma pharmacokinetics and risk for myopathy. Interestingly, little is known regarding the molecular determinants of statin distribution into skeletal muscle and its relevance to toxicity. We sought to identify statin transporters in human skeletal muscle and determine their impact on statin toxicity in vitro. We demonstrate that the uptake transporter OATP2B1 (human organic anion transporting polypeptide 2B1) and the efflux transporters, multidrug resistance-associated protein (MRP)1, MRP4, and MRP5 are expressed on the sarcolemmal membrane of human skeletal muscle fibers and that atorvastatin and rosuvastatin are substrates of these transporters when assessed using a heterologous expression system. In an in vitro model of differentiated, primary human skeletal muscle myoblast cells, we demonstrate basal membrane expression and drug efflux activity of MRP1, which contributes to reducing intracellular statin accumulation. Furthermore, we show that expression of human OATP2B1 in human skeletal muscle myoblast cells by adenoviral vectors increases intracellular accumulation and toxicity of statins and such effects were abrogated when cells overexpressed MRP1. These results identify key membrane transporters as modulators of skeletal muscle statin exposure and toxicity.

Skeletal muscle atrophy in bioengineered skeletal muscle: a new model system.

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Lee, Peter H U; Vandenburgh, Herman H

2013-10-01

Skeletal muscle atrophy has been well characterized in various animal models, and while certain pathways that lead to disuse atrophy and its associated functional deficits have been well studied, available drugs to counteract these deficiencies are limited. An ex vivo tissue-engineered skeletal muscle offers a unique opportunity to study skeletal muscle physiology in a controlled in vitro setting. Primary mouse myoblasts isolated from adult muscle were tissue engineered into bioartificial muscles (BAMs) containing hundreds of aligned postmitotic muscle fibers expressing sarcomeric proteins. When electrically stimulated, BAMs generated measureable active forces within 2-3 days of formation. The maximum isometric tetanic force (Po) increased for ∼3 weeks to 2587±502 μN/BAM and was maintained at this level for greater than 80 days. When BAMs were reduced in length by 25% to 50%, muscle atrophy occurred in as little as 6 days. Length reduction resulted in significant decreases in Po (50.4%), mean myofiber cross-sectional area (21.7%), total protein synthesis rate (22.0%), and noncollagenous protein content (6.9%). No significant changes occurred in either the total metabolic activity or protein degradation rates. This study is the first in vitro demonstration that length reduction alone can induce skeletal muscle atrophy, and establishes a novel in vitro model for the study of skeletal muscle atrophy.

Chiral Orientation of Skeletal Muscle Cells Requires Rigid Substrate

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Ninghao Zhu

2017-06-01

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

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

International Nuclear Information System (INIS)

Sassoli, Chiara; Nosi, Daniele; Tani, Alessia; Chellini, Flaminia; Mazzanti, Benedetta; Quercioli, Franco; Zecchi-Orlandini, Sandra; Formigli, Lucia

2014-01-01

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 + 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

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.

Abelson tyrosine-protein kinase 2 Regulates Myoblast Proliferation and Controls Muscle Fiber Length

OpenAIRE

Burden, Steven; Lee, Jennifer

2017-01-01

Muscle fiber length is nearly uniform within a muscle but widely different among muscles. Here, we show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm and other muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of available myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but expansion of the diaphragm ...

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

DEFF Research Database (Denmark)

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

2013-01-01

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

Activation of estrogen response elements is mediated both via estrogen and muscle contractions in rat skeletal muscle myotubes

DEFF Research Database (Denmark)

Wiik, A.; Hellsten, Ylva; Berthelson, P.

2009-01-01

is ER independent. The muscle contraction-induced transactivation of ERE and increase in ERbeta mRNA were instead found to be MAP kinase (MAPK) dependent. This study demonstrates for the first time that muscle contractions have a similar functional effect as estrogen in skeletal muscle myotubes, causing......The aim of the present study was to investigate the activation of estrogen response elements (EREs) by estrogen and muscle contractions in rat myotubes in culture and to assess whether the activation is dependent on the estrogen receptors (ERs). In addition, the effect of estrogen and contraction...... on the mRNA levels of ERalpha and ERbeta was studied to determine the functional consequence of the transactivation. Myoblasts were isolated from rat skeletal muscle and transfected with a vector consisting of sequences of EREs coupled to the gene for luciferase. The transfected myoblasts were...

Identification of gene expression modifications in myostatin-stimulated myoblasts

International Nuclear Information System (INIS)

Yang Wei; Zhang Yong; Ma Guoda; Zhao Xinyi; Chen Yan; Zhu Dahai

2005-01-01

Myostatin belongs to the transforming growth factor beta superfamily and has been shown to function as an inhibitor of skeletal muscle proliferation and differentiation. To gain insight into the molecular mechanisms of myostatin function during myogenesis, differential display reverse transcription PCR was employed to identify altered gene expressions associated with myostatin inhibitory function in chicken fetal myoblasts (CFMs). In this work, we have identified seven up-regulated and 12 down-regulated genes in myostatin stimulated CFMs. Those genes are involved in myogenic differentiation, cell architecture, energy metabolism, signal transduction, and apoptosis. The down-regulation of muscle creatine kinase B, troponin C, and myosin regulatory light chain is in agreement with the myostatin negative role in myocyte differentiation. In addition, the expression alteration of skeletal muscle-specific cardiac ankyrin repeat protein and the bcl-2 related anti-apoptotic protein Nr-13 suggests possible unique roles for myostatin in regulating myogenesis by controlling cofactors participated transcriptional regulation and apoptosis

Defective myoblasts identified in Duchenne muscular dystrophy.

OpenAIRE

Blau, H M; Webster, C; Pavlath, G K

1983-01-01

A defect in the proliferative capacity of satellite cells, mononucleated precursors of mature muscle fibers, was found in clonal analyses of cells cultured from Duchenne muscular dystrophy (DMD) patients. The total yield of myoblasts per gram of muscle biopsy was decreased to 5% of normal. Of the DMD myoblast clones obtained, a large proportion contained a morphological class of flat distended cells that had an increased generation time and ceased to proliferate beyond 100-1,000 cells but cou...

Cardiac cell therapy: overexpression of connexin43 in skeletal myoblasts and prevention of ventricular arrhythmias

NARCIS (Netherlands)

Fernandes, Sarah; van Rijen, Harold V. M.; Forest, Virginie; Evain, Stéphane; Leblond, Anne-Laure; Mérot, Jean; Charpentier, Flavien; de Bakker, Jacques M. T.; Lemarchand, Patricia

2009-01-01

Cell-based therapies have great potential for the treatment of cardiovascular diseases. Recently, using a transgenic mouse model Roell et al. reported that cardiac engraftment of connexin43 (Cx43)-overexpressing myoblasts in vivo prevents post-infarct arrhythmia, a common cause of death in patients

The role of Na+/K+-ATPase during chick skeletal myogenesis.

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Taissa Neustadt Oliveira

Full Text Available The formation of a vertebrate skeletal muscle fiber involves a series of sequential and interdependent events that occurs during embryogenesis. One of these events is myoblast fusion which has been widely studied, yet not completely understood. It was previously shown that during myoblast fusion there is an increase in the expression of Na+/K+-ATPase. This fact prompted us to search for a role of the enzyme during chick in vitro skeletal myogenesis. Chick myogenic cells were treated with the Na+/K+-ATPase inhibitor ouabain in four different concentrations (0.01-10 μM and analyzed. Our results show that 0.01, 0.1 and 1 μM ouabain did not induce changes in cell viability, whereas 10 μM induced a 45% decrease. We also observed a reduction in the number and thickness of multinucleated myotubes and a decrease in the number of myoblasts after 10 μM ouabain treatment. We tested the involvement of MEK-ERK and p38 signaling pathways in the ouabain-induced effects during myogenesis, since both pathways have been associated with Na+/K+-ATPase. The MEK-ERK inhibitor U0126 alone did not alter cell viability and did not change ouabain effect. The p38 inhibitor SB202190 alone or together with 10 μM ouabain did not alter cell viability. Our results show that the 10 μM ouabain effects in myofiber formation do not involve the MEK-ERK or the p38 signaling pathways, and therefore are probably related to the pump activity function of the Na+/K+-ATPase.

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.

Abelson tyrosine-protein kinase 2 regulates myoblast proliferation and controls muscle fiber length

OpenAIRE

Lee, Jennifer K; Hallock, Peter T; Burden, Steven J

2017-01-01

Muscle fiber length is nearly uniform within a muscle but widely different among different muscles. We show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm, intercostal and levator auris muscles but not limb muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of myoblasts and leading to increased myoblast fusion. Abl2 acts in myobla...

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.

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

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Sindhu Subramaniam

Full Text Available 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

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

IGF-II-mediated downregulation of peroxisome proliferator-activated receptor-γ coactivator-1α in myoblast cells involves PI3K/Akt/FoxO1 signaling pathway.

Science.gov (United States)

Mu, Xiaoyu; Qi, Weihong; Liu, Yunzhang; Zhou, Jianfeng; Li, Yun; Rong, Xiaozhi; Lu, Ling

2017-08-01

Insulin-like growth factor II (IGF-II) can stimulate myogenesis and is critically involved in skeletal muscle differentiation. The presence of negative regulators of this process, however, is not well explored. Here, we showed that in myoblast cells, IGF-II negatively regulated peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mRNA expression, while constitutive expression of PGC-1α induced myoblast differentiation. These results suggest that the negative regulation of PGC-1α by IGF-II may act as a negative feedback mechanism in IGF-II-induced myogenic differentiation. Reporter assays demonstrated that IGF-II suppresses the basal PGC-1α promoter activity. Blocking the IGF-II signaling pathway increased the endogenous PGC-1α levels. In addition, pharmacological inhibition of PI3 kinase activity prevented the downregulation of PGC-1α but the activation of mTOR was not required for this process. Importantly, further analysis showed that forkhead transcription factor FoxO1 contributes to mediating the effects of IGF-II on PGC-1 promoter activity. These findings indicate that IGF-II reduces PGC-1α expression in skeletal muscle cells through a mechanism involving PI3K-Akt-FoxO1 but not p38 MAPK or Erk1/2 MAPK pathways.

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

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Michael Caruso

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

The Recent Understanding of the Neurotrophin's Role in Skeletal Muscle Adaptation

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Kunihiro Sakuma

2011-01-01

Full Text Available This paper summarizes the various effects of neurotrophins in skeletal muscle and how these proteins act as potential regulators of the maintenance, function, and regeneration of skeletal muscle fibers. Increasing evidence suggests that this family of neurotrophic factors influence not only the survival and function of innervating motoneurons but also the development and differentiation of myoblasts and muscle fibers. Muscle contractions (e.g., exercise produce BDNF mRNA and protein in skeletal muscle, and the BDNF seems to play a role in enhancing glucose metabolism and may act for myokine to improve various brain disorders (e.g., Alzheimer's disease and major depression. In adults with neuromuscular disorders, variations in neurotrophin expression are found, and the role of neurotrophins under such conditions is beginning to be elucidated. This paper provides a basis for a better understanding of the role of these factors under such pathological conditions and for treatment of human neuromuscular disease.

Increased skeletal muscle capillarization enhances insulin sensitivity

DEFF Research Database (Denmark)

Åkerström, Thorbjörn; Laub, Lasse; Vedel, Kenneth

2014-01-01

Increased skeletal muscle capillarization is associated with improved glucose tolerance and insulin sensitivity. However, a possible causal relationship has not previously been identified. We therefore investigated whether increased skeletal muscle capillarization increases insulin sensitivity....... Skeletal muscle specific angiogenesis was induced by adding the α1-adrenergic receptor antagonist Prazosin to the drinking water of Sprague Dawley rats (n=33) while 34 rats served as controls. Insulin sensitivity was measured ≥40 h after termination of the 3-week Prazosin treatment, which ensured...... that Prazosin was cleared from the blood stream. Whole-body insulin sensitivity was measured in conscious, unrestrained rats by hyperinsulinemic euglycemic clamp. Tissue specific insulin sensitivity was assessed by administration of 2-deoxy-[(3)H]-Glucose during the plateau phase of the clamp. Whole...

Sirtuin-3 (Sirt3) regulates skeletal muscle metabolism and insulin signaling via altered mitochondrial oxidation and reactive oxygen species production

DEFF Research Database (Denmark)

Jing, Enxuan; Emanuelli, Brice; Hirschey, Matthew D

2011-01-01

Sirt3 is a member of the sirtuin family of protein deacetylases that is localized in mitochondria and regulates mitochondrial function. Sirt3 expression in skeletal muscle is decreased in models of type 1 and type 2 diabetes and regulated by feeding, fasting, and caloric restriction. Sirt3 knockout...... mice exhibit decreased oxygen consumption and develop oxidative stress in skeletal muscle, leading to JNK activation and impaired insulin signaling. This effect is mimicked by knockdown of Sirt3 in cultured myoblasts, which exhibit reduced mitochondrial oxidation, increased reactive oxygen species......, activation of JNK, increased serine and decreased tyrosine phosphorylation of IRS-1, and decreased insulin signaling. Thus, Sirt3 plays an important role in diabetes through regulation of mitochondrial oxidation, reactive oxygen species production, and insulin resistance in skeletal muscle....

Abelson tyrosine-protein kinase 2 regulates myoblast proliferation and controls muscle fiber length

Science.gov (United States)

Lee, Jennifer K; Hallock, Peter T

2017-01-01

Muscle fiber length is nearly uniform within a muscle but widely different among different muscles. We show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm, intercostal and levator auris muscles but not limb muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but as a consequence of expansion of the diaphragm muscle, the diaphragm central tendon is reduced in size, likely contributing to reduced stamina of Abl2 mutant mice. Ectopic muscle islands, each composed of myofibers of uniform length and orientation, form within the central tendon of Abl2+/− mice. Specialized tendon cells, resembling tendon cells at myotendinous junctions, form at the ends of these muscle islands, suggesting that myofibers induce differentiation of tendon cells, which reciprocally regulate myofiber length and orientation. PMID:29231808

Abelson tyrosine-protein kinase 2 regulates myoblast proliferation and controls muscle fiber length.

Science.gov (United States)

Lee, Jennifer K; Hallock, Peter T; Burden, Steven J

2017-12-12

Muscle fiber length is nearly uniform within a muscle but widely different among different muscles. We show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm, intercostal and levator auris muscles but not limb muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but as a consequence of expansion of the diaphragm muscle, the diaphragm central tendon is reduced in size, likely contributing to reduced stamina of Abl2 mutant mice. Ectopic muscle islands, each composed of myofibers of uniform length and orientation, form within the central tendon of Abl2 +/- mice. Specialized tendon cells, resembling tendon cells at myotendinous junctions, form at the ends of these muscle islands, suggesting that myofibers induce differentiation of tendon cells, which reciprocally regulate myofiber length and orientation.

HGF potentiates extracellular matrix-driven migration of human myoblasts: involvement of matrix metalloproteinases and MAPK/ERK pathway.

Science.gov (United States)

González, Mariela Natacha; de Mello, Wallace; Butler-Browne, Gillian S; Silva-Barbosa, Suse Dayse; Mouly, Vincent; Savino, Wilson; Riederer, Ingo

2017-10-10

The hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration. We evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting. We demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin. We demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving

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.

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

International Nuclear Information System (INIS)

Retamales, A.; Zuloaga, R.; Valenzuela, C.A.; Gallardo-Escarate, C.; Molina, A.; Valdés, J.A.

2015-01-01

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

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.

Ionizing radiation increases primary cilia incidence and induces multiciliation in C2C12 myoblasts

Czech Academy of Sciences Publication Activity Database

Filipová, A.; Diaz-Garcia, D.; Bezrouk, A.; Čížková, D.; Havelek, R.; Vávrová, J.; Dayanithi, Govindan; Řezáčová, M.

2015-01-01

Roč. 39, č. 8 (2015), s. 943-953 ISSN 1065-6995 Institutional support: RVO:68378041 Keywords : cell line * ionizing radiation * multiple cilia * myoblast * primary cilium * serum starvation stress Subject RIV: FP - Other Medical Disciplines Impact factor: 1.663, year: 2015

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

International Nuclear Information System (INIS)

Cambier, Linda; Pomies, Pascal

2011-01-01

Highlights: → The cytoskeleton-associated protein, smALP, is expressed in differentiated skeletal muscle. → smALP is translocated from the cytoplasm to the nucleus of C2C12 myoblasts upon induction of myogenesis. → The differentiation-dependent nuclear translocation of smALP occurs in parallel with the nuclear accumulation of myogenin. → The LIM domain of smALP is essential for the nuclear accumulation of the protein. → 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.

Myogenic Precursors from iPS Cells for Skeletal Muscle Cell Replacement Therapy

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Isart Roca

2015-01-01

Full Text Available The use of adult myogenic stem cells as a cell therapy for skeletal muscle regeneration has been attempted for decades, with only moderate success. Myogenic progenitors (MP made from induced pluripotent stem cells (iPSCs are promising candidates for stem cell therapy to regenerate skeletal muscle since they allow allogenic transplantation, can be produced in large quantities, and, as compared to adult myoblasts, present more embryonic-like features and more proliferative capacity in vitro, which indicates a potential for more self-renewal and regenerative capacity in vivo. Different approaches have been described to make myogenic progenitors either by gene overexpression or by directed differentiation through culture conditions, and several myopathies have already been modeled using iPSC-MP. However, even though results in animal models have shown improvement from previous work with isolated adult myoblasts, major challenges regarding host response have to be addressed and clinically relevant transplantation protocols are lacking. Despite these challenges we are closer than we think to bringing iPSC-MP towards clinical use for treating human muscle disease and sporting injuries.

Isolation, culture and biological characteristics of multipotent porcine skeletal muscle satellite cells.

Science.gov (United States)

Yang, Jinjuan; Liu, Hao; Wang, Kunfu; Li, Lu; Yuan, Hongyi; Liu, Xueting; Liu, Yingjie; Guan, Weijun

2017-12-01

Skeletal muscle has a huge regenerative potential for postnatal muscle growth and repair, which mainly depends on a kind of muscle progenitor cell population, called satellite cell. Nowadays, the majority of satellite cells were obtained from human, mouse, rat and other animals but rarely from pig. In this article, the porcine skeletal muscle satellite cells were isolated and cultured in vitro. The expression of surface markers of satellite cells was detected by immunofluorescence and RT-PCR assays. The differentiation capacity was assessed by inducing satellite cells into adipocytes, myoblasts and osteoblasts. The results showed that satellite cells isolated from porcine tibialis anterior were subcultured up to 12 passages and were positive for Pax7, Myod, c-Met, desmin, PCNA and NANOG but were negative for Myogenin. Satellite cells were also induced to differentiate into adipocytes, osteoblasts and myoblasts, respectively. These findings indicated that porcine satellite cells possess similar biological characteristics of stem cells, which may provide theoretical basis and experimental evidence for potential therapeutic application in the treatment of dystrophic muscle and other muscle injuries.

Insulin Increases Ceramide Synthesis in Skeletal Muscle

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M. E. Hansen

2014-01-01

Full Text Available Aims. The purpose of this study was to determine the effect of insulin on ceramide metabolism in skeletal muscle. Methods. Skeletal muscle cells were treated with insulin with or without palmitate for various time periods. Lipids (ceramides and TAG were isolated and gene expression of multiple biosynthetic enzymes were quantified. Additionally, adult male mice received daily insulin injections for 14 days, followed by muscle ceramide analysis. Results. In muscle cells, insulin elicited an increase in ceramides comparable to palmitate alone. This is likely partly due to an insulin-induced increase in expression of multiple enzymes, particularly SPT2, which, when knocked down, prevented the increase in ceramides. In mice, 14 days of insulin injection resulted in increased soleus ceramides, but not TAG. However, insulin injections did significantly increase hepatic TAG compared with vehicle-injected animals. Conclusions. This study suggests that insulin elicits an anabolic effect on sphingolipid metabolism in skeletal muscle, resulting in increased ceramide accumulation. These findings reveal a potential mechanism of the deleterious consequences of the hyperinsulinemia that accompanies insulin resistance and suggest a possible novel therapeutic target to mitigate its effects.

Role of an inward rectifier K+ current and of hyperpolarization in human myoblast fusion

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Liu, J-H; Bijlenga, P; Fischer-Lougheed, J; Occhiodoro, T; Kaelin, A; Bader, C R; Bernheim, L

1998-01-01

The role of K+ channels and membrane potential in myoblast fusion was evaluated by examining resting membrane potential and timing of expression of K+ currents at three stages of differentiation of human myogenic cells: undifferentiated myoblasts, fusion-competent myoblasts (FCMBs), and freshly formed myotubes. Two K+ currents contribute to a hyperpolarization of myoblasts prior to fusion: IK(NI), a non-inactivating delayed rectifier, and IK(IR), an inward rectifier. IK(NI) density is low in undifferentiated myoblasts, increases in FCMBs and declines in myotubes. On the other hand, IK(IR) is expressed in 28 % of the FCMBs and in all myotubes. IK(IR) is reversibly blocked by Ba2+ or Cs+. Cells expressing IK(IR) have resting membrane potentials of −65 mV. A block by Ba2+ or Cs+ induces a depolarization to a voltage determined by IK(NI) (−32 mV). Cs+ and Ba2+ ions reduce myoblast fusion. It is hypothesized that the IK(IR)-mediated hyperpolarization allows FCMBs to recruit Na+, K+ and T-type Ca2+ channels which are present in these cells and would otherwise be inactivated. FCMBs, rendered thereby capable of firing action potentials, could amplify depolarizing signals and may accelerate fusion. PMID:9705997

Intercellular adhesion molecule-1 augments myoblast adhesion and fusion through homophilic trans-interactions.

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Pizza, Francis X; Martin, Ryan A; Springer, Evan M; Leffler, Maxwell S; Woelmer, Bryce R; Recker, Isaac J; Leaman, Douglas W

2017-07-11

The overall objective of the study was to identify mechanisms through which intercellular adhesion molecule-1 (ICAM-1) augments the adhesive and fusogenic properties of myogenic cells. Hypotheses were tested using cultured myoblasts and fibroblasts, which do not constitutively express ICAM-1, and myoblasts and fibroblasts forced to express full length ICAM-1 or a truncated form lacking the cytoplasmic domain of ICAM-1. ICAM-1 mediated myoblast adhesion and fusion were quantified using novel assays and cell mixing experiments. We report that ICAM-1 augments myoblast adhesion to myoblasts and myotubes through homophilic trans-interactions. Such adhesive interactions enhanced levels of active Rac in adherent and fusing myoblasts, as well as triggered lamellipodia, spreading, and fusion of myoblasts through the signaling function of the cytoplasmic domain of ICAM-1. Rac inhibition negated ICAM-1 mediated lamellipodia, spreading, and fusion of myoblasts. The fusogenic property of ICAM-1-ICAM-1 interactions was restricted to myogenic cells, as forced expression of ICAM-1 by fibroblasts did not augment their fusion to ICAM-1+ myoblasts/myotubes. We conclude that ICAM-1 augments myoblast adhesion and fusion through its ability to self-associate and initiate Rac-mediated remodeling of the actin cytoskeleton.

Live cell imaging reveals marked variability in myoblast proliferation and fate

Science.gov (United States)

2013-01-01

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

The Satellite Cell Niche Regulates the Balance between Myoblast Differentiation and Self-Renewal via p53.

Science.gov (United States)

Flamini, Valentina; Ghadiali, Rachel S; Antczak, Philipp; Rothwell, Amy; Turnbull, Jeremy E; Pisconti, Addolorata

2018-03-13

Satellite cells are adult muscle stem cells residing in a specialized niche that regulates their homeostasis. How niche-generated signals integrate to regulate gene expression in satellite cell-derived myoblasts is poorly understood. We undertook an unbiased approach to study the effect of the satellite cell niche on satellite cell-derived myoblast transcriptional regulation and identified the tumor suppressor p53 as a key player in the regulation of myoblast quiescence. After activation and proliferation, a subpopulation of myoblasts cultured in the presence of the niche upregulates p53 and fails to differentiate. When satellite cell self-renewal is modeled ex vivo in a reserve cell assay, myoblasts treated with Nutlin-3, which increases p53 levels in the cell, fail to differentiate and instead become quiescent. Since both these Nutlin-3 effects are rescued by small interfering RNA-mediated p53 knockdown, we conclude that a tight control of p53 levels in myoblasts regulates the balance between differentiation and return to quiescence. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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.

Myoblast fusion in Drosophila

International Nuclear Information System (INIS)

Haralalka, Shruti; Abmayr, Susan M.

2010-01-01

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.

Deficient leukemia inhibitory factor signaling in muscle precursor cells from patients with type 2 diabetes

DEFF Research Database (Denmark)

Broholm, Christa; Brandt, Claus; Schultz, Ninna S

2012-01-01

The cytokine leukemia-inhibitory factor (LIF) is expressed by skeletal muscle and induces proliferation of muscle precursor cells, an important feature of skeletal muscle maintenance and repair. We hypothesized that muscle precursor cells from patients with type 2 diabetes had a deficient response...... nor proliferation rate was affected. In conclusion, although LIF and LIFR proteins were increased in muscle tissue and myoblasts from diabetic patients, LIF signaling and LIF-stimulated cell proliferation were impaired in diabetic myoblasts, suggesting a novel mechanism by which muscle function......RNA knockdown of suppressor of cytokine signaling (SOCS)3 in myoblast cultures established from healthy individuals and patients with type 2 diabetes. Myoblast proliferation rate was assessed by bromodeoxyuridine incorporation. LIF and LIFR proteins were increased in both muscle tissue and cultured myoblasts...

Isolation of human foetal myoblasts and its application for microencapsulation

Science.gov (United States)

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

2008-01-01

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

Phosphoinositide 3-kinase/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.

Science.gov (United States)

Lim, Jeong A; Woo, Joo Hong; Kim, Hye Sun

2008-09-01

In this study, it was found that undifferentiated myoblasts were more vulnerable to menadione-induced oxidative stress than differentiated myotubes. Cell death occurred with a relatively low concentration of menadione in myoblasts compared to myotubes. With the same concentration of menadione, the Bcl-2/Bax ratio decreased and nuclei containing condensed chromatin were observed in myoblasts to a greater extent than in myotubes. However, myotubes became increasingly susceptible to menadione when phosphoinositide 3-kinase (PI3-K) was blocked by pre-incubation with LY294002, a PI3-K inhibitor. Actually, PI3-K activity was reduced by menadione in myoblasts but not in myotubes. In addition, the phosphorylation of Akt, a downstream effector of PI3-K, was inhibited in myoblasts by menadione but increased in myotubes. Both LY294002 and API-2, an Akt inhibitor, decreased the Bcl-2/Bax ratio in menadione-exposed myotubes. These results suggest that the differential activity of PI3-K/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.

Use of Flow Focusing Technique for Microencapsulation of Myoblasts.

Science.gov (United States)

Ciriza, J; Saenz del Burgo, L; Hernández, R M; Orive, G; Pedraz, J L

2017-01-01

Alginate cell microencapsulation implies the immobilization of cells within a polymeric membrane that allows the bidirectional diffusion of nutrients and oxygen inside the microcapsules and the release of waste and therapeutic molecules outside them. This technology has been applied to several cell types and it has been extensively described with pancreatic islets. However, other cells such as myoblasts are being currently studied and showing high interest. Moreover, different systems and approaches have been developed for cell encapsulation such as electrostatic extrusion and Flow focusing technology. When Flow focusing technology is applied for myoblast encapsulation, several factors should be considered, such as the pressure, the flow of the system, or the diameter size of the nebulizer, which will determine the final diameter size and shape of the microcapsules containing the myoblasts. Finally, viability of encapsulated myoblasts needs to be assessed before further studies are performed.

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

Energy Technology Data Exchange (ETDEWEB)

Song, Young Joon [Department of Biological Sciences, College of Natural Science, Inha University, 253 Yonghyun-dong, Nam-Gu, Incheon, Korea, 402-751 (Korea, Republic of); Lee, Hansol, E-mail: hlee@inha.ac.kr [Department of Biological Sciences, College of Natural Science, Inha University, 253 Yonghyun-dong, Nam-Gu, Incheon, Korea, 402-751 (Korea, Republic of)

2010-02-15

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

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

International Nuclear Information System (INIS)

Song, Young Joon; Lee, Hansol

2010-01-01

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

Improved Cell Culture Method for Growing Contracting Skeletal Muscle Models

Science.gov (United States)

Marquette, Michele L.; Sognier, Marguerite A.

2013-01-01

An improved method for culturing immature muscle cells (myoblasts) into a mature skeletal muscle overcomes some of the notable limitations of prior culture methods. The development of the method is a major advance in tissue engineering in that, for the first time, a cell-based model spontaneously fuses and differentiates into masses of highly aligned, contracting myotubes. This method enables (1) the construction of improved two-dimensional (monolayer) skeletal muscle test beds; (2) development of contracting three-dimensional tissue models; and (3) improved transplantable tissues for biomedical and regenerative medicine applications. With adaptation, this method also offers potential application for production of other tissue types (i.e., bone and cardiac) from corresponding precursor cells.

Expression of Pannexin 1 and Pannexin 3 during skeletal muscle development, regeneration, and Duchenne muscular dystrophy.

Science.gov (United States)

Pham, Tammy L; St-Pierre, Marie-Eve; Ravel-Chapuis, Aymeric; Parks, Tara E C; Langlois, Stéphanie; Penuela, Silvia; Jasmin, Bernard J; Cowan, Kyle N

2018-05-10

Pannexin 1 (Panx1) and Pannexin 3 (Panx3) are single membrane channels recently implicated in myogenic commitment, as well as myoblast proliferation and differentiation in vitro. However, their expression patterns during skeletal muscle development and regeneration had yet to be investigated. Here, we show that Panx1 levels increase during skeletal muscle development becoming highly expressed together with Panx3 in adult skeletal muscle. In adult mice, Panx1 and Panx3 were differentially expressed in fast- and slow-twitch muscles. We also report that Panx1/PANX1 and Panx3/PANX3 are co-expressed in mouse and human satellite cells, which play crucial roles in skeletal muscle regeneration. Interestingly, Panx1 and Panx3 levels were modulated in muscle degeneration/regeneration, similar to the pattern seen during skeletal muscle development. As Duchenne muscular dystrophy is characterized by skeletal muscle degeneration and impaired regeneration, we next used mild and severe mouse models of this disease and found a significant dysregulation of Panx1 and Panx3 levels in dystrophic skeletal muscles. Together, our results are the first demonstration that Panx1 and Panx3 are differentially expressed amongst skeletal muscle types with their levels being highly modulated during skeletal muscle development, regeneration, and dystrophy. These findings suggest that Panx1 and Panx3 channels may play important and distinct roles in healthy and diseased skeletal muscles. © 2018 Wiley Periodicals, Inc.

Secreted Protein Acidic and Rich in Cysteine (SPARC) in Human Skeletal Muscle

Science.gov (United States)

Jørgensen, Louise H.; Petersson, Stine J.; Sellathurai, Jeeva; Andersen, Ditte C.; Thayssen, Susanne; Sant, Dorte J.; Jensen, Charlotte H.; Schrøder, Henrik D.

2009-01-01

Secreted protein acidic and rich in cysteine (SPARC)/osteonectin is expressed in different tissues during remodeling and repair, suggesting a function in regeneration. Several gene expression studies indicated that SPARC was expressed in response to muscle damage. Studies on myoblasts further indicated a function of SPARC in skeletal muscle. We therefore found it of interest to study SPARC expression in human skeletal muscle during development and in biopsies from Duchenne and Becker muscular dystrophy and congenital muscular dystrophy, congenital myopathy, inclusion body myositis, and polymyositis patients to analyze SPARC expression in a selected range of inherited and idiopathic muscle wasting diseases. SPARC-positive cells were observed both in fetal and neonatal muscle, and in addition, fetal myofibers were observed to express SPARC at the age of 15–16 weeks. SPARC protein was detected in the majority of analyzed muscle biopsies (23 of 24), mainly in mononuclear cells of which few were pax7 positive. Myotubes and regenerating myofibers also expressed SPARC. The expression-degree seemed to reflect the severity of the lesion. In accordance with these in vivo findings, primary human-derived satellite cells were found to express SPARC both during proliferation and differentiation in vitro. In conclusion, this study shows SPARC expression both during muscle development and in regenerating muscle. The expression is detected both in satellite cells/myoblasts and in myotubes and muscle fibers, indicating a role for SPARC in the skeletal muscle compartment. (J Histochem Cytochem 57:29–39, 2009) PMID:18796407

The effect of low intensity shockwave treatment (Li-SWT) on human myoblasts and mouse skeletal muscle

DEFF Research Database (Denmark)

Hansen, Lise K; Schrøder, Henrik D; Lund, Lars

2017-01-01

, proliferation and differentiation were tested. Cardiotoxin induced injury was created in tibialis anterior muscles of 28 mice, and two days later, the lesions were treated with 500 impulses of Li-SWT on one of the legs. The treatment was repeated every third day of the period and ended on day 14 after...... incremental effect on expression of pro-angiogenic genes. However, we found no changes in the number of PAX7 positive cells or blood vessel density in Li-SWT treated and control muscle. Furthermore, Li-SWT in the selected doses did not decrease survival, proliferation or differentiation of myoblasts in vitro....

Obscurin Depletion Impairs Organization of Skeletal Muscle in Developing Zebrafish Embryos

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Maide Ö. Raeker

2011-01-01

Full Text Available During development, skeletal myoblasts differentiate into myocytes and skeletal myotubes with mature contractile structures that are precisely oriented with respect to surrounding cells and tissues. Establishment of this highly ordered structure requires reciprocal interactions between the differentiating myocytes and the surrounding extracellular matrix to form correctly positioned and well-organized attachments from the skeletal muscle to the bony skeleton. Using the developing zebrafish embryo as a model, we examined the relationship between new myofibril assembly and the organization of the membrane domains involved in cell-extracellular matrix interactions. We determined that depletion of obscurin, a giant muscle protein, resulted in irregular cell morphology and disturbed extracellular matrix organization during skeletal muscle development. The resulting impairment of myocyte organization was associated with disturbance of the internal architecture of the myocyte suggesting that obscurin participates in organizing the internal structure of the myocyte and translating those structural cues to surrounding cells and tissues.

Obscurin Depletion Impairs Organization of Skeletal Muscle in Developing Zebrafish Embryos

Science.gov (United States)

Raeker, Maide Ö.; Russell, Mark W.

2011-01-01

During development, skeletal myoblasts differentiate into myocytes and skeletal myotubes with mature contractile structures that are precisely oriented with respect to surrounding cells and tissues. Establishment of this highly ordered structure requires reciprocal interactions between the differentiating myocytes and the surrounding extracellular matrix to form correctly positioned and well-organized attachments from the skeletal muscle to the bony skeleton. Using the developing zebrafish embryo as a model, we examined the relationship between new myofibril assembly and the organization of the membrane domains involved in cell-extracellular matrix interactions. We determined that depletion of obscurin, a giant muscle protein, resulted in irregular cell morphology and disturbed extracellular matrix organization during skeletal muscle development. The resulting impairment of myocyte organization was associated with disturbance of the internal architecture of the myocyte suggesting that obscurin participates in organizing the internal structure of the myocyte and translating those structural cues to surrounding cells and tissues. PMID:22190853

Tissue-Engineered Skeletal Muscle Organoids for Reversible Gene Therapy

Science.gov (United States)

Vandenburgh, Herman; DelTatto, Michael; Shansky, Janet; Lemaire, Julie; Chang, Albert; Payumo, Francis; Lee, Peter; Goodyear, Amy; Raven, Latasha

1996-01-01

Genetically modified murine skeletal myoblasts were tissue engineered in vitro into organ-like structures (organoids) containing only postmitotic myofibers secreting pharmacological levels of recombinant human growth hormone (rhGH). Subcutaneous organoid Implantation under tension led to the rapid and stable appearance of physiological sera levels of rhGH for up to 12 weeks, whereas surgical removal led to its rapid disappearance. Reversible delivery of bioactive compounds from postimtotic cells in tissue engineered organs has several advantages over other forms of muscle gene therapy.

Phosphorylation of Lbx1 controls lateral myoblast migration into the limb.

Science.gov (United States)

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

2017-10-15

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

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

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

2011-01-01

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

Neonatal epicardial-derived progenitors aquire myogenic traits in skeletal muscle, but not cardiac muscle

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Andersen, Ditte C; Jensen, Charlotte H; Skovrind, Ida

2016-01-01

heart missing regenerative signals essential for directed differentiation of EPDCs. Herein, we aimed to evaluate the myogenic potential of neonatal EPDCs in adult and neonatal mouse myocardium, as well as in skeletal muscle. The two latter tissues have an intrinsic capability to develop and regenerate......, in contrast to the adult heart. METHODS: Highly purified mouse EPDCs were transplanted into damaged neonatal and adult myocardium as well as regenerating skeletal muscle. Co-cultures with skeletal myoblasts were used to distinguish fusion independent myogenic conversion. RESULTS: No donor EPDC...... that EPDCs may be more myogenic than previously anticipated. But, the heart may lack factors for induction of myogenesis of EPDCs, a scenario that should be taken into consideration when aiming for repair of damaged myocardium by stem cell transplantation....

Injectable skeletal muscle matrix hydrogel promotes neovascularization and muscle cell infiltration in a hindlimb ischemia model

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JA DeQuach

2012-06-01

Full Text Available Peripheral artery disease (PAD currently affects approximately 27 million patients in Europe and North America, and if untreated, may progress to the stage of critical limb ischemia (CLI, which has implications for amputation and potential mortality. Unfortunately, few therapies exist for treating the ischemic skeletal muscle in these conditions. Biomaterials have been used to increase cell transplant survival as well as deliver growth factors to treat limb ischemia; however, existing materials do not mimic the native skeletal muscle microenvironment they are intended to treat. Furthermore, no therapies involving biomaterials alone have been examined. The goal of this study was to develop a clinically relevant injectable hydrogel derived from decellularized skeletal muscle extracellular matrix and examine its potential for treating PAD as a stand-alone therapy by studying the material in a rat hindlimb ischemia model. We tested the mitogenic activity of the scaffold’s degradation products using an in vitro assay and measured increased proliferation rates of smooth muscle cells and skeletal myoblasts compared to collagen. In a rat hindlimb ischemia model, the femoral artery was ligated and resected, followed by injection of 150 µL of skeletal muscle matrix or collagen 1 week post-injury. We demonstrate that the skeletal muscle matrix increased arteriole and capillary density, as well as recruited more desmin-positive and MyoD-positive cells compared to collagen. Our results indicate that this tissue-specific injectable hydrogel may be a potential therapy for treating ischemia related to PAD, as well as have potential beneficial effects on restoring muscle mass that is typically lost in CLI.

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

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

2011-01-01

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

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

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Sebastian Frese

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

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

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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.

Irisin is a pro-myogenic factor that induces skeletal muscle hypertrophy and rescues denervation-induced atrophy.

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Reza, Musarrat Maisha; Subramaniyam, Nathiya; Sim, Chu Ming; Ge, Xiaojia; Sathiakumar, Durgalakshmi; McFarlane, Craig; Sharma, Mridula; Kambadur, Ravi

2017-10-24

Exercise induces expression of the myokine irisin, which is known to promote browning of white adipose tissue and has been shown to mediate beneficial effects following exercise. Here we show that irisin induces expression of a number of pro-myogenic and exercise response genes in myotubes. Irisin increases myogenic differentiation and myoblast fusion via activation of IL6 signaling. Injection of irisin in mice induces significant hypertrophy and enhances grip strength of uninjured muscle. Following skeletal muscle injury, irisin injection improves regeneration and induces hypertrophy. The effects of irisin on hypertrophy are due to activation of satellite cells and enhanced protein synthesis. In addition, irisin injection rescues loss of skeletal muscle mass following denervation by enhancing satellite cell activation and reducing protein degradation. These data suggest that irisin functions as a pro-myogenic factor in mice.

Loss of MyoD and Myf5 in Skeletal Muscle Stem Cells Results in Altered Myogenic Programming and Failed Regeneration

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Masakazu Yamamoto

2018-03-01

Full Text Available Summary: MyoD and Myf5 are fundamental regulators of skeletal muscle lineage determination in the embryo, and their expression is induced in satellite cells following muscle injury. MyoD and Myf5 are also expressed by satellite cell precursors developmentally, although the relative contribution of historical and injury-induced expression to satellite cell function is unknown. We show that satellite cells lacking both MyoD and Myf5 (double knockout [dKO] are maintained with aging in uninjured muscle. However, injured muscle fails to regenerate and dKO satellite cell progeny accumulate in damaged muscle but do not undergo muscle differentiation. dKO satellite cell progeny continue to express markers of myoblast identity, although their myogenic programming is labile, as demonstrated by dramatic morphological changes and increased propensity for non-myogenic differentiation. These data demonstrate an absolute requirement for either MyoD or Myf5 in muscle regeneration and indicate that their expression after injury stabilizes myogenic identity and confers the capacity for muscle differentiation. : In this article, Goldhamer and colleagues show that loss of both MyoD and Myf5 in skeletal muscle satellite cells results in regenerative failure following injury. Satellite cell progeny accumulate in injured muscle and continue to express markers of myoblast identity, but do not undergo muscle differentiation, and exhibit a propensity for non-myogenic differentiation. Keywords: skeletal muscle regeneration, muscle stem cell programming, muscle differentiation, satellite cell, MyoD, Myf5, adipogenesis, fibrosis, conditional knockout, Cre/loxP

Deletion of the Ste20-like kinase SLK in skeletal muscle results in a progressive myopathy and muscle weakness.

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Pryce, Benjamin R; Al-Zahrani, Khalid N; Dufresne, Sébastien; Belkina, Natalya; Labrèche, Cédrik; Patino-Lopez, Genaro; Frenette, Jérôme; Shaw, Stephen; Sabourin, Luc A

2017-02-02

The Ste20-like kinase, SLK, plays an important role in cell proliferation and cytoskeletal remodeling. In fibroblasts, SLK has been shown to respond to FAK/Src signaling and regulate focal adhesion turnover through Paxillin phosphorylation. Full-length SLK has also been shown to be essential for embryonic development. In myoblasts, the overexpression of a dominant negative SLK is sufficient to block myoblast fusion. In this study, we crossed the Myf5-Cre mouse model with our conditional SLK knockout model to delete SLK in skeletal muscle. A thorough analysis of skeletal muscle tissue was undertaken in order to identify defects in muscle development caused by the lack of SLK. Isometric force analysis was performed on adult knockout mice and compared to age-matched wild-type mice. Furthermore, cardiotoxin injections were performed followed by immunohistochemistry for myogenic markers to assess the efficiency muscle regeneration following SLK deletion. We show here that early deletion of SLK from the myogenic lineage does not markedly impair skeletal muscle development but delays the regenerative process. Interestingly, adult mice (~6 months) display an increase in the proportion of central nuclei and increased p38 activation. Furthermore, mice as young as 3 months old present with decreased force generation, suggesting that the loss of SLK impairs myofiber stability and function. Assessment of structural components revealed aberrant localization of focal adhesion proteins, such as FAK and paxillin. Our data show that the loss of SLK results in unstable myofibers resulting in a progressive myopathy. Additionally, the loss of SLK resulted in a delay in muscle regeneration following cardiotoxin injections. Our results show that SLK is dispensable for muscle development and regeneration but is required for myofiber stability and optimal force generation.

Establishment of bipotent progenitor cell clone from rat skeletal muscle.

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Murakami, Yousuke; Yada, Erica; Nakano, Shin-ichi; Miyagoe-Suzuki, Yuko; Hosoyama, Tohru; Matsuwaki, Takashi; Yamanouchi, Keitaro; Nishihara, Masugi

2011-12-01

The present study describes the isolation, cloning and characterization of adipogenic progenitor cells from rat skeletal muscle. Among the obtained 10 clones, the most highly adipogenic progenitor, 2G11 cells, were further characterized. In addition to their adipogenicity, 2G11 cells retain myogenic potential as revealed by formation of multinucleated myotubes when co-cultured with myoblasts. 2G11 cells were resistant to an inhibitory effect of basic fibroblast growth factor on adipogenesis, while adipogenesis of widely used preadipogenic cell line, 3T3-L1 cells, was suppressed almost completely by the same treatment. In vivo transplantation experiments revealed that 2G11 cells are able to possess both adipogenicity and myogenicity in vivo. These results indicate the presence of bipotent progenitor cells in rat skeletal muscle, and suggest that such cells may contribute to ectopic fat formation in skeletal muscle. © 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.

Analysis of differential lipofection efficiency in primary and established myoblasts.

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Pampinella, Francesca; Lechardeur, Delphine; Zanetti, Elena; MacLachlan, Ian; Benharouga, Mohammed; Lukacs, Gergely L; Vitiello, Libero

2002-02-01

In this study we have compared the process of lipid-mediated transfection in primary and established myoblasts, in an attempt to elucidate the mechanisms responsible for the scarce transfectability of the former. We determined the metabolic stability of cytoplasmically injected and lipofected DNA in primary and established myoblasts and carried out a comparative time course analysis of luciferase reporter-gene expression and DNA stability. The efficiency of the transcription-translation machinery of the two cell types was compared by intranuclear injection of naked plasmid DNA encoding luciferase. Subcellular colocalization of fluorescein-labeled lipopolyplexes with specific endosomal and lysosomal markers was performed by confocal microscopy to monitor the intracellular trafficking of plasmid DNA during transfection. The metabolic stability of plasmid DNA was similar in primary and established myoblasts after both lipofection and cytoplasmic injection. In both cell types, lipofection had no detectable effect on the rate of cell proliferation. Confocal analysis showed that nuclear translocation of transfected DNA coincided with localization in a compartment devoid of endosome- or lysosome-specific marker proteins. The residency time of plasmid DNA in this compartment differed for primary and established myoblasts. Our findings suggest that the lower transfectability of primary myoblasts is mostly due to a difference in the intracellular delivery pathway that correlates with more rapid delivery of internalized complex to the lysosomal compartment.

[Human myoblast culture as muscle stem cells in medical and biological studies].

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Terekhov, S M; Krokhina, T B; Shishkin, S S; Krakhmaleva, I N; Zakharov, S F; Ershova, E S

2001-01-01

The method for obtaining human myoblast culture has been modified to consider the specific histological localization of the satellite cells as well as their growth properties; the cultivation conditions have been selected to grow up to 150000 cells/cm2. At high densities, the cells remain mononuclear and preserve their typical myoblast morphology as well as the capacity for fusion and the formation of myotubes. By contrast to fibroblasts, up to 80% of the cells in the myoblast culture were positive in the acid phosphatase test, which indicates their stem nature. The obtained myoblast cultures were used in the clinical tests of cell-mediated gene therapy of Duchenne's muscular dystrophy as well as in the bioassay for the effects of biologically active compounds.

Type II iodothyronine deiodinase provides intracellular 3,5,3′-triiodothyronine to normal and regenerating mouse skeletal muscle

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Marsili, Alessandro; Tang, Dan; Harney, John W.; Singh, Prabhat; Zavacki, Ann Marie; Dentice, Monica; Salvatore, Domenico

2011-01-01

The FoxO3-dependent increase in type II deiodinase (D2), which converts the prohormone thyroxine (T4) to 3,5,3′-triiodothyronine (T3), is required for normal mouse skeletal muscle differentiation and regeneration. This implies a requirement for an increase in D2-generated intracellular T3 under these conditions, which has not been directly demonstrated despite the presence of D2 activity in skeletal muscle. We directly show that D2-mediated T4-to-T3 conversion increases during differentiation in C2C12 myoblast and primary cultures of mouse neonatal skeletal muscle precursor cells, and that blockade of D2 eliminates this. In adult mice given 125I-T4 and 131I-T3, the intracellular 125I-T3/131I-T3 ratio is significantly higher than in serum in both the D2-expressing cerebral cortex and the skeletal muscle of wild-type, but not D2KO, mice. In D1-expressing liver and kidney, the 125I-T3/131I-T3 ratio does not differ from that in serum. Hypothyroidism increases D2 activity, and in agreement with this, the difference in 125I-T3/131I-T3 ratio is increased further in hypothyroid wild-type mice but not altered in the D2KO. Notably, in wild-type but not in D2KO mice, the muscle production of 125I-T3 is doubled after skeletal muscle injury. Thus, D2-mediated T4-to-T3 conversion generates significant intracellular T3 in normal mouse skeletal muscle, with the increased T3 required for muscle regeneration being provided by increased D2 synthesis, not by T3 from the circulation. PMID:21771965

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.

Disruption of myoblast alignment by highly motile rhabdomyosarcoma cell in tissue structure.

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Li, Menglu; Nagamori, Eiji; Kino-Oka, Masahiro

2017-02-01

Rhabdomyosarcoma (RMS) is a highly malignant tumor type of skeletal muscle origin, hallmarked by local invasion. Interaction between invasive tumor cells and normal cells plays a major role in tumor invasion and metastasis. Culturing tumor cells in a three-dimensional (3D) model can translate tumor malignancy relevant cell-cell interaction. To mimic tumor heterogeneity in vitro, a co-culture system consisting of a malignant embryonal rhabdomyosarcoma (ERMS) cell line RD and a normal human skeletal muscle myoblast (HSMM) cell line was established by cell sheet technology. Various ratios of RDs to HSMMs were employed to understand the quantitative effect on intercellular interactions. Disruption of sheet structure was observed in heterogeneous cell sheets having a low ratio of RDs to HSMMs, whereas homogeneous HSMM or RD sheets maintained intact structure. Deeper exploration of dynamic tumor cell behavior inside HSMM sheets revealed that HSMM cell alignment was disrupted by highly motile RDs. This study demonstrated that RMS cells are capable of compromising their surrounding environment through induced decay of HSMMs alignment in a cell-based 3D system. This suggests that muscle disruption might be a major consequence of RMS cell invasion into muscles, which could be a promising target to preventing tumor invasion. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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

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

2000-01-01

The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight.

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

International Nuclear Information System (INIS)

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

2015-01-01

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

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

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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

Effects of creatine and its analog, β-guanidinopropionic acid, on the differentiation of and nucleoli in myoblasts.

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Ohira, Yoshinobu; Matsuoka, Yoshikazu; Kawano, Fuminori; Ogura, Akihiko; Higo, Yoko; Ohira, Takashi; Terada, Masahiro; Oke, Yoshihiko; Nakai, Naoya

2011-01-01

The effects of supplementation with creatine (Cr) and its analog, β-guanidinopropionic acid (β-GPA), on the differentiation of myoblasts and the numbers of nucleoli were studied in C2C12 cells. The cells were cultured in differentiation medium for 4 d. Then Cr (1 mM) or β-GPA (1 mM) was added to the cells, and the mixture was cultured for an additional 2 d. Although the number of myotubes was not different among the groups, myotube diameters and nuclear numbers in myotubes were increased by Cr and β-GPA treatment respectively. The expression of differentiation marker proteins, myogenin, and the myosine heavy chain, was increased in the β-GPA group. Supplementation with β-GPA also increased the percentage of p21 (inhibitor for cell cycle progression)-positive myoblasts. Supplementation with Cr inhibited the decrease in nucleoli numbers, whereas β-GPA increased nucleolar sizes in the myotubes. These results suggest that β-GPA supplementation stimulated the differentiation of myoblasts into multi-nucleated myotubes through induction of p21 expression.

Transcriptional profiling identifies differentially expressed genes in developing turkey skeletal muscle

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Velleman Sandra G

2011-03-01

Full Text Available Abstract Background Skeletal muscle growth and development from embryo to adult consists of a series of carefully regulated changes in gene expression. Understanding these developmental changes in agriculturally important species is essential to the production of high quality meat products. For example, consumer demand for lean, inexpensive meat products has driven the turkey industry to unprecedented production through intensive genetic selection. However, achievements of increased body weight and muscle mass have been countered by an increased incidence of myopathies and meat quality defects. In a previous study, we developed and validated a turkey skeletal muscle-specific microarray as a tool for functional genomics studies. The goals of the current study were to utilize this microarray to elucidate functional pathways of genes responsible for key events in turkey skeletal muscle development and to compare differences in gene expression between two genetic lines of turkeys. To achieve these goals, skeletal muscle samples were collected at three critical stages in muscle development: 18d embryo (hyperplasia, 1d post-hatch (shift from myoblast-mediated growth to satellite cell-modulated growth by hypertrophy, and 16wk (market age from two genetic lines: a randombred control line (RBC2 maintained without selection pressure, and a line (F selected from the RBC2 line for increased 16wk body weight. Array hybridizations were performed in two experiments: Experiment 1 directly compared the developmental stages within genetic line, while Experiment 2 directly compared the two lines within each developmental stage. Results A total of 3474 genes were differentially expressed (false discovery rate; FDR Conclusions The current study identified gene pathways and uncovered novel genes important in turkey muscle growth and development. Future experiments will focus further on several of these candidate genes and the expression and mechanism of action of

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

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

2016-01-29

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

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

International Nuclear Information System (INIS)

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

2016-01-01

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

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

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

2014-01-01

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

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.

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.

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

International Nuclear Information System (INIS)

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

2013-01-01

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

Effect of heat stress on contractility of tissue-engineered artificial skeletal muscle.

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Takagi, Shunya; Nakamura, Tomohiro; Fujisato, Toshia

2018-01-23

The effects of heat stress on tissue like skeletal muscle have been widely studied. However, the mechanism responsible for the effect of heat stress is still unclear. A useful experimental tissue model is necessary because muscle function in cell culture may differ from native muscle and measuring its contractility is difficult. We previously reported three-dimensional tissue-engineered artificial skeletal muscle (TEM) that can be easily set in a measurement apparatus for quantitative evaluation of contractility. We have now applied TEM to the investigation of heat stress. We analyzed contractility immediately after thermal exposure at 39 °C for 24 or 48 h to evaluate the acute effects and after thermal exposure followed by normal culture to evaluate the aftereffects. Peak twitch contractile force and time-to-peak twitch were used as contractile parameters. Heat stress increased the TCF in the early stage (1 week) after normal culture; the TCF decreased temporarily in the middle to late stages (2-3 weeks). These results suggest that heat stress may affect both myoblast fusion and myotube differentiation in the early stage of TEM culture, but not myotube maturation in the late stage. The TCF increase rate with thermal exposure was significantly higher than that without thermal exposure. Although detailed analysis at the molecular level is necessary for further investigation, our artificial skeletal muscle may be a promising tool for heat stress investigation.

TGF-β's delay skeletal muscle progenitor cell differentiation in an isoform-independent manner

International Nuclear Information System (INIS)

Schabort, Elske J.; Merwe, Mathilde van der; Loos, Benjamin; Moore, Frances P.; Niesler, Carola U.

2009-01-01

Satellite cells are a quiescent heterogenous population of mononuclear stem and progenitor cells which, once activated, differentiate into myotubes and facilitate skeletal muscle repair or growth. The Transforming Growth Factor-β (TGF-β) superfamily members are elevated post-injury and their importance in the regulation of myogenesis and wound healing has been demonstrated both in vitro and in vivo. Most studies suggest a negative role for TGF-β on satellite cell differentiation. However, none have compared the effect of these three isoforms on myogenesis in vitro. This is despite known isoform-specific effects of TGF-β1, -β2 and -β3 on wound repair in other tissues. In the current study we compared the effect of TGF-β1, -β2 and -β3 on proliferation and differentiation of the C2C12 myoblast cell-line. We found that, irrespective of the isoform, TGF-β increased proliferation of C2C12 cells by changing the cellular localisation of PCNA to promote cell division and prevent cell cycle exit. Concomitantly, TGF-β1, -β2 and -β3 delayed myogenic commitment by increasing MyoD degradation and decreasing myogenin expression. Terminal differentiation, as measured by a decrease in myosin heavy chain (MHC) expression, was also delayed. These results demonstrate that TGF-β promotes proliferation and delays differentiation of C2C12 myoblasts in an isoform-independent manner

Hypoxia induces adipogenic differentitation of myoblastic cell lines

Energy Technology Data Exchange (ETDEWEB)

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

2010-09-03

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

PEDF-derived peptide promotes skeletal muscle regeneration through its mitogenic effect on muscle progenitor cells.

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Ho, Tsung-Chuan; Chiang, Yi-Pin; Chuang, Chih-Kuang; Chen, Show-Li; Hsieh, Jui-Wen; Lan, Yu-Wen; Tsao, Yeou-Ping

2015-08-01

In response injury, intrinsic repair mechanisms are activated in skeletal muscle to replace the damaged muscle fibers with new muscle fibers. The regeneration process starts with the proliferation of satellite cells to give rise to myoblasts, which subsequently differentiate terminally into myofibers. Here, we investigated the promotion effect of pigment epithelial-derived factor (PEDF) on muscle regeneration. We report that PEDF and a synthetic PEDF-derived short peptide (PSP; residues Ser(93)-Leu(112)) induce satellite cell proliferation in vitro and promote muscle regeneration in vivo. Extensively, soleus muscle necrosis was induced in rats by bupivacaine, and an injectable alginate gel was used to release the PSP in the injured muscle. PSP delivery was found to stimulate satellite cell proliferation in damaged muscle and enhance the growth of regenerating myofibers, with complete regeneration of normal muscle mass by 2 wk. In cell culture, PEDF/PSP stimulated C2C12 myoblast proliferation, together with a rise in cyclin D1 expression. PEDF induced the phosphorylation of ERK1/2, Akt, and STAT3 in C2C12 myoblasts. Blocking the activity of ERK, Akt, or STAT3 with pharmacological inhibitors attenuated the effects of PEDF/PSP on the induction of C2C12 cell proliferation and cyclin D1 expression. Moreover, 5-bromo-2'-deoxyuridine pulse-labeling demonstrated that PEDF/PSP stimulated primary rat satellite cell proliferation in myofibers in vitro. In summary, we report for the first time that PSP is capable of promoting the regeneration of skeletal muscle. The signaling mechanism involves the ERK, AKT, and STAT3 pathways. These results show the potential utility of this PEDF peptide for muscle regeneration. Copyright © 2015 the American Physiological Society.

Rac1 Activation Caused by Membrane Translocation of a Guanine Nucleotide Exchange Factor in Akt2-Mediated Insulin Signaling in Mouse Skeletal Muscle.

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Nobuyuki Takenaka

Full Text Available Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4, which is translocated to the plasma membrane following insulin stimulation. Several lines of evidence suggested that the protein kinase Akt2 plays a key role in this insulin action. The small GTPase Rac1 has also been implicated as a regulator of insulin-stimulated GLUT4 translocation, acting downstream of Akt2. However, the mechanisms whereby Akt2 regulates Rac1 activity remain obscure. The guanine nucleotide exchange factor FLJ00068 has been identified as a direct regulator of Rac1 in Akt2-mediated signaling, but its characterization was performed mostly in cultured myoblasts. Here, we provide in vivo evidence that FLJ00068 indeed acts downstream of Akt2 as a Rac1 regulator by using mouse skeletal muscle. Small interfering RNA knockdown of FLJ00068 markedly diminished GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated mutant of either phosphoinositide 3-kinase or Akt2. Additionally, insulin and these constitutively activated mutants caused the activation of Rac1 as shown by immunofluorescent microscopy using a polypeptide probe specific to activated Rac1 in isolated gastrocnemius muscle fibers and frozen sections of gastrocnemius muscle. This Rac1 activation was also abrogated by FLJ00068 knockdown. Furthermore, we observed translocation of FLJ00068 to the cell periphery following insulin stimulation in cultured myoblasts. Localization of FLJ00068 in the plasma membrane in insulin-stimulated, but not unstimulated, myoblasts and mouse gastrocnemius muscle was further affirmed by subcellular fractionation and subsequent immunoblotting. Collectively, these results strongly support a critical role of FLJ00068 in Akt2-mediated Rac1 activation in mouse skeletal muscle insulin signaling.

Primary skeletal muscle cells cultured on gelatin bead microcarriers develop structural and biochemical features characteristic of adult skeletal muscle.

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Kubis, Hans-Peter; Scheibe, Renate J; Decker, Brigitte; Hufendiek, Karsten; Hanke, Nina; Gros, Gerolf; Meissner, Joachim D

2016-04-01

A primary skeletal muscle cell culture, in which myoblasts derived from newborn rabbit hindlimb muscles grow on gelatin bead microcarriers in suspension and differentiate into myotubes, has been established previously. In the course of differentiation and beginning spontaneous contractions, these multinucleated myotubes do not detach from their support. Here, we describe the development of the primary myotubes with respect to their ultrastructural differentiation. Scanning electron microscopy reveals that myotubes not only grow around the surface of one carrier bead but also attach themselves to neighboring carriers, forming bridges between carriers. Transmission electron microscopy demonstrates highly ordered myofibrils, T-tubules, and sarcoplasmic reticulum. The functionality of the contractile apparatus is evidenced by contractile activity that occurs spontaneously or can be elicited by electrostimulation. Creatine kinase activity increases steadily until day 20 of culture. Regarding the expression of isoforms of myosin heavy chains (MHC), we could demonstrate that from day 16 on, no non-adult MHC isoform mRNAs are present. Instead, on day 28 the myotubes express predominantly adult fast MHCIId/x mRNA and protein. This MHC pattern resembles that of fast muscles of adult rabbits. In contrast, primary myotubes grown on matrigel-covered culture dishes express substantial amounts of non-adult MHC protein even on day 21. To conclude, primary myotubes grown on microcarriers in their later stages exhibit many features of adult skeletal muscle and characteristics of fast type II fibers. Thus, the culture represents an excellent model of adult fast skeletal muscle, for example, when investigating molecular mechanisms of fast-to-slow fiber-type transformation. © 2015 International Federation for Cell Biology.

An Antibody Blocking Activin Type II Receptors Induces Strong Skeletal Muscle Hypertrophy and Protects from Atrophy

Science.gov (United States)

Minetti, Giulia C.; Sheppard, KellyAnn; Ibebunjo, Chikwendu; Feige, Jerome N.; Hartmann, Steffen; Brachat, Sophie; Rivet, Helene; Koelbing, Claudia; Morvan, Frederic; Hatakeyama, Shinji

2014-01-01

The myostatin/activin type II receptor (ActRII) pathway has been identified to be critical in regulating skeletal muscle size. Several other ligands, including GDF11 and the activins, signal through this pathway, suggesting that the ActRII receptors are major regulatory nodes in the regulation of muscle mass. We have developed a novel, human anti-ActRII antibody (bimagrumab, or BYM338) to prevent binding of ligands to the receptors and thus inhibit downstream signaling. BYM338 enhances differentiation of primary human skeletal myoblasts and counteracts the inhibition of differentiation induced by myostatin or activin A. BYM338 prevents myostatin- or activin A-induced atrophy through inhibition of Smad2/3 phosphorylation, thus sparing the myosin heavy chain from degradation. BYM338 dramatically increases skeletal muscle mass in mice, beyond sole inhibition of myostatin, detected by comparing the antibody with a myostatin inhibitor. A mouse version of the antibody induces enhanced muscle hypertrophy in myostatin mutant mice, further confirming a beneficial effect on muscle growth beyond myostatin inhibition alone through blockade of ActRII ligands. BYM338 protects muscles from glucocorticoid-induced atrophy and weakness via prevention of muscle and tetanic force losses. These data highlight the compelling therapeutic potential of BYM338 for the treatment of skeletal muscle atrophy and weakness in multiple settings. PMID:24298022

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

NARCIS (Netherlands)

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

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,

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

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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.

Generation of a vascularized organoid using skeletal muscle as the inductive source.

LENUS (Irish Health Repository)

Messina, Aurora

2005-09-01

The technology required for creating an in vivo microenvironment and a neovasculature that can grow with and service new tissue is lacking, precluding the possibility of engineering complex three-dimensional organs. We have shown that when an arterio-venous (AV) loop is constructed in vivo in the rat groin, and placed inside a semisealed chamber, an extensive functional vasculature is generated. To test whether this unusually angiogenic environment supports the survival and growth of implanted tissue or cells, we inserted various preparations of rat and human skeletal muscle. We show that after 6 weeks incubation of muscle tissue, the chamber filled with predominantly well-vascularized recipient-derived adipose tissue, but some new donor-derived skeletal muscle and connective tissue were also evident. When primary cultured myoblasts were inserted into the chamber with the AV loop, they converted to mature striated muscle fibers. Furthermore, we identify novel adipogenesis-inducing properties of skeletal muscle. This represents the first report of a specific three-dimensional tissue grown on its own vascular supply.

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

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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.

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 mut...... genetic analysis demonstrates thus that the function of Rac in myoblast fusion is evolutionarily conserved from insects to mammals and that Cdc42, a molecule hitherto not implicated in myoblast fusion, is essential for the fusion of murine myoblasts....

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

Science.gov (United States)

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

2013-02-08

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. Copyright © 2013 Elsevier Inc. All rights reserved.

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

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

2007-07-15

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

[Molecular mechanism for ET-1-induced insulin resistance in skeletal muscle cells].

Science.gov (United States)

Horinouchi, Takahiro; Mazaki, Yuichi; Terada, Koji; Miwa, Soichi

2018-01-01

Insulin resistance is a condition where the sensitivity to insulin of the tissues expressing insulin receptor (InsR) is decreased due to a functional disturbance of InsR-mediated intracellular signaling. Insulin promotes the entry of glucose into the tissues and skeletal muscle is the most important tissue responsible for the insulin's action of decreasing blood glucose levels. Endothelin-1 (ET-1), a potent vasoconstrictor and pro-inflammatory peptide, induces insulin resistance through a direct action on skeletal muscle. However, the signaling pathways of ET-1-induced insulin resistance in skeletal muscle remain unclear. Here we show molecular mechanism underlying the inhibitory effect of ET-1 on insulin-stimulated Akt phosphorylation and glucose uptake in myotubes of rat L6 skeletal muscle cell line. mRNA expression levels of differentiation marker genes, MyoD and myogenin, were increased during L6 myoblasts differentiation into myotubes. Some of myotubes possessed the ability to spontaneously contract. In myotubes, insulin promoted Akt phosphorylation at Thr 308 and Ser 473 , and [ 3 H]-labelled 2-deoxy-D-glucose ([ 3 H]2-DG) uptake. The insulin-facilitated Akt phosphorylation and [ 3 H]2-DG uptake were inhibited by ET-1. The inhibitory effect of ET-1 was counteracted by blockade of ET type A receptor (ET A R), inhibition of G q/11 protein, and siRNA knockdown of G protein-coupled receptor kinase 2 (GRK2). The exogenously overexpressed GRK2 directly bound to endogenous Akt and their association was facilitated by ET-1. In summary, activation of ET A R with ET-1 inhibits insulin-induced Akt phosphorylation and [ 3 H]2-DG uptake in a G q/11 protein- and GRK2-dependent manner in skeletal muscle. These findings indicate that ET A R and GRK2 are potential targets for insulin resistance.

Lactate dehydrogenase regulation in aged skeletal muscle: Regulation by anabolic steroids and functional overload.

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Washington, Tyrone A; Healey, Julie M; Thompson, Raymond W; Lowe, Larry L; Carson, James A

2014-09-01

Aging alters the skeletal muscle response to overload-induced growth. The onset of functional overload is characterized by increased myoblast proliferation and an altered muscle metabolic profile. The onset of functional overload is associated with increased energy demands that are met through the interconversion of lactate and pyruvate via the activity of lactate dehydrogenase (LDH). Testosterone targets many of the processes activated at the onset of functional overload. However, the effect of aging on this metabolic plasticity at the onset of functional overload and how anabolic steroid administration modulates this response is not well understood. The purpose of this study was to determine if aging would alter overload-induced LDH activity and expression at the onset of functional overload and whether anabolic steroid administration would modulate this response. Five-month and 25-month male Fischer 344xF1 BRN were given nandrolone decanoate (ND) or sham injections for 14days and then the plantaris was functionally overloaded (OV) for 3days by synergist ablation. Aging reduced muscle LDH-A & LDH-B activity 70% (pyoung muscle. Our study provides evidence that aging alters aspects of skeletal muscle metabolic plasticity normally induced by overload and anabolic steroid administration. Copyright © 2014 Elsevier Inc. All rights reserved.

Membrane glycoproteins of differentiating skeletal muscle cells

International Nuclear Information System (INIS)

Miller, K.R.; Remy, C.N.; Smith, P.B.

1987-01-01

The composition of N-linked glycoprotein oligosaccharides was studied in myoblasts and myotubes of the C2 muscle cell line. Oligosaccharides were radioactively labelled for 15 hr with [ 3 H] mannose and plasma membranes isolated. Ten glycopeptides were detected by SDS-PAGE and fluorography. The extent of labelling was 4-6 fold greater in myoblasts vs myotubes. A glycopeptide of Mr > 100,000 was found exclusively in myoblast membranes. Lectin chromatography revealed that the proportion of tri-, tetranntenary, biantennary and high mannose chains was similar throughout differentiation. The high mannose chain fraction was devoid of hybrid chains. The major high mannose chain contained nine mannose residues. The higher level of glycopeptide labelling in myoblasts vs myotubes corresponded to a 5-fold greater rate of protein synthesis. Pulse-chase experiments were used to follow the synthesis of the Dol-oligosaccharides. Myoblasts and myotubes labelled equivalently the glucosylated tetradecasaccharide but myoblasts labelled the smaller intermediates 3-4 greater than myotubes. Myoblasts also exhibited a 2-3 fold higher Dol-P dependent glycosyl transferase activity for chain elongation and Dol-sugar synthesis. Together these results show that the degree of protein synthesis and level of Dol-P are contributing factors in the higher capacity of myoblasts to produce N-glycoproteins compared to myotubes

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

KAUST Repository

Ballarino, Monica; Cazzella, Valentina; D'Andrea, Daniel; Grassi, Luigi; Bisceglie, Lavinia; Cipriano, Andrea; Santini, Tiziana; Pinnarò , Chiara; Morlando, Mariangela; Tramontano, Anna; Bozzoni, Irene

2014-01-01

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.

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.

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

Science.gov (United States)

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

2016-06-07

Duchenne muscular dystrophy (DMD) remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs). Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our "chemical-compound-based" strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFβ signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological "dual-SMAD" inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form "rescued" multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human "DMD-in-a-dish" model using hiPSC-based disease modeling. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2016-10-21

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 C₂C 12 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.

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

Science.gov (United States)

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

2013-12-01

Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the gene for G-protein coupled receptor 56 (GPR56) was transiently up-regulated during the early fusion of human myoblasts. Human mutations in the gene for GPR56 cause the disease bilateral frontoparietal polymicrogyria; however, the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR56 in skeletal muscle. GPR56(-/-) myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, a loss of GPR56 expression in muscle cells results in decreases or delays in the expression of myogenic differentiation 1, myogenin and nuclear factor of activated T-cell (NFAT)c2. Our data suggest that these abnormalities result from decreased GPR56-mediated serum response element and NFAT signalling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase compared to wild-type. In agreement with these findings, clinical data from 13 bilateral frontoparietal polymicrogyria patients revealed mild serum creatine kinase increase in only two patients. In summary, targeted disruption of GPR56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR56 knockout mice and human patients suggests that other factors may compensate for the lack of this G-protein coupled receptor during muscle development and that the motor delay observed in these patients is likely not a result of primary muscle abnormalities. © 2013 FEBS.

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

International Nuclear Information System (INIS)

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

2016-01-01

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

Bioreactor perfusion system for the long-term maintenance of tissue-engineered skeletal muscle organoids

Science.gov (United States)

Chromiak, J. A.; Shansky, J.; Perrone, C.; Vandenburgh, H. H.

1998-01-01

Three-dimensional skeletal muscle organ-like structures (organoids) formed in tissue culture by fusion of proliferating myoblasts into parallel networks of long, unbranched myofibers provide an in vivo-like model for examining the effects of growth factors, tension, and space flight on muscle cell growth and metabolism. To determine the feasibility of maintaining either avian or mammalian muscle organoids in a commercial perfusion bioreactor system, we measured metabolism, protein turnover. and autocrine/paracrine growth factor release rates. Medium glucose was metabolized at a constant rate in both low-serum- and serum-free media for up to 30 d. Total organoid noncollagenous protein and DNA content decreased approximately 22-28% (P skeletal muscle growth factors prostaglandin F2alpha (PGF2alpha) and insulin-like growth factor-1 (IGF-1) could be measured accurately in collected media fractions, even after storage at 37 degrees C for up to 10 d. In contrast, creatine kinase activity (a marker of cell damage) in collected media fractions was unreliable. These results provide initial benchmarks for long-term ex vivo studies of tissue-engineered skeletal muscle.

Exercise training and return to a well-balanced diet activate the neuregulin 1/ErbB pathway in skeletal muscle of obese rats

Science.gov (United States)

Ennequin, Gaël; Boisseau, Nathalie; Caillaud, Kevin; Chavanelle, Vivien; Gerbaix, Maude; Metz, Lore; Etienne, Monique; Walrand, Stéphane; Masgrau, Aurélie; Guillet, Christelle; Courteix, Daniel; Niu, Airu; Li, Yi-Ping; Capel, Fréderic; Sirvent, Pascal

2015-01-01

Some studies suggest that the signalling pathway of neuregulin 1 (NRG1), a protein involved in the regulation of skeletal muscle metabolism, could be altered by nutritional and exercise interventions. We hypothesized that diet-induced obesity could lead to alterations of the NRG1 signalling pathway and that chronic exercise could improve NRG1 signalling in rat skeletal muscle. To test this hypothesis, male Wistar rats received a high fat/high sucrose (HF/HS) diet for 16 weeks. At the end of this period, NRG1 and ErbB expression/activity in skeletal muscle was assessed. The obese rats then continued the HF/HS diet or were switched to a well-balanced diet. Moreover, in both groups, half of the animals also performed low intensity treadmill exercise training. After another 8 weeks, NRG1 and ErbB expression/activity in skeletal muscle were tested again. The 16 week HF/HS diet induced obesity, but did not significantly affect the NRG1/ErbB signalling pathway in rat skeletal muscle. Conversely, after the switch to a well-balanced diet, NRG1 cleavage ratio and ErbB4 amount were increased. Chronic exercise training also promoted NRG1 cleavage, resulting in increased ErbB4 phosphorylation. This result was associated with increased protein expression and phosphorylation ratio of the metalloprotease ADAM17, which is involved in NRG1 shedding. Similarly, in vitro stretch-induced activation of ADAM17 in rat myoblasts induced NRG1 cleavage and ErbB4 activation. These results show that low intensity endurance training and well-balanced diet activate the NRG1-ErbB4 pathway, possibly via the metalloprotease ADAM17, in skeletal muscle of diet-induced obese rats. PMID:25820551

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

Science.gov (United States)

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

2016-03-15

The dire effects of cancer-induced cachexia undermine treatment and contribute to decreased survival rates. Therapeutic options for this syndrome are limited, and therefore efforts to identify signs of precachexia in cancer patients are necessary for early intervention. The applications of molecular and functional imaging that would enable a whole-body "holistic" approach to this problem may lead to new insights and advances for diagnosis and treatment of this syndrome. Here we have developed a myoblast optical reporter system with the purpose of identifying early cachectic events. We generated a myoblast cell line expressing a dual tdTomato:GFP construct that was grafted onto the muscle of mice-bearing human pancreatic cancer xenografts to provide noninvasive live imaging of events associated with cancer-induced cachexia (i.e., weight loss). Real-time optical imaging detected a strong tdTomato fluorescent signal from skeletal muscle grafts in mice with weight losses of only 1.2% to 2.7% and tumor burdens of only approximately 79 to 170 mm(3). Weight loss in cachectic animals was also associated with a depletion of lipid, cholesterol, valine, and alanine levels, which may provide informative biomarkers of cachexia. Taken together, our findings demonstrate the utility of a reporter system that is capable of tracking tumor-induced weight loss, an early marker of cachexia. Future studies incorporating resected tissue from human pancreatic ductal adenocarcinoma into a reporter-carrying mouse may be able to provide a risk assessment of cachexia, with possible implications for therapeutic development. ©2015 American Association for Cancer Research.

Transdifferentiation of myoblasts into osteoblasts - possible use for bone therapy.

Science.gov (United States)

Lin, Daphne P L; Carnagarin, Revathy; Dharmarajan, Arun; Dass, Crispin R

2017-12-01

Transdifferentiation is defined as the conversion of one cell type to another and is an ever-expanding field with a growing number of cells found to be capable of such a process. To date, the fact remains that there are limited treatment options for fracture healing, osteoporosis and bone repair post-destruction by bone tumours. Hence, this review focuses on the transdifferentiation of myoblast to osteoblast as a means to further understand the transdifferentiation process and to investigate a potential therapeutic option if successful. The potent osteoinductive effects of the bone morphogenetic protein-2 are largely implicated in the transdifferentiation of myoblast to osteoblast. Bone morphogenetic protein-2-induced activation of the Smad1 protein ultimately results in JunB synthesis, the first transcriptional step in myoblast dedifferentiation. The upregulation of the activating protein-1 binding activity triggers the transcription of the runt-related transcription factor 2 gene, a transcription factor that plays a major role in osteoblast differentiation. This potential transdifferentiation treatment may be utilised for dental implants, fracture healing, osteoporosis and bone repair post-destruction by bone tumours. © 2017 Royal Pharmaceutical Society.

CD36 is required for myoblast fusion during myogenic differentiation

International Nuclear Information System (INIS)

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

2012-01-01

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

CD36 is required for myoblast fusion during myogenic differentiation

Energy Technology Data Exchange (ETDEWEB)

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

2012-11-02

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

A systems-based investigation into vitamin D and skeletal muscle repair, regeneration, and hypertrophy.

Science.gov (United States)

Owens, Daniel J; Sharples, Adam P; Polydorou, Ioanna; Alwan, Nura; Donovan, Timothy; Tang, Jonathan; Fraser, William D; Cooper, Robert G; Morton, James P; Stewart, Claire; Close, Graeme L

2015-12-15

Skeletal muscle is a direct target for vitamin D. Observational studies suggest that low 25[OH]D correlates with functional recovery of skeletal muscle following eccentric contractions in humans and crush injury in rats. However, a definitive association is yet to be established. To address this gap in knowledge in relation to damage repair, a randomised, placebo-controlled trial was performed in 20 males with insufficient concentrations of serum 25(OH)D (45 ± 25 nmol/l). Prior to and following 6 wk of supplemental vitamin D3 (4,000 IU/day) or placebo (50 mg of cellulose), participants performed 20 × 10 damaging eccentric contractions of the knee extensors, with peak torque measured over the following 7 days of recovery. Parallel experimentation using isolated human skeletal muscle-derived myoblast cells from biopsies of 14 males with low serum 25(OH)D (37 ± 11 nmol/l) were subjected to mechanical wound injury, which enabled corresponding in vitro studies of muscle repair, regeneration, and hypertrophy in the presence and absence of 10 or 100 nmol 1α,25(OH)2D3. Supplemental vitamin D3 increased serum 25(OH)D and improved recovery of peak torque at 48 h and 7 days postexercise. In vitro, 10 nmol 1α,25(OH)2D3 improved muscle cell migration dynamics and resulted in improved myotube fusion/differentiation at the biochemical, morphological, and molecular level together with increased myotube hypertrophy at 7 and 10 days postdamage. Together, these preliminary data are the first to characterize a role for vitamin D in human skeletal muscle regeneration and suggest that maintaining serum 25(OH)D may be beneficial for enhancing reparative processes and potentially for facilitating subsequent hypertrophy. Copyright © 2015 the American Physiological Society.

Requirement of myomaker-mediated stem cell fusion for skeletal muscle hypertrophy.

Science.gov (United States)

Goh, Qingnian; Millay, Douglas P

2017-02-10

Fusion of skeletal muscle stem/progenitor cells is required for proper development and regeneration, however the significance of this process during adult muscle hypertrophy has not been explored. In response to muscle overload after synergist ablation in mice, we show that myomaker, a muscle specific membrane protein essential for myoblast fusion, is activated mainly in muscle progenitors and not myofibers. We rendered muscle progenitors fusion-incompetent through genetic deletion of myomaker in muscle stem cells and observed a complete reduction of overload-induced hypertrophy. This blunted hypertrophic response was associated with a reduction in Akt and p70s6k signaling and protein synthesis, suggesting a link between myonuclear accretion and activation of pro-hypertrophic pathways. Furthermore, fusion-incompetent muscle exhibited increased fibrosis after muscle overload, indicating a protective role for normal stem cell activity in reducing myofiber strain associated with hypertrophy. These findings reveal an essential contribution of myomaker-mediated stem cell fusion during physiological adult muscle hypertrophy.

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

Directory of Open Access Journals (Sweden)

Antti Siltanen

2011-04-01

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

Brain and muscle Arnt-like 1 promotes skeletal muscle regeneration through satellite cell expansion

Energy Technology Data Exchange (ETDEWEB)

Chatterjee, Somik [Center for Diabetes Research, Department of Medicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Yin, Hongshan [Center for Diabetes Research, Department of Medicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Department of Cardiovascular Medicine, Third Affiliated Hospital, Hebei Medical University, Shijiazhuang 050051, Hebei (China); Nam, Deokhwa [Center for Diabetes Research, Department of Medicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Li, Yong [Department of Pediatric Surgery, Center for Stem Cell Research and Regenerative Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030 (United States); Ma, Ke, E-mail: kma@houstonmethodist.org [Center for Diabetes Research, Department of Medicine, Houston Methodist Research Institute, Houston, TX 77030 (United States)

2015-02-01

Circadian clock is an evolutionarily conserved timing mechanism governing diverse biological processes and the skeletal muscle possesses intrinsic functional clocks. Interestingly, although the essential clock transcription activator, Brain and muscle Arnt-like 1 (Bmal1), participates in maintenance of muscle mass, little is known regarding its role in muscle growth and repair. In this report, we investigate the in vivo function of Bmal1 in skeletal muscle regeneration using two muscle injury models. Bmal1 is highly up-regulated by cardiotoxin injury, and its genetic ablation significantly impairs regeneration with markedly suppressed new myofiber formation and attenuated myogenic induction. A similarly defective regenerative response is observed in Bmal1-null mice as compared to wild-type controls upon freeze injury. Lack of satellite cell expansion accounts for the regeneration defect, as Bmal1{sup −/−} mice display significantly lower satellite cell number with nearly abolished induction of the satellite cell marker, Pax7. Furthermore, satellite cell-derived primary myoblasts devoid of Bmal1 display reduced growth and proliferation ex vivo. Collectively, our results demonstrate, for the first time, that Bmal1 is an integral component of the pro-myogenic response that is required for muscle repair. This mechanism may underlie its role in preserving adult muscle mass and could be targeted therapeutically to prevent muscle-wasting diseases. - Highlights: • Bmal1 is highly inducible by muscle injury and myogenic stimuli. • Genetic ablation of Bmal1 significantly impairs muscle regeneration. • Bmal1 promotes satellite cell expansion during muscle regeneration. • Bmal1-deficient primary myoblasts display attenuated growth and proliferation.

Brain and muscle Arnt-like 1 promotes skeletal muscle regeneration through satellite cell expansion

International Nuclear Information System (INIS)

Chatterjee, Somik; Yin, Hongshan; Nam, Deokhwa; Li, Yong; Ma, Ke

2015-01-01

Circadian clock is an evolutionarily conserved timing mechanism governing diverse biological processes and the skeletal muscle possesses intrinsic functional clocks. Interestingly, although the essential clock transcription activator, Brain and muscle Arnt-like 1 (Bmal1), participates in maintenance of muscle mass, little is known regarding its role in muscle growth and repair. In this report, we investigate the in vivo function of Bmal1 in skeletal muscle regeneration using two muscle injury models. Bmal1 is highly up-regulated by cardiotoxin injury, and its genetic ablation significantly impairs regeneration with markedly suppressed new myofiber formation and attenuated myogenic induction. A similarly defective regenerative response is observed in Bmal1-null mice as compared to wild-type controls upon freeze injury. Lack of satellite cell expansion accounts for the regeneration defect, as Bmal1 −/− mice display significantly lower satellite cell number with nearly abolished induction of the satellite cell marker, Pax7. Furthermore, satellite cell-derived primary myoblasts devoid of Bmal1 display reduced growth and proliferation ex vivo. Collectively, our results demonstrate, for the first time, that Bmal1 is an integral component of the pro-myogenic response that is required for muscle repair. This mechanism may underlie its role in preserving adult muscle mass and could be targeted therapeutically to prevent muscle-wasting diseases. - Highlights: • Bmal1 is highly inducible by muscle injury and myogenic stimuli. • Genetic ablation of Bmal1 significantly impairs muscle regeneration. • Bmal1 promotes satellite cell expansion during muscle regeneration. • Bmal1-deficient primary myoblasts display attenuated growth and proliferation

Water-Hydrogel Binding Affinity Modulates Freeze-Drying-Induced Micropore Architecture and Skeletal Myotube Formation.

Science.gov (United States)

Rich, Max H; Lee, Min Kyung; Marshall, Nicholas; Clay, Nicholas; Chen, Jinrong; Mahmassani, Ziad; Boppart, Marni; Kong, Hyunjoon

2015-08-10

Freeze-dried hydrogels are increasingly used to create 3D interconnected micropores that facilitate biomolecular and cellular transports. However, freeze-drying is often plagued by variance in micropore architecture based on polymer choice. We hypothesized that water-polymer binding affinity plays a significant role in sizes and numbers of micropores formed through freeze-drying, influencing cell-derived tissue quality. Poly(ethylene glycol)diacrylate (PEGDA) hydrogels with alginate methacrylate (AM) were used due to AM's higher binding affinity for water than PEGDA. PEGDA-AM hydrogels with larger AM concentrations resulted in larger sizes and numbers of micropores than pure PEGDA hydrogels, attributed to the increased mass of water binding to the PEGDA-AM gel. Skeletal myoblasts loaded in microporous PEGDA-AM hydrogels were active to produce 3D muscle-like tissue, while those loaded in pure PEGDA gels were localized on the gel surface. We propose that this study will be broadly useful in designing and improving the performance of various microporous gels.

Ductile electroactive biodegradable hyperbranched polylactide copolymers enhancing myoblast differentiation.

Science.gov (United States)

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

2015-12-01

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

Ursolic acid increases skeletal muscle and brown fat and decreases diet-induced obesity, glucose intolerance and fatty liver disease.

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Steven D Kunkel

Full Text Available Skeletal muscle Akt activity stimulates muscle growth and imparts resistance to obesity, glucose intolerance and fatty liver disease. We recently found that ursolic acid increases skeletal muscle Akt activity and stimulates muscle growth in non-obese mice. Here, we tested the hypothesis that ursolic acid might increase skeletal muscle Akt activity in a mouse model of diet-induced obesity. We studied mice that consumed a high fat diet lacking or containing ursolic acid. In skeletal muscle, ursolic acid increased Akt activity, as well as downstream mRNAs that promote glucose utilization (hexokinase-II, blood vessel recruitment (Vegfa and autocrine/paracrine IGF-I signaling (Igf1. As a result, ursolic acid increased skeletal muscle mass, fast and slow muscle fiber size, grip strength and exercise capacity. Interestingly, ursolic acid also increased brown fat, a tissue that shares developmental origins with skeletal muscle. Consistent with increased skeletal muscle and brown fat, ursolic acid increased energy expenditure, leading to reduced obesity, improved glucose tolerance and decreased hepatic steatosis. These data support a model in which ursolic acid reduces obesity, glucose intolerance and fatty liver disease by increasing skeletal muscle and brown fat, and suggest ursolic acid as a potential therapeutic approach for obesity and obesity-related illness.

Estrogens enhance myoblast differentiation in facioscapulohumeral muscular dystrophy by antagonizing DUX4 activity

OpenAIRE

Teveroni, Emanuela; Pellegrino, Marsha; Sacconi, Sabrina; Calandra, Patrizia; Cascino, Isabella; Farioli-Vecchioli, Stefano; Puma, Angela; Garibaldi, Matteo; Morosetti, Roberta; Tasca, Giorgio; Ricci, Enzo; Trevisan, Carlo Pietro; Galluzzi, Giuliana; Pontecorvi, Alfredo; Crescenzi, Marco

2017-01-01

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is characterized by extreme variability in symptoms, with females being less severely affected than males and presenting a higher proportion of asymptomatic carriers. The sex-related factors involved in the disease are not known. Here, we have utilized myoblasts isolated from FSHD patients (FSHD myoblasts) to investigate the effect of estrogens on muscle properties. Our results demonstrated that...

Advancements in stem cells treatment of skeletal muscle wasting

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mirella emeregalli

2014-02-01

Full Text Available Muscular dystrophies (MDs are a heterogeneous group of inherited disorders, in which progressive muscle wasting and weakness is often associated with exhaustion of muscle regeneration potential. Although physiological properties of skeletal muscle tissue are now well known, no treatments are effective for these diseases. Muscle regeneration was attempted by means transplantation of myogenic cells (from myoblast to embryonic stem cells and also by interfering with the malignant processes that originate in pathological tissues, such as uncontrolled fibrosis and inflammation. Taking into account the advances in the isolation of new subpopulation of stem cells and in the creation of artificial stem cell niches, we discuss how these emerging technologies offer great promises for therapeutic approaches to muscle diseases and muscle wasting associated with aging.

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.)

Creating Interactions between Tissue-Engineered Skeletal Muscle and the Peripheral Nervous System.

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Smith, Alec S T; Passey, Samantha L; Martin, Neil R W; Player, Darren J; Mudera, Vivek; Greensmith, Linda; Lewis, Mark P

2016-01-01

Effective models of mammalian tissues must allow and encourage physiologically (mimetic) correct interactions between co-cultured cell types in order to produce culture microenvironments as similar as possible to those that would normally occur in vivo. In the case of skeletal muscle, the development of such a culture model, integrating multiple relevant cell types within a biomimetic scaffold, would be of significant benefit for investigations into the development, functional performance, and pathophysiology of skeletal muscle tissue. Although some work has been published regarding the behaviour of in vitro muscle models co-cultured with organotypic slices of CNS tissue or with stem cell-derived neurospheres, little investigation has so far been made regarding the potential to maintain isolated motor neurons within a 3D biomimetic skeletal muscle culture platform. Here, we review the current state of the art for engineering neuromuscular contacts in vitro and provide original data detailing the development of a 3D collagen-based model for the co-culture of primary muscle cells and motor neurons. The devised culture system promotes increased myoblast differentiation, forming arrays of parallel, aligned myotubes on which areas of nerve-muscle contact can be detected by immunostaining for pre- and post-synaptic proteins. Quantitative RT-PCR results indicate that motor neuron presence has a positive effect on myotube maturation, suggesting neural incorporation influences muscle development and maturation in vitro. The importance of this work is discussed in relation to other published neuromuscular co-culture platforms along with possible future directions for the field. © 2016 S. Karger AG, Basel.

Leukemia inhibitory factor increases glucose uptake in mouse skeletal muscle

DEFF Research Database (Denmark)

Brandt, Nina; O'Neill, Hayley M; Kleinert, Maximilian

2015-01-01

INTRODUCTION: Members of the interleukin-6 (IL-6) family, IL-6 and ciliary neurotrophic factor (CNTF) have been shown to increase glucose uptake and fatty acid oxidation in skeletal muscle. However, the metabolic effects of another family member, leukemia inhibitory factor (LIF), are not well...

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

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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.

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

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

2011-04-29

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

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

International Nuclear Information System (INIS)

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

2011-01-01

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

Study of muscle cell dedifferentiation after skeletal muscle injury of mice with a Cre-Lox system.

Science.gov (United States)

Mu, Xiaodong; Peng, Hairong; Pan, Haiying; Huard, Johnny; Li, Yong

2011-02-03

Dedifferentiation of muscle cells in the tissue of mammals has yet to be observed. One of the challenges facing the study of skeletal muscle cell dedifferentiation is the availability of a reliable model that can confidentially distinguish differentiated cell populations of myotubes and non-fused mononuclear cells, including stem cells that can coexist within the population of cells being studied. In the current study, we created a Cre/Lox-β-galactosidase system, which can specifically tag differentiated multinuclear myotubes and myotube-generated mononuclear cells based on the activation of the marker gene, β-galactosidase. By using this system in an adult mouse model, we found that β-galactosidase positive mononuclear cells were generated from β-galactosidase positive multinuclear myofibers upon muscle injury. We also demonstrated that these mononuclear cells can develop into a variety of different muscle cell lineages, i.e., myoblasts, satellite cells, and muscle derived stem cells. These novel findings demonstrated, for the first time, that cellular dedifferentiation of skeletal muscle cells actually occurs in mammalian skeletal muscle following traumatic injury in vivo.

The effect of caffeine on skeletal muscle anabolic signaling and hypertrophy.

Science.gov (United States)

Moore, Timothy M; Mortensen, Xavier M; Ashby, Conrad K; Harris, Alexander M; Kump, Karson J; Laird, David W; Adams, Aaron J; Bray, Jeremy K; Chen, Ting; Thomson, David M

2017-06-01

Caffeine is a widely consumed stimulant with the potential to enhance physical performance through multiple mechanisms. However, recent in vitro findings have suggested that caffeine may block skeletal muscle anabolic signaling through AMP-activated protein kinase (AMPK)-mediated inhibition of mechanistic target of rapamycin (mTOR) signaling pathway. This could negatively affect protein synthesis and the capacity for muscle growth. The primary purpose of this study was to assess the effect of caffeine on in vivo AMPK and mTOR pathway signaling, protein synthesis, and muscle growth. In cultured C2C12 muscle cells, physiological levels of caffeine failed to impact mTOR activation or myoblast proliferation or differentiation. We found that caffeine administration to mice did not significantly enhance the phosphorylation of AMPK or inhibit signaling proteins downstream of mTOR (p70S6k, S6, or 4EBP1) or protein synthesis after a bout of electrically stimulated contractions. Skeletal muscle-specific knockout of LKB1, the primary AMPK activator in skeletal muscle, on the other hand, eliminated AMPK activation by contractions and enhanced S6k, S6, and 4EBP1 activation before and after contractions. In rats, the addition of caffeine did not affect plantaris hypertrophy induced by the tenotomy of the gastrocnemius and soleus muscles. In conclusion, caffeine administration does not impair skeletal muscle load-induced mTOR signaling, protein synthesis, or muscle hypertrophy.

Productive infection of human skeletal muscle cells by pandemic and seasonal influenza A(H1N1 viruses.

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Marion Desdouits

Full Text Available Besides the classical respiratory and systemic symptoms, unusual complications of influenza A infection in humans involve the skeletal muscles. Numerous cases of acute myopathy and/or rhabdomyolysis have been reported, particularly following the outbreak of pandemic influenza A(H1N1 in 2009. The pathogenesis of these influenza-associated myopathies (IAM remains unkown, although the direct infection of muscle cells is suspected. Here, we studied the susceptibility of cultured human primary muscle cells to a 2009 pandemic and a 2008 seasonal influenza A(H1N1 isolate. Using cells from different donors, we found that differentiated muscle cells (i. e. myotubes were highly susceptible to infection by both influenza A(H1N1 isolates, whereas undifferentiated cells (i. e. myoblasts were partially resistant. The receptors for influenza viruses, α2-6 and α2-3 linked sialic acids, were detected on the surface of myotubes and myoblasts. Time line of viral nucleoprotein (NP expression and nuclear export showed that the first steps of the viral replication cycle could take place in muscle cells. Infected myotubes and myoblasts exhibited budding virions and nuclear inclusions as observed by transmission electron microscopy and correlative light and electron microscopy. Myotubes, but not myoblasts, yielded infectious virus progeny that could further infect naive muscle cells after proteolytic treatment. Infection led to a cytopathic effect with the lysis of muscle cells, as characterized by the release of lactate dehydrogenase. The secretion of proinflammatory cytokines by muscle cells was not affected following infection. Our results are compatible with the hypothesis of a direct muscle infection causing rhabdomyolysis in IAM patients.

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

Efficient direct conversion of human fibroblasts into myogenic lineage induced by co-transduction with MYCL and MYOD1.

Science.gov (United States)

Wakao, Junko; Kishida, Tsunao; Fumino, Shigehisa; Kimura, Koseki; Yamamoto, Kenta; Kotani, Shin-Ichiro; Mizushima, Katsura; Naito, Yuji; Yoshikawa, Toshikazu; Tajiri, Tatsuro; Mazda, Osam

2017-06-24

The skeletal muscle consists of contractile myofibers and plays essential roles for maintenance of body posture, movement, and metabolic regulation. During the development and regeneration of the skeletal muscle tissue, the myoblasts fuse into multinucleated myotubes that subsequently form myofibers. Transplantation of myoblasts may make possible a novel regenerative therapy against defects or dysfunction of the skeletal muscle. It is reported that rodent fibroblasts are converted into myoblast-like cells and fuse to form syncytium after forced expression of exogenous myogenic differentiation 1 (MYOD1) that is a key transcription factor for myoblast differentiation. But human fibroblasts are less efficiently converted into myoblasts and rarely fused by MYOD1 alone. Here we found that transduction of v-myc avian myelocytomatosis viral oncogene lung carcinoma derived homolog (MYCL) gene in combination with MYOD1 gene induced myoblast-like phenotypes in human fibroblasts more strongly than MYOD1 gene alone. The rate of conversion was approximately 90%. The directly converted myoblasts (dMBs) underwent fusion in an ERK5 pathway-dependent manner. The dMBs also formed myofiber-like structure in vivo after an inoculation into mice at the subcutaneous tissue. The present results strongly suggest that the combination of MYCL plus MYOD1 may promote direct conversion of human fibroblasts into functional myoblasts that could potentially be used for regenerative therapy for muscle diseases and congenital muscle defects. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Institute of Scientific and Technical Information of China (English)

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.

The role of hyperplasia on the increase of skeletal muscle

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Victor Hugo Maciel Meloni

2005-06-01

Full Text Available Skeletal muscle hypertrophy is resulted from the individual increase of the fiber cross-sectional area. This adaptative phenomenon is normally observed in the muscle tissue submitted to a regimen of physical exercises, like strength training. The degree of muscular hypertrophy is directly related to the type of exercise and its intensity. Strength training normally produces a hypertrophy of greater magnitude when compared to other types of physical exercise. However, it is possible that there is another adaptive mechanism contributing for increasing skeletal muscle size. This mechanism is called hyperplasia, and can be defined as an increase in the cells, or fibers, number in the muscle. This brief review aims to verify the role of hyperplasia in the increase of skeletal muscle size. RESUMO A hipertrofia do músculo esquelético é resultado do aumento individual da área transversal da fibra. Este fenômeno adaptativo é comumente observado no tecido muscular submetido à um regime de exercícios físicos, como o treinamento de força. O grau de hipertrofia muscular está diretamente relacionado ao tipo de exercício e sua intensidade. O treinamento de força normalmente produz uma hipertrofia de maior magnitude, quando comparada aos outros tipos de exercício físico. Todavia, é provável que haja outro mecanismo adaptativo contribuindo para a hipertrofia do músculo esquelético. Este mecanismo chama-se hiperplasia, e pode ser traduzida por um aumento no número de células, ou fibras musculares em relação ao número original. Este breve resumo tem por objetivo verificar qual é o papel da hiperplasia na hipertrofia do músculo esquelético.

Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy

Science.gov (United States)

Powell, C.; Shansky, J.; Del Tatto, M.; Forman, D. E.; Hennessey, J.; Sullivan, K.; Zielinski, B. A.; Vandenburgh, H. H.

1999-01-01

Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

The adipokine leptin increases skeletal muscle mass and significantly alters skeletal muscle miRNA expression profile in aged mice

International Nuclear Information System (INIS)

Hamrick, Mark W.; Herberg, Samuel; Arounleut, Phonepasong; He, Hong-Zhi; Shiver, Austin; Qi, Rui-Qun; Zhou, Li; Isales, Carlos M.

2010-01-01

Research highlights: → Aging is associated with muscle atrophy and loss of muscle mass, known as the sarcopenia of aging. → We demonstrate that age-related muscle atrophy is associated with marked changes in miRNA expression in muscle. → Treating aged mice with the adipokine leptin significantly increased muscle mass and the expression of miRNAs involved in muscle repair. → Recombinant leptin therapy may therefore be a novel approach for treating age-related muscle atrophy. -- Abstract: Age-associated loss of muscle mass, or sarcopenia, contributes directly to frailty and an increased risk of falls and fractures among the elderly. Aged mice and elderly adults both show decreased muscle mass as well as relatively low levels of the fat-derived hormone leptin. Here we demonstrate that loss of muscle mass and myofiber size with aging in mice is associated with significant changes in the expression of specific miRNAs. Aging altered the expression of 57 miRNAs in mouse skeletal muscle, and many of these miRNAs are now reported to be associated specifically with age-related muscle atrophy. These include miR-221, previously identified in studies of myogenesis and muscle development as playing a role in the proliferation and terminal differentiation of myogenic precursors. We also treated aged mice with recombinant leptin, to determine whether leptin therapy could improve muscle mass and alter the miRNA expression profile of aging skeletal muscle. Leptin treatment significantly increased hindlimb muscle mass and extensor digitorum longus fiber size in aged mice. Furthermore, the expression of 37 miRNAs was altered in muscles of leptin-treated mice. In particular, leptin treatment increased the expression of miR-31 and miR-223, miRNAs known to be elevated during muscle regeneration and repair. These findings suggest that aging in skeletal muscle is associated with marked changes in the expression of specific miRNAs, and that nutrient-related hormones such as leptin

The adipokine leptin increases skeletal muscle mass and significantly alters skeletal muscle miRNA expression profile in aged mice

Energy Technology Data Exchange (ETDEWEB)

Hamrick, Mark W., E-mail: mhamrick@mail.mcg.edu [Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Department of Orthopaedic Surgery, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Herberg, Samuel; Arounleut, Phonepasong [Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Department of Orthopaedic Surgery, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); He, Hong-Zhi [Henry Ford Immunology Program, Henry Ford Health System, Detroit, MI (United States); Department of Dermatology, Henry Ford Health System, Detroit, MI (United States); Shiver, Austin [Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Department of Orthopaedic Surgery, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Qi, Rui-Qun [Henry Ford Immunology Program, Henry Ford Health System, Detroit, MI (United States); Department of Dermatology, Henry Ford Health System, Detroit, MI (United States); Zhou, Li [Henry Ford Immunology Program, Henry Ford Health System, Detroit, MI (United States); Department of Dermatology, Henry Ford Health System, Detroit, MI (United States); Department of Internal Medicine, Henry Ford Health System, Detroit, MI (United States); Isales, Carlos M. [Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Department of Orthopaedic Surgery, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); others, and

2010-09-24

Research highlights: {yields} Aging is associated with muscle atrophy and loss of muscle mass, known as the sarcopenia of aging. {yields} We demonstrate that age-related muscle atrophy is associated with marked changes in miRNA expression in muscle. {yields} Treating aged mice with the adipokine leptin significantly increased muscle mass and the expression of miRNAs involved in muscle repair. {yields} Recombinant leptin therapy may therefore be a novel approach for treating age-related muscle atrophy. -- Abstract: Age-associated loss of muscle mass, or sarcopenia, contributes directly to frailty and an increased risk of falls and fractures among the elderly. Aged mice and elderly adults both show decreased muscle mass as well as relatively low levels of the fat-derived hormone leptin. Here we demonstrate that loss of muscle mass and myofiber size with aging in mice is associated with significant changes in the expression of specific miRNAs. Aging altered the expression of 57 miRNAs in mouse skeletal muscle, and many of these miRNAs are now reported to be associated specifically with age-related muscle atrophy. These include miR-221, previously identified in studies of myogenesis and muscle development as playing a role in the proliferation and terminal differentiation of myogenic precursors. We also treated aged mice with recombinant leptin, to determine whether leptin therapy could improve muscle mass and alter the miRNA expression profile of aging skeletal muscle. Leptin treatment significantly increased hindlimb muscle mass and extensor digitorum longus fiber size in aged mice. Furthermore, the expression of 37 miRNAs was altered in muscles of leptin-treated mice. In particular, leptin treatment increased the expression of miR-31 and miR-223, miRNAs known to be elevated during muscle regeneration and repair. These findings suggest that aging in skeletal muscle is associated with marked changes in the expression of specific miRNAs, and that nutrient

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

Science.gov (United States)

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

2012-09-01

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

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

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Louise H Jørgensen

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

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

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

2009-06-01

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

Acute exercise induces biphasic increase in respiratory mRNA in skeletal muscle

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Ikeda, Shin-ichi; Kizaki, Takako; Haga, Shukoh; Ohno, Hideki; Takemasa, Tohru

2008-01-01

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) promotes the expression of oxidative enzymes in skeletal muscle. We hypothesized that activation of the p38 MAPK (mitogen-activated protein kinase) in response to exercise was associated with exercise-induced PGC-1α and respiratory enzymes expression and aimed to demonstrate this under the physiological level. We subjected mice to a single bout of treadmill running and found that the exercise induced a biphasic increase in the expression of respiratory enzymes mRNA. The second phase of the increase was accompanied by an increase in PGC-1α protein, but the other was not. Administration of SB203580 (SB), an inhibitor of p38 MAPK, suppressed the increase in PGC-1α expression and respiratory enzymes mRNA in both phases. These data suggest that p38 MAPK is associated with the exercise-induced expression of PGC-1α and biphasic increase in respiratory enzyme mRNAs in mouse skeletal muscle under physiological conditions

Skeletal myocyte hypertrophy requires mTOR kinase activity and S6K1

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Park, In-Hyun; Erbay, Ebru; Nuzzi, Paul; Chen Jie

2005-01-01

The protein kinase mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and growth, with the ribosomal subunit S6 kinase 1 (S6K1) as one of the key downstream signaling effectors. A critical role of mTOR signaling in skeletal muscle differentiation has been identified recently, and an unusual regulatory mechanism independent of mTOR kinase activity and S6K1 is revealed. An mTOR pathway has also been reported to regulate skeletal muscle hypertrophy, but the regulatory mechanism is not completely understood. Here, we report the investigation of mTOR's function in insulin growth factor I (IGF-I)-induced C2C12 myotube hypertrophy. Added at a later stage when rapamycin no longer had any effect on normal myocyte differentiation, rapamycin completely blocked myocyte hypertrophy as measured by myotube diameter. Importantly, a concerted increase of average myonuclei per myotube was observed in IGF-I-stimulated myotubes, which was also inhibited by rapamycin added at a time when it no longer affected normal differentiation. The mTOR protein level, its catalytic activity, its phosphorylation on Ser2448, and the activity of S6K1 were all found increased in IGF-I-stimulated myotubes compared to unstimulated myotubes. Using C2C12 cells stably expressing rapamycin-resistant forms of mTOR and S6K1, we provide genetic evidence for the requirement of mTOR and its downstream effector S6K1 in the regulation of myotube hypertrophy. Our results suggest distinct mTOR signaling mechanisms in different stages of skeletal muscle development: While mTOR regulates the initial myoblast differentiation in a kinase-independent and S6K1-independent manner, the hypertrophic function of mTOR requires its kinase activity and employs S6K1 as a downstream effector

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

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

2017-07-01

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

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

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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. Copyright © 2012 John Wiley & Sons, Ltd.

Myoblast sensitivity and fibroblast insensitivity to osteogenic conversion by BMP-2 correlates with the expression of Bmpr-1a

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North Kathryn N

2009-05-01

Full Text Available Abstract Background Osteoblasts are considered to primarily arise from osseous progenitors within the periosteum or bone marrow. We have speculated that cells from local soft tissues may also take on an osteogenic phenotype. Myoblasts are known to adopt a bone gene program upon treatment with the osteogenic bone morphogenetic proteins (BMP-2,-4,-6,-7,-9, but their osteogenic capacity relative to other progenitor types is unclear. We further hypothesized that the sensitivity of cells to BMP-2 would correlate with BMP receptor expression. Methods We directly compared the BMP-2 sensitivity of myoblastic murine cell lines and primary cells with osteoprogenitors from osseous tissues and fibroblasts. Fibroblasts forced to undergo myogenic conversion by transduction with a MyoD-expressing lentiviral vector (LV-MyoD were also examined. Outcome measures included alkaline phosphatase expression, matrix mineralization, and expression of osteogenic genes (alkaline phosphatase, osteocalcin and bone morphogenetic protein receptor-1A as measured by quantitative PCR. Results BMP-2 induced a rapid and robust osteogenic response in myoblasts and osteoprogenitors, but not in fibroblasts. Myoblasts and osteoprogenitors grown in osteogenic media rapidly upregulated Bmpr-1a expression. Chronic BMP-2 treatment resulted in peak Bmpr-1a expression at day 6 before declining, suggestive of a negative feedback mechanism. In contrast, fibroblasts expressed low levels of Bmpr-1a that was only weakly up-regulated by BMP-2 treatment. Bioinformatics analysis confirmed the presence of myogenic responsive elements in the proximal promoter region of human and murine BMPR-1A/Bmpr-1a. Forced myogenic gene expression in fibroblasts was associated with a significant increase in Bmpr-1a expression and a synergistic increase in the osteogenic response to BMP-2. Conclusion These data demonstrate the osteogenic sensitivity of muscle progenitors and provide a mechanistic insight into the

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

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Sellathurai, Jeeva; Nielsen, Joachim; Hejbøl, Eva Kildall

2016-01-01

-PCR, immunocytochemistry and western blot. RESULTS AND CONCLUSIONS: We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET......, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch...... and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β...

Myostatin-deficiency in mice increases global gene expression at the Dlk1-Dio3 locus in the skeletal muscle.

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Hitachi, Keisuke; Tsuchida, Kunihiro

2017-01-24

Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle growth and development. Myostatin inhibition leads to increased skeletal muscle mass in mammals; hence, myostatin is considered a potential therapeutic target for skeletal muscle wasting. However, downstream molecules of myostatin in the skeletal muscle have not been fully elucidated. Here, we identified the Dlk1-Dio3 locus at the mouse chromosome 12qF1, also called as the callipyge locus in sheep, as a novel downstream target of myostatin. In skeletal muscle of myostatin knockout mice, the expression of mature miRNAs at the Dlk1-Dio3 locus was significantly increased. The increased miRNA levels are caused by the transcriptional activation of the Dlk1-Dio3 locus, because a significant increase in the primary miRNA transcript was observed in myostatin knockout mice. In addition, we found increased expression of coding and non-coding genes (Dlk1, Gtl2, Rtl1/Rtl1as, and Rian) at the Dlk1-Dio3 locus in myostatin-deficient skeletal muscle. Moreover, epigenetic changes, associated with the regulation of the Dlk1-Dio3 locus, were observed in myostatin knockout mice. Taken together, this is the first report demonstrating the role of myostatin in regulating the Dlk1-Dio3 (the callipyge) locus in the skeletal muscle.

L-Citrulline Supplementation-Increased Skeletal Muscle PGC-1α Expression is Associated With Exercise Performance and Increased Skeletal Muscle Weight.

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Villareal, Myra O; Matsukawa, Toshiya; Isoda, Hiroko

2018-05-24

L-citrulline has recently been reported as a more effective supplement for promoting intracellular NO production compared to L-arginine. Here, the effect of L-citrulline on skeletal muscle and its influence on exercise performance were investigated. The underlying mechanism of its effect, specifically on the expression of skeletal muscle peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), was also elucidated. Six-week-old ICR mice were orally supplemented with L-citrulline (250 mg kg -1 ) daily, and their performance in weight-loaded swimming exercise every other day for 15 days, was evaluated. In addition, mice muscles were weighed and evaluated for the expression of PGC-1α and PGC-1α-regulated genes. Mice orally supplemented with L-citrulline had significantly higher gastrocnemius and biceps femoris muscle mass. Although not statistically significant, L-citrulline prolonged the swimming time to exhaustion. PGC-1α upregulation was associated with vascular endothelial growth factor α (VEGFα) and insulin-like growth factor 1 (IGF1) upregulation. VEGFα and IGF1 are important for angiogenesis and muscle growth, respectively, and are regulated by PGC-1α. Treatment with L-NAME, a nitric oxide synthesis inhibitor, suppressed the L-citrulline-induced PGC-1α upregulation in-vitro. Supplementation with L-citrulline upregulates skeletal muscle PGC-1α levels resulting to higher skeletal muscle weight that improves time to exhaustion during exercise. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Biodistribution studies of 99mTc-labeled myoblasts in a murine model of muscular dystrophy

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Colombo, F.R.; Torrente, Y.; Casati, R.; Benti, R.; Corti, S.; Salani, S.; D'Angelo, M.G.; DeLiso, A.; Scarlato, G.; Bresolin, N.; Gerundini, P.

2001-01-01

The purpose of this study was twofold: first, to evaluate the myoblast labeling of various 99m Tc complexes and to select the complex that best accomplishes this labeling, and second to evaluate the biodistribution of myoblasts labeled with this complex using mice with MDX muscular dystrophy (the murine homologue of Duchenne's muscular dystrophy). The following ligands were used to prepare the corresponding 99m Tc complexes: hexakis-methoxy-isobutyl-isonitrile (MIBI), bis(2-ethoxyethyl)diphosphinoethane (Tf), (RR,SS)-4,8-diaza-3,6,6,9-tetramethyl-undecane-2,10-dione-bisoxime (HM-PAO), bis(N-ethyl)dithiocarbamate (NEt), and bis(N-ethoxy, N-ethyl)dithiocarbamate (NOEt). One million murine myoblasts were incubated for 30-60 minutes with 5 mCi of each of the 99mTc complexes prepared from the above ligands. Viability was assessed by microscopic counting after trypan blue staining, and the radioactivity absorbed in the cells was measured after centrifugation. The compound with the highest uptake in cellular pellets was [ 99m Tc]N-NOEt. The biodistribution of myoblasts labeled with this complex was evaluated after intraaortic injection in dystrophic mice. Such an approach has the potential of effecting widespread gene transfer through the bloodstream to muscles lacking dystrophin

Complex interactions between human myoblasts and the surrounding 3D fibrin-based matrix.

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Stéphane Chiron

Full Text Available Anchorage of muscle cells to the extracellular matrix is crucial for a range of fundamental biological processes including migration, survival and differentiation. Three-dimensional (3D culture has been proposed to provide a more physiological in vitro model of muscle growth and differentiation than routine 2D cultures. However, muscle cell adhesion and cell-matrix interplay of engineered muscle tissue remain to be determined. We have characterized cell-matrix interactions in 3D muscle culture and analyzed their consequences on cell differentiation. Human myoblasts were embedded in a fibrin matrix cast between two posts, cultured until confluence, and then induced to differentiate. Myoblasts in 3D aligned along the longitudinal axis of the gel. They displayed actin stress fibers evenly distributed around the nucleus and a cortical mesh of thin actin filaments. Adhesion sites in 3D were smaller in size than in rigid 2D culture but expression of adhesion site proteins, including α5 integrin and vinculin, was higher in 3D compared with 2D (p<0.05. Myoblasts and myotubes in 3D exhibited thicker and ellipsoid nuclei instead of the thin disk-like shape of the nuclei in 2D (p<0.001. Differentiation kinetics were faster in 3D as demonstrated by higher mRNA concentrations of α-actinin and myosin. More important, the elastic modulus of engineered muscle tissues increased significantly from 3.5 ± 0.8 to 7.4 ± 4.7 kPa during proliferation (p<0.05 and reached 12.2 ± 6.0 kPa during differentiation (p<0.05, thus attesting the increase of matrix stiffness during proliferation and differentiation of the myocytes. In conclusion, we reported modulations of the adhesion complexes, the actin cytoskeleton and nuclear shape in 3D compared with routine 2D muscle culture. These findings point to complex interactions between muscle cells and the surrounding matrix with dynamic regulation of the cell-matrix stiffness.

Oxidative stress induces caveolin 1 degradation and impairs caveolae functions in skeletal muscle cells.

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Alexis Mougeolle

Full Text Available Increased level of oxidative stress, a major actor of cellular aging, impairs the regenerative capacity of skeletal muscle and leads to the reduction in the number and size of muscle fibers causing sarcopenia. Caveolin 1 is the major component of caveolae, small membrane invaginations involved in signaling and endocytic trafficking. Their role has recently expanded to mechanosensing and to the regulation of oxidative stress-induced pathways. Here, we increased the amount of reactive oxidative species in myoblasts by addition of hydrogen peroxide (H2O2 at non-toxic concentrations. The expression level of caveolin 1 was significantly decreased as early as 10 min after 500 μM H2O2 treatment. This reduction was not observed in the presence of a proteasome inhibitor, suggesting that caveolin 1 was rapidly degraded by the proteasome. In spite of caveolin 1 decrease, caveolae were still able to assemble at the plasma membrane. Their functions however were significantly perturbed by oxidative stress. Endocytosis of a ceramide analog monitored by flow cytometry was significantly diminished after H2O2 treatment, indicating that oxidative stress impaired its selective internalization via caveolae. The contribution of caveolae to the plasma membrane reservoir has been monitored after osmotic cell swelling. H2O2 treatment increased membrane fragility revealing that treated cells were more sensitive to an acute mechanical stress. Altogether, our results indicate that H2O2 decreased caveolin 1 expression and impaired caveolae functions. These data give new insights on age-related deficiencies in skeletal muscle.

Primary skeletal muscle myoblasts from chronic heart failure patients exhibit loss of anti-inflammatory and proliferative activity

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Sente, T.; Berendoncks, A.M. Van; Jonckheere, A.I.; Rodenburg, R.J.T.; Lauwers, P.; Hoof, V. Van; Wouters, A.; Lardon, F.; Hoymans, V.Y.; Vrints, C.J.

2016-01-01

BACKGROUND: Peripheral skeletal muscle wasting is a common finding with adverse effects in chronic heart failure (HF). Whereas its clinical relevance is beyond doubt, the underlying pathophysiological mechanisms are not yet fully elucidated. We aimed to introduce and characterize the primary culture

Proliferation of Human Primary Myoblasts Is Associated with Altered Energy Metabolism in Dependence on Ageing In Vivo and In Vitro

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Reedik Pääsuke

2016-01-01

Full Text Available Background. Ageing is associated with suppressed regenerative potential of muscle precursor cells due to decrease of satellite cells and suppressive intramuscular milieu on their activation, associated with ageing-related low-grade inflammation. The aim of the study was to characterize the function of oxidative phosphorylation (OXPHOS, glycolysis, adenylate kinase (AK, and creatine kinase (CK mediated systems in young and older individuals. Materials and Methods. Myoblasts were cultivated from biopsies taken by transcutaneous conchotomy from vastus lateralis muscle in young (20–29 yrs, n=7 and older (70–79 yrs, n=7 subjects. Energy metabolism was assessed in passages 2 to 6 by oxygraphy and enzyme analysis. Results. In myoblasts of young and older subjects the rate of OXPHOS decreased during proliferation from passages 2 to 6. The total activities of CK and AK decreased. Myoblasts of passage 2 cultivated from young muscle showed higher rate of OXPHOS and activities of CK and AK compared to myoblasts from older subjects while hexokinase and pyruvate kinase were not affected by ageing. Conclusions. Proliferation of myoblasts in vitro is associated with downregulation of OXPHOS and energy storage and transfer systems. Ageing in vivo exerts an impact on satellite cells which results in altered metabolic profile in favour of the prevalence of glycolytic pathways over mitochondrial OXPHOS of myoblasts.

Inhibition of Na+ channel currents in rat myoblasts by 4-aminopyridine

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Lu Boxun; Liu Linyun; Liao Lei; Zhang Zhihong; Mei Yanai

2005-01-01

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

Exercise increases TBC1D1 phosphorylation in human skeletal muscle

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Jessen, Niels; An, Ding; Lihn, Aina S.; Nygren, Jonas; Hirshman, Michael F.; Thorell, Anders

2011-01-01

Exercise and weight loss are cornerstones in the treatment and prevention of type 2 diabetes, and both interventions function to increase insulin sensitivity and glucose uptake into skeletal muscle. Studies in rodents demonstrate that the underlying mechanism for glucose uptake in muscle involves site-specific phosphorylation of the Rab-GTPase-activating proteins AS160 (TBC1D4) and TBC1D1. Multiple kinases, including Akt and AMPK, phosphorylate TBC1D1 and AS160 on distinct residues, regulating their activity and allowing for GLUT4 translocation. In contrast to extensive rodent-based studies, the regulation of AS160 and TBC1D1 in human skeletal muscle is not well understood. In this study, we determined the effects of dietary intervention and a single bout of exercise on TBC1D1 and AS160 site-specific phosphorylation in human skeletal muscle. Ten obese (BMI 33.4 ± 2.4, M-value 4.3 ± 0.5) subjects were studied at baseline and after a 2-wk dietary intervention. Muscle biopsies were obtained from the subjects in the resting (basal) state and immediately following a 30-min exercise bout (70% V̇o2 max). Muscle lysates were analyzed for AMPK activity and Akt phosphorylation and for TBC1D1 and AS160 phosphorylation on known or putative AMPK and Akt sites as follows: AS160 Ser711 (AMPK), TBC1D1 Ser231 (AMPK), TBC1D1 Ser660 (AMPK), TBC1D1 Ser700 (AMPK), and TBC1D1 Thr590 (Akt). The diet intervention that consisted of a major shift in the macronutrient composition resulted in a 4.2 ± 0.4 kg weight loss (P < 0.001) and a significant increase in insulin sensitivity (M value 5.6 ± 0.6), but surprisingly, there was no effect on expression or phosphorylation of any of the muscle-signaling proteins. Exercise increased muscle AMPKα2 activity but did not increase Akt phosphorylation. Exercise increased phosphorylation on AS160 Ser711, TBC1D1 Ser231, and TBC1D1 Ser660 but had no effect on TBC1D1 Ser700. Exercise did not increase TBC1D1 Thr590 phosphorylation or TBC1D1/AS160 PAS

Increased Stiffness in Aged Skeletal Muscle Impairs Muscle Progenitor Cell Proliferative Activity.

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Grégory Lacraz

Full Text Available Skeletal muscle aging is associated with a decreased regenerative potential due to the loss of function of endogenous stem cells or myogenic progenitor cells (MPCs. Aged skeletal muscle is characterized by the deposition of extracellular matrix (ECM, which in turn influences the biomechanical properties of myofibers by increasing their stiffness. Since the stiffness of the MPC microenvironment directly impacts MPC function, we hypothesized that the increase in muscle stiffness that occurs with aging impairs the behavior of MPCs, ultimately leading to a decrease in regenerative potential.We showed that freshly isolated individual myofibers from aged mouse muscles contain fewer MPCs overall than myofibers from adult muscles, with fewer quiescent MPCs and more proliferative and differentiating MPCs. We observed alterations in cultured MPC behavior in aged animals, where the proliferation and differentiation of MPCs were lower and higher, respectively. These alterations were not linked to the intrinsic properties of aged myofibers, as shown by the similar values for the cumulative population-doubling values and fusion indexes. However, atomic force microscopy (AFM indentation experiments revealed a nearly 4-fold increase in the stiffness of the MPC microenvironment. We further showed that the increase in stiffness is associated with alterations to muscle ECM, including the accumulation of collagen, which was correlated with higher hydroxyproline and advanced glycation end-product content. Lastly, we recapitulated the impaired MPC behavior observed in aging using a hydrogel substrate that mimics the stiffness of myofibers.These findings provide novel evidence that the low regenerative potential of aged skeletal muscle is independent of intrinsic MPC properties but is related to the increase in the stiffness of the MPC microenvironment.

Heavy ion irradiation induces autophagy in irradiated C2C12 myoblasts and their bystander cells

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Hino, Mizuki; Tajika, Yuki; Hamada, Nobuyuki

2010-01-01

Autophagy is one of the major processes involved in the degradation of intracellular materials. Here, we examined the potential impact of heavy ion irradiation on the induction of autophagy in irradiated C2C12 mouse myoblasts and their non-targeted bystander cells. In irradiated cells, ultrastructural analysis revealed the accumulation of autophagic structures at various stages of autophagy (id est (i.e.) phagophores, autophagosomes and autolysosomes) within 20 min after irradiation. Multivesicular bodies (MVBs) and autolysosomes containing MVBs (amphisomes) were also observed. Heavy ion irradiation increased the staining of microtubule-associated protein 1 light chain 3 and LysoTracker Red (LTR). Such enhanced staining was suppressed by an autophagy inhibitor 3-methyladenine. In addition to irradiated cells, bystander cells were also positive with LTR staining. Altogether, these results suggest that heavy ion irradiation induces autophagy not only in irradiated myoblasts but also in their bystander cells. (author)

Telmisartan ameliorates insulin sensitivity by activating the AMPK/SIRT1 pathway in skeletal muscle of obese db/db mice

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Shiota Asuka

2012-11-01

Full Text Available Abstract Background Telmisartan is a well-established angiotensin II type 1 receptor blocker that improves insulin sensitivity in animal models of obesity and insulin resistance, as well as in humans. Telmisartan has been reported to function as a partial agonist of the peroxisome proliferator-activated receptor (PPAR γ, which is also targeted by the nicotinamide adenine dinucleotide (NAD-dependent deacetylase (SIRT1. Here, we investigated the pathways through which telmisartan acts on skeletal muscle, in vitro as well as in vivo. Methods Nine-week-old male db/db mice were fed a 60% high-fat diet, with orally administrated either vehicle (carboxymethyl-cellulose, CMC, 5 mg/kg telmisartan, or 5 mg/kg telmisartan and 1 mg/kg GW9662, a selective irreversible antagonist of PPARγ, for 5 weeks. Effects of telmisartan on Sirt1 mRNA, AMPK phosphorylation, and NAD+/NADH ratio were determined in C2C12 cultured myocytes. Results and discussion Telmisartan treatment improved insulin sensitivity in obese db/db mice fed a high-fat diet and led to reduction in the size of hypertrophic pancreatic islets in these mice. Moreover, in vitro treatment with telmisartan led to increased expression of Sirt1 mRNA in C2C12 skeletal muscle cells; the increase in Sirt1 mRNA in telmisartan-treated C2C12 myoblasts occurred concomitantly with an increase in AMPK phosphorylation, an increase in NAD+/NADH ratio, and increases in the mRNA levels of PGC1α, FATP1, ACO, and GLUT4. Conclusions Our results indicate that telmisartan acts through a PPARγ-independent pathway, but at least partially exerts its effects by acting directly on skeletal muscle AMPK/SIRT1 pathways.

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

2006-01-01

a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have shown that mechanical signals transmitted through the C2C12 cells interaction with laminin cause an increase in cellular differentiation. This signaling results in an increase in the number of myotubes formed in the cultures...

Skeletal muscle protein synthesis and the abundance of the mRNA translation initiation repressor PDCD4 are inversely regulated by fasting and refeeding in rats.

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Zargar, Sana; Moreira, Tracy S; Samimi-Seisan, Helena; Jeganathan, Senthure; Kakade, Dhanshri; Islam, Nushaba; Campbell, Jonathan; Adegoke, Olasunkanmi A J

2011-06-01

Optimal skeletal muscle mass is vital to human health, because defects in muscle protein metabolism underlie or exacerbate human diseases. The mammalian target of rapamycin complex 1 is critical in the regulation of mRNA translation and protein synthesis. These functions are mediated in part by the ribosomal protein S6 kinase 1 (S6K1) through mechanisms that are poorly understood. The tumor suppressor programmed cell death 4 (PDCD4) has been identified as a novel substrate of S6K1. Here, we examined 1) the expression of PDCD4 in skeletal muscle and 2) its regulation by feed deprivation (FD) and refeeding. Male rats (~100 g; n = 6) were subjected to FD for 48 h; some rats were refed for 2 h. FD suppressed muscle fractional rates of protein synthesis and Ser(67) phosphorylation of PDCD4 (-50%) but increased PDCD4 abundance (P muscle fractional rates of protein synthesis and reduced PDCD4 abundance relative to FD. Finally, when myoblasts were grown in amino acid- and serum-free medium, phenylalanine incorporation into proteins in cells depleted of PDCD4 more than doubled the values in cells with a normal level of PDCD4 (P skeletal muscle in parallel with the reduction of the abundance of this mRNA translation inhibitor.

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

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Suzuki Erika

2011-07-01

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

Biodistribution studies of {sup 99m}Tc-labeled myoblasts in a murine model of muscular dystrophy

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Colombo, F.R. E-mail: colombof@policlinico.mi.it; Torrente, Y.; Casati, R.; Benti, R.; Corti, S.; Salani, S.; D' Angelo, M.G.; DeLiso, A.; Scarlato, G.; Bresolin, N.; Gerundini, P

2001-11-01

The purpose of this study was twofold: first, to evaluate the myoblast labeling of various {sup 99m}Tc complexes and to select the complex that best accomplishes this labeling, and second to evaluate the biodistribution of myoblasts labeled with this complex using mice with MDX muscular dystrophy (the murine homologue of Duchenne's muscular dystrophy). The following ligands were used to prepare the corresponding {sup 99m}Tc complexes: hexakis-methoxy-isobutyl-isonitrile (MIBI), bis(2-ethoxyethyl)diphosphinoethane (Tf), (RR,SS)-4,8-diaza-3,6,6,9-tetramethyl-undecane-2,10-dione-bisoxime (HM-PAO), bis(N-ethyl)dithiocarbamate (NEt), and bis(N-ethoxy, N-ethyl)dithiocarbamate (NOEt). One million murine myoblasts were incubated for 30-60 minutes with 5 mCi of each of the 99mTc complexes prepared from the above ligands. Viability was assessed by microscopic counting after trypan blue staining, and the radioactivity absorbed in the cells was measured after centrifugation. The compound with the highest uptake in cellular pellets was [{sup 99m}Tc]N-NOEt. The biodistribution of myoblasts labeled with this complex was evaluated after intraaortic injection in dystrophic mice. Such an approach has the potential of effecting widespread gene transfer through the bloodstream to muscles lacking dystrophin.

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

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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.

Specific association of growth-associated protein 43 with calcium release units in skeletal muscles of lower vertebrates

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G.A. Caprara

2014-10-01

Full Text Available Growth-associated protein 43 (GAP43, is a strictly conserved protein among vertebrates implicated in neuronal development and neurite branching. Since GAP43 structure contains a calmodulin-binding domain, this protein is able to bind calmodulin and gather it nearby membrane network, thus regulating cytosolic calcium and consequently calcium-dependent intracellular events. Even if for many years GAP43 has been considered a neuronal-specific protein, evidence from different laboratories described its presence in myoblasts, myotubes and adult skeletal muscle fibers. Data from our laboratory showed that GAP43 is localized between calcium release units (CRUs and mitochondria in mammalian skeletal muscle suggesting that, also in skeletal muscle, this protein can be a key player in calcium/calmodulin homeostasis. However, the previous studies could not clearly distinguish between a mitochondrion- or a triad-related positioning of GAP43. To solve this question, the expression and localization of GAP43 was studied in skeletal muscle of Xenopus and Zebrafish known to have triads located at the level of the Z-lines and mitochondria not closely associated with them. Western blotting and immunostaining experiments revealed the expression of GAP43 also in skeletal muscle of lower vertebrates (like amphibians and fishes, and that the protein is localized closely to the triad junction. Once more, these results and GAP43 structural features, support an involvement of the protein in the dynamic intracellular Ca2+ homeostasis, a common conserved role among the different species.

Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts

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

2011-07-01

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

Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts

International Nuclear Information System (INIS)

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

2011-01-01

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

Exercise training and return to a well-balanced diet activate the neuregulin 1/ErbB pathway in skeletal muscle of obese rats.

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Ennequin, Gaël; Boisseau, Nathalie; Caillaud, Kevin; Chavanelle, Vivien; Gerbaix, Maude; Metz, Lore; Etienne, Monique; Walrand, Stéphane; Masgrau, Aurélie; Guillet, Christelle; Courteix, Daniel; Niu, Airu; Li, Yi-Ping; Capel, Fréderic; Sirvent, Pascal

2015-06-15

associated with increased protein expression and phosphorylation ratio of the metalloprotease ADAM17, which is involved in NRG1 shedding. Similarly, in vitro stretch-induced activation of ADAM17 in rat myoblasts induced NRG1 cleavage and ErbB4 activation. These results show that low intensity endurance training and well-balanced diet activate the NRG1-ErbB4 pathway, possibly via the metalloprotease ADAM17, in skeletal muscle of diet-induced obese rats. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Skeletal muscle-specific expression of PGC-1α-b, an exercise-responsive isoform, increases exercise capacity and peak oxygen uptake.

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Miki Tadaishi

Full Text Available Maximal oxygen uptake (VO(2max predicts mortality and is associated with endurance performance. Trained subjects have a high VO(2max due to a high cardiac output and high metabolic capacity of skeletal muscles. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α, a nuclear receptor coactivator, promotes mitochondrial biogenesis, a fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training increases PGC-1α in skeletal muscle, PGC-1α-mediated changes may contribute to the improvement of exercise capacity and VO(2max. There are three isoforms of PGC-1α mRNA. PGC-1α-b protein, whose amino terminus is different from PGC-1α-a protein, is a predominant PGC-1α isoform in response to exercise. We investigated whether alterations of skeletal muscle metabolism by overexpression of PGC-1α-b in skeletal muscle, but not heart, would increase VO(2max and exercise capacity.Transgenic mice showed overexpression of PGC-1α-b protein in skeletal muscle but not in heart. Overexpression of PGC-1α-b promoted mitochondrial biogenesis 4-fold, increased the expression of fatty acid transporters, enhanced angiogenesis in skeletal muscle 1.4 to 2.7-fold, and promoted exercise capacity (expressed by maximum speed by 35% and peak oxygen uptake by 20%. Across a broad range of either the absolute exercise intensity, or the same relative exercise intensities, lipid oxidation was always higher in the transgenic mice than wild-type littermates, suggesting that lipid is the predominant fuel source for exercise in the transgenic mice. However, muscle glycogen usage during exercise was absent in the transgenic mice.Increased mitochondrial biogenesis, capillaries, and fatty acid transporters in skeletal muscles may contribute to improved exercise capacity via an increase in fatty acid utilization. Increases in PGC-1α-b protein or function might be a useful strategy for sedentary subjects to perform exercise

Endurance training increases the efficiency of rat skeletal muscle mitochondria.

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Zoladz, Jerzy A; Koziel, Agnieszka; Woyda-Ploszczyca, Andrzej; Celichowski, Jan; Jarmuszkiewicz, Wieslawa

2016-10-01

Endurance training enhances mitochondrial oxidative capacity, but its effect on mitochondria functioning is poorly understood. In the present study, the influence of an 8-week endurance training on the bioenergetic functioning of rat skeletal muscle mitochondria under different assay temperatures (25, 35, and 42 °C) was investigated. The study was performed on 24 adult 4-month-old male Wistar rats, which were randomly assigned to either a treadmill training group (n = 12) or a sedentary control group (n = 12). In skeletal muscles, endurance training stimulated mitochondrial biogenesis and oxidative capacity. In isolated mitochondria, endurance training increased the phosphorylation rate and elevated levels of coenzyme Q. Moreover, a decrease in mitochondrial uncoupling, including uncoupling protein-mediated proton leak, was observed after training, which could explain the increased reactive oxygen species production (in nonphosphorylating mitochondria) and enhanced oxidative phosphorylation efficiency. At all studied temperatures, endurance training significantly augmented H2O2 production (and coenzyme Q reduction level) in nonphosphorylating mitochondria and decreased H2O2 production (and coenzyme Q reduction level) in phosphorylating mitochondria. Endurance training magnified the hyperthermia-induced increase in oxidative capacity and attenuated the hyperthermia-induced decline in oxidative phosphorylation efficiency and reactive oxygen species formation of nonphosphorylating mitochondria via proton leak enhancement. Thus, endurance training induces both quantitative and qualitative changes in muscle mitochondria that are important for cell signaling as well as for maintaining muscle energy homeostasis, especially at high temperatures.

Comparative Analyses between Skeletal Muscle miRNAomes from Large White and Min Pigs Revealed MicroRNAs Associated with Postnatal Muscle Hypertrophy.

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Sheng, Xihui; Wang, Ligang; Ni, Hemin; Wang, Lixian; Qi, Xiaolong; Xing, Shuhan; Guo, Yong

2016-01-01

The molecular mechanism regulated by microRNAs (miRNAs) that underlies postnatal hypertrophy of skeletal muscle is complex and remains unclear. Here, the miRNAomes of longissimus dorsi muscle collected at five postnatal stages (60, 120, 150, 180, and 210 days after birth) from Large White (commercial breed) and Min pigs (indigenous breed of China) were analyzed by Illumina sequencing. We identified 734 miRNAs comprising 308 annotated miRNAs and 426 novel miRNAs, of which 307 could be considered pig-specific. Comparative analysis between two breeds suggested that 60 and 120 days after birth were important stages for skeletal muscle hypertrophy and intramuscular fat accumulation. A total of 263 miRNAs were significantly differentially expressed between two breeds at one or more developmental stages. In addition, the differentially expressed miRNAs between every two adjacent developmental stages in each breed were determined. Notably, ssc-miR-204 was significantly more highly expressed in Min pig skeletal muscle at all postnatal stages compared with its expression in Large White pig skeletal muscle. Based on gene ontology and KEGG pathway analyses of its predicted target genes, we concluded that ssc-miR-204 may exert an impact on postnatal hypertrophy of skeletal muscle by regulating myoblast proliferation. The results of this study will help in elucidating the mechanism underlying postnatal hypertrophy of skeletal muscle modulated by miRNAs, which could provide valuable information for improvement of pork quality and human myopathy.

Gene expression deregulation in postnatal skeletal muscle of TK2 deficient mice reveals a lower pool of proliferating myogenic progenitor cells.

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João A Paredes

Full Text Available Loss of thymidine kinase 2 (TK2 causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA depletion syndrome (MDS in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue.

Gene expression deregulation in postnatal skeletal muscle of TK2 deficient mice reveals a lower pool of proliferating myogenic progenitor cells.

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Paredes, João A; Zhou, Xiaoshan; Höglund, Stefan; Karlsson, Anna

2013-01-01

Loss of thymidine kinase 2 (TK2) causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA) depletion syndrome (MDS) in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue.

α-Syntrophin is involved in the survival signaling pathway in myoblasts under menadione-induced oxidative stress.

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Lim, Jeong-A; Choi, Su Jin; Moon, Jae Yun; Kim, Hye Sun

2016-05-15

Dystrophin-deficient muscle is known to be more vulnerable to oxidative stress, but not much is known about the signaling pathway(s) responsible for this phenomenon. α-Syntrophin, a component of the dystrophin-glycoprotein complex, can function as a scaffold protein because of its multiple protein interaction domains. In this study, we investigated the role of α-syntrophin in C2 myoblasts under menadione-induced oxidative stress. We found that the protein level of α-syntrophin was elevated when cells were exposed to menadione. To investigate the function of α-syntrophin during oxidative stress, we established α-syntrophin-overexpressing and knockdown cell lines. The α-syntrophin-overexpressing cells were resistant to the menadione-induced oxidative stress. In addition, survival signalings such as protein kinase B (Akt) phosphorylation and the Bcl-2/BAX ratio were increased in these cells. On the other hand, apoptotic signals such as cleavage of caspase-3 and poly ADP ribose polymerase (PARP) were increased in the α-syntrophin knockdown cells. Furthermore, Ca(2+)influx, which is known to increase when cells are exposed to oxidative stress, decreased in the α-syntrophin-overexpressing cells, but increased in the knockdown cells. These results suggest that α-syntrophin plays a pivotal role in the survival pathway triggered by menadione-induced oxidative stress in cultured myoblasts. Copyright © 2016 Elsevier Inc. All rights reserved.

A New In Vitro Co-Culture Model Using Magnetic Force-Based Nanotechnology

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Takanari, Hiroki; Miwa, Keiko; Fu, Xianming; Nakai, Junichi; Ito, Akira; Ino, Kousuke; Honda, Hiroyuki; Tonomura, Wataru; Konishi, Satoshi; Opthof, Tobias; van der Heyden, Marcel Ag; Kodama, Itsuo; Lee, Jong-Kook

2016-01-01

Skeletal myoblast (SkMB) transplantation has been conducted as a therapeutic strategy for severe heart failure. However, arrhythmogenicity following transplantation remains unsolved. We developed an in vitro model of myoblast transplantation with "patterned" or "randomly-mixed" co-culture of SkMBs

Radiation-induced increase in the release of amino acids by isolated, perfused skeletal muscle

International Nuclear Information System (INIS)

Schwenen, M.

1989-01-01

Local exposure of the hindquarter of the rat to 15Gy of gamma-radiation resulted, 4-6h after irradiation, in increased release of amino acids by the isolated, perfused hindquarter preparation, 70% of which is skeletal muscle. This increase in release involves not only alanine and glutamine, but also those amino acids not metabolized by muscle and, therefore, released in proportion to their occurrence in muscle proteins. Because metabolic parameters and content of energy-rich phosphate compounds in muscle remain unchanged, it is unlikely that general cellular damage is the underlying cause of the radiation-induced increase in amino acid release. The findings strongly favour the hypothesis that increased availability of amino acids results from enhanced protein break-down in skeletal muscle which has its onset shortly after irradiation. This radiation-induced disturbance in protein metabolism might be one of the pathogenetic factors in the aetiology of radiation myopathy. (author)

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

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

2017-04-01

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

Is increase in bone mineral content caused by increase in skeletal muscle mass/strength in adult patients with GH-treated GH deficiency?

DEFF Research Database (Denmark)

Klefter, Oliver; Feldt-Rasmussen, Ulla

2009-01-01

to a muscle modulating effect, and if treatment with GH would primarily increase muscle mass and strength with a secondary increase in BMD/BMC, thus supporting the present physiological concept that mass and strength of bones are mainly determined by dynamic loads from the skeletal muscles. METHOD: We...... performed a systematic literature analysis, including 51 clinical trials published between 1996 and 2008, which had studied the development in muscle mass, muscle strength, BMD, and/or BMC in GH-treated adult GHD patients. RESULTS: GH therapy had an anabolic effect on skeletal muscle. The largest increase...... in muscle mass occurred during the first 12 months of therapy. Most trials measuring BMD/BMC reported significant increases from baseline values. The significant increases in BMD/BMC occurred after 12-18 months of treatment, i.e. usually later than the increases in muscle parameters. Only seven trials...

Microfluidic-enhanced 3D bioprinting of aligned myoblast-laden hydrogels leads to functionally organized myofibers in vitro and in vivo.

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Costantini, Marco; Testa, Stefano; Mozetic, Pamela; Barbetta, Andrea; Fuoco, Claudia; Fornetti, Ersilia; Tamiro, Francesco; Bernardini, Sergio; Jaroszewicz, Jakub; Święszkowski, Wojciech; Trombetta, Marcella; Castagnoli, Luisa; Seliktar, Dror; Garstecki, Piotr; Cesareni, Gianni; Cannata, Stefano; Rainer, Alberto; Gargioli, Cesare

2017-07-01

We present a new strategy for the fabrication of artificial skeletal muscle tissue with functional morphologies based on an innovative 3D bioprinting approach. The methodology is based on a microfluidic printing head coupled to a co-axial needle extruder for high-resolution 3D bioprinting of hydrogel fibers laden with muscle precursor cells (C2C12). To promote myogenic differentiation, we formulated a tailored bioink with a photocurable semi-synthetic biopolymer (PEG-Fibrinogen) encapsulating cells into 3D constructs composed of aligned hydrogel fibers. After 3-5 days of culture, the encapsulated myoblasts started migrating and fusing, forming multinucleated myotubes within the 3D bioprinted fibers. The obtained myotubes showed high degree of alignment along the direction of hydrogel fiber deposition, further revealing maturation, sarcomerogenesis, and functionality. Following subcutaneous implantation in the back of immunocompromised mice, bioprinted constructs generated organized artificial muscle tissue in vivo. Finally, we demonstrate that our microfluidic printing head allows to design three dimensional multi-cellular assemblies with an exquisite compartmentalization of the encapsulated cells. Our results demonstrate an enhanced myogenic differentiation with the formation of parallel aligned long-range myotubes. The approach that we report here represents a robust and valid candidate for the fabrication of macroscopic artificial muscle to scale up skeletal muscle tissue engineering for human clinical application. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

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

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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.

Increased peripheral vascular disease risk progressively constrains perfusion adaptability in the skeletal muscle microcirculation

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Butcher, Joshua T.; Frisbee, Stephanie J.; Olfert, I. Mark; Chantler, Paul D.; Tabone, Lawrence E.; d'Audiffret, Alexandre C.; Shrader, Carl D.; Goodwill, Adam G.; Stapleton, Phoebe A.; Brooks, Steven D.; Brock, Robert W.; Lombard, Julian H.

2015-01-01

To determine the impact of progressive elevations in peripheral vascular disease (PVD) risk on microvascular function, we utilized eight rat models spanning “healthy” to “high PVD risk” and used a multiscale approach to interrogate microvascular function and outcomes: healthy: Sprague-Dawley rats (SDR) and lean Zucker rats (LZR); mild risk: SDR on high-salt diet (HSD) and SDR on high-fructose diet (HFD); moderate risk: reduced renal mass-hypertensive rats (RRM) and spontaneously hypertensive rats (SHR); high risk: obese Zucker rats (OZR) and Dahl salt-sensitive rats (DSS). Vascular reactivity and biochemical analyses demonstrated that even mild elevations in PVD risk severely attenuated nitric oxide (NO) bioavailability and caused progressive shifts in arachidonic acid metabolism, increasing thromboxane A2 levels. With the introduction of hypertension, arteriolar myogenic activation and adrenergic constriction were increased. However, while functional hyperemia and fatigue resistance of in situ skeletal muscle were not impacted with mild or moderate PVD risk, blood oxygen handling suggested an increasingly heterogeneous perfusion within resting and contracting skeletal muscle. Analysis of in situ networks demonstrated an increasingly stable and heterogeneous distribution of perfusion at arteriolar bifurcations with elevated PVD risk, a phenomenon that was manifested first in the distal microcirculation and evolved proximally with increasing risk. The increased perfusion distribution heterogeneity and loss of flexibility throughout the microvascular network, the result of the combined effects on NO bioavailability, arachidonic acid metabolism, myogenic activation, and adrenergic constriction, may represent the most accurate predictor of the skeletal muscle microvasculopathy and poor health outcomes associated with chronic elevations in PVD risk. PMID:26702145

Prior AICAR stimulation increases insulin sensitivity in mouse skeletal muscle in an AMPK-dependent manner

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Kjøbsted, Rasmus; Treebak, Jonas Thue; Fentz, Joachim

2015-01-01

Acute exercise increases glucose uptake in skeletal muscle by an insulin-independent mechanism. In the period after exercise insulin sensitivity to increase glucose uptake is enhanced. The molecular mechanisms underpinning this phenomenon are poorly understood, but appear to involve an increased ...

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

International Nuclear Information System (INIS)

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

2012-01-01

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

A New In Vitro Co-Culture Model Using Magnetic Force-Based Nanotechnology

NARCIS (Netherlands)

Takanari, Hiroki; Miwa, Keiko; Fu, Xian Ming; Nakai, Junichi; Ito, Akira; Ino, Kousuke; Honda, Hiroyuki; Tonomura, Wataru; Konishi, Satoshi; Opthof, Tobias; van der Heyden, Marcel A G; Kodama, Itsuo; Lee, Jong Kook

2016-01-01

Skeletal myoblast (SkMB) transplantation has been conducted as a therapeutic strategy for severe heart failure. However, arrhythmogenicity following transplantation remains unsolved. We developed an in vitro model of myoblast transplantation with “patterned” or “randomly-mixed” co-culture of SkMBs

Serum Is Not Necessary for Prior Pharmacological Activation of AMPK to Increase Insulin Sensitivity of Mouse Skeletal Muscle

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Nicolas O. Jørgensen

2018-04-01

Full Text Available Exercise, contraction, and pharmacological activation of AMP-activated protein kinase (AMPK by 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR have all been shown to increase muscle insulin sensitivity for glucose uptake. Intriguingly, improvements in insulin sensitivity following contraction of isolated rat and mouse skeletal muscle and prior AICAR stimulation of isolated rat skeletal muscle seem to depend on an unknown factor present in serum. One study recently questioned this requirement of a serum factor by showing serum-independency with muscle from old rats. Whether a serum factor is necessary for prior AICAR stimulation to increase insulin sensitivity of mouse skeletal muscle is not known. Therefore, we investigated the necessity of serum for this effect of AICAR in mouse skeletal muscle. We found that the ability of prior AICAR stimulation to improve insulin sensitivity of mouse skeletal muscle did not depend on the presence of serum during AICAR stimulation. Although prior AICAR stimulation did not enhance proximal insulin signaling, insulin-stimulated phosphorylation of Tre-2/BUB2/CDC16- domain family member 4 (TBC1D4 Ser711 was greater in prior AICAR-stimulated muscle compared to all other groups. These results imply that the presence of a serum factor is not necessary for prior AMPK activation by AICAR to enhance insulin sensitivity of mouse skeletal muscle.

Insulin increases phosphorylation of mitochondrial proteins in human skeletal muscle in vivo

DEFF Research Database (Denmark)

Zhao, Xiaolu; Bak, Steffen; Pedersen, Andreas James Thestrup

2014-01-01

, we investigated the effect of insulin on the phosphorylation of mitochondrial proteins in human skeletal muscle in vivo. Using a combination of TiO2 phosphopeptide-enrichment, HILIC fractionation, and LC−MS/MS, we compared the phosphoproteomes of isolated mitochondria from skeletal muscle samples...... obtained from healthy individuals before and after 4 h of insulin infusion. In total, we identified 207 phosphorylation sites in 95 mitochondrial proteins. Of these phosphorylation sites, 45% were identified in both basal and insulin-stimulated samples. Insulin caused a 2-fold increase in the number...... of different mitochondrial phosphopeptides (87 ± 7 vs 40 ± 7, p = 0.015) and phosphoproteins (46 ± 2 vs 26 ± 3, p = 0.005) identified in each mitochondrial preparation. Almost half of the mitochondrial phosphorylation sites (n = 94) were exclusively identified in the insulin-stimulated state and included...

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

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

2015-12-30

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

SPARC Interacts with Actin in Skeletal Muscle in Vitro and in Vivo

DEFF Research Database (Denmark)

Jørgensen, Louise H; Jepsen, Pia Lørup; Boysen, Anders

2017-01-01

to actin. This interaction is present in regenerating myofibers of patients with Duchenne muscular dystrophy, polymyositis, and compartment syndrome. Analysis of the α-, β-, and γ-actin isoforms in SPARC knockout myoblasts reveals a changed expression pattern with dominance of γ-actin. In SPARC knockout......The cytoskeleton is an integral part of skeletal muscle structure, and reorganization of the cytoskeleton occurs during various modes of remodeling. We previously found that the extracellular matrix protein secreted protein acidic and rich in cysteine (SPARC) is up-regulated and expressed...... intracellularly in developing muscle, during regeneration and in myopathies, which together suggests that SPARC might serve a specific role within muscle cells. Using co-immunoprecipitation combined with mass spectrometry and verified by staining for direct protein-protein interaction, we find that SPARC binds...

Overexpression of PGC-1α Increases Fatty Acid Oxidative Capacity of Human Skeletal Muscle Cells

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Nataša Nikolić

2012-01-01

Full Text Available We investigated the effects of PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α overexpression on the oxidative capacity of human skeletal muscle cells ex vivo. PGC-1α overexpression increased the oxidation rate of palmitic acid and mRNA expression of genes regulating lipid metabolism, mitochondrial biogenesis, and function in human myotubes. Basal and insulin-stimulated deoxyglucose uptake were decreased, possibly due to upregulation of PDK4 mRNA. Expression of fast fiber-type gene marker (MHCIIa was decreased. Compared to skeletal muscle in vivo, PGC-1α overexpression increased expression of several genes, which were downregulated during the process of cell isolation and culturing. In conclusion, PGC-1α overexpression increased oxidative capacity of cultured myotubes by improving lipid metabolism, increasing expression of genes involved in regulation of mitochondrial function and biogenesis, and decreasing expression of MHCIIa. These results suggest that therapies aimed at increasing PGC-1α expression may have utility in treatment of obesity and obesity-related diseases.

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

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Soo-Yeon Lee

2018-01-01

Conclusion: BG enhances myoblast differentiation and myotube hypertrophy by activating Akt/mTOR/p70S6k axis. Thus, our study demonstrates that BG has promising potential to treat or prevent muscle loss related to aging or other pathological conditions, such as diabetes.

Exercise Training-Induced Adaptations Associated with Increases in Skeletal Muscle Glycogen Content

Science.gov (United States)

Manabe, Yasuko; Gollisch, Katja S.C.; Holton, Laura; Kim, Young–Bum; Brandauer, Josef; Fujii, Nobuharu L.; Hirshman, Michael F.; Goodyear, Laurie J.

2012-01-01

Chronic exercise training results in numerous skeletal muscle adaptations, including increases in insulin sensitivity and glycogen content. To understand the mechanism for increased muscle glycogen, we studied the effects of exercise training on glycogen regulatory proteins in rat skeletal muscle. Female Sprague Dawley rats performed voluntary wheel running for 1, 4, or 7 weeks. After 7 weeks of training, insulin-stimulated glucose uptake was increased in epitrochlearis muscle. Compared to sedentary control rats, muscle glycogen did not change after 1 week of training, but increased significantly after 4 and 7 weeks. The increases in muscle glycogen were accompanied by elevated glycogen synthase activity and protein expression. To assess the regulation of glycogen synthase, we examined its major activator, protein phosphatase 1 (PP1), and its major deactivator, glycogen synthase kinase 3 (GSK3). Consistent with glycogen synthase activity, PP1 activity was unchanged after 1 week of training but significantly increased after 4 and 7 weeks of training. Protein expression of RGL(GM), another regulatory PP1 subunit, significantly decreased after 4 and 7 weeks of training. Unlike PP1, GSK3 phosphorylation did not follow the pattern of glycogen synthase activity. The ~40% decrease in GSK-3α phosphorylation after 1 week of exercise training persisted until 7 weeks and may function as a negative feedback to elevated glycogen. Our findings suggest that exercise training-induced increases in muscle glycogen content could be regulated by multiple mechanisms including enhanced insulin sensitivity, glycogen synthase expression, allosteric activation of glycogen synthase and PP1activity. PMID:23206309

Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy.

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Rancourt, Ann; Dufresne, Sébastien S; St-Pierre, Guillaume; Lévesque, Julie-Christine; Nakamura, Haruka; Kikuchi, Yodai; Satoh, Masahiko S; Frenette, Jérôme; Sato, Sachiko

2018-06-12

The muscle membrane, sarcolemma, must be firmly attached to the basal lamina. The failure of proper attachment results in muscle injury, which is the underlying cause of Duchenne muscular dystrophy (DMD), in which mutations in the dystrophin gene disrupts the firm adhesion. In patients with DMD, even moderate contraction causes damage, leading to progressive muscle degeneration. The damaged muscles are repaired through myogenesis. Consequently, myogenesis is highly active in patients with DMD, and the repeated activation of myogenesis leads to the exhaustion of the myogenic stem cells. Therefore, approaches to reducing the risk of the exhaustion are to develop a treatment that strengthens the interaction between the sarcolemma and the basal lamina and increases the efficiency of the myogenesis. Galectin-3 is an oligosaccharide-binding protein and is known to be involved in cell-cell interactions and cell-matrix interactions. Galectin-3 is expressed in myoblasts and skeletal muscle, although its function in muscle remains elusive. In this study, we found evidence that galectin-3 and the monosaccharide N-acetylglucosamine, which increases the synthesis of binding partners (oligosaccharides) of galectin-3, promote myogenesis in vitro. Moreover, in the mdx mouse model of DMD, treatment with N-acetylglucosamine increased muscle-force production. The results suggest that treatment with N-acetylglucosamine might mitigate the burden of DMD.-Rancourt, A., Dufresne, S. S., St-Pierre, G., Lévesque, J.-C., Nakamura, H., Kikuchi, Y., Satoh, M. S., Frenette, J., Sato, S. Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy.

Discovery of a Mammalian Splice Variant of Myostatin That Stimulates Myogenesis

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Jeanplong, Ferenc; Falconer, Shelley J.; Oldham, Jenny M.; Thomas, Mark; Gray, Tarra S.; Hennebry, Alex; Matthews, Kenneth G.; Kemp, Frederick C.; Patel, Ketan; Berry, Carole; Nicholas, Gina; McMahon, Christopher D.

2013-01-01

Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over-expression increased the abundance of MyoD, Myogenin and MRF4 proteins (Pmyostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product. PMID:24312578

Satellite cell proliferation in adult skeletal muscle

Science.gov (United States)

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

1995-01-01

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

Biphasic regulation of intracellular calcium by gemfibrozil contributes to inhibiting L6 myoblast differentiation: implications for clinical myotoxicity.

Science.gov (United States)

Liu, Aiming; Yang, Julin; Gonzalez, Frank J; Cheng, Gary Q; Dai, Renke

2011-02-18

Gemfibrozil is the most myotoxic fibrate drug commonly used for dyslipidemia, but the mechanism is poorly understood. The current study revealed that gemfibrozil inhibits myoblast differentiation through the regulation of intracellular calcium ([Ca(2+)]i) as revealed in L6 myoblasts by use of laser scan confocal microscopy and flow cytometry using Fluo-4 AM as a probe. Gemfibrozil at 20-400 μM, could regulate [Ca(2+)]i in L6 cells in a biphasic manner, and sustained reduction was observed when the concentration reached 200 μM. Inhibition of L6 differentiation by gemfibrozil was concentration-dependent with maximal effect noted between 200 and 400 μM, as indicated by creatine kinase activities and the differentiation index, respectively. In differentiating L6 myoblasts, gemfibrozil at concentrations below 400 μM led to no significant signs of apoptosis or cytotoxicity, whereas differentiation, inhibited by 200 μM gemfibrozil, was only partially recovered. A good correlation was noted between gemfibrozil concentrations that regulate [Ca(2+)]i and inhibit L6 myoblasts differentiation, and both are within the range of total serum concentrations found in the clinic. These data suggest a potential pharmacodynamic effect of gemfibrozil on myogenesis as a warning sign, in addition to the complex pharmacokinetic interactions. It is also noteworthy that mobilization of [Ca(2+)]i by gemfibrozil may trigger complex biological responses besides myocyte differentiation. Information revealed in this study explores the mechanism of gemfibrozil-induced myotoxicity through the regulation of intracellular calcium.

Antifibrotic effects of Smad4 small interfering RNAs in injured skeletal muscle after acute contusion.

Science.gov (United States)

Li, H; Chen, J; Chen, S; Zhang, Q; Chen, S

2011-10-01

Muscle injuries are common musculoskeletal problems encountered in sports medicine clinics. In this study, we examined the effect of lentivirus-mediated small interfering RNA (siRNA) targeting Smad4 on the suppression of the fibrosis in injured skeletal muscles. We found that Smad4-siRNA could efficiently knock down the expression of Smad4 in the C2C12 myoblast cells and in the contunded mice gastrocnemius muscle. The expression of mRNA level of Smad4 decreased to 11% and 49% compared to the control group, respectively, and the expression of protein level decreased to 13% and 57% respectively. Moreover, the lentivirus-mediated siRNA was stably transfected only into the skeletal muscle and not into the liver of the animals. In contunded mice gastrocnemius, the collagenous and vimentin-positive area in the Smad4 siRNA group reduced to 36% and 37% compared to the control group, respectively. Furthermore, compared to the scrambled Smad4 siRNA-injected mice and PBS control-injected mice, the muscle function of the mice injected with lentivirus-mediated Smad4 siRNA improved in terms of both fast-twitch and tetanic strength (P<0.05). The results suggest that the gene therapy of inhibiting Smad4 by lentivirus-mediated siRNA could be a useful approach to prevent scar tissue formation and improve the function of injured skeletal muscle. © Georg Thieme Verlag KG Stuttgart · New York.

Topologically Micropatterned Collagen and Poly(ε-caprolactone) Struts Fabricated Using the Poly(vinyl alcohol) Fibrillation/Leaching Process To Develop Efficiently Engineered Skeletal Muscle Tissue.

Science.gov (United States)

Kim, Minseong; Kim, WonJin; Kim, GeunHyung

2017-12-20

Optimally designed three-dimensional (3D) biomedical scaffolds for skeletal muscle tissue regeneration pose significant research challenges. Currently, most studies on scaffolds focus on the two-dimensional (2D) surface structures that are patterned in the micro-/nanoscales with various repeating sizes and shapes to induce the alignment of myoblasts and myotube formation. The 2D patterned surface clearly provides effective analytical results of pattern size and shape of the myoblast alignment and differentiation. However, it is inconvenient in terms of the direct application for clinical usage due to the limited thickness and 3D shapeability. Hence, the present study suggests an innovative hydrogel or synthetic structure that consists of uniaxially surface-patterned cylindrical struts for skeleton muscle regeneration. The alignment of the pattern on the hydrogel (collagen) and poly(ε-caprolactone) struts was attained with the fibrillation of poly(vinyl alcohol) and the leaching process. Various cell culture results indicate that the C2C12 cells on the micropatterned collagen structure were fully aligned, and that a significantly high level of myotube formation was achieved when compared to the collagen structures that were not treated with the micropatterning process.

Modulation of mitochondrial bioenergetics in a skeletal muscle cell line model of mitochondrial toxicity

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William Dott

2014-01-01

Full Text Available Mitochondrial toxicity is increasingly being implicated as a contributing factor to many xenobiotic-induced organ toxicities, including skeletal muscle toxicity. This has necessitated the need for predictive in vitro models that are able to sensitively detect mitochondrial toxicity of chemical entities early in the research and development process. One such cell model involves substituting galactose for glucose in the culture media. Since cells cultured in galactose are unable to generate sufficient ATP from glycolysis they are forced to rely on mitochondrial oxidative phosphorylation for ATP generation and consequently are more sensitive to mitochondrial perturbation than cells grown in glucose. The aim of this study was to characterise cellular growth, bioenergetics and mitochondrial toxicity of the L6 rat skeletal muscle cell line cultured in either high glucose or galactose media. L6 myoblasts proliferated more slowly when cultured in galactose media, although they maintained similar levels of ATP. Galactose cultured L6 cells were significantly more sensitive to classical mitochondrial toxicants than glucose-cultured cells, confirming the cells had adapted to galactose media. Analysis of bioenergetic function with the XF Seahorse extracellular flux analyser demonstrated that oxygen consumption rate (OCR was significantly increased whereas extracellular acidification rate (ECAR, a measure of glycolysis, was decreased in cells grown in galactose. Mitochondria operated closer to state 3 respiration and had a lower mitochondrial membrane potential and basal mitochondrial O2·– level compared to cells in the glucose model. An antimycin A (AA dose response revealed that there was no difference in the sensitivity of OCR to AA inhibition between glucose and galactose cells. Importantly, cells in glucose were able to up-regulate glycolysis, while galactose cells were not. These results confirm that L6 cells are able to adapt to growth in a

Anti-skeletal muscle atrophy effect of Oenothera odorata root extract via reactive oxygen species-dependent signaling pathways in cellular and mouse model.

Science.gov (United States)

Lee, Yong-Hyeon; Kim, Wan-Joong; Lee, Myung-Hun; Kim, Sun-Young; Seo, Dong-Hyun; Kim, Han-Sung; Gelinsky, Michael; Kim, Tack-Joong

2016-01-01

Skeletal muscle atrophy can be defined as a decrease of muscle volume caused by injury or lack of use. This condition is associated with reactive oxygen species (ROS), resulting in various muscular disorders. We acquired 2D and 3D images using micro-computed tomography in gastrocnemius and soleus muscles of sciatic-denervated mice. We confirmed that sciatic denervation-small animal model reduced muscle volume. However, the intraperitoneal injection of Oenothera odorata root extract (EVP) delayed muscle atrophy compared to a control group. We also investigated the mechanism of muscle atrophy's relationship with ROS. EVP suppressed expression of SOD1, and increased expression of HSP70, in both H2O2-treated C2C12 myoblasts and sciatic-denervated mice. Moreover, EVP regulated apoptotic signals, including caspase-3, Bax, Bcl-2, and ceramide. These results indicate that EVP has a positive effect on reducing the effect of ROS on muscle atrophy.

Interleukin-6 markedly decreases skeletal muscle protein turnover and increases nonmuscle amino acid utilization in healthy individuals

DEFF Research Database (Denmark)

van Hall, Gerrit; Steensberg, Adam; Fischer, Christian

2008-01-01

CONTEXT: IL-6 is a key modulator of immune function and suggested to be involved in skeletal muscle wasting as seen in sepsis. OBJECTIVE: Our objective was to determine the role of IL-6 in human in vivo systemic and skeletal muscle amino acid metabolism and protein turnover. SUBJECTS AND METHODS...... synthesis was more suppressed than breakdown, causing a small increase in net muscle protein breakdown. Furthermore, rhIL-6 decreased arterial amino acid concentration with 20-40%, despite the increase net release from muscle. CONCLUSIONS: We demonstrated that IL-6 profoundly alters amino acid turnover....... A substantial decrease in plasma amino acids was observed with a concomitant 50% decrease in muscle protein turnover, however, modest increase in net muscle degradation. We hypothesize that the profound reduction in muscle protein turnover and modest increase in net degradation are primarily caused...

Patterning Muscles Using Organizers: Larval Muscle Templates and Adult Myoblasts Actively Interact to Pattern the Dorsal Longitudinal Flight Muscles of Drosophila

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Roy, Sudipto; VijayRaghavan, K.

1998-01-01

Pattern formation in muscle development is often mediated by special cells called muscle organizers. During metamorphosis in Drosophila, a set of larval muscles function as organizers and provide scaffolding for the development of the dorsal longitudinal flight muscles. These organizers undergo defined morphological changes and dramatically split into templates as adult fibers differentiate during pupation. We have investigated the cellular mechanisms involved in the use of larval fibers as templates. Using molecular markers that label myoblasts and the larval muscles themselves, we show that splitting of the larval muscles is concomitant with invasion by imaginal myoblasts and the onset of differentiation. We show that the Erect wing protein, an early marker of muscle differentiation, is not only expressed in myoblasts just before and after fusion, but also in remnant larval nuclei during muscle differentiation. We also show that interaction between imaginal myoblasts and larval muscles is necessary for transformation of the larval fibers. In the absence of imaginal myoblasts, the earliest steps in metamorphosis, such as the escape of larval muscles from histolysis and changes in their innervation, are normal. However, subsequent events, such as the splitting of these muscles, fail to progress. Finally, we show that in a mutant combination, null for Erect wing function in the mesoderm, the splitting of the larval muscles is aborted. These studies provide a genetic and molecular handle for the understanding of mechanisms underlying the use of muscle organizers in muscle patterning. Since the use of such organizers is a common theme in myogenesis in several organisms, it is likely that many of the processes that we describe are conserved. PMID:9606206

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

International Nuclear Information System (INIS)

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

2012-01-01

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

Is increase in bone mineral content caused by increase in skeletal muscle mass/strength in adult patients with GH-treated GH deficiency? A systematic literature analysis

DEFF Research Database (Denmark)

Klefter, O.; Feldt-Rasmussen, U.

2009-01-01

to a muscle modulating effect, and if treatment with GH would primarily increase muscle mass and strength with a secondary increase in BMD/BMC, thus supporting the present physiological concept that mass and strength of bones are mainly determined by dynamic loads from the skeletal muscles. METHOD: We...... performed a systematic literature analysis, including 51 clinical trials published between 1996 and 2008, which had studied the development in muscle mass, muscle strength, BMD, and/or BMC in GH-treated adult GHD patients. RESULTS: GH therapy had an anabolic effect on skeletal muscle. The largest increase...... in muscle mass occurred during the first 12 months of therapy. Most trials measuring BMD/BMC reported significant increases from baseline values. The significant increases in BMD/BMC occurred after 12-18 months of treatment, i.e. usually later than the increases in muscle parameters. Only seven trials...

Dual oxidase maturation factor 1 (DUOXA1) overexpression increases reactive oxygen species production and inhibits murine muscle satellite cell differentiation.

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Sandiford, Shelley D E; Kennedy, Karen A M; Xie, Xiaojun; Pickering, J Geoffrey; Li, Shawn S C

2014-01-11

Dual oxidase maturation factor 1 (DUOXA1) has been associated with the maturation of the reactive oxygen species (ROS) producing enzyme, dual oxidase 1 (DUOX1) in the adult thyroid. However, ROS have also been implicated in the development of several tissues. We found that activated muscle satellite cells and primary myoblasts isolated from mice express robust levels of DUOXA1 and that its levels are altered as cells differentiate. To determine whether DUOXA1 levels affect muscle differentiation, we used an adenoviral construct (pCMV5-DUOXA1-GFP) to drive constitutive overexpression of this protein in primary myoblasts. High levels of DUOXA1 throughout myogenesis resulted in enhanced H2O2 production, fusion defects, reduced expression of early (myogenin) and late (myosin heavy chain) markers of differentiation, and elevated levels of apoptosis compared to control cells infected with an empty adenoviral vector (pCMV5-GFP). DUOXA1 knockdown (using a DUOXA1 shRNA construct) resulted in enhanced differentiation compared to cells subjected to a control shRNA, and subjecting DUOXA1 overexpressing cells to siRNAs targeting DUOX1 or apoptosis signal-regulating kinase 1 (ASK1) rescued the phenotype. This study represents the first to demonstrate the importance of DUOXA1 in skeletal muscle myoblasts and that DUOXA1 overexpression in muscle stem cells induces apoptosis and inhibits differentiation through DUOX1 and ASK1.

Stem Cell Antigen-1 in Skeletal Muscle Function

OpenAIRE

Bernstein, Harold S.; Samad, Tahmina; Cholsiripunlert, Sompob; Khalifian, Saami; Gong, Wenhui; Ritner, Carissa; Aurigui, Julian; Ling, Vivian; Wilschut, Karlijn J.; Bennett, Stephen; Hoffman, Julien; Oishi, Peter

2013-01-01

Stem cell antigen-1 (Sca-1) is a member of the Ly-6 multigene family encoding highly homologous, glycosyl-phosphatidylinositol-anchored membrane proteins. Sca-1 is expressed on muscle-derived stem cells and myogenic precursors recruited to sites of muscle injury. We previously reported that inhibition of Sca-1 expression stimulated myoblast proliferation in vitro and regulated the tempo of muscle repair in vivo. Despite its function in myoblast expansion during muscle repair, a role for Sca-1...

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

Czech Academy of Sciences Publication Activity Database

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

2015-01-01

Roč. 39, č. 5 (2015), s. 340-350 ISSN 0191-3123 R&D Projects: GA ČR(CZ) GB14-36804G; GA MŠk(CZ) LH13028 Institutional support: RVO:67985823 Keywords : cristae * Fiji * image analysis * mitochondrial disorders * myoblasts * ultrastructure Subject RIV: EA - Cell Biology Impact factor: 0.828, year: 2015

Signals of Ezh2, Src, and Akt Involve in Myostatin-Pax7 Pathways Regulating the Myogenic Fate Determination during the Sheep Myoblast Proliferation and Differentiation

Science.gov (United States)

Li, Li; Liu, Ruizao; Zhang, Li; Zhao, Fuping; Lu, Jian; Zhang, Xiaoning; Du, Lixin

2015-01-01

Myostatin and Pax7 have been well documented individually, however, the mechanism by which Myostatin regulates Pax7 is seldom reported. Here, based on muscle transcriptome analysis in Texel (Myostatin mutant) and Ujumqin (wild type) sheep across the five fetal stages, we constructed and examined the Myostatin-Pax7 pathways in muscle. Then we validated the signals by RNAi in the proliferating and differentiating sheep myoblasts in vitro at mRNA, protein, and cell morphological levels. We reveal that Myostatin signals to Pax7 at least through Ezh2, Src, and Akt during the sheep myoblast proliferation and differentiation. Other signals such as p38MAPK, mTOR, Erk1/2, Wnt, Bmp2, Smad, Tgfb1, and p21 are most probably involved in the Myostatin-affected myogenic events. Myostatin knockdown significantly reduces the counts of nucleus and myotube, but not the fusion index of myoblasts during cell differentiation. In addition, findings also indicate that Myostatin is required for normal myogenic differentiation of the sheep myoblasts, which is different from the C2C12 myoblasts. We expand the regulatory network of Myostatin-Pax7 pathways and first illustrate that Myostatin as a global regulator participates in the epigenetic events involved in myogenesis, which contributes to understand the molecular mechanism of Myostatin in regulation of myogenesis. PMID:25811841

Increased Muscular 5α-Dihydrotestosterone in Response to Resistance Training Relates to Skeletal Muscle Mass and Glucose Metabolism in Type 2 Diabetic Rats.

Directory of Open Access Journals (Sweden)

Naoki Horii

Full Text Available Regular resistance exercise induces skeletal muscle hypertrophy and improvement of glycemic control in type 2 diabetes patients. Administration of dehydroepiandrosterone (DHEA, a sex steroid hormone precursor, increases 5α-dihydrotestosterone (DHT synthesis and is associated with improvements in fasting blood glucose level and skeletal muscle hypertrophy. Therefore, the aim of this study was to investigate whether increase in muscle DHT levels, induced by chronic resistance exercise, can contribute to skeletal muscle hypertrophy and concomitant improvement of muscular glucose metabolism in type 2 diabetic rats. Male 20-week-old type 2 diabetic rats (OLETF were randomly divided into 3 groups: sedentary control, resistance training (3 times a week on alternate days for 8 weeks, or resistance training with continuous infusion of a 5α-reductase inhibitor (n = 8 each group. Age-matched, healthy nondiabetic Long-Evans Tokushima Otsuka (LETO rats (n = 8 were used as controls. The results indicated that OLETF rats showed significant decrease in muscular DHEA, free testosterone, DHT levels, and protein expression of steroidogenic enzymes, with loss of skeletal muscle mass and hyperglycemia, compared to that of LETO rats. However, 8-week resistance training in OLETF rats significantly increased the levels of muscle sex steroid hormones and protein expression of steroidogenic enzymes with a concomitant increase in skeletal muscle mass, improved fasting glucose level, and insulin sensitivity index. Moreover, resistance training accelerated glucose transporter-4 (GLUT-4 translocation and protein kinase B and C-ζ/λ phosphorylation. Administering the 5α-reductase inhibitor in resistance-trained OLETF rats resulted in suppression of the exercise-induced effects on skeletal muscle mass, fasting glucose level, insulin sensitivity index, and GLUT-4 signaling, with a decline in muscular DHT levels. These findings suggest that resistance training

Effects of curcumin and ursolic acid on the mitochondrial coupling efficiency and hydrogen peroxide emission of intact skeletal myoblasts.

Science.gov (United States)

Tueller, Daniel J; Harley, Jackson S; Hancock, Chad R

2017-10-21

Curcumin may improve blood glucose management, but the mechanism is not fully established. We demonstrated that curcumin (40 μM) reduced the mitochondrial coupling efficiency (percentage of oxygen consumption coupled to ATP synthesis) of intact skeletal muscle cells. A 30-minute pretreatment with curcumin reduced mitochondrial coupling efficiency by 17.0 ± 0.4% relative to vehicle (p Curcumin pretreatment also decreased the rate of hydrogen peroxide emission by 43 ± 13% compared to vehicle (p curcumin revealed a 40 ± 4% increase in the rate of oxygen consumption upon curcumin administration (p curcumin-pretreated cells after permeabilization of cell membranes (p > 0.7). The interaction between curcumin and ursolic acid, another natural compound that may improve blood glucose management, was also examined. Pretreatment with ursolic acid (0.12 μM) increased the mitochondrial coupling efficiency of intact cells by 4.1 ± 1.1% relative to vehicle (p curcumin when the two compounds were used in combination. The observed changes to mitochondrial coupling efficiency and hydrogen peroxide emission were consistent with the established effects of curcumin on blood glucose control. Our findings also show that changes to mitochondrial coupling efficiency after curcumin pretreatment may go undetected unless cells are assessed in the intact condition. Copyright © 2017 Elsevier Inc. All rights reserved.

Biomimetic elastomeric, conductive and biodegradable polycitrate-based nanocomposites for guiding myogenic differentiation and skeletal muscle regeneration.

Science.gov (United States)

Du, Yuzhang; Ge, Juan; Li, Yannan; Ma, Peter X; Lei, Bo

2018-03-01

Artificial muscle-like biomaterials have gained tremendous interests owing to their broad applications in regenerative medicine, wearable devices, bioelectronics and artificial intelligence. Unfortunately, key challenges are still existed for current materials, including biomimetic viscoelasticity, biocompatibility and biodegradation, multifunctionality. Herein, for the first time, we develop highly elastomeric, conductive and biodegradable poly (citric acid-octanediol-polyethylene glycol)(PCE)-graphene (PCEG) nanocomposites, and demonstrate their applications in myogenic differentiation and guiding skeletal muscle tissue regeneration. In PCEG nanocomposites, PCE provides the biomimetic elastomeric behavior, and the addition of reduced graphene oxide (RGO) endows the enhanced mechanical strength and conductivity. The highly elastomeric behavior, significantly enhanced modulus (400%-800%), strength (200%-300%) of PCEG nanocomposites with controlled biodegradability and electrochemical conductivity were achieved. The myoblasts proliferation and myogenic differentiation were significantly improved by PCEG nanocomposite. Significantly high in vivo biocompatibility of PCEG nanocomposites was observed when implanted in the subcutaneous tissue for 4 weeks in rats. PCEG nanocomposites could significantly enhance the muscle fibers and blood vessels formation in vivo in a skeletal muscle lesion model of rat. This study may provide a novel strategy to develop multifunctional elastomeric nanocomposites with high biocompatibility for potential soft tissue regeneration and stretchable bioelectronic devices. Copyright © 2017 Elsevier Ltd. All rights reserved.

Skeletal imaging composition

International Nuclear Information System (INIS)

Vanduzee, B.F.; Degenhardt, C.R.

1983-01-01

This invention is based on the discovery that the adjustment of pH, within a particular range, during the process of manufacturing lyophilized diphosphonate-containing skeletal imaging kits yields a kit which produces a technetium skeletal imaging agent with superior imaging properties. This increased performance is manifested through faster blood clearance and higher skeletal uptake of the technetium imaging agent. The process for producing a dry-powder imaging kit comprises the steps of: preparing a solution of a diphosphonate carrier, stannous reductant, and a stabilizer in water; adjusting the pH to between 5.5 and 6.5; and lyophilizing the solution

Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy.

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Siemionow, M; Cwykiel, J; Heydemann, A; Garcia-Martinez, J; Siemionow, K; Szilagyi, E

2018-04-01

Over the past decade different stem cell (SC) based approaches were tested to treat Duchenne Muscular Dystrophy (DMD), a lethal X-linked disorder caused by mutations in dystrophin gene. Despite research efforts, there is no curative therapy for DMD. Allogeneic SC therapies aim to restore dystrophin in the affected muscles; however, they are challenged by rejection and limited engraftment. Thus, there is a need to develop new more efficacious SC therapies. Chimeric Cells (CC), created via ex vivo fusion of donor and recipient cells, represent a promising therapeutic option for tissue regeneration and Vascularized Composite Allotransplantation (VCA) due to tolerogenic properties that eliminate the need for lifelong immunosuppression. This proof of concept study tested feasibility of myoblast fusion for Dystrophin Expressing. Chimeric Cell (DEC) therapy through in vitro characterization and in vivo assessment of engraftment, survival, and efficacy in the mdx mouse model of DMD. Murine DEC were created via ex vivo fusion of normal (snj) and dystrophin-deficient (mdx) myoblasts using polyethylene glycol. Efficacy of myoblast fusion was confirmed by flow cytometry and dystrophin immunostaining, while proliferative and myogenic differentiation capacity of DEC were assessed in vitro. Therapeutic effect after DEC transplant (0.5 × 10 6 ) into the gastrocnemius muscle (GM) of mdx mice was assessed by muscle functional tests. At 30 days post-transplant dystrophin expression in GM of injected mdx mice increased to 37.27 ± 12.1% and correlated with improvement of muscle strength and function. Our study confirmed feasibility and efficacy of DEC therapy and represents a novel SC based approach for treatment of muscular dystrophies.

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

Activation of muscarinic M-1 cholinoceptors by curcumin to increase glucose uptake into skeletal muscle isolated from Wistar rats.

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Cheng, Tse-Chou; Lin, Chian-Shiung; Hsu, Chih-Chieh; Chen, Li-Jen; Cheng, Kai-Chun; Cheng, Juei-Tang

2009-11-20

Curcumin, an active principle contained in rhizome of Curcuma longa, has been mentioned to show merit for diabetes through its anti-oxidative and anti-inflammatory properties. In the present study, we found that curcumin caused a concentration-dependent increase of glucose uptake into skeletal muscle isolated from Wistar rats. This action was inhibited by pirenzepine at concentration enough to block muscarinic M-1 cholinoceptor (M(1)-mAChR). In radioligand binding assay, the binding of [(3)H]-pirenzepine was also displaced by curcumin in a concentration-dependent manner. In the presence of inhibitors for PLC-PI3K pathway, either U73122 (phospholipase C inhibitor) or LY294002 (phosphoinositide 3-kinase inhibitor), curcumin-stimulated glucose uptake into skeletal muscle was markedly reduced. In Western blotting analysis, the membrane protein level of glucose transporter 4 (GLUT4) increased by curcumin was also reversed by blockade of M(1)-mAChR or PLC-PI3K pathway in a same manner. In conclusion, the obtained results suggest that curcumin can activate M(1)-mAChR at concentrations lower than to scavenge free radicals for increase of glucose uptake into skeletal muscle through PLC-PI3-kinase pathway.

Skeletal muscle performance and ageing.

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

2018-02-01

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

Fasting Increases Human Skeletal Muscle Net Phenylalanine Release and This Is Associated with Decreased mTOR Signaling

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Vendelbo, Mikkel Holm; Møller, Andreas Buch; Christensen, Britt; Nellemann, Birgitte; Clasen, Berthil Frederik Forrest; Nair, K. Sreekumaran; Jørgensen, Jens Otto Lunde; Jessen, Niels; Møller, Niels

2014-01-01

Aim Fasting is characterised by profound changes in energy metabolism including progressive loss of body proteins. The underlying mechanisms are however unknown and we therefore determined the effects of a 72-hour-fast on human skeletal muscle protein metabolism and activation of mammalian target of rapamycin (mTOR), a key regulator of cell growth. Methods Eight healthy male volunteers were studied twice: in the postabsorptive state and following 72 hours of fasting. Regional muscle amino acid kinetics was measured in the forearm using amino acid tracers. Signaling to protein synthesis and breakdown were assessed in skeletal muscle biopsies obtained during non-insulin and insulin stimulated conditions on both examination days. Results Fasting significantly increased forearm net phenylalanine release and tended to decrease phenylalanine rate of disappearance. mTOR phosphorylation was decreased by ∼50% following fasting, together with reduced downstream phosphorylation of 4EBP1, ULK1 and rpS6. In addition, the insulin stimulated increase in mTOR and rpS6 phosphorylation was significantly reduced after fasting indicating insulin resistance in this part of the signaling pathway. Autophagy initiation is in part regulated by mTOR through ULK1 and fasting increased expression of the autophagic marker LC3B-II by ∼30%. p62 is degraded during autophagy but was increased by ∼10% during fasting making interpretation of autophagic flux problematic. MAFbx and MURF1 ubiquitin ligases remained unaltered after fasting indicating no change in protesomal protein degradation. Conclusions Our results show that during fasting increased net phenylalanine release in skeletal muscle is associated to reduced mTOR activation and concomitant decreased downstream signaling to cell growth. PMID:25020061

Fasting increases human skeletal muscle net phenylalanine release and this is associated with decreased mTOR signaling.

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Mikkel Holm Vendelbo

Full Text Available Fasting is characterised by profound changes in energy metabolism including progressive loss of body proteins. The underlying mechanisms are however unknown and we therefore determined the effects of a 72-hour-fast on human skeletal muscle protein metabolism and activation of mammalian target of rapamycin (mTOR, a key regulator of cell growth.Eight healthy male volunteers were studied twice: in the postabsorptive state and following 72 hours of fasting. Regional muscle amino acid kinetics was measured in the forearm using amino acid tracers. Signaling to protein synthesis and breakdown were assessed in skeletal muscle biopsies obtained during non-insulin and insulin stimulated conditions on both examination days.Fasting significantly increased forearm net phenylalanine release and tended to decrease phenylalanine rate of disappearance. mTOR phosphorylation was decreased by ∼50% following fasting, together with reduced downstream phosphorylation of 4EBP1, ULK1 and rpS6. In addition, the insulin stimulated increase in mTOR and rpS6 phosphorylation was significantly reduced after fasting indicating insulin resistance in this part of the signaling pathway. Autophagy initiation is in part regulated by mTOR through ULK1 and fasting increased expression of the autophagic marker LC3B-II by ∼30%. p62 is degraded during autophagy but was increased by ∼10% during fasting making interpretation of autophagic flux problematic. MAFbx and MURF1 ubiquitin ligases remained unaltered after fasting indicating no change in protesomal protein degradation.Our results show that during fasting increased net phenylalanine release in skeletal muscle is associated to reduced mTOR activation and concomitant decreased downstream signaling to cell growth.

Discovery of a mammalian splice variant of myostatin that stimulates myogenesis.

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Ferenc Jeanplong

Full Text Available Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over-expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P<0.05, which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a

Increased Muscular 5α-Dihydrotestosterone in Response to Resistance Training Relates to Skeletal Muscle Mass and Glucose Metabolism in Type 2 Diabetic Rats

OpenAIRE

Horii, Naoki; Sato, Koji; Mesaki, Noboru; Iemitsu, Motoyuki

2016-01-01

Regular resistance exercise induces skeletal muscle hypertrophy and improvement of glycemic control in type 2 diabetes patients. Administration of dehydroepiandrosterone (DHEA), a sex steroid hormone precursor, increases 5?-dihydrotestosterone (DHT) synthesis and is associated with improvements in fasting blood glucose level and skeletal muscle hypertrophy. Therefore, the aim of this study was to investigate whether increase in muscle DHT levels, induced by chronic resistance exercise, can co...

Modulation of Protein Quality Control and Proteasome to Autophagy Switch in Immortalized Myoblasts from Duchenne Muscular Dystrophy Patients

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Marion Wattin

2018-01-01

Full Text Available The maintenance of proteome integrity is of primary importance in post-mitotic tissues such as muscle cells; thus, protein quality control mechanisms must be carefully regulated to ensure their optimal efficiency, a failure of these processes being associated with various muscular disorders. Duchenne muscular dystrophy (DMD is one of the most common and severe forms of muscular dystrophies and is caused by mutations in the dystrophin gene. Protein quality control modulations have been diversely observed in degenerating muscles of patients suffering from DMD or in animal models of the disease. In this study, we investigated whether modulations of protein quality control mechanisms already pre-exist in undifferentiated myoblasts originating from DMD patients. We report for the first time that the absence of dystrophin in human myoblasts is associated with protein aggregation stress characterized by an increase of protein aggregates. This stress is combined with BAG1 to BAG3 switch, NFκB activation and up-regulation of BAG3/HSPB8 complexes that ensure preferential routing of misfolded/aggregated proteins to autophagy rather than to deficient 26S proteasome. In this context, restoration of pre-existing alterations of protein quality control processes might represent an alternative strategy for DMD therapies.

Skeletal muscle contraction-induced vasodilation in the microcirculation.

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Hong, Kwang-Seok; Kim, Kijeong

2017-10-01

Maximal whole body exercise leads skeletal muscle blood flow to markedly increase to match metabolic demands, a phenomenon termed exercise hyperaemia that is accomplished by increasing vasodilation. However, local vasodilatory mechanisms in response to skeletal muscle contraction remain uncertain. This review highlights metabolic vasodilators released from contracting skeletal muscle, endothelium, or blood cells. As a considerable skeletal muscle vasodilation potentially results in hypotension, sympathetic nerve activity needs to be augmented to elevate cardiac output and blood pressure during dynamic exercise. However, since the enhanced sympathetic vasoconstriction restrains skeletal muscle blood flow, intramuscular arteries have an indispensable ability to blunt sympathetic activity for exercise hyperaemia. In addition, we discuss that mechanical compression of the intramuscular vasculature contributes to causing the initial phase of increasing vasodilation following a single muscle contraction. We have also chosen to focus on conducted (or ascending) electrical signals that evoke vasodilation of proximal feed arteries to elevate blood flow in the microcirculation of skeletal muscle. Endothelial hyperpolarization originating within distal arterioles ascends into the proximal feed arteries, thereby increasing total blood flow in contracting skeletal muscle. This brief review summarizes molecular mechanisms underlying the regulation of skeletal muscle blood flow to a single or sustained muscle contraction.

Skeletal Muscle Differentiation on a Chip Shows Human Donor Mesoangioblasts' Efficiency in Restoring Dystrophin in a Duchenne Muscular Dystrophy Model.

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Serena, Elena; Zatti, Susi; Zoso, Alice; Lo Verso, Francesca; Tedesco, F Saverio; Cossu, Giulio; Elvassore, Nicola

2016-12-01

: Restoration of the protein dystrophin on muscle membrane is the goal of many research lines aimed at curing Duchenne muscular dystrophy (DMD). Results of ongoing preclinical and clinical trials suggest that partial restoration of dystrophin might be sufficient to significantly reduce muscle damage. Different myogenic progenitors are candidates for cell therapy of muscular dystrophies, but only satellite cells and pericytes have already entered clinical experimentation. This study aimed to provide in vitro quantitative evidence of the ability of mesoangioblasts to restore dystrophin, in terms of protein accumulation and distribution, within myotubes derived from DMD patients, using a microengineered model. We designed an ad hoc experimental strategy to miniaturize on a chip the standard process of muscle regeneration independent of variables such as inflammation and fibrosis. It is based on the coculture, at different ratios, of human dystrophin-positive myogenic progenitors and dystrophin-negative myoblasts in a substrate with muscle-like physiological stiffness and cell micropatterns. Results showed that both healthy myoblasts and mesoangioblasts restored dystrophin expression in DMD myotubes. However, mesoangioblasts showed unexpected efficiency with respect to myoblasts in dystrophin production in terms of the amount of protein produced (40% vs. 15%) and length of the dystrophin membrane domain (210-240 µm vs. 40-70 µm). These results show that our microscaled in vitro model of human DMD skeletal muscle validated previous in vivo preclinical work and may be used to predict efficacy of new methods aimed at enhancing dystrophin accumulation and distribution before they are tested in vivo, reducing time, costs, and variability of clinical experimentation. This study aimed to provide in vitro quantitative evidence of the ability of human mesoangioblasts to restore dystrophin, in terms of protein accumulation and distribution, within myotubes derived from

Evidence for the involvement of the CXCL12 system in the adaptation of skeletal muscles to physical exercise.

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Puchert, Malte; Adams, Volker; Linke, Axel; Engele, Jürgen

2016-09-01

The chemokine CXCL12 and its primary receptor, CXCR4, not only promote developmental myogenesis, but also muscle regeneration. CXCL12 chemoattracts CXCR4-positive satellite cells/blood-borne progenitors to the injured muscle, promotes myoblast fusion, partially with existing myofibers, and induces angiogenesis in regenerating muscles. Interestingly, the mechanisms underlying muscle regeneration are in part identical to those involved in muscular adaptation to intensive physical exercise. These similarities now prompted us to determine whether physical exercise would impact the CXCL12 system in skeletal muscle. We found that CXCL12 and CXCR4 are upregulated in the gastrocnemius muscle of rats that underwent a four-week period of constrained daily running exercise on a treadmill. Double-staining experiments confirmed that CXCL12 and CXCR4 are predominantly expressed in MyHC-positive muscle fibers. Moreover, these training-dependent increases in CXCL12 and CXCR4 expression also occurred in rats with surgical coronary artery occlusion, implying that the muscular CXCL12 system is still active in skeletal myopathy resulting from chronic heart failure. Expression of the second CXCL12 receptor, CXCR7, which presumably acts as a scavenger receptor in muscle, was not affected by training. Attempts to dissect the molecular events underlying the training-dependent effects of CXCL12 revealed that the CXCL12-CXCR4 axis activates anabolic mTOR-p70S6K signaling and prevents upregulation of the catabolic ubiquitin ligase MurF-1 in C2C12 myotubes, eventually increasing myotube diameters. Together, these findings point to a pivotal role of the CXCL12-CXCR4 axis in exercise-induced muscle maintenance and/or growth. Copyright © 2016 Elsevier Inc. All rights reserved.

Endogenous Sonic Hedgehog limits inflammation and angiogenesis in the ischaemic skeletal muscle of mice.

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Caradu, Caroline; Guy, Alexandre; James, Chloé; Reynaud, Annabel; Gadeau, Alain-Pierre; Renault, Marie-Ange

2018-04-01

Hedgehog (Hh) signalling has been shown to be re-activated in ischaemic tissues and participate in ischaemia-induced angiogenesis. Sonic Hedgehog (Shh) is upregulated by more than 80-fold in the ischaemic skeletal muscle, however its specific role in ischaemia-induced angiogenesis has not yet been fully investigated. The purpose of the present study was to investigate the role of endogenous Shh in ischaemia-induced angiogenesis. To this aim, we used inducible Shh knock-out (KO) mice and unexpectedly found that capillary density was significantly increased in re-generating muscle of Shh deficient mice 5 days after hind limb ischaemia was induced, demonstrating that endogenous Shh does not promote angiogenesis but more likely limits it. Myosin and MyoD expression were equivalent in Shh deficient mice and control mice, indicating that endogenous Shh is not required for ischaemia-induced myogenesis. Additionally, we observed a significant increase in macrophage infiltration in the ischaemic muscle of Shh deficient mice. Our data indicate that this was due to an increase in chemokine expression by myoblasts in the setting of impaired Hh signalling, using tissue specific Smoothened conditional KO mice. The increased macrophage infiltration in mice deficient for Hh signalling in myocytes was associated with increased VEGFA expression and a transiently increased angiogenesis, demonstrating that Shh limits inflammation and angiogenesis indirectly by signalling to myocytes. Although ectopic administration of Shh has previously been shown to promote ischaemia-induced angiogenesis, the present study reveals that endogenous Shh does not promote ischaemia-induced angiogenesis. On the contrary, the absence of Shh leads to aberrant ischaemic tissue inflammation and a transiently increased angiogenesis.

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

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Konigsberg, M; Pérez, V I; Ríos, C; Liu, Y; Lee, S; Shi, Y; Van Remmen, H

2013-01-01

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.

A role for Insulin-like growth factor 2 in specification of the fast skeletal muscle fibre

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Ting Tao

2007-06-01

Full Text Available Abstract Background Fibre type specification is a poorly understood process beginning in embryogenesis in which skeletal muscle myotubes switch myosin-type to establish fast, slow and mixed fibre muscle groups with distinct function. Growth factors are required to establish slow fibres; it is unknown how fast twitch fibres are specified. Igf-2 is an embryonically expressed growth factor with established in vitro roles in skeletal muscle. Its localisation and role in embryonic muscle differentiation had not been established. Results Between E11.5 and E15.5 fast Myosin (FMyHC localises to secondary myotubes evenly distributed throughout the embryonic musculature and gradually increasing in number so that by E15.5 around half contain FMyHC. The Igf-2 pattern closely correlates with FMyHC from E13.5 and peaks at E15.5 when over 90% of FMyHC+ myotubes also contain Igf-2. Igf-2 lags FMyHC and it is absent from muscle myotubes until E13.5. Igf-2 strongly down-regulates by E17.5. A striking feature of the FMyHC pattern is its increased heterogeneity and attenuation in many fibres from E15.5 to day one after birth (P1. Transgenic mice (MIG which express Igf-2 in all of their myotubes, have increased FMyHC staining, a higher proportion of FMyHC+ myotubes and loose their FMyHC staining heterogeneity. In Igf-2 deficient mice (MatDi FMyHC+ myotubes are reduced to 60% of WT by E15.5. In vitro, MIG induces a 50% excess of FMyHC+ and a 30% reduction of SMHyC+ myotubes in C2 cells which can be reversed by Igf-2-targeted ShRNA resulting in 50% reduction of FMyHC. Total number of myotubes was not affected. Conclusion In WT embryos the appearance of Igf-2 in embryonic myotubes lags FMyHC, but by E15.5 around 45% of secondary myotubes contain both proteins. Forced expression of Igf-2 into all myotubes causes an excess, and absence of Igf-2 suppresses, the FMyHC+ myotube component in both embryonic muscle and differentiated myoblasts. Igf-2 is thus required, not for

Dual roles of palladin protein in in vitro myogenesis: inhibition of early induction but promotion of myotube maturation.

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Ngoc-Uyen-Nhi Nguyen

Full Text Available Palladin is a microfilament-associated phosphoprotein whose function in skeletal muscle has rarely been studied. Therefore, we investigate whether myogenesis is influenced by the depletion of palladin expression known to interfere with the actin cytoskeleton dynamic required for skeletal muscle differentiation. The inhibition of palladin in C2C12 myoblasts leads to precocious myogenic differentiation with a concomitant reduction in cell apoptosis. This premature myogenesis is caused, in part, by an accelerated induction of p21, myogenin, and myosin heavy chain, suggesting that palladin acts as a negative regulator in early differentiation phases. Paradoxically, palladin-knockdown myoblasts are unable to differentiate terminally, despite their ability to perform some initial steps of differentiation. Cells with attenuated palladin expression form thinner myotubes with fewer myonuclei compared to those of the control. It is noteworthy that a negative regulator of myogenesis, myostatin, is activated in palladin-deficient myotubes, suggesting the palladin-mediated impairment of late-stage myogenesis. Additionally, overexpression of 140-kDa palladin inhibits myoblast differentiation while 200-kDa and 90-kDa palladin-overexpressed cells display an enhanced differentiation rate. Together, our data suggest that palladin might have both positive and negative roles in maintaining the proper skeletal myogenic differentiation in vitro.

Heterokaryon analysis of muscle differentiation: regulation of the postmitotic state.

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Clegg, C H; Hauschka, S D

1987-08-01

MM14 mouse myoblasts withdraw irreversibly from the cell cycle and become postmitotic within a few hours of being deprived of fibroblast growth factor (Clegg, C. H., T. A. Linkhart, B. B. Olwin, and S. D. Hauschka, 1987, J. Cell Biol., 105:949-956). To examine the mechanisms that may regulate this developmental state of skeletal muscle, we tested the mitogen responsiveness of various cell types after their polyethylene glycol-mediated fusion with post-mitotic myocytes. Heterokaryons containing myocytes and quiescent nonmyogenic cells such as 3T3, L cell, and a differentiation-defective myoblast line (DD-1) responded to mitogen-rich medium by initiating DNA synthesis. Myonuclei replicated DNA and reexpressed thymidine kinase. In contrast, (myocyte x G1 myoblast) heterokaryons failed to replicate DNA in mitogen-rich medium and became postmitotic. This included cells with a nuclear ratio of three myoblasts to one myocyte. Proliferation dominance in (myocyte x 3T3 cell) and (myocyte x DD-1) heterokaryons was conditionally regulated by the timing of mitogen treatment; such cells became postmitotic when mitogen exposure was delayed for as little as 6 h after cell fusion. In addition, (myocyte x DD-1) heterokaryons expressed a muscle-specific trait and lost epidermal growth factor receptors when they became postmitotic. These results demonstrate that DNA synthesis is not irreversibly blocked in skeletal muscle; myonuclei readily express proliferation-related functions when provided with a mitogenic signal. Rather, myocyte-specific repression of DNA synthesis in heterokaryons argues that the postmitotic state of skeletal muscle is regulated by diffusible factors that inhibit processes of cellular mitogenesis.

Potentiation of cGMP signaling increases oxygen delivery and oxidative metabolism in contracting skeletal muscle of older but not young humans

DEFF Research Database (Denmark)

Nyberg, Michael Permin; Piil, Peter Bergmann; Egelund, Jon

2015-01-01

regulation remain unresolved. Cyclic guanosine monophosphate (cGMP) is one of the main second messengers that mediate smooth muscle vasodilation and alterations in cGMP signaling could, therefore, be one mechanism by which skeletal muscle perfusion is impaired with advancing age. The current study aimed...... to evaluate the effect of inhibiting the main enzyme involved in cGMP degradation, phosphodiesterase 5 (PDE5), on blood flow and O2 delivery in contracting skeletal muscle of young and older humans. A group of young (23 ± 1 years) and a group of older (72 ± 2 years) male human subjects performed submaximal...... in the older subjects correlated with the increase in leg O2 uptake (r (2) = 0.843). These findings suggest an insufficient O2 delivery to the contracting skeletal muscle of aged individuals and that reduced cGMP availability is a novel mechanism underlying impaired skeletal muscle perfusion with advancing age....

Increase in relative skeletal muscle mass over time and its inverse association with metabolic syndrome development: a 7-year retrospective cohort study.

Science.gov (United States)

Kim, Gyuri; Lee, Seung-Eun; Jun, Ji Eun; Lee, You-Bin; Ahn, Jiyeon; Bae, Ji Cheol; Jin, Sang-Man; Hur, Kyu Yeon; Jee, Jae Hwan; Lee, Moon-Kyu; Kim, Jae Hyeon

2018-02-05

Skeletal muscle mass was negatively associated with metabolic syndrome prevalence in previous cross-sectional studies. The aim of this study was to investigate the impact of baseline skeletal muscle mass and changes in skeletal muscle mass over time on the development of metabolic syndrome in a large population-based 7-year cohort study. A total of 14,830 and 11,639 individuals who underwent health examinations at the Health Promotion Center at Samsung Medical Center, Seoul, Korea were included in the analyses of baseline skeletal muscle mass and those changes from baseline over 1 year, respectively. Skeletal muscle mass was estimated by bioelectrical impedance analysis and was presented as a skeletal muscle mass index (SMI), a body weight-adjusted appendicular skeletal muscle mass value. Using Cox regression models, hazard ratio for developing metabolic syndrome associated with SMI values at baseline or changes of SMI over a year was analyzed. During 7 years of follow-up, 20.1% of subjects developed metabolic syndrome. Compared to the lowest sex-specific SMI tertile at baseline, the highest sex-specific SMI tertile showed a significant inverse association with metabolic syndrome risk (adjusted hazard ratio [AHR] = 0.61, 95% confidence interval [CI] 0.54-0.68). Furthermore, compared with SMI changes metabolic syndrome development were 0.87 (95% CI 0.78-0.97) for 0-1% changes and 0.67 (0.56-0.79) for > 1% changes in SMI over 1 year after additionally adjusting for baseline SMI and glycometabolic parameters. An increase in relative skeletal muscle mass over time has a potential preventive effect on developing metabolic syndrome, independently of baseline skeletal muscle mass and glycometabolic parameters.

Thimerosal-induced apoptosis in mouse C2C12 myoblast cells occurs through suppression of the PI3K/Akt/survivin pathway.

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Wen-Xue Li

Full Text Available BACKGROUND: Thimerosal, a mercury-containing preservative, is one of the most widely used preservatives and found in a variety of biological products. Concerns over its possible toxicity have reemerged recently due to its use in vaccines. Thimerosal has also been reported to be markedly cytotoxic to neural tissue. However, little is known regarding thimerosal-induced toxicity in muscle tissue. Therefore, we investigated the cytotoxic effect of thimerosal and its possible mechanisms on mouse C2C12 myoblast cells. METHODOLOGY/PRINCIPAL FINDINGS: The study showed that C2C12 myoblast cells underwent inhibition of proliferation and apoptosis after exposure to thimerosal (125-500 nM for 24, 48 and 72 h. Thimerosal caused S phase arrest and induced apoptosis as assessed by flow cytometric analysis, Hoechst staining and immunoblotting. The data revealed that thimerosal could trigger the leakage of cytochrome c from mitochondria, followed by cleavage of caspase-9 and caspase-3, and that an inhibitor of caspase could suppress thimerosal-induced apoptosis. Thimerosal inhibited the phosphorylation of Akt(ser473 and survivin expression. Wortmannin, a PI3K inhibitor, inhibited Akt activity and decreased survivin expression, resulting in increased thimerosal-induced apoptosis in C2C12 cells, while the activation of PI3K/Akt pathway by mIGF-I (50 ng/ml increased the expression of survivin and attenuated apoptosis. Furthermore, the inhibition of survivin expression by siRNA enhanced thimerosal-induced cell apoptosis, while overexpression of survivin prevented thimerosal-induced apoptosis. Taken together, the data show that the PI3K/Akt/survivin pathway plays an important role in the thimerosal-induced apoptosis in C2C12 cells. CONCLUSIONS/SIGNIFICANCE: Our results suggest that in C2C12 myoblast cells, thimerosal induces S phase arrest and finally causes apoptosis via inhibition of PI3K/Akt/survivin signaling followed by activation of the mitochondrial apoptotic

A novel approach for in vitro meat production.

Science.gov (United States)

Pandurangan, Muthuraman; Kim, Doo Hwan

2015-07-01

The present review describes the possibility of in vitro meat production with the help of advanced co-culturing methods. In vitro meat production method could be a possible alternative for the conventional meat production. Originally, the research on in vitro meat production was initiated by the National Aeronautics and Space Administration (NASA) for space voyages. The required key qualities for accepting in vitro meat for consumption would be good efficiency ratio, increased protein synthesis rate in skeletal muscles, and mimicking the conventional meat qualities. In vitro culturing of meat is possible with the use of skeletal muscle tissue engineering, stem cell, cell co-culture, and tissue culture methods. Co-culture of myoblast and fibroblast is believed as one of the major techniques for in vitro meat production. In our lab, we have co-cultured myoblast and fibroblast. We believe that a billion pounds of in vitro meat could be produced from one animal for consumption. However, we require a great deal of research on in vitro meat production.

Tissue Engineered Skeletal Myofibers can Directly "Sense" Gravitational Force Changes

Science.gov (United States)

Vandenburgh, Herman H.; Shansky, J.; DelTatto, M.; Lee, Peter; Meir, J.

1999-01-01

Long-term manned space flight requires a better understanding of skeletal muscle atrophy resulting from microgravity. Atrophy most likely results from changes at both the systemic level (e.g. decreased circulating growth hormone, increased circulating glucocorticoids) and locally (e.g. decreased myofiber resting tension). Differentiated skeletal myofibers in tissue culture have provided a model system over the last decade for gaining a better understanding of the interactions of exogenous growth factors, endogenous growth factors, and muscle fiber tension in regulating protein turnover rates and muscle cell growth. Tissue engineering these cells into three dimensional bioartificial muscle (BAM) constructs has allowed us to extend their use to Space flight studies for the potential future development of countermeasures. Embryonic avian muscle cells were isolated and BAMs tissue engineered as described previously. The myoblasts proliferate and fuse into aligned postmitotic myofibers after ten to fourteen days in vitro. A cylindrical muscle-like structure containing several thousand myofibers is formed which is approximately 30 mm in length, 2-3 mm in diameter, and attached at each end. For the Space Shuttle experiments, the BAMs were transferred to 55 mL bioreactor cartridges (6 BAMs/cartridge). At Kennedy Space Center, the cartridges were mounted in two Space Tissue Loss (STL) Modules (three to four cartridges per Module) and either maintained as ground controls or loaded in a Mid-Deck locker of the Space Shuttle. The BAM cartridges were continuously perfused during the experiment at 1.5 mL/ min with tissue culture medium. Eighteen BAMs were flown for nine days on Mission STS66 while eighteen BAMs served as ground controls. The complete experiment was repeated on Mission STS77 with twenty four BAMs in each group. BAMs could be maintained in a healthy state for at least 30 days in the perfusion bioreactor cartridges. The BAM muscle fibers directly detected both the

Detection of satellite cells during skeletal muscle wound healing in rats: time-dependent expressions of Pax7 and MyoD in relation to wound age.

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Tian, Zhi-Ling; Jiang, Shu-Kun; Zhang, Miao; Wang, Meng; Li, Jiao-Yong; Zhao, Rui; Wang, Lin-Lin; Li, Shan-Shan; Liu, Min; Zhang, Meng-Zhou; Guan, Da-Wei

2016-01-01

The study was focused on time-dependent expressions of paired-box transcription factor 7 (Pax7) and myoblast determination protein (MyoD) during skeletal muscle wound healing. An animal model of skeletal muscle contusion was established in 40 Sprague-Dawley male rats. Samples were taken at 1, 3, 5, 7, 9, 13, 17, and 21 days after injury, respectively (five rats in each posttraumatic interval). Five rats were employed as control. By morphometric analysis, the data based on the number of Pax7(+)/MyoD(-), Pax7(+)/MyoD(+), and Pax7(-)/MyoD(+) cells were highly correlated with the wound age. Pax7 and MyoD expressions were upregulated after injury by Western blot and quantitative real-time PCR assays. The relative quantity of Pax7 protein peaked at 5 days after injury, which was >1.13, and decreased thereafter. Similarly, the relative quantity of MyoD mRNA expression peaked at 3 days after injury, which was >2.59. The relative quantity of Pax7 protein >0.73 or mRNA expression >2.38 or the relative quantity of MyoD protein >1.33 suggested a wound age of 3 to 7 days. The relative quantity of MyoD mRNA expression >2.02 suggested a wound age of 1 to 7 days post-injury. In conclusion, the expressions of Pax7 and MyoD are upregulated in a time-dependent manner during skeletal muscle wound healing, suggesting that Pax7 and MyoD may be potential markers for wound age estimation in skeletal muscle.

Reduced Dnmt3a increases Gdf5 expression with suppressed satellite cell differentiation and impaired skeletal muscle regeneration.

Science.gov (United States)

Hatazawa, Yukino; Ono, Yusuke; Hirose, Yuma; Kanai, Sayaka; Fujii, Nobuharu L; Machida, Shuichi; Nishino, Ichizo; Shimizu, Takahiko; Okano, Masaki; Kamei, Yasutomi; Ogawa, Yoshihiro

2018-03-01

DNA methylation is an epigenetic mechanism regulating gene expression. In this study, we observed that DNA methyltransferase 3a (Dnmt3a) expression is decreased after muscle atrophy. We made skeletal muscle-specific Dnmt3a-knockout (Dnmt3a-KO) mice. The regeneration capacity after muscle injury was markedly decreased in Dnmt3a-KO mice. Diminished mRNA and protein expression of Dnmt3a were observed in skeletal muscles as well as in satellite cells, which are important for muscle regeneration, in Dnmt3a-KO mice. Dnmt3a-KO satellite cell showed smaller in size (length/area), suggesting suppressed myotube differentiation. Microarray analysis of satellite cells showed that expression of growth differentiation factor 5 (Gdf5) mRNA was markedly increased in Dnmt3a-KO mice. The DNA methylation level of the Gdf5 promoter was markedly decreased in Dnmt3a-KO satellite cells. In addition, DNA methylation inhibitor azacytidine treatment increased Gdf5 expression in wild-type satellite cells, suggesting Gdf5 expression is regulated by DNA methylation. Also, we observed increased inhibitor of differentiation (a target of Gdf5) mRNA expression in Dnmt3a-KO satellite cells. Thus, Dnmt3a appears to regulate satellite cell differentiation via DNA methylation. This mechanism may play a role in the decreased regeneration capacity during atrophy such as in aged sarcopenia.-Hatazawa, Y., Ono, Y., Hirose, Y., Kanai, S., Fujii, N. L., Machida, S., Nishino, I., Shimizu, T., Okano, M., Kamei, Y., Ogawa, Y. Reduced Dnmt3a increases Gdf5 expression with suppressed satellite cell differentiation and impaired skeletal muscle regeneration.

Andrographolide attenuates skeletal muscle dystrophy in mdx mice and increases efficiency of cell therapy by reducing fibrosis.

Science.gov (United States)

Cabrera, Daniel; Gutiérrez, Jaime; Cabello-Verrugio, Claudio; Morales, Maria Gabriela; Mezzano, Sergio; Fadic, Ricardo; Casar, Juan Carlos; Hancke, Juan L; Brandan, Enrique

2014-01-01

Duchenne muscular dystrophy (DMD) is characterized by the absence of the cytoskeletal protein dystrophin, muscle wasting, increased transforming growth factor type beta (TGF-β) signaling, and fibrosis. At the present time, the only clinically validated treatments for DMD are glucocorticoids. These drugs prolong muscle strength and ambulation of patients for a short term only and have severe adverse effects. Andrographolide, a bicyclic diterpenoid lactone, has traditionally been used for the treatment of colds, fever, laryngitis, and other infections with no or minimal side effects. We determined whether andrographolide treatment of mdx mice, an animal model for DMD, affects muscle damage, physiology, fibrosis, and efficiency of cell therapy. mdx mice were treated with andrographolide for three months and skeletal muscle histology, creatine kinase activity, and permeability of muscle fibers were evaluated. Fibrosis and TGF-β signaling were evaluated by indirect immunofluorescence and Western blot analyses. Muscle strength was determined in isolated skeletal muscles and by a running test. Efficiency of cell therapy was determined by grafting isolated skeletal muscle satellite cells onto the tibialis anterior of mdx mice. mdx mice treated with andrographolide exhibited less severe muscular dystrophy than untreated dystrophic mice. They performed better in an exercise endurance test and had improved muscle strength in isolated muscles, reduced skeletal muscle impairment, diminished fibrosis and a significant reduction in TGF-β signaling. Moreover, andrographolide treatment of mdx mice improved grafting efficiency upon intramuscular injection of dystrophin-positive satellite cells. These results suggest that andrographolide could be used to improve quality of life in individuals with DMD.

Increased mitochondrial substrate sensitivity in skeletal muscle of patients with type 2 diabetes

DEFF Research Database (Denmark)

Larsen, S; Stride, N; Hey-Mogensen, Martin

2011-01-01

AIMS/HYPOTHESIS: Mitochondrial respiration has been linked to insulin resistance. We studied mitochondrial respiratory capacity and substrate sensitivity in patients with type 2 diabetes (patients), and obese and lean control participants. METHODS: Mitochondrial respiration was measured.......4). Substrate sensitivity for octanoyl-carnitine did not differ between groups. CONCLUSIONS/INTERPRETATION: Increased mitochondrial substrate sensitivity is seen in skeletal muscle from type 2 diabetic patients and is confined to non-lipid substrates. Respiratory capacity per mitochondrion is not decreased...... and maximal oxygen uptake (VO2) were also determined. Insulin sensitivity was determined with the isoglycaemic-hyperinsulinaemic clamp technique. RESULTS: Insulin sensitivity was different (p

Skeletal scintigraphic changes in osteoporosis treated with sodium fluoride: concise communication

International Nuclear Information System (INIS)

Schulz, E.E.; Libanati, C.R.; Farley, S.M.; Kirk, G.A.; Baylink, D.J.

1984-01-01

An appendicular skeletal response to sodium fluoride (NaF) was detected by total skeletal scintigrams. Twelve postmenopausal osteoporotic women were treated with NaF (88 mg/day) and calcium (1500 mg/day). Total skeletal scintigrams were obtained before and during treatment. Within 4 to 21 mo (mean: 8.3), all 12 patients showed new areas of increased uptake corresponding to metaphyseal regions and short bones of the appendicular skeleton. Nine patients showed an increase in serum alkaline phosphatase activity, which was attributed to an increase in the skeletal isoenzyme. Seven of 12 patients developed bone pain in one or more of the regions of increased uptake. This study establishes that the skeletal scintigram is a sensitive index of the peripheral skeletal response to NaF

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

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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.

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

International Nuclear Information System (INIS)

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

2013-01-01

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

The CDM Superfamily Protein MBC Directs Myoblast Fusion through a Mechanism That Requires Phosphatidylinositol 3,4,5-Triphosphate Binding but Is Independent of Direct Interaction with DCrk▿§

Science.gov (United States)

Balagopalan, Lakshmi; Chen, Mei-Hui; Geisbrecht, Erika R.; Abmayr, Susan M.

2006-01-01

myoblast city (mbc), a member of the CDM superfamily, is essential in the Drosophila melanogaster embryo for fusion of myoblasts into multinucleate fibers. Using germ line clones in which both maternal and zygotic contributions were eliminated and rescue of the zygotic loss-of-function phenotype, we established that mbc is required in the fusion-competent subset of myoblasts. Along with its close orthologs Dock180 and CED-5, MBC has an SH3 domain at its N terminus, conserved internal domains termed DHR1 and DHR2 (or “Docker”), and C-terminal proline-rich domains that associate with the adapter protein DCrk. The importance of these domains has been evaluated by the ability of MBC mutations and deletions to rescue the mbc loss-of-function muscle phenotype. We demonstrate that the SH3 and Docker domains are essential. Moreover, ethyl methanesulfonate-induced mutations that change amino acids within the MBC Docker domain to residues that are conserved in other CDM family members nevertheless eliminate MBC function in the embryo, which suggests that these sites may mediate interactions specific to Drosophila MBC. A functional requirement for the conserved DHR1 domain, which binds to phosphatidylinositol 3,4,5-triphosphate, implicates phosphoinositide signaling in myoblast fusion. Finally, the proline-rich C-terminal sites mediate strong interactions with DCrk, as expected. These sites are not required for MBC to rescue the muscle loss-of-function phenotype, however, which suggests that MBC's role in myoblast fusion can be carried out independently of direct DCrk binding. PMID:17030600

Feasibility of dual reporter gene in rat myoblast cell line using human sodium iodide symporter (hNIS) and enhanced green fluorescent protein (EGFP) gene

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Lee, Yong Jin; Lee, You La; Ahn, Sohn Joo; Choi, Chang Ik; Lee, Sang Woo; Ahn, Byeong Cheol; Lee, Jae Tae [School of Medicine, Kyungpook National University, Daegu (Korea, Republic of)

2007-07-01

To develop a non-invasive combined imaging method of gamma camera and optical imaging to assess rat myoblast cell line, H9c2, we constructed retrovirus containing hNIS and EGFP gene, and transfected to rat myoblast cell and monitored hNIS and EGFP expression. Rat myoblast cell line, H9C2, was transfected with hNIS and EGFP gene using retrovirus (H9C2-NG). The expression of hNIS and EGFP gene was determined by RT-PCR and fluorescence microscopy, respectively. The uptake and efflux of I-125 were measured in the transfected and wild type cell lines. Each cell line was injected to 4 flank sites (H9c2: 1X107 or 2X107, H9C2-NG: 1X107 or 2X107) in nude mouse. Scintigraphic image was performed at 3h, 1 day after H9C2 and H9C2-NG cell inoculation. We performed gamma camera and animal PET imaging to evaluate NIS expression. Also, GFP image obtained using optical imaging system. The expression of hNIS and EGFP gene was confirmed by RT-PCR. In iodide uptake, H9C2-NG cells accumulated 274.52.2 pmol/ mg protein at 30 min. But wild type cell line did not uptake iodide. In fluorescent microscopy, H9C2-NG cells were highly fluorescent than that of H9C2 cells. In iodide efflux study, 50% of radioactivity flowed out during the first 10min. Scintigraphy showed increased uptake of Tc-99m in H9c2-NG than in H9C2 for 1 day. Also, H9C2-NG cells showed high signal-to-background fluorescent spots in animal body. In this study, NIS and EGFP reporter gene were successfully transfected by a retrovirus in myoblast cell line, and the transfected cell can be easily visualized in vivo. These results suggest that NIS and EGFP gene has an excellent feasibility as a reporter gene, and it can be used to monitor cell trafficking for monitoring.

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.

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

Directory of Open Access Journals (Sweden)

Swarnali Acharyya

2010-08-01

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

Culture Conditions Affect Expression of DUX4 in FSHD Myoblasts

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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.

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.

Increased expression of Myosin binding protein H in the skeletal muscle of amyotrophic lateral sclerosis patients

KAUST Repository

Conti, Antonio

2014-01-01

Amyotrophic lateral sclerosis (ALS) is a severe and fatal neurodegenerative disease of still unknown pathogenesis. Recent findings suggest that the skeletal muscle may play an active pathogenetic role. To investigate ALS\\'s pathogenesis and to seek diagnostic markers, we analyzed skeletal muscle biopsies with the differential expression proteomic approach. We studied skeletal muscle biopsies from healthy controls (CN), sporadic ALS (sALS), motor neuropathies (MN) and myopathies (M). Pre-eminently among several differentially expressed proteins, Myosin binding protein H (MyBP-H) expression in ALS samples was anomalously high. MyBP-H is a component of the thick filaments of the skeletal muscle and has strong affinity for myosin, but its function is still unclear. High MyBP-H expression level was associated with abnormal expression of Rho kinase 2 (ROCK2), LIM domain kinase 1 (LIMK1) and cofilin2, that might affect the actin-myosin interaction. We propose that MyBP-H expression level serves, as a putative biomarker in the skeletal muscle, to discriminate ALS from motor neuropathies, and that it signals the onset of dysregulation in actin-myosin interaction; this in turn might contribute to the pathogenesis of ALS. © 2013 Elsevier B.V.

Activation of the skeletal alpha-actin promoter during muscle regeneration.

Science.gov (United States)

Marsh, D R; Carson, J A; Stewart, L N; Booth, F W

1998-11-01

Little is known concerning promoter regulation of genes in regenerating skeletal muscles. In young rats, recovery of muscle mass and protein content is complete within 21 days. During the initial 5-10 days of regeneration, mRNA abundance for IGF-I, myogenin and MyoD have been shown to be dramatically increased. The skeletal alpha-actin promoter contains E box and serum response element (SRE) regulatory regions which are directly or indirectly activated by myogenin (or MyoD) and IGF-I proteins, respectively. We hypothesized that the skeletal alpha-actin promoter activity would increase during muscle regeneration, and that this induction would occur before muscle protein content returned to normal. Total protein content and the percentage content of skeletal alpha-actin protein was diminished at 4 and 8 days and re-accumulation had largely occurred by 16 days post-bupivacaine injection. Skeletal alpha-actin mRNA per whole muscle was decreased at day 8, and thereafter returned to control values. During regeneration at day 8, luciferase activity (a reporter of promoter activity) directed by -424 skeletal alpha-actin and -99 skeletal alpha-actin promoter constructs was increased by 700% and 250% respectively; however, at day 16, skeletal alpha-actin promoter activities were similar to control values. Thus, initial activation of the skeletal alpha-actin promoter is associated with regeneration of skeletal muscle, despite not being sustained during the later stages of regrowth. The proximal SRE of the skeletal alpha-actin promoter was not sufficient to confer a regeneration-induced promoter activation, despite increased serum response factor protein binding to this regulatory element in electrophoretic mobility shift assays. Skeletal alpha-actin promoter induction during regeneration is due to a combination of regulatory elements, at least including the SRE and E box.

Reduced Notch signalling leads to postnatal skeletal muscle hypertrophy in Pofut1cax/cax mice.

Science.gov (United States)

Al Jaam, Bilal; Heu, Katy; Pennarubia, Florian; Segelle, Alexandre; Magnol, Laetitia; Germot, Agnès; Legardinier, Sébastien; Blanquet, Véronique; Maftah, Abderrahman

2016-09-01

Postnatal skeletal muscle growth results from the activation of satellite cells and/or an increase in protein synthesis. The Notch signalling pathway maintains satellite cells in a quiescent state, and once activated, sustains their proliferation and commitment towards differentiation. In mammals, POFUT1-mediated O-fucosylation regulates the interactions between NOTCH receptors and ligands of the DELTA/JAGGED family, thus initiating the activation of canonical Notch signalling. Here, we analysed the consequences of downregulated expression of the Pofut1 gene on postnatal muscle growth in mutant Pofut1(cax/cax) (cax, compact axial skeleton) mice and differentiation of their satellite cell-derived myoblasts (SCDMs). Pofut1(cax/cax) mice exhibited muscle hypertrophy, no hyperplasia and a decrease in satellite cell numbers compared with wild-type C3H mice. In agreement with these observations, Pofut1(cax/cax) SCDMs differentiated earlier concomitant with reduced Pax7 expression and decrease in PAX7(+)/MYOD(-) progenitor cells. In vitro binding assays showed a reduced interaction of DELTA-LIKE 1 ligand (DLL1) with NOTCH receptors expressed at the cell surface of SCDMs, leading to a decreased Notch signalling as seen by the quantification of cleaved NICD and Notch target genes. These results demonstrated that POFUT1-mediated O-fucosylation of NOTCH receptors regulates myogenic cell differentiation and affects postnatal muscle growth in mice. © 2016 The Authors.

Low skeletal muscle mass is associated with increased hospital expenditure in patients undergoing cancer surgery of the alimentary tract

NARCIS (Netherlands)

J.L.A. van Vugt (Jeroen); S. Büttner (Stefan); S. Levolger (S.); R.R.J. Coebergh van den Braak (Robert); M. Suker (Mustafa); M.P. Gaspersz (Marcia); R.W.F. de Bruin (Ron); C. Verhoef (Kees); Van Eijck, C.H.C. (Casper H. C.); Bossche, N. (Niek); B. Groot Koerkamp (Bas); J.N.M. IJzermans (Jan)

2017-01-01

textabstractBackground: Low skeletal muscle mass is associated with poor postoperative outcomes in cancer patients. Furthermore, it is associated with increased healthcare costs in the United States. We investigated its effect on hospital expenditure in a Western-European healthcare system, with

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

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Knight James DR

2012-03-01

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

Gain-of-function R225W mutation in human AMPKgamma(3 causing increased glycogen and decreased triglyceride in skeletal muscle.

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Sheila R Costford

Full Text Available BACKGROUND: AMP-activated protein kinase (AMPK is a heterotrimeric enzyme that is evolutionarily conserved from yeast to mammals and functions to maintain cellular and whole body energy homeostasis. Studies in experimental animals demonstrate that activation of AMPK in skeletal muscle protects against insulin resistance, type 2 diabetes and obesity. The regulatory gamma(3 subunit of AMPK is expressed exclusively in skeletal muscle; however, its importance in controlling overall AMPK activity is unknown. While evidence is emerging that gamma subunit mutations interfere specifically with AMP activation, there remains some controversy regarding the impact of gamma subunit mutations. Here we report the first gain-of-function mutation in the muscle-specific regulatory gamma(3 subunit in humans. METHODS AND FINDINGS: We sequenced the exons and splice junctions of the AMPK gamma(3 gene (PRKAG3 in 761 obese and 759 lean individuals, identifying 87 sequence variants including a novel R225W mutation in subjects from two unrelated families. The gamma(3 R225W mutation is homologous in location to the gamma(2R302Q mutation in patients with Wolf-Parkinson-White syndrome and to the gamma(3R225Q mutation originally linked to an increase in muscle glycogen content in purebred Hampshire Rendement Napole (RN- pigs. We demonstrate in differentiated muscle satellite cells obtained from the vastus lateralis of R225W carriers that the mutation is associated with an approximate doubling of both basal and AMP-activated AMPK activities. Moreover, subjects bearing the R225W mutation exhibit a approximately 90% increase of skeletal muscle glycogen content and a approximately 30% decrease in intramuscular triglyceride (IMTG. CONCLUSIONS: We have identified for the first time a mutation in the skeletal muscle-specific regulatory gamma(3 subunit of AMPK in humans. The gamma(3R225W mutation has significant functional effects as demonstrated by increases in basal and AMP

Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress.

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Zhang, Qiang; Liu, Jianing; Chen, Shulan; Liu, Jing; Liu, Lijuan; Liu, Guirong; Wang, Fang; Jiang, Wenxin; Zhang, Caixia; Wang, Shuangyu; Yuan, Xiao

2016-04-01

It is well recognized that mandibular growth, which is caused by a variety of functional appliances, is considered to be the result of both neuromuscular and skeletal adaptations. Accumulating evidence has demonstrated that apoptosis plays an important role in the adaptation of skeletal muscle function. However, the underlying mechanism of apoptosis that is induced by stretch continues to be incompletely understood. Endoplasmic reticulum stress (ERS), a newly defined signaling pathway, initiates apoptosis. This study seeks to determine if caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress in myoblast and its underlying mechanism. Apoptosis was assessed by Hochest staining, DAPI staining and annexin V binding and PI staining. ER chaperones, such as GRP78, CHOP and caspase-12, were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Furthermore, caspase-12 inhibitor was used to value the mechanism of the caspase-12 pathway. Apoptosis of myoblast, which is subjected to cyclic stretch, was observed in a time-dependent manner. We found that GRP78 mRNA and protein were significantly increased and CHOP and caspase-12 were activated in myoblast that was exposed to cyclic stretch. Caspase-12 inhibition reduced stretch-induced apoptosis, and caspase-12 activated caspase-3 to induce apoptosis. We concluded that caspase-12 played an important role in stretch-induced apoptosis that is associated by endoplasmic reticulum stress by activating caspase-3.

Predictors Associated with Increase in Skeletal Muscle Mass after Sustained Virological Response in Chronic Hepatitis C Treated with Direct Acting Antivirals

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Kazunori Yoh

2017-10-01

Full Text Available Aims: We aimed to examine changes in skeletal muscle mass in chronic hepatitis C (CHC patients undergoing interferon (IFN-free direct acting antivirals (DAAs therapy who achieved sustained virological response (SVR. Patients and methods: A total of 69 CHC patients treated with DAAs were analyzed. We compared the changes in skeletal muscle index (SMI using bio-impedance analysis at baseline and SMI at SVR. SMI was calculated as the sum of skeletal muscle mass in upper and lower extremities divided by height squared (cm2/m2. Further, we identified pretreatment parameters contributing to the increased SMI at SVR. Results: SMI in males at baseline ranged from 6.73 to 9.08 cm2/m2 (median, 7.65 cm2/m2, while that in females ranged from 4.45 to 7.27 cm2/m2 (median, 5.81 cm2/m2. At SVR, 36 patients (52.2% had increased SMI as compared with baseline. In the univariate analysis, age (p = 0.0392, hyaluronic acid (p = 0.0143, and branched-chain amino acid to tyrosine ratio (BTR (p = 0.0024 were significant pretreatment factors linked to increased SMI at SVR. In the multivariate analysis, only BTR was an independent predictor linked to the increased SMI at SVR (p = 0.0488. Conclusion: Pretreatment BTR level can be helpful for predicting increased SMI after SVR in CHC patients undergoing IFN-free DAAs therapy.

Cold acclimation increases mitochondrial oxidative capacity without inducing mitochondrial uncoupling in goldfish white skeletal muscle

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Reinaldo Sousa Dos Santos

2012-11-01

Goldfish have been used for cold acclimation studies, which have focused on changes in glycolytic and oxidative enzymes or alterations in lipid composition in skeletal muscle. Here we examine the effects of cold acclimation on the functional properties of isolated mitochondria and permeabilized fibers from goldfish white skeletal muscle, focusing on understanding the types of changes that occur in the mitochondrial respiratory states. We observed that cold acclimation promoted a significant increase in the mitochondrial oxygen consumption rates. Western blot analysis showed that UCP3 was raised by ∼1.5-fold in cold-acclimated muscle mitochondria. Similarly, we also evidenced a rise in the adenine nucleotide translocase content in cold-acclimated muscle mitochondria compared to warm-acclimated mitochondria (0.96±0.05 vs 0.68±0.02 nmol carboxyatractyloside mg−1 protein. This was followed by a 2-fold increment in the citrate synthase activity, which suggests a higher mitochondrial content in cold-acclimated goldfish. Even with higher levels of UCP3 and ANT, the effects of activator (palmitate and inhibitors (carboxyatractyloside and GDP on mitochondrial parameters were similar in both warm- and cold-acclimated goldfish. Thus, we propose that cold acclimation in goldfish promotes an increase in functional oxidative capacity, with higher mitochondrial content without changes in the mitochondrial uncoupling pathways.

The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes

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Giebelstein, J; Poschmann, G; Højlund, K

2012-01-01

The molecular mechanisms underlying insulin resistance in skeletal muscle are incompletely understood. Here, we aimed to obtain a global picture of changes in protein abundance in skeletal muscle in obesity and type 2 diabetes, and those associated with whole-body measures of insulin action....

Increased expression of Myosin binding protein H in the skeletal muscle of amyotrophic lateral sclerosis patients.

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Conti, Antonio; Riva, Nilo; Pesca, Mariasabina; Iannaccone, Sandro; Cannistraci, Carlo V; Corbo, Massimo; Previtali, Stefano C; Quattrini, Angelo; Alessio, Massimo

2014-01-01

Amyotrophic lateral sclerosis (ALS) is a severe and fatal neurodegenerative disease of still unknown pathogenesis. Recent findings suggest that the skeletal muscle may play an active pathogenetic role. To investigate ALS's pathogenesis and to seek diagnostic markers, we analyzed skeletal muscle biopsies with the differential expression proteomic approach. We studied skeletal muscle biopsies from healthy controls (CN), sporadic ALS (sALS), motor neuropathies (MN) and myopathies (M). Pre-eminently among several differentially expressed proteins, Myosin binding protein H (MyBP-H) expression in ALS samples was anomalously high. MyBP-H is a component of the thick filaments of the skeletal muscle and has strong affinity for myosin, but its function is still unclear. High MyBP-H expression level was associated with abnormal expression of Rho kinase 2 (ROCK2), LIM domain kinase 1 (LIMK1) and cofilin2, that might affect the actin-myosin interaction. We propose that MyBP-H expression level serves, as a putative biomarker in the skeletal muscle, to discriminate ALS from motor neuropathies, and that it signals the onset of dysregulation in actin-myosin interaction; this in turn might contribute to the pathogenesis of ALS. © 2013 Elsevier B.V. All rights reserved.

NF-κB–YY1–miR-29 Regulatory Circuitry in Skeletal Myogenesis and Rhabdomyosarcoma

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Wang, Huating; Garzon, Ramiro; Sun, Hao; Ladner, Katherine J.; Singh, Ravi; Dahlman, Jason; Cheng, Alfred; Hall, Brett M.; Qualman, Stephen J.; Chandler, Dawn S.; Croce, Carlo M.; Guttridge, Denis C.

2008-01-01

SUMMARY Studies support the importance of microRNAs in physiological and pathological processes. Here we describe the regulation and function of miR-29 in myogenesis and Rhabdomyosarcoma (RMS). Results demonstrate that in myoblasts miR-29 is repressed by NF-κB acting through YY1 and the Polycomb. During myogenesis, NF-κB and YY1 downregulation causes derepression of miR-29, which in turn accelerates differentiation by targeting its repressor YY1. However, in RMS cells and primary tumors that possess impaired differentiation, miR-29 is epigenetically silenced by an activated NF-κB-YY1 pathway. Reconstitution of miR-29 in RMS in mice inhibits tumor growth and stimulates differentiation, suggesting that miR-29 acts as a tumor suppressor through its pro-myogenic function. Together, results identify a NF-κB–YY1–miR-29 regulatory circuit whose disruption may contribute to RMS. SIGNIFICANCE MicroRNAs regulate skeletal myogenesis, but their impact in muscle diseases is not well understood. Here we describe miR-29 as an enhancer of myogenic differentiation and a suppressor of RMS. We find that miR-29 exists in a regulatory circuit involving NF-κB and YY1. In myoblasts NF-B acts through YY1 to epigenetically suppress miR-29, while during differentiation miR-29 is induced to facilitate myogenesis by a negative feedback on YY1. Significantly, RMS tumors lose miR-29 due to an elevation in NF-B and YY1, and readjustment of miR-29 levels in RMS stimulates differentiation. Thus, myogenesis is dependent on NF-κB–YY1–miR-29 circuitry whose dysfunction may contribute to RMS pathogenesis. Such findings offer potential avenues for the diagnosis and treatment of muscle relevant cancers. PMID:18977326

Physical exercise in aging human skeletal muscle increases mitochondrial calcium uniporter expression levels and affects mitochondria dynamics.

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Zampieri, Sandra; Mammucari, Cristina; Romanello, Vanina; Barberi, Laura; Pietrangelo, Laura; Fusella, Aurora; Mosole, Simone; Gherardi, Gaia; Höfer, Christian; Löfler, Stefan; Sarabon, Nejc; Cvecka, Jan; Krenn, Matthias; Carraro, Ugo; Kern, Helmut; Protasi, Feliciano; Musarò, Antonio; Sandri, Marco; Rizzuto, Rosario

2016-12-01

Age-related sarcopenia is characterized by a progressive loss of muscle mass with decline in specific force, having dramatic consequences on mobility and quality of life in seniors. The etiology of sarcopenia is multifactorial and underlying mechanisms are currently not fully elucidated. Physical exercise is known to have beneficial effects on muscle trophism and force production. Alterations of mitochondrial Ca 2+ homeostasis regulated by mitochondrial calcium uniporter (MCU) have been recently shown to affect muscle trophism in vivo in mice. To understand the relevance of MCU-dependent mitochondrial Ca 2+ uptake in aging and to investigate the effect of physical exercise on MCU expression and mitochondria dynamics, we analyzed skeletal muscle biopsies from 70-year-old subjects 9 weeks trained with either neuromuscular electrical stimulation (ES) or leg press. Here, we demonstrate that improved muscle function and structure induced by both trainings are linked to increased protein levels of MCU Ultrastructural analyses by electron microscopy showed remodeling of mitochondrial apparatus in ES-trained muscles that is consistent with an adaptation to physical exercise, a response likely mediated by an increased expression of mitochondrial fusion protein OPA1. Altogether these results indicate that the ES-dependent physiological effects on skeletal muscle size and force are associated with changes in mitochondrial-related proteins involved in Ca 2+ homeostasis and mitochondrial shape. These original findings in aging human skeletal muscle confirm the data obtained in mice and propose MCU and mitochondria-related proteins as potential pharmacological targets to counteract age-related muscle loss. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Stem cell antigen-1 in skeletal muscle function.

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Bernstein, Harold S; Samad, Tahmina; Cholsiripunlert, Sompob; Khalifian, Saami; Gong, Wenhui; Ritner, Carissa; Aurigui, Julian; Ling, Vivian; Wilschut, Karlijn J; Bennett, Stephen; Hoffman, Julien; Oishi, Peter

2013-08-15

Stem cell antigen-1 (Sca-1) is a member of the Ly-6 multigene family encoding highly homologous, glycosyl-phosphatidylinositol-anchored membrane proteins. Sca-1 is expressed on muscle-derived stem cells and myogenic precursors recruited to sites of muscle injury. We previously reported that inhibition of Sca-1 expression stimulated myoblast proliferation in vitro and regulated the tempo of muscle repair in vivo. Despite its function in myoblast expansion during muscle repair, a role for Sca-1 in normal, post-natal muscle has not been thoroughly investigated. We systematically compared Sca-1-/- (KO) and Sca-1+/+ (WT) mice and hindlimb muscles to elucidate the tissue, contractile, and functional effects of Sca-1 in young and aging animals. Comparison of muscle volume, fibrosis, myofiber cross-sectional area, and Pax7+ myoblast number showed little differences between ages or genotypes. Exercise protocols, however, demonstrated decreased stamina in KO versus WT mice, with young KO mice achieving results similar to aging WT animals. In addition, KO mice did not improve with practice, while WT animals demonstrated conditioning over time. Surprisingly, myomechanical analysis of isolated muscles showed that KO young muscle generated more force and experienced less fatigue. However, KO muscle also demonstrated incomplete relaxation with fatigue. These findings suggest that Sca-1 is necessary for muscle conditioning with exercise, and that deficient conditioning in Sca-1 KO animals becomes more pronounced with age.

Skeletal and reticuloendothelial imaging in osteopetrosis: case report

International Nuclear Information System (INIS)

Park, H.M.; Lambertus, J.

1977-01-01

Skeletal and reticuloendothelial images, using Tc-99m HEDP and Tc-99m sulfur colloid, respectively, were obtained from two adult patients with osteopetrosis. Skeletal images demonstrated increased activity in multiple fracture sites, in mandibular osteomyelitis, in ends of splayed long bones adjacent to joints, and in the epiphyseal ends of short tubular bones. The remainder of the skeleton involved with osteopetrosis showed no generalized increased uptake of Tc-99m HEDP. These findings indicate that metabolic activity in this disease is abnormally increased in the usual areas of bone growth but appears normal elsewhere. Reticuloendothelial imaging showed an almost total lack of activity in the axial and peripheral skeletal marrow space. Anemia, however, was only moderate in these patients. Skeletal scintigraphy may be useful to evaluate the presence and extent of the frequent complications of osteopetrosis, namely fractures and osteomyelitis

Skeletal muscle and fetal alcohol spectrum disorder.

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Myrie, Semone B; Pinder, Mark A

2018-04-01

Skeletal muscle is critical for mobility and many metabolic functions integral to survival and long-term health. Alcohol can affect skeletal muscle physiology and metabolism, which will have immediate and long-term consequences on health. While skeletal muscle abnormalities, including morphological, biochemical, and functional impairments, are well-documented in adults that excessively consume alcohol, there is a scarcity of information about the skeletal muscle in the offspring prenatally exposed to alcohol ("prenatal alcohol exposure"; PAE). This minireview examines the available studies addressing skeletal muscle abnormalities due to PAE. Growth restriction, fetal alcohol myopathy, and abnormalities in the neuromuscular system, which contribute to deficits in locomotion, are some direct, immediate consequences of PAE on skeletal muscle morphology and function. Long-term health consequences of PAE-related skeletal abnormalities include impaired glucose metabolism in the skeletal muscle, resulting in glucose intolerance and insulin resistance, leading to an increased risk of type 2 diabetes. In general, there is limited information on the morphological, biochemical, and functional features of skeletal abnormalities in PAE offspring. There is a need to understand how PAE affects muscle growth and function at the cellular level during early development to improve the immediate and long-term health of offspring suffering from PAE.

Quantitative skeletal scintiscanning

International Nuclear Information System (INIS)

Haushofer, R.

1982-01-01

330 patients were examined by skeletal scintiscanning with sup(99m)Tc pyrophosphate and sup(99m)methylene diphosphonate in the years between 1977 and 1979. Course control examinations were carried out in 12 patients. The collective of patients presented with primary skeletal tumours, metastases, inflammatory and degenerative skeletal diseases. Bone scintiscanning combined with the ''region of interest'' technique was found to be an objective and reproducible technique for quantitative measurement of skeletal radioactivity concentrations. The validity of nuclear skeletal examinations can thus be enhanced as far as diagnosis, course control, and differential diagnosis are concerned. Quantitative skeletal scintiscanning by means of the ''region of interest'' technique has opened up a new era in skeletal diagnosis by nuclear methods. (orig./MG) [de

Expression profiling of skeletal muscle following acute and chronic β2-adrenergic stimulation: implications for hypertrophy, metabolism and circadian rhythm

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Lynch Gordon S

2009-09-01

Full Text Available Abstract Background Systemic administration of β-adrenoceptor (β-AR agonists has been found to induce skeletal muscle hypertrophy and significant metabolic changes. In the context of energy homeostasis, the importance of β-AR signaling has been highlighted by the inability of β1-3-AR-deficient mice to regulate energy expenditure and susceptibility to diet induced obesity. However, the molecular pathways and gene expression changes that initiate and maintain these phenotypic modulations are poorly understood. Therefore, the aim of this study was to identify differential changes in gene expression in murine skeletal muscle associated with systemic (acute and chronic administration of the β2-AR agonist formoterol. Results Skeletal muscle gene expression (from murine tibialis anterior was profiled at both 1 and 4 hours following systemic administration of the β2-AR agonist formoterol, using Illumina 46K mouse BeadArrays. Illumina expression profiling revealed significant expression changes in genes associated with skeletal muscle hypertrophy, myoblast differentiation, metabolism, circadian rhythm, transcription, histones, and oxidative stress. Differentially expressed genes relevant to the regulation of muscle mass and metabolism (in the context of the hypertrophic phenotype were further validated by quantitative RT-PCR to examine gene expression in response to both acute (1-24 h and chronic administration (1-28 days of formoterol at multiple timepoints. In terms of skeletal muscle hypertrophy, attenuation of myostatin signaling (including differential expression of myostatin, activin receptor IIB, phospho-Smad3 etc was observed following acute and chronic administration of formoterol. Acute (but not chronic administration of formoterol also significantly induced the expression of genes involved in oxidative metabolism, including hexokinase 2, sorbin and SH3 domain containing 1, and uncoupling protein 3. Interestingly, formoterol

Nitric oxide increases cyclic GMP levels, AMP-activated protein kinase (AMPK)alpha1-specific activity and glucose transport in human skeletal muscle

DEFF Research Database (Denmark)

Deshmukh, A S; Long, Y C; de Castro Barbosa, T

2010-01-01

-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) would increase intracellular cyclic GMP (cGMP) levels and promote glucose transport. METHODS: Skeletal muscle strips were prepared from vastus lateralis muscle biopsies obtained from seven healthy men. Muscle strips were incubated in the absence or presence...... of 5 mmol/l spermine NONOate or 120 nmol/l insulin. The L6 muscle cells were treated with spermine NONOate (20 micromol/l) and incubated in the absence or presence of insulin (120 nmol/l). The direct effect of spermine NONOate and insulin on glucose transport, cGMP levels and signal transduction...... was determined. RESULTS: In human skeletal muscle, spermine NONOate increased glucose transport 2.4-fold (p GMP levels (80-fold, p

Regulation of the skeletal muscle blood flow in humans

DEFF Research Database (Denmark)

Mortensen, Stefan; Saltin, Bengt

2014-01-01

In humans, skeletal muscle blood flow is regulated by an interaction between several locally formed vasodilators including nitric oxide (NO) and prostaglandins. In plasma, ATP is a potent vasodilator that stimulates the formation of NO and prostaglandins and very importantly can offset local...... concentration does not increase during exercise. In the skeletal muscle interstitium, there is a marked increase in the concentration of ATP and adenosine and this increase is tightly coupled to the increase in blood flow. The sources of interstitial ATP and adenosine are thought to be skeletal muscle cells...... hyperaemia whereas the role of ATP remains uncertain due to lack of specific purinergic receptor blockers for human use. The purpose of this review is to address the interaction between vasodilator systems and to discuss the multiple proposed roles of ATP in human skeletal muscle blood flow regulation...

Actovegin, a non-prohibited drug increases oxidative capacity in human skeletal muscle

DEFF Research Database (Denmark)

Søndergård, Stine D; Dela, Flemming; Helge, Jørn W

2016-01-01

Actovegin, a deproteinized haemodialysate of calf blood, is suggested to have ergogenic properties, but this potential effect has never been investigated in human skeletal muscle. To investigate this purported ergogenic effect, we measured the mitochondrial respiratory capacity in permeabilized h...

Meat Science and Muscle Biology Symposium: manipulating meat tenderness by increasing the turnover of intramuscular connective tissue.

Science.gov (United States)

Purslow, P P; Archile-Contreras, A C; Cha, M C

2012-03-01

Controlled reduction of the connective tissue contribution to cooked meat toughness is an objective that would have considerable financial impact in terms of added product value. The amount of intramuscular connective tissue in a muscle appears connected to its in vivo function, so reduction of the overall connective tissue content is not thought to be a viable target. However, manipulation of the state of maturity of the collagenous component is a biologically viable target; by increasing connective tissue turnover, less mature structures can be produced that are functional in vivo but more easily broken down on cooking at temperatures above 60°C, thus improving cooked meat tenderness. Recent work using cell culture models of fibroblasts derived from muscle and myoblasts has identified a range of factors that alter the activity of the principal enzymes responsible for connective tissue turnover, the matrix metalloproteinases (MMP). Fibroblasts cultured from 3 different skeletal muscles from the same animal show different cell proliferation and MMP activity, which may relate to the different connective tissue content and architecture in functionally different muscles. Expression of MMP by fibroblasts is increased by vitamins that can counter the negative effects of oxidative stress on new collagen synthesis. Preliminary work using in situ zymography of myotubes in culture also indicates increased MMP activity in the presence of epinephrine and reactive oxidative species. Comparison of the relative changes in MMP expression from muscle cells vs. fibroblasts shows that myoblasts are more responsive to a range of stimuli. Muscle cells are likely to produce more of the total MMP in muscle tissue as a whole, and the expression of latent forms of the enzymes (i.e., pro-MMP) may vary between oxidative and glycolytic muscle fibers within the same muscle. The implication is that the different muscle fiber composition of different muscles eaten as meat may influence the

Early de novo DNA methylation and prolonged demethylation in the muscle lineage.

Science.gov (United States)

Tsumagari, Koji; Baribault, Carl; Terragni, Jolyon; Varley, Katherine E; Gertz, Jason; Pradhan, Sirharsa; Badoo, Melody; Crain, Charlene M; Song, Lingyun; Crawford, Gregory E; Myers, Richard M; Lacey, Michelle; Ehrlich, Melanie

2013-03-01

Myogenic cell cultures derived from muscle biopsies are excellent models for human cell differentiation. We report the first comprehensive analysis of myogenesis-specific DNA hyper- and hypo-methylation throughout the genome for human muscle progenitor cells (both myoblasts and myotubes) and skeletal muscle tissue vs. 30 non-muscle samples using reduced representation bisulfite sequencing. We also focused on four genes with extensive hyper- or hypo-methylation in the muscle lineage (PAX3, TBX1, MYH7B/MIR499 and OBSCN) to compare DNA methylation, DNaseI hypersensitivity, histone modification, and CTCF binding profiles. We found that myogenic hypermethylation was strongly associated with homeobox or T-box genes and muscle hypomethylation with contractile fiber genes. Nonetheless, there was no simple relationship between differential gene expression and myogenic differential methylation, rather only for subsets of these genes, such as contractile fiber genes. Skeletal muscle retained ~30% of the hypomethylated sites but only ~3% of hypermethylated sites seen in myogenic progenitor cells. By enzymatic assays, skeletal muscle was 2-fold enriched globally in genomic 5-hydroxymethylcytosine (5-hmC) vs. myoblasts or myotubes and was the only sample type enriched in 5-hmC at tested myogenic hypermethylated sites in PAX3/CCDC140 andTBX1. TET1 and TET2 RNAs, which are involved in generation of 5-hmC and DNA demethylation, were strongly upregulated in myoblasts and myotubes. Our findings implicate de novo methylation predominantly before the myoblast stage and demethylation before and after the myotube stage in control of transcription and co-transcriptional RNA processing. They also suggest that, in muscle, TET1 or TET2 are involved in active demethylation and in formation of stable 5-hmC residues.

Morphofunctional and Biochemical Approaches for Studying Mitochondrial Changes during Myoblasts Differentiation

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Elena Barbieri

2011-01-01

Full Text Available This study describes mitochondrial behaviour during the C2C12 myoblast differentiation program and proposes a proteomic approach to mitochondria integrated with classical morphofunctional and biochemical analyses. Mitochondrial ultrastructure variations were determined by transmission electron microscopy; mitochondrial mass and membrane potential were analysed by Mitotracker Green and JC-1 stains and by epifluorescence microscope. Expression of PGC1 , NRF1 , and Tfam genes controlling mitochondrial biogenesis was studied by real-time PCR. The mitochondrial functionality was tested by cytochrome c oxidase activity and COXII expression. Mitochondrial proteomic profile was also performed. These assays showed that mitochondrial biogenesis and activity significantly increase in differentiating myotubes. The proteomic profile identifies 32 differentially expressed proteins, mostly involved in oxidative metabolism, typical of myotubes formation. Other notable proteins, such as superoxide dismutase (MnSOD, a cell protection molecule, and voltage-dependent anion-selective channel protein (VDAC1 involved in the mitochondria-mediated apoptosis, were found to be regulated by the myogenic process. The integration of these approaches represents a helpful tool for studying mitochondrial dynamics, biogenesis, and functionality in comparative surveys on mitochondrial pathogenic or senescent satellite cells.

Effect of experimental hyperthyroidism on protein turnover in skeletal and cardiac muscle.

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Carter, W J; Van Der Weijden Benjamin, W S; Faas, F H

1980-10-01

Since experimental hyperthyroidism reduces skeletal muscle mass while simultaneously increasing cardiac muscle mass, the effect of hyperthyroidism on muscle protein degradation was compared in skeletal and cardiac muscle. Pulse-labeling studies using (3H) leucine and (14C) carboxyl labeled aspartate and glutamate were carried out. Hyperthyroidism caused a 25%-29% increase in protein breakdown in both sarcoplasmic and myofibrillar fractions of skeletal muscle. Increased muscle protein degradation may be a major factor in the development of skeletal muscle wasting and weakness in hyperthyroidism. In contrast, protein breakdown appeared to be reduced 22% in the sarcoplasmic fraction of hyperthyroid heart muscle and was unchanged in the myofibrillar fraction. Possible reasons for the contrasting effects of hyperthyroidism on skeletal and cardiac muscle include increased sensitivity of the hyperthyroid heart to catecholamines, increased cardiac work caused by the hemodynamic effects of hyperthyroidism, and a different direct effect of thyroid hormone at the nuclear level in cardiac as opposed to skeletal muscle.

Polyunsaturated fatty acids acutely affect triacylglycerol-derived skeletal muscle fatty acid uptake and increases postprandial insulin sensitivity

NARCIS (Netherlands)

Jans, Anneke; Konings, Ellen; Goossens, Gijs H.; Bouwman, Freek G.; Moors, Chantalle C.; Boekschoten, Mark; Afman, Lydia; Muller, Michael; Mariman, Edwin C.; Blaak, Ellen E.

2012-01-01

Dietary fat quality may influence skeletal muscle lipid handling and fat accumulation, thereby modulating insulin sensitivity. Objective: To examine acute effects of meals with various fatty acid (FA) compositions on skeletal muscle FA handling and postprandial insulin sensitivity in obese insulin

Induced skeletal mutations

International Nuclear Information System (INIS)

Selby, P.B.

1979-01-01

This paper describes a large-scale experiment that, by means of breeding tests, confirmed that many dominant skeletal mutations are induced by large-dose radiation exposure. The author also discusses: (1) the major advantages and disadvantages of the skeletal method in improving estimates of genetic hazard to man; (2) future uses of the skeletal method; (3) direct estimation of risk beyond the first generation using the skeletal method; and (4) the possibility of using the skeletal method as a quick and easy screen for chemical mutagens

Physical activity opposes the age-related increase in skeletal muscle and plasma endothelin-1 levels and normalizes plasma endothelin-1 levels in individuals with essential hypertension.

Science.gov (United States)

Nyberg, M; Mortensen, S P; Hellsten, Y

2013-03-01

Endothelin-1 has potent constrictor and proliferative activity in vascular smooth muscle, and essential hypertension and aging are associated with increased endothelin-1-mediated vasoconstrictor tone. The aim of this study was to investigate the effect of physical activity, hypertension and age on endothelin-1 levels in plasma and skeletal muscle and endothelin receptors in skeletal muscle in human subjects. In study 1, normotensive (46 ± 1 years, n = 11) and hypertensive (47 ± 1 years, n = 10) subjects were studied before and after 8 weeks of aerobic exercise training. In study 2, young (23 ± 1 years, n = 8), older lifelong sedentary (66 ± 2 years, n = 8) and older lifelong endurance-trained (62 ± 2 years, n = 8) subjects were studied in a cross-sectional design. Skeletal muscle and plasma endothelin-1 levels were increased with age and plasma endothelin-1 levels were higher in hypertensive than normotensive individuals. Eight weeks of exercise training normalized plasma endothelin-1 levels in the hypertensive subjects and increased the protein expression of the ET(A) receptor in skeletal muscle of normotensive subjects. Similarly, individuals that had performed lifelong physical activity had similar plasma and muscle endothelin-1 levels as the young controls and had higher ET(A) receptor levels. Our findings suggest that aerobic exercise training opposes the age-related increase in skeletal muscle and plasma endothelin-1 levels and normalizes plasma endothelin-1 levels in individuals with essential hypertension. This effect may explain some of the beneficial effects of training on the cardiovascular system in older and hypertensive subjects. © 2012 The Authors Acta Physiologica © 2012 Scandinavian Physiological Society.

Measurement of skeletal muscle collagen breakdown by microdialysis

DEFF Research Database (Denmark)

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

2011-01-01

Exercise increases the synthesis of collagen in the extracellular matrix of skeletal muscle. Breakdown of skeletal muscle collagen has not yet been determined because of technical limitations. The purpose of the present study was to use local sampling to determine skeletal muscle collagen breakdown...... collagen breakdown 17–21 h post-exercise, and our measurement of OHP using GC–MS was in agreement with traditional assays....

Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

International Nuclear Information System (INIS)

Takegahara, Yuki; Yamanouchi, Keitaro; Nakamura, Katsuyuki; Nakano, Shin-ichi; Nishihara, Masugi

2014-01-01

Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. - Highlights: • We examined the effects of pre- and mature adipocytes on myogenesis in vitro. • Preadipocytes and mature adipocytes affect myoblast fusion. • Preadipocytes play an important role in maintaining skeletal muscle mass. • Mature adipocytes lead to muscle deterioration observed in skeletal muscle pathologies

Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

Energy Technology Data Exchange (ETDEWEB)

Takegahara, Yuki; Yamanouchi, Keitaro, E-mail: akeita@mail.ecc.u-tokyo.ac.jp; Nakamura, Katsuyuki; Nakano, Shin-ichi; Nishihara, Masugi

2014-05-15

Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. - Highlights: • We examined the effects of pre- and mature adipocytes on myogenesis in vitro. • Preadipocytes and mature adipocytes affect myoblast fusion. • Preadipocytes play an important role in maintaining skeletal muscle mass. • Mature adipocytes lead to muscle deterioration observed in skeletal muscle pathologies.

MicroRNA-27a Is Induced by Leucine and Contributes to Leucine-Induced Proliferation Promotion in C2C12 Cells

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Guangmang Liu

2013-07-01

Full Text Available Leucine, a branched chain amino acid, is well known to stimulate protein synthesis in skeletal muscle. However, the role of leucine in myoblast proliferation remains unclear. In this study, we found that leucine could promote proliferation of C2C12 cells. Moreover, expressions of miR-27a and myostatin (a bona fide target of miR-27a were upregulated and downregulated, respectively, following leucine treatment. We also found that miR-27a loss-of-function by transfection of a miR-27a inhibitor suppressed the promotion of myoblast proliferation caused by leucine. Our results suggest that miR-27a is induced by leucine and contributes to leucine-induced proliferation promotion of myoblast.

TNF Inhibits Notch-1 in Skeletal Muscle Cells by Ezh2 and DNA Methylation Mediated Repression: Implications in Duchenne Muscular Dystrophy

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Acharyya, Swarnali; Sharma, Sudarshana M.; Cheng, Alfred S.; Ladner, Katherine J.; He, Wei; Kline, William; Wang, Huating; Ostrowski, Michael C.; Huang, Tim H.; Guttridge, Denis C.

2010-01-01

Background Classical NF-κB signaling functions as a negative regulator of skeletal myogenesis through potentially multiple mechanisms. The inhibitory actions of TNFα on skeletal muscle differentiation are mediated in part through sustained NF-κB activity. In dystrophic muscles, NF-κB activity is compartmentalized to myofibers to inhibit regeneration by limiting the number of myogenic progenitor cells. This regulation coincides with elevated levels of muscle derived TNFα that is also under IKKβ and NF-κB control. Methodology/Principal Findings Based on these findings we speculated that in DMD, TNFα secreted from myotubes inhibits regeneration by directly acting on satellite cells. Analysis of several satellite cell regulators revealed that TNFα is capable of inhibiting Notch-1 in satellite cells and C2C12 myoblasts, which was also found to be dependent on NF-κB. Notch-1 inhibition occurred at the mRNA level suggesting a transcriptional repression mechanism. Unlike its classical mode of action, TNFα stimulated the recruitment of Ezh2 and Dnmt-3b to coordinate histone and DNA methylation, respectively. Dnmt-3b recruitment was dependent on Ezh2. Conclusions/Significance We propose that in dystrophic muscles, elevated levels of TNFα and NF-κB inhibit the regenerative potential of satellite cells via epigenetic silencing of the Notch-1 gene. PMID:20814569

GSK3β is increased in adipose tissue and skeletal muscle from women with gestational diabetes where it regulates the inflammatory response.

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Martha Lappas

Full Text Available Infection and inflammation, through their ability to increase pro-inflammatory cytokines and chemokines and adhesion molecules, are thought to play a central role in the pathophysiology of insulin resistance and type 2 diabetes. Recent studies have shown that glycogen synthase kinase 3 (GSK3 plays a central role in regulating this inflammation. There are, however, no studies on the role of GSK3 in pregnancies complicated by gestational diabetes mellitus (GDM. Thus, the aims of this study were (i to determine whether GSK3 is increased in adipose tissue and skeletal muscle from women with GDM; and (ii to investigate the effect of GSK3 inhibition on inflammation in the presence of inflammation induced by bacterial endotoxin lipopolysaccharide (LPS or the pro-inflammatory cytokine IL-1β. Human omental adipose tissue and skeletal muscle were obtained from normal glucose tolerant (NGT women and BMI-matched women with diet-control GDM at the time of Caesarean section. Western blotting was performed to determine GSK3 protein expression. Tissue explants were performed to determine the effect of the GSK3 inhibitor CHIR99021 on markers of inflammation. When compared to women with NGT, omental adipose tissue and skeletal muscle obtained from women with diet-controlled GDM had significantly higher GSK3β activity as evidenced by a decrease in the expression of GSK3β phosphorylated at serine 9. The GSK3 inhibitor CHIR99021 significantly reduced the gene expression and secretion of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6; the pro-inflammatory chemokines IL-8 and MCP-1; and the adhesion molecules ICAM-1 and VCAM-1 in tissues stimulated with LPS or IL-1β. In conclusion, GSK3 activity is increased in GDM adipose tissue and skeletal muscle and regulates infection- and inflammation-induced pro-inflammatory mediators.

Role of skeletal muscle in ear development.

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Rot, Irena; Baguma-Nibasheka, Mark; Costain, Willard J; Hong, Paul; Tafra, Robert; Mardesic-Brakus, Snjezana; Mrduljas-Djujic, Natasa; Saraga-Babic, Mirna; Kablar, Boris

2017-10-01

The current paper is a continuation of our work described in Rot and Kablar, 2010. Here, we show lists of 10 up- and 87 down-regulated genes obtained by a cDNA microarray analysis that compared developing Myf5-/-:Myod-/- (and Mrf4-/-) petrous part of the temporal bone, containing middle and inner ear, to the control, at embryonic day 18.5. Myf5-/-:Myod-/- fetuses entirely lack skeletal myoblasts and muscles. They are unable to move their head, which interferes with the perception of angular acceleration. Previously, we showed that the inner ear areas most affected in Myf5-/-:Myod-/- fetuses were the vestibular cristae ampullaris, sensitive to angular acceleration. Our finding that the type I hair cells were absent in the mutants' cristae was further used here to identify a profile of genes specific to the lacking cell type. Microarrays followed by a detailed consultation of web-accessible mouse databases allowed us to identify 6 candidate genes with a possible role in the development of the inner ear sensory organs: Actc1, Pgam2, Ldb3, Eno3, Hspb7 and Smpx. Additionally, we searched for human homologues of the candidate genes since a number of syndromes in humans have associated inner ear abnormalities. Mutations in one of our candidate genes, Smpx, have been reported as the cause of X-linked deafness in humans. Our current study suggests an epigenetic role that mechanical, and potentially other, stimuli originating from muscle, play in organogenesis, and offers an approach to finding novel genes responsible for altered inner ear phenotypes.

Skeletal Muscle Angiogenesis and Its Relation to Insulin Sensitivity

DEFF Research Database (Denmark)

Lindqvist, Anna Maria Charlotte K

mediator of angiogenesis) are reduced in insulin resistant individuals. Exercise training can improve skeletal muscle capillarization and the angiogenic potential and physical activity has also been proven to enhance muscle insulin sensitivity. Increased skeletal muscle capillarization is associated......) or by overexpression of VEGF-A in the tibialis anterior muscle (transfection; study II) and the effect of the increased muscle capillarization on muscle insulin sensitivity was examined. In study I skeletal muscle specific angiogenesis was induced by administering an α1-adrenergic antagonist (prazosin) to healthy...

[Molecular mechanisms of skeletal muscle hypertrophy].

Science.gov (United States)

Astratenkova, I V; Rogozkin, V A

2014-06-01

Enzymes Akt, AMPK, mTOR, S6K and PGC-1a coactivator take part in skeletal muscles in the regulation of synthesis of proteins. The expression of these proteins is regulated by growth factors, hormones, nutrients, mechanical loading and leads to an increase in muscle mass and skeletal muscle hypertrophy. The review presents the results of studies published in the past four years, which expand knowledge on the effects of various factors on protein synthesis in skeletal muscle. The attention is focused on the achievements that reveal and clarify the signaling pathways involved in the regulation of protein synthesis in skeletal muscle. The central place is taken by mTOR enzyme which controls and regulates the main stages of the cascade of reactions of muscle proteins providing synthesis in the conditions of human life. coactivator PGC-1a.

p38 MAPK activation and H3K4 trimethylation is decreased by lactate in vitro and high intensity resistance training in human skeletal muscle.

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Lena Willkomm

Full Text Available Exercise induces adaptation of skeletal muscle by acutely modulating intracellular signaling, gene expression, protein turnover and myogenic activation of skeletal muscle stem cells (Satellite cells, SCs. Lactate (La-induced metabolic stimulation alone has been shown to modify SC proliferation and differentiation. Although the mechanistic basis remains elusive, it was demonstrated that La affects signaling via p38 mitogen activated protein kinase (p38 MAPK which might contribute to trimethylation of histone 3 lysine 4 (H3K4me3 known to regulate satellite cell proliferation and differentiation. We investigated the effects of La on p38 MAPK and H3K4me3 in a model of activated SCs. Differentiating C2C12 myoblasts were treated with La (20 mM and samples analysed using qRT-PCR, immunofluorescence, and western blotting. We determined a reduction of p38 MAPK phosphorylation, decreased H3K4me3 and reduced expression of Myf5, myogenin, and myosin heavy chain (MHC leading to decreased differentiation of La-treated C2C12 cells after 5 days of repeated La treatment. We further investigated whether this regulatory pathway would be affected in human skeletal muscle by the application of two different resistance exercise regimes (RE associated with distinct metabolic demands and blood La accumulation. Muscle biopsies were obtained 15, 30 min, 1, 4, and 24 h post exercise after moderate intensity RE (STD vs. high intensity RE (HIT. Consistent with in vitro results, reduced p38 phosphorylation and blunted H3K4me3 were also observed upon metabolically demanding HIT RE in human skeletal muscle. Our data provide evidence that La-accumulation acutely affects p38 MAPK signaling, gene expression and thereby cell differentiation and adaptation in vitro, and likely in vivo.

p38 MAPK activation and H3K4 trimethylation is decreased by lactate in vitro and high intensity resistance training in human skeletal muscle.

Science.gov (United States)

Willkomm, Lena; Gehlert, Sebastian; Jacko, Daniel; Schiffer, Thorsten; Bloch, Wilhelm

2017-01-01

Exercise induces adaptation of skeletal muscle by acutely modulating intracellular signaling, gene expression, protein turnover and myogenic activation of skeletal muscle stem cells (Satellite cells, SCs). Lactate (La)-induced metabolic stimulation alone has been shown to modify SC proliferation and differentiation. Although the mechanistic basis remains elusive, it was demonstrated that La affects signaling via p38 mitogen activated protein kinase (p38 MAPK) which might contribute to trimethylation of histone 3 lysine 4 (H3K4me3) known to regulate satellite cell proliferation and differentiation. We investigated the effects of La on p38 MAPK and H3K4me3 in a model of activated SCs. Differentiating C2C12 myoblasts were treated with La (20 mM) and samples analysed using qRT-PCR, immunofluorescence, and western blotting. We determined a reduction of p38 MAPK phosphorylation, decreased H3K4me3 and reduced expression of Myf5, myogenin, and myosin heavy chain (MHC) leading to decreased differentiation of La-treated C2C12 cells after 5 days of repeated La treatment. We further investigated whether this regulatory pathway would be affected in human skeletal muscle by the application of two different resistance exercise regimes (RE) associated with distinct metabolic demands and blood La accumulation. Muscle biopsies were obtained 15, 30 min, 1, 4, and 24 h post exercise after moderate intensity RE (STD) vs. high intensity RE (HIT). Consistent with in vitro results, reduced p38 phosphorylation and blunted H3K4me3 were also observed upon metabolically demanding HIT RE in human skeletal muscle. Our data provide evidence that La-accumulation acutely affects p38 MAPK signaling, gene expression and thereby cell differentiation and adaptation in vitro, and likely in vivo.

Use of 5-Bromodeoxyuridine and irradiation for the estimation of the myoblast and myocyte content of primary rat heart cell cultures

International Nuclear Information System (INIS)

Masse, M.J.O.; Harary, I.

1980-01-01

A method for killing dividing cells was adapted for the elimination of dividing heart muscle cells (myoblasts) in cultures. We have used this method to demonstrate their presence and to estimate their number as well as the number of nondividing heart muscle cells (myocytes) in the neo-natal rat heart. Cells were cultivated in BUdR (5-bromodeoxyuridine) 10 -4 M for 3 days and then irradiated with long uv light. The selective elimination of dividing cells led to a loss of myosin Ca 2+ -activated ATPase in the cultures. The percent of ATPase left after irradiation was 32% of the control in cultures derived from 1-day postnatal rats and 48% in cultures from 4-day postnatal rats. This reflects an in vivo shift of myoblasts to myocytes in the muscle cell population as the rat ages

Retrospective review to determine the utility of follow-up skeletal surveys in child abuse evaluations when the initial skeletal survey is normal

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Kachelmeyer Andrea

2011-09-01

Full Text Available Abstract Objective The AAP recommends that a follow-up skeletal survey be obtained for all children Methods A retrospective review of radiology records from September 1, 1998 - January 31, 2007 was conducted. Suspected victims of child abuse who were Results Forty-seven children had a negative initial skeletal survey and were included for analysis. The mean age was 6.9 months (SD 5.7; the mean number of days between skeletal surveys was 18.7 (SD 10.1 Four children (8.5% had signs of healing bone trauma on a follow-up skeletal survey. Three of these children (75% had healing rib fractures and one child had a healing proximal humerus fracture. The findings on the follow-up skeletal survey yielded forensically important information in all 4 cases and strengthened the diagnosis of non-accidental trauma. Conclusion 8.5 percent of children with negative initial skeletal surveys had forensically important findings on follow-up skeletal survey that increased the certainty of the diagnosis of non-accidental trauma. A follow-up skeletal survey can be useful even when the initial skeletal survey is negative.

Effect of Eicosapentaenoic Acid and Docosahexaenoic Acid on Myogenesis and Mitochondrial Biosynthesis during Murine Skeletal Muscle Cell Differentiation

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Tun-Yun Hsueh

2018-03-01

Full Text Available Polyunsaturated fatty acids are important nutrients for human health, especially omega-3 fatty acids such as eicosapentaenoic acid (EPA and docosahexaenoic acid (DHA, which have been found to play positive roles in the prevention of various diseases. However, previous studies have reported that excessive omega-3 fatty acids supplement during pregnancy caused side effects such as slower neural transmission times and postnatal growth restriction. In this study, we investigated the effect of EPA and DHA on mitochondrial function and gene expression in C2C12 myoblasts during skeletal muscle differentiation. C2C12 myoblasts were cultured to confluency and then treated with differentiation medium that contained fatty acids (50-µM EPA and DHA. After 72 h of myogenic differentiation, mRNA was collected, and gene expression was analyzed by real-time PCR. Microscopy was used to examine cell morphology following treatment with fatty acids. The effect of EPA and DHA on cellular oxygen consumption was measured using a Seahorse XF24 Analyzer. Cells treated with fatty acids had fewer myotubes formed (P ≤ 0.05 compared with control cells. The expression of the genes related to myogenesis was significantly lower (P ≤ 0.05 in cells treated with fatty acids, compared with control cells. Genes associated with adipogenesis had higher (P ≤ 0.05 expression after treatment with fatty acids. Also, the mitochondrial biogenesis decreased with lower (P ≤ 0.05 gene expression and lower (P ≤ 0.05 mtDNA/nDNA ratio in cells treated with fatty acids compared with control cells. However, the expression of genes related to peroxisome biosynthesis was higher (P ≤ 0.05 in cells treated with fatty acids. Moreover, fatty-acid treatment reduced (P ≤ 0.05 oxygen consumption rate under oligomycin-inhibited (reflecting proton leak and uncoupled conditions. Our data imply that fatty acids might reduce myogenesis and increase adipogenesis in myotube formation. Fatty acids

Exercise increases sphingoid base-1-phosphate levels in human blood and skeletal muscle in a time- and intensity-dependent manner

DEFF Research Database (Denmark)

Baranowski, Marcin; Błachnio-Zabielska, Agnieszka U; Charmas, Małgorzata

2015-01-01

PURPOSE: Sphingosine-1-phosphate (S1P) regulates cardiovascular function and plays an important role in muscle biology. We have previously reported that cycling exercise increased plasma S1P. Here, we investigated the effect of exercise duration and intensity on plasma and skeletal muscle S1P lev...

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

Science.gov (United States)

2013-01-01

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

The MSX1 homeoprotein recruits G9a methyltransferase to repressed target genes in myoblast cells.

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Jingqiang Wang

Full Text Available Although the significance of lysine modifications of core histones for regulating gene expression is widely appreciated, the mechanisms by which these modifications are incorporated at specific regulatory elements during cellular differentiation remains largely unknown. In our previous studies, we have shown that in developing myoblasts the Msx1 homeoprotein represses gene expression by influencing the modification status of chromatin at its target genes. We now show that genomic binding by Msx1 promotes enrichment of the H3K9me2 mark on repressed target genes via recruitment of G9a histone methyltransferase, the enzyme responsible for catalyzing this histone mark. Interaction of Msx1 with G9a is mediated via the homeodomain and is required for transcriptional repression and regulation of cellular differentiation, as well as enrichment of the H3K9me2 mark in proximity to Msx1 binding sites on repressed target genes in myoblast cells as well as the developing limb. We propose that regulation of chromatin status by Msx1 recruitment of G9a and other histone modifying enzymes to regulatory regions of target genes represents an important means of regulating the gene expression during development.

CD90-positive cells, an additional cell population, produce laminin α2 upon transplantation to dy3k/dy3k mice

International Nuclear Information System (INIS)

Fukada, So-ichiro; Yamamoto, Yukiko; Segawa, Masashi; Sakamoto, Kenta; Nakajima, Mari; Sato, Masaki; Morikawa, Daisuke; Uezumi, Akiyoshi; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi; Tsujikawa, Kazutake; Yamamoto, Hiroshi

2008-01-01

Laminin α2 is a component of skeletal and cardiac muscle basal lamina. A defect of the laminin α2 chain leads to severe congenital muscular dystrophy (MDC1A) in humans and dy/dy mice. Myogenic cells including myoblasts, myotubes, and myofibers in skeletal muscle are a possible source of the laminin α2 chain, and myogenic cells are thus proposed as a cell source for congenital muscular dystrophy therapy. However, we observed production of laminin α2 in non-myogenic cells of normal mice, and we could enrich these laminin α2-producing cells in CD90 + cell fractions. Intriguingly, the number of CD90 + cells increased dramatically during skeletal muscle regeneration in mice. This fraction did not include myogenic cells but exhibited a fibroblast-like phenotype. Moreover, these cells were resident in skeletal muscle, not derived from bone marrow. Finally, the production of laminin α2 in CD90 + cells was not dependent on fusion with myogenic cells. Thus, CD90 + cells are a newly identified additional cell fraction that increased during skeletal muscle regeneration in vivo and could be another cell source for therapy for lama2-deficient muscular dystrophy

Deletion of skeletal muscle SOCS3 prevents insulin resistance in obesity

DEFF Research Database (Denmark)

Beck Jørgensen, Sebastian; O'Neill, Hayley M; Sylow, Lykke

2013-01-01

Obesity is associated with chronic low-grade inflammation that contributes to defects in energy metabolism and insulin resistance. Suppressor of cytokine signaling (SOCS)-3 expression is increased in skeletal muscle of obese humans. SOCS3 inhibits leptin signaling in the hypothalamus and insulin...... of hyperinsulinemia and insulin resistance because of enhanced skeletal muscle insulin receptor substrate 1 (IRS1) and Akt phosphorylation that resulted in increased skeletal muscle glucose uptake. These data indicate that skeletal muscle SOCS3 does not play a critical role in regulating muscle development or energy...... expenditure, but it is an important contributing factor for inhibiting insulin sensitivity in obesity. Therapies aimed at inhibiting SOCS3 in skeletal muscle may be effective in reversing obesity-related glucose intolerance and insulin resistance....

Physical activity opposes the age-related increase in skeletal muscle and plasma endothelin-1 levels and normalizes plasma endothelin-1 levels in individuals with essential hypertension

DEFF Research Database (Denmark)

Nyberg, Michael Permin; Mortensen, Stefan Peter; Hellsten, Ylva

2013-01-01

performed lifelong physical activity had similar plasma and muscle endothelin-1 levels as the young controls and had higher ET(A) receptor levels. CONCLUSION: Our findings suggest that aerobic exercise training opposes the age-related increase in skeletal muscle and plasma endothelin-1 levels and normalizes......AIMS: Endothelin-1 has potent constrictor and proliferative activity in vascular smooth muscle, and essential hypertension and aging are associated with increased endothelin-1-mediated vasoconstrictor tone. The aim of this study was to investigate the effect of physical activity, hypertension...... and age on endothelin-1 levels in plasma and skeletal muscle and endothelin receptors in skeletal muscle in human subjects. METHODS: In study 1, normotensive (46 ± 1 years, n = 11) and hypertensive (47 ± 1 years, n = 10) subjects were studied before and after 8 weeks of aerobic exercise training. In study...

Skeletal muscle inflammation and insulin resistance in obesity

Science.gov (United States)

Wu, Huaizhu; Ballantyne, Christie M.

2017-01-01

Obesity is associated with chronic inflammation, which contributes to insulin resistance and type 2 diabetes mellitus. Under normal conditions, skeletal muscle is responsible for the majority of insulin-stimulated whole-body glucose disposal; thus, dysregulation of skeletal muscle metabolism can strongly influence whole-body glucose homeostasis and insulin sensitivity. Increasing evidence suggests that inflammation occurs in skeletal muscle in obesity and is mainly manifested by increased immune cell infiltration and proinflammatory activation in intermyocellular and perimuscular adipose tissue. By secreting proinflammatory molecules, immune cells may induce myocyte inflammation, adversely regulate myocyte metabolism, and contribute to insulin resistance via paracrine effects. Increased influx of fatty acids and inflammatory molecules from other tissues, particularly visceral adipose tissue, can also induce muscle inflammation and negatively regulate myocyte metabolism, leading to insulin resistance. PMID:28045398

Method of cell transplantation promoting the organization of intraarterial thrombus.

Science.gov (United States)

Hirano, Koji; Shimono, Takatsugu; Imanaka-Yoshida, Kyoko; Miyamoto, Keiichi; Fujinaga, Kazuya; Kajimoto, Masaki; Miyake, Yoichiro; Nishikawa, Masakatsu; Yoshida, Toshimichi; Uchida, Atsumasa; Shimpo, Hideto; Yada, Isao; Hirata, Hitoshi

2005-08-30

Endovascular aortic repairs have been developed as less invasive treatments for aortic aneurysms. Some aneurismal cavities, however, remain without organization, causing a re-expansion of the aneurysms. We studied cell transplantation into the aneurismal sac to promote the organization of thrombus for the complete healing of aneurysms. Skin fibroblasts and skeletal myoblasts were isolated from rats for cell transplantation. An intraarterial thrombus model was made by ligation of the carotid artery. Culture medium (medium group, n=11), collagen gel (gel group, n=11), fibroblasts with collagen gel (F group, n=15), myoblasts with collagen gel (M group, n=12), or mixture of fibroblasts and myoblasts with collagen gel (F+M group, n=14) were injected into the thrombus. After 28 days, histologically, the arterial lumens of the F and M groups were partly filled with fibrous tissues, whereas in the F+M group organization was almost completed and luminal sizes diminished. Immunohistochemical staining demonstrated that alpha-smooth muscle actin-positive cells were more abundantly contained in the organized area of the F+M group than in the other groups. We also analyzed cellular function in vitro with immunofluorescence; coculture of fibroblasts and myoblasts showed that the fraction of alpha-smooth muscle actin-positive fibroblasts increased. This phenomenon accounts for the rapid organization of thrombus in the F+M group in vivo. Cell transplantation accelerated thrombus organization. Especially, myoblasts enhanced differentiation of fibroblasts into myofibroblasts, contributing to rapid thrombus organization. Cell transplantation into unorganized spaces seems applicable to endovascular treatment of aneurysms.

Skeletal blood flow: implications for bone-scan interpretation

International Nuclear Information System (INIS)

Charkes, N.D.

1980-01-01

The dispersion of the skeleton throughout the body and its complex vascular anatomy require indirect methods for the measurement of skeletal blood flow. The results of one such method, compartmental analysis of skeletal tracer kinetics, are presented. The assumptions underlying the models were tested in animals and found to be in agreement with experimental observations. Based upon the models and the experimental results, inferences concerning bone-scan interpretation can be drawn: decreased cardiac output produces low-contrast (technically poor) scans; decreased skeletal flow produces photon-deficient lesions; increase of cardiac output or of generalized systemic blood flow is undetectable 1 to 2 h after dose; increased local skeletal blood flow results from disturbance of the bone microvasculature and can occur from neurologic (sympatholytic) disorders or in association with focal abnormalities that also incite the formation of reactive bone (e.g., metastasis, fracture, etc.). Mathematical solutions of tracer kinetic data thus become relevant to bone-scan interpretation

Cardiac, Skeletal, and smooth muscle mitochondrial respiration

DEFF Research Database (Denmark)

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

2014-01-01

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

Anticancer activity of biologically synthesized silver and gold nanoparticles on mouse myoblast cancer cells and their toxicity against embryonic zebrafish

International Nuclear Information System (INIS)

Ramachandran, Rajan; Krishnaraj, Chandran; Sivakumar, Allur Subramaniyan; Prasannakumar, Palaniappan; Abhay Kumar, V.K.; Shim, Kwan Seob; Song, Chul-Gyu; Yun, Soon-Il

2017-01-01

The aim of this study was to evaluate the anticancer activity of bioinspired silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) against mouse myoblast cancer cells (C 2 C 12 ). Both AgNPs and AuNPs were biologically synthesized using Spinacia oleracea Linn., aqueous leaves extract. UV–Vis. spectrophotometer, high resolution-transmission electron microscopy (HR-TEM), field emission-scanning electron microscopy (FE-SEM) and X-ray diffraction (XRD) studies supported the successful synthesis of AgNPs and AuNPs. Both these NPs have shown cytotoxicity against C 2 C 12 cells even at very low concentration (5 μg/mL). Acridine orange/Ethidium bromide (AO/EB) dual staining confirmed the apoptotic morphological features. The levels of caspase enzymes (caspase-3 and caspase-7) were significantly up-regulated in NPs treated myoblast cells than the plant extract. Furthermore, in zebrafish embryo toxicity study, AgNPs showed 100% mortality at 3 μg/mL concentration while AuNPs exhibited the same at much higher concentration (300 mg/mL). Taken together, these results provide a preliminary guidance for the development of biomaterials based drugs to fight against the fatal diseases for example cancer. - Highlights: • Anticancer activity was done for the first time against mouse myoblast cells. • AgNPs showed 100% growth inhibition against C 2 C 12 cells at 20 μg/mL concentration. • AO/EB dual staining and caspase assays confirmed the apoptotic features. • Nanoparticles treated embryos showed yolk sac edema and tail malformation. • AgNPs were found to be more toxic to embryonic zebrafishes than the AuNPs.

Anticancer activity of biologically synthesized silver and gold nanoparticles on mouse myoblast cancer cells and their toxicity against embryonic zebrafish

Energy Technology Data Exchange (ETDEWEB)

Ramachandran, Rajan [Centre for Advanced Studies in Botany, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu (India); Krishnaraj, Chandran [Department of Food Science & Technology, College of Agriculture & Life Sciences, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); M/s. Eureka Forbes Ltd, R & D Centre, Kudlu, Bangalore (India); Sivakumar, Allur Subramaniyan [Department of Animal Biotechnology, Chonbuk National University, Jeonju 54896 (Korea, Republic of); Prasannakumar, Palaniappan [Advanced Biomedical Imaging Center, Department of Electronic Engineering, Chonbuk National University, Jeonju 54896 (Korea, Republic of); Abhay Kumar, V.K. [M/s. Eureka Forbes Ltd, R & D Centre, Kudlu, Bangalore (India); Shim, Kwan Seob [Department of Animal Biotechnology, Chonbuk National University, Jeonju 54896 (Korea, Republic of); Song, Chul-Gyu [Advanced Biomedical Imaging Center, Department of Electronic Engineering, Chonbuk National University, Jeonju 54896 (Korea, Republic of); Yun, Soon-Il, E-mail: siyun@jbnu.ac.kr [Department of Food Science & Technology, College of Agriculture & Life Sciences, Chonbuk National University, Jeonju 561-756 (Korea, Republic of)

2017-04-01

The aim of this study was to evaluate the anticancer activity of bioinspired silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) against mouse myoblast cancer cells (C{sub 2}C{sub 12}). Both AgNPs and AuNPs were biologically synthesized using Spinacia oleracea Linn., aqueous leaves extract. UV–Vis. spectrophotometer, high resolution-transmission electron microscopy (HR-TEM), field emission-scanning electron microscopy (FE-SEM) and X-ray diffraction (XRD) studies supported the successful synthesis of AgNPs and AuNPs. Both these NPs have shown cytotoxicity against C{sub 2}C{sub 12} cells even at very low concentration (5 μg/mL). Acridine orange/Ethidium bromide (AO/EB) dual staining confirmed the apoptotic morphological features. The levels of caspase enzymes (caspase-3 and caspase-7) were significantly up-regulated in NPs treated myoblast cells than the plant extract. Furthermore, in zebrafish embryo toxicity study, AgNPs showed 100% mortality at 3 μg/mL concentration while AuNPs exhibited the same at much higher concentration (300 mg/mL). Taken together, these results provide a preliminary guidance for the development of biomaterials based drugs to fight against the fatal diseases for example cancer. - Highlights: • Anticancer activity was done for the first time against mouse myoblast cells. • AgNPs showed 100% growth inhibition against C{sub 2}C{sub 12} cells at 20 μg/mL concentration. • AO/EB dual staining and caspase assays confirmed the apoptotic features. • Nanoparticles treated embryos showed yolk sac edema and tail malformation. • AgNPs were found to be more toxic to embryonic zebrafishes than the AuNPs.

Intense resistance exercise induces early and transient increases in ryanodine receptor 1 phosphorylation in human skeletal muscle.

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Sebastian Gehlert

Full Text Available BACKGROUND: While ryanodine receptor 1 (RyR1 critically contributes to skeletal muscle contraction abilities by mediating Ca²⁺ion oscillation between sarcoplasmatic and myofibrillar compartments, AMP-activated protein kinase (AMPK senses contraction-induced energetic stress by phosphorylation at Thr¹⁷². Phosphorylation of RyR1 at serine²⁸⁴³ (pRyR1Ser²⁸⁴³ results in leaky RyR1 channels and impaired Ca²⁺homeostasis. Because acute resistance exercise exerts decreased contraction performance in skeletal muscle, preceded by high rates of Ca²⁺-oscillation and energetic stress, intense myofiber contractions may induce increased RyR1 and AMPK phosphorylation. However, no data are available regarding the time-course and magnitude of early RyR1 and AMPK phosphorylation in human myofibers in response to acute resistance exercise. PURPOSE: Determine the effects and early time-course of resistance exercise on pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² in type I and II myofibers. METHODS: 7 male subjects (age 23±2 years, height: 185±7 cm, weight: 82±5 kg performed 3 sets of 8 repetitions of maximum eccentric knee extensions. Muscle biopsies were taken at rest, 15, 30 and 60 min post exercise. pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² levels were determined by western blot and semi-quantitative immunohistochemistry techniques. RESULTS: While total RyR1 and total AMPK levels remained unchanged, RyR1 was significantly more abundant in type II than type I myofibers. pRyR1Ser²⁸⁴³ increased 15 min and peaked 30 min (p<0.01 post exercise in both myofiber types. Type I fibers showed relatively higher increases in pRyR1Ser²⁸⁴³ levels than type II myofibers and remained elevated up to 60 min post resistance exercise (p<0.05. pAMPKThr¹⁷² also increased 15 to 30 min post exercise (p<0.01 in type I and II myofibers and in whole skeletal muscle. CONCLUSION: Resistance exercise induces acutely increased pRyR1Ser²⁸⁴³ and

FDG-PET/CT in Skeletal Muscle: Pitfalls and Pathologies.

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Parida, Girish Kumar; Roy, Shambo Guha; Kumar, Rakesh

2017-07-01

FDG-PET/CT is an integral part of modern-day practice of medicine. By detecting increased cellular metabolism, FDG-PET/CT can help us detect infection, inflammatory disorders, or tumors, and also help us in prognostication of patients. However, one of the most important challenges is to correctly differentiate the abnormal uptake that is potentially pathologic from the physiological uptake. So while interpreting a PET/CT, one must be aware of normal biodistribution and different physiological variants of FDG uptake. Skeletal muscles constitute a large part of our body mass and one of the major users of glucose. Naturally, they are often the site of increased FDG uptake in a PET study. We as a nuclear medicine physician must be aware of all the pitfalls of increased skeletal muscle uptake to differentiate between physiological and pathologic causes. In this review, we have discussed the different causes and patterns of physiological FDG uptake in skeletal muscles. This knowledge of normal physiological variants of FDG uptake in the skeletal muscles is essential for differentiating pathologic uptake from the physiological ones. Also, we reviewed the role of FDG-PET/CT in various benign and malignant diseases involving skeletal muscle. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel in vitro model for studying quiescence and activation of primary isolated human myoblasts

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Sellathurai, Jeeva; Cheedipudi, Sirisha; Dhawan, Jyotsna

2013-01-01

term exponentially proliferating cultures normally used for in vitro studies. Human myoblasts cultured through many passages inevitably consist of a mixture of proliferating and non-proliferating cells, while cells activated from G0 are in a synchronously proliferating phase, and therefore may...... be a better model for in vivo proliferating satellite cells. Furthermore, the temporal propagation of proliferation in these synchronized cultures resembles the pattern seen in vivo during regeneration. We therefore present this culture model as a useful and novel condition for molecular analysis...

Skeletal Muscle-specific G Protein-coupled Receptor Kinase 2 Ablation Alters Isolated Skeletal Muscle Mechanics and Enhances Clenbuterol-stimulated Hypertrophy.

Science.gov (United States)

Woodall, Benjamin P; Woodall, Meryl C; Luongo, Timothy S; Grisanti, Laurel A; Tilley, Douglas G; Elrod, John W; Koch, Walter J

2016-10-14

GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2 fl/fl ) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2 fl/fl mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a β 2 -adrenergic receptor (β 2 AR) agonist, was significantly enhanced in MLC-Cre:GRK2 fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β 2 AR-induced hypertrophy. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Skeletal Muscle-specific G Protein-coupled Receptor Kinase 2 Ablation Alters Isolated Skeletal Muscle Mechanics and Enhances Clenbuterol-stimulated Hypertrophy*

Science.gov (United States)

Woodall, Benjamin P.; Woodall, Meryl C.; Luongo, Timothy S.; Grisanti, Laurel A.; Tilley, Douglas G.; Elrod, John W.; Koch, Walter J.

2016-01-01

GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2fl/fl) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2fl/fl mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a β2-adrenergic receptor (β2AR) agonist, was significantly enhanced in MLC-Cre:GRK2fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β2AR-induced hypertrophy. PMID:27566547

Structural, biochemical, cellular, and functional changes in skeletal muscle extracellular matrix with aging

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Kragstrup, T W; Kjaer, M; Mackey, A L

2011-01-01

The extracellular matrix (ECM) of skeletal muscle is critical for force transmission and for the passive elastic response of skeletal muscle. Structural, biochemical, cellular, and functional changes in skeletal muscle ECM contribute to the deterioration in muscle mechanical properties with aging....... Structural changes include an increase in the collagen concentration, a change in the elastic fiber system, and an increase in fat infiltration of skeletal muscle. Biochemical changes include a decreased turnover of collagen with potential accumulation of enzymatically mediated collagen cross...

PGC-1α regulates alanine metabolism in muscle cells.

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Hatazawa, Yukino; Qian, Kun; Gong, Da-Wei; Kamei, Yasutomi

2018-01-01

The skeletal muscle is the largest organ in the human body, depositing energy as protein/amino acids, which are degraded in catabolic conditions such as fasting. Alanine is synthesized and secreted from the skeletal muscle that is used as substrates of gluconeogenesis in the liver. During fasting, the expression of PGC-1α, a transcriptional coactivator of nuclear receptors, is increased in the liver and regulates gluconeogenesis. In the present study, we observed increased mRNA expression of PGC-1α and alanine aminotransferase 2 (ALT2) in the skeletal muscle during fasting. In C2C12 myoblast cells overexpressing PGC-1α, ALT2 expression was increased concomitant with an increased alanine level in the cells and medium. In addition, PGC-1α, along with nuclear receptor ERR, dose-dependently enhanced the ALT2 promoter activity in reporter assay using C2C12 cells. In the absence of glucose in the culture medium, mRNA levels of PGC-1α and ALT2 increased. Endogenous PGC-1α knockdown in C2C12 cells reduced ALT2 gene expression level, induced by the no-glucose medium. Taken together, in the skeletal muscle, PGC-1α activates ALT2 gene expression, and alanine production may play roles in adaptation to fasting.

Generalized skeletal pathology: Results of radionuclide studies

International Nuclear Information System (INIS)

Fueger, G.F.; Aigner, R.

1987-01-01

Generalized pathological changes may involve the skeleton systematically (bone tissue, bone marrow) or at multiple sites involving destruction or infiltration. Appropriate radionuclide studies include total-body bone or bone marrow scintigraphy, absorptiometry (osteodensitometry) and the 24 h whole-body retention measurement. Established radioindicators are 99m-Tc-(hydroxy)methylendiphosphonate (HMDP or MDP) and 99m-Tc-human serumalbumin-nanocolloid. Absorptiometry of the forearm, extended by computer-assisted transaxial tomography, may be expected to prove as the most efficient method of bone density measurement. The 24 h whole-body retention measurement is useful for the diagnosis and follow-up of metabolic and endocrine osteopathies, if the very same osteotropic 99m-Tc-chelate is used. Whole-body bone scintigraphy today is one of the most important radionuclide studies for diagnosis and follow-up of skeletal metastases. Scintigraphy provides evidence of skeletal metastases several months earlier than radiological examinations. In about 40 percent of patients with cancer of the prostate, scintigraphy provided positive findings of skeletal metastases in the absence of both pain and increased levels of phosphatase. In patients with a history of malignancy, 60 percent of solitary findings on skeletal scintigraphy are metastases. The frequency of false negative findings obtained by whole-body skeletal scintigraphy are metastases. The frequency of false negative findings obtained by whole-body skeletal scintigraphy ranges from 2 to 4%. Compared to skeletal scintigraphy, bone marrow scintigraphy frequently yields significant additional findings in cases of plasmocytoma, histiocytoma, lymphoma and haemoblastoses. (orig.) [de

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

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

2015-11-27

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

DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation

International Nuclear Information System (INIS)

Chen, Zirong; Jin, Guorong; Lin, Shuibin; Lin, Xiumei; Gu, Yumei; Zhu, Yujuan; Hu, Chengbin; Zhang, Qingjiong; Wu, Lizi; Shen, Huangxuan

2012-01-01

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

ALDH2 restores exhaustive exercise-induced mitochondrial dysfunction in skeletal muscle

International Nuclear Information System (INIS)

Zhang, Qiuping; Zheng, Jianheng; Qiu, Jun; Wu, Xiahong; Xu, Yangshuo; Shen, Weili; Sun, Mengwei

2017-01-01

Background: Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is highly expressed in heart and skeletal muscles, and is the major enzyme that metabolizes acetaldehyde and toxic aldehydes. The cardioprotective effects of ALDH2 during cardiac ischemia/reperfusion injury have been recognized. However, less is known about the function of ALDH2 in skeletal muscle. This study was designed to evaluate the effect of ALDH2 on exhaustive exercise-induced skeletal muscle injury. Methods: We created transgenic mice expressing ALDH2 in skeletal muscles. Male wild-type C57/BL6 (WT) and ALDH2 transgenic mice (ALDH2-Tg), 8-weeks old, were challenged with exhaustive exercise for 1 week to induce skeletal muscle injury. Animals were sacrificed 24 h post-exercise and muscle tissue was excised. Results: ALDH2-Tg mice displayed significantly increased treadmill exercise capacity compared to WT mice. Exhaustive exercise caused an increase in mRNA levels of the muscle atrophy markers, Atrogin-1 and MuRF1, and reduced mitochondrial biogenesis and fusion in WT skeletal muscles; these effects were attenuated in ALDH2-Tg mice. Exhaustive exercise also enhanced mitochondrial autophagy pathway activity, including increased conversion of LC3-I to LC3-II and greater expression of Beclin1 and Bnip3; the effects of which were mitigated by ALDH2 overexpression. In addition, ALDH2-Tg reversed the increase of an oxidative stress biomarker (4-hydroxynonenal) and decreased levels of mitochondrial antioxidant proteins, including manganese superoxide dismutase and NAD(P)H:quinone oxidoreductase 1, in skeletal muscle induced by exhaustive exercise. Conclusion: ALDH2 may reverse skeletal muscle mitochondrial dysfunction due to exhaustive exercise by regulating mitochondria dynamic remodeling and enhancing the quality of mitochondria. - Highlights: • Skeletal muscle ALDH2 expression and activity declines during exhaustive exercise. • ALDH2 overexpression enhances physical performance and restores muscle

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

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Yasunari Matsuzaka

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

Exercise increases the plasma membrane content of the Na+ -K+ pump and its mRNA in rat skeletal muscles.

Science.gov (United States)

Tsakiridis, T; Wong, P P; Liu, Z; Rodgers, C D; Vranic, M; Klip, A

1996-02-01

Muscle fibers adapt to ionic challenges of exercise by increasing the plasma membrane Na+-K+ pump activity. Chronic exercise training has been shown to increase the total amount of Na+-K+ pumps present in skeletal muscle. However, the mechanism of adaptation of the Na+-K+ pump to an acute bout of exercise has not been determined, and it is not known whether it involves alterations in the content of plasma membrane pump subunits. Here we examine the effect of 1 h of treadmill running (20 m/min, 10% grade) on the subcellular distribution and expression of Na+-K+ pump subunits in rat skeletal muscles. Red type I and IIa (red-I/IIa) and white type IIa and IIb (white-IIa/IIb) hindlimb muscles from resting and exercised female Sprague-Dawley rats were removed for subcellular fractionation. By homogenization and gradient centrifugation, crude membranes and purified plasma membranes were isolated and subjected to gel electrophoresis and immunoblotting by using pump subunit-specific antibodies. Furthermore, mRNA was isolated from specific red type I (red-I) and white type IIb (white-IIb) muscles and subjected to Northern blotting by using subunit-specific probes. In both red-I/IIa and white-IIa/IIb muscles, exercise significantly raised the plasma membrane content of the alpha1-subunit of the pump by 64 +/- 24 and 55 +/- 22%, respectively (P < 0.05), and elevated the alpha2-polypeptide by 43 +/- 22 and 94 +/- 39%, respectively (P < 0.05). No significant effect of exercise could be detected on the amount of these subunits in an internal membrane fraction or in total membranes. In addition, exercise significantly increased the alpha1-subunit mRNA in red-I muscle (by 50 +/- 7%; P < 0.05) and the beta2-subunit mRNA in white-IIb muscles (by 64 +/- 19%; P < 0.01), but the alpha2- and beta1-mRNA levels were unaffected in this time period. We conclude that increased presence of alpha1- and alpha2-polypeptides at the plasma membrane and subsequent elevation of the alpha1- and beta2

Symmetric increased skeletal muscular uptake of 18F fluoro-deoxyglucose: a clue for the diagnosis of Graves' disease

International Nuclear Information System (INIS)

Santhosh, Sampath; Mittal, Bhagwant Rai; Kashyap, Raghava; Bhattacharya, Anish; Singh, Baljinder

2011-01-01

18 F fluoro-deoxyglucose (FDG) uptake in the thyroid and thymus is well reported in patients with Grave's disease. Incidental skeletal muscle uptake has also been reported in other non-musculoskeletal (benign and malignant) pathologies. We report a patient of Grave's disease showing symmetrical skeletal muscle uptake but no thyroidal or thymus uptake of FDG. (author)

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

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Luciana Miato Gonçalves Silva

Full Text Available 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.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.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.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 effect of ATP synthesis may

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

Science.gov (United States)

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

1999-01-01

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

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 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.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.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.

Extrarenal potassium adaptation: role of skeletal muscle

International Nuclear Information System (INIS)

Blachley, J.D.; Crider, B.P.; Johnson, J.H.

1986-01-01

Following the ingestion of a high-potassium-content diet for only a few days, the plasma potassium of rats rises only modestly in response to a previously lethal dose of potassium salts. This acquired tolerance, termed potassium adaptation, is principally the result of increased capacity to excrete potassium into the urine. However, a substantial portion of the acute potassium dose is not immediately excreted and is apparently translocated into cells. Previous studies have failed to show an increase in the content of potassium of a variety of tissues from such animals. Using 86 Rb as a potassium analogue, we have shown that the skeletal muscle of potassium-adapted rats takes up significantly greater amounts of potassium in vivo in response to an acute challenge than does that of control animals. Furthermore, the same animals exhibit greater efflux of 86 Rb following the termination of the acute infusion. We have also shown that the Na+-K+-ATPase activity and ouabain-binding capacity of skeletal muscle microsomes are increased by the process of potassium adaptation. We conclude that skeletal muscle is an important participant in potassium adaptation and acts to temporarily buffer acute increases in the extracellular concentration of potassium

Upon the triple phase skeletal scintigraphy in traumatology

International Nuclear Information System (INIS)

Spitz, W.

1988-01-01

A broadly established indication catalogue for skeletal scintigraphy in traumatology is resulting from about 1500 skeletal scans. Aside from the exclusion of any osseous lesion, from the differentiation of uncertain X-ray findings, from the determination of the extent of osseous lesions in polytraumatic conditions and from the assessment of the relative fracture age, the follow-up after trauma and therapeutical intervention, the demonstration of battered child syndromes and of soft tissue lesions are of special importance with regard to these topics. For all that, the high sensitivity of the 3-phase skeletal scintigraphy for every enhancement of osseous turnover represents the elementary prerequisite for the employment of this non-invasive technique as an ideal screening method in traumatological diagnostics. The experiences from the past years have resulted in an increased frequency of skeletal scintigraphic studies to a similarly high level, as it is already established in the majority of institutions with respect to oncological problems, In the development of efficient and cost favourable diagnostic strategies with only little burden to the patient, skeletal scintigraphy will in future play an important role within the palette of modern skeletal diagnostics in traumatology. (orig.) [de

Ammonia lowering reverses sarcopenia of cirrhosis by restoring skeletal muscle proteostasis.

Science.gov (United States)

Kumar, Avinash; Davuluri, Gangarao; Silva, Rafaella Nascimento E; Engelen, Marielle P K J; Ten Have, Gabrie A M; Prayson, Richard; Deutz, Nicolaas E P; Dasarathy, Srinivasan

2017-06-01

Sarcopenia or skeletal muscle loss is a frequent, potentially reversible complication in cirrhosis that adversely affects clinical outcomes. Hyperammonemia is a consistent abnormality in cirrhosis that results in impaired skeletal muscle protein synthesis and breakdown (proteostasis). Despite the availability of effective ammonia-lowering therapies, whether lowering ammonia restores proteostasis and increases muscle mass is unknown. Myotube diameter, protein synthesis, and molecular responses in C2C12 murine myotubes to withdrawal of ammonium acetate following 24-hour exposure to 10 mM ammonium acetate were complemented by in vivo studies in the hyperammonemic portacaval anastomosis rat and sham-operated, pair-fed Sprague-Dawley rats treated with ammonia-lowering therapy by l-ornithine l-aspartate and rifaximin orally for 4 weeks. We observed reduced myotube diameter, impaired protein synthesis, and increased autophagy flux in response to hyperammonemia, which were partially reversed following 24-hour and 48-hour withdrawal of ammonium acetate. Consistently, 4 weeks of ammonia-lowering therapy resulted in significant lowering of blood and skeletal muscle ammonia, increase in lean body mass, improved grip strength, higher skeletal muscle mass and diameter, and an increase in type 2 fibers in treated compared to untreated portacaval anastomosis rats. The increased skeletal muscle myostatin expression, reduced mammalian target of rapamycin complex 1 function, and hyperammonemic stress response including autophagy markers normally found in portacaval anastomosis rats were reversed by treatment with ammonia-lowering therapy. Despite significant improvement, molecular and functional readouts were not completely reversed by ammonia-lowering measures. Ammonia-lowering therapy results in improvement in skeletal muscle phenotype and function and molecular perturbations of hyperammonemia; these preclinical studies complement previous studies on ammonia-induced skeletal muscle

Glucocorticoids and inhibition of bone formation induced by skeletal unloading

International Nuclear Information System (INIS)

Halloran, B.P.; Bikle, D.D.; Cone, C.M.; Morey-Holton, E.

1988-01-01

Skeletal unloading or loss of normal weight bearing in the growing animal inhibits bone formation and reduces bone calcium. To determine whether the inhibition of bone formation induced by skeletal unloading is a consequence of an increase in plasma glucocorticoids and/or an increase in bone sensitivity to glucocorticoids, the authors measured plasma corticosterone throughout the day in unloaded and normally loaded rats (hindlimb elevation model) and examined the effect of adrenalectomy on the response of bone to skeletal unloading. Plasma corticosterone levels were similar in normally loaded and unloaded rats at all times. Skeletal unloading in sham-adrenalectomized animals reduced tibial and vertebral calcium by 11.5 and 11.1%, respectively, and in adrenalectomized animals by 15.3 and 20.3%, respectively. Uptake of 45 Ca and [ 3 H]proline in the tibia was reduced by 8 and 14%, respectively, in the sham-adrenalectomized animals and by 13 and 19% in the adrenalectomized animals. Bone formation and apposition rates were reduced to the same level in sham- and adrenalectomized animals. These results suggest that the inhibition of bone formation induced by skeletal unloading is not a consequence of increased plasma glucocorticoids or an increase in bone sensitivity to the glucocorticoids but, rather, point to a local mediator in bone that senses mechanical load and transmits that information to the bone-forming cells directly

A metabolic link to skeletal muscle wasting and regeneration

Directory of Open Access Journals (Sweden)

René eKoopman

2014-02-01

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

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

Long-term rates of mitochondrial protein synthesis are increased in mouse skeletal muscle with high-fat feeding regardless of insulin-sensitizing treatment.

Science.gov (United States)

Newsom, Sean A; Miller, Benjamin F; Hamilton, Karyn L; Ehrlicher, Sarah E; Stierwalt, Harrison D; Robinson, Matthew M

2017-11-01

Skeletal muscle mitochondrial protein synthesis is regulated in part by insulin. The development of insulin resistance with diet-induced obesity may therefore contribute to impairments to protein synthesis and decreased mitochondrial respiration. Yet the impact of diet-induced obesity and insulin resistance on mitochondrial energetics is controversial, with reports varying from decreases to increases in mitochondrial respiration. We investigated the impact of changes in insulin sensitivity on long-term rates of mitochondrial protein synthesis as a mechanism for changes to mitochondrial respiration in skeletal muscle. Insulin resistance was induced in C57BL/6J mice using 4 wk of a high-fat compared with a low-fat diet. For 8 additional weeks, diets were enriched with pioglitazone to restore insulin sensitivity compared with nonenriched control low-fat or high-fat diets. Skeletal muscle mitochondrial protein synthesis was measured using deuterium oxide labeling during weeks 10-12 High-resolution respirometry was performed using palmitoyl-l-carnitine, glutamate+malate, and glutamate+malate+succinate as substrates for mitochondria isolated from quadriceps. Mitochondrial protein synthesis and palmitoyl- l-carnitine oxidation were increased in mice consuming a high-fat diet, regardless of differences in insulin sensitivity with pioglitazone treatment. There was no effect of diet or pioglitazone treatment on ADP-stimulated respiration or H 2 O 2 emission using glutamate+malate or glutamate+malate+succinate. The results demonstrate no impairments to mitochondrial protein synthesis or respiration following induction of insulin resistance. Instead, mitochondrial protein synthesis was increased with a high-fat diet and may contribute to remodeling of the mitochondria to increase lipid oxidation capacity. Mitochondrial adaptations with a high-fat diet appear driven by nutrient availability, not intrinsic defects that contribute to insulin resistance. Copyright © 2017 the

Signalling and the control of skeletal muscle size

International Nuclear Information System (INIS)

Otto, Anthony; Patel, Ketan

2010-01-01

Skeletal muscle is highly adaptive to environmental stimuli and can alter its mass accordingly. This tissue is almost unique in that it can increase its size through two distinct mechanisms. It can grow through a cellular process mediated by cell fusion, or it can increase its size simply by increasing its protein content. Understanding how these processes are regulated is crucial for the development of potential therapies against debilitating skeletal muscle wasting diseases. Two key signalling molecules, Insulin like Growth Factor (IGF) and GDF-8/myostatin, have emerged in recent years to be potent regulators of skeletal muscle size. In this review we bring together recent data highlighting the important and novel aspects of both molecules and their signalling pathways, culminating in a discussion of the cellular and tissue phenotypic outcomes of their stimulation or antagonism. We emphasise the complex regulatory mechanisms and discuss the temporal and spatial differences that control their action, understanding of which is crucial to further their use as potential therapeutic targets.

Signalling and the control of skeletal muscle size

Energy Technology Data Exchange (ETDEWEB)

Otto, Anthony [School of Biological Sciences, Hopkins Building, University of Reading, Whiteknights Campus, Reading, Berkshire, RG6 6UB (United Kingdom); Patel, Ketan, E-mail: ketan.patel@reading.ac.uk [School of Biological Sciences, Hopkins Building, University of Reading, Whiteknights Campus, Reading, Berkshire, RG6 6UB (United Kingdom)

2010-11-01

Skeletal muscle is highly adaptive to environmental stimuli and can alter its mass accordingly. This tissue is almost unique in that it can increase its size through two distinct mechanisms. It can grow through a cellular process mediated by cell fusion, or it can increase its size simply by increasing its protein content. Understanding how these processes are regulated is crucial for the development of potential therapies against debilitating skeletal muscle wasting diseases. Two key signalling molecules, Insulin like Growth Factor (IGF) and GDF-8/myostatin, have emerged in recent years to be potent regulators of skeletal muscle size. In this review we bring together recent data highlighting the important and novel aspects of both molecules and their signalling pathways, culminating in a discussion of the cellular and tissue phenotypic outcomes of their stimulation or antagonism. We emphasise the complex regulatory mechanisms and discuss the temporal and spatial differences that control their action, understanding of which is crucial to further their use as potential therapeutic targets.

Leucine stimulation of skeletal muscle protein synthesis

International Nuclear Information System (INIS)

Layman, D.K.; Grogan, C.K.

1986-01-01

Previous work in this laboratory has demonstrated a stimulatory effect of leucine on skeletal muscle protein synthesis measured in vitro during catabolic conditions. Studies in other laboratories have consistently found this effect in diaphragm muscle, however, studies examining effects on nitrogen balance or with in vivo protein synthesis in skeletal muscle are equivocal. This experiment was designed to determine the potential of leucine to stimulate skeletal muscle protein synthesis in vivo. Male Sprague-Dawley rats weighing 200 g were fasted for 12 hrs, anesthetized, a jugular cannula inserted, and protein synthesis measured using a primed continuous infusion of 14 C-tyrosine. A plateau in specific activity was reached after 30 to 60 min and maintained for 3 hrs. The leucine dose consisted of a 240 umole priming dose followed by a continuous infusion of 160 umoles/hr. Leucine infusion stimulated protein synthesis in the soleus muscle (28%) and in the red (28%) and white portions (12%) of the gastrocnemius muscle compared with controls infused with only tyrosine. The increased rates of protein synthesis were due to increased incorporation of tyrosine into protein and to decreased specific activity of the free tyrosine pool. These data indicate that infusion of leucine has the potential to stimulate in vivo protein synthesis in skeletal muscles

Skeletal muscle glucose uptake during exercise

DEFF Research Database (Denmark)

Rose, Adam John; Richter, Erik

2005-01-01

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

Radiation injury to skeletal muscle

International Nuclear Information System (INIS)

Persons, C.C.M.; Wondergem, J.; Leer, J.W.H.

1997-01-01

Radiotherapy of neoplasia has increased the mean life expectancy of cancer patients. On the other hand, more reports are published on morbidity of the treatment with regard to normal tissue. Studies on skeletal muscle injury specifically are scarce, but many clinical long term follow-up studies make note of side effects as muscle atrophy, fibrosis and limited function. Furthermore it is suggested that skeletal muscles of children are more prone to radiation injury than those of adult subjects. Effects of radiation on skeletal muscle were studied in rats. On hind limb of young (100 g) and adult (350 g) rats was irradiated with single doses (15-30 Gy), while the other served as control. Follow-up was up to 12 months post treatment. Muscular function in young rats was decreased significantly at 6 months post irradiation, but did not further decrease in the following 6 months. The amount of collagen, on the other hand, was not increased at 6 months, but became highly elevated at 12 months past treatment. This suggests that at 6 months, impaired muscular function may not be explained by increased fibrotic tissues. This is an agreement with results obtained in adult rats, where function was also impaired, without concomitant increase in collagen. In an earlier study, mitochondrial oxygen consumption was dose dependently decreased after irradiation, at 12 months, but not at 6 months post treatment. Furthermore, myosin-actin interaction was measured in skinned fibers. The first results of this study indicate changes in the interaction of contraction proteins, as early as 6 months post treatment. (authors)

Effect of extraluminal ATP application on vascular tone and blood flow in skeletal muscle

DEFF Research Database (Denmark)

Nyberg, Michael Permin; Al-Khazraji, Baraa K; Mortensen, Stefan P

2013-01-01

During skeletal muscle contractions, the concentration of ATP increases in muscle interstitial fluid as measured by microdialysis probes. This increase is associated with the magnitude of blood flow, suggesting that interstitial ATP may be important for contraction-induced vasodilation. However...... studied. The rat gluteus maximus skeletal muscle model was used to study changes in local skeletal muscle hemodynamics. Superfused ATP at concentrations found during muscle contractions (1-10 µM) increased blood flow by up to 400%. In this model, the underlying mechanism was also examined by inhibition...... in interstitial ATP concentrations increases muscle blood flow, indicating that the contraction-induced increase in skeletal muscle interstitial [ATP] is important for exercise hyperemia. The vasodilator effect of ATP application is mediated by NO and prostanoid formation....

Lipolysis in Skeletal Muscle

DEFF Research Database (Denmark)

Serup, Annette Karen Lundbeck

chemical structure of DAG. We took advantage of the fact that insulin sensitivity is increased after exercise, and that mice knocked out (KO) of HSL accumulate DAG after exercise, and measured insulin stimulated glucose uptake after treadmill running in skeletal muscle from HSL KO mice and wildtype control...

PGC-1α-mediated adaptations in skeletal muscle

DEFF Research Database (Denmark)

Olesen, Jesper; Kiilerich, Kristian; Pilegaard, Henriette

2010-01-01

multiple pathways and functions underline the potential importance of PGC-1alpha in skeletal muscle adaptations in humans. The absence of exercise-induced PGC-1alpha-mediated gene regulation during a physical inactive lifestyle is suggested to lead to reduced oxidative capacity of skeletal muscle...... involved in angiogenesis and the anti-oxidant defence as well as to affect expression of inflammatory markers. Exercise increases PGC-1alpha transcription and potentially PGC-1alpha activity through post-translational modifications, and concomitant PGC-1alpha-mediated gene regulation is suggested...... to be an underlying mechanism for adaptations in skeletal muscle, when exercise is repeated. The current review presents some of the key findings in PGC-1alpha-mediated regulation of metabolically related, anti-oxidant and inflammatory proteins in skeletal muscle in the basal state and in response to exercise...

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.

Exhaustive Training Increases Uncoupling Protein 2 Expression and Decreases Bcl-2/Bax Ratio in Rat Skeletal Muscle

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W. Y. Liu

2013-01-01

Full Text Available This work investigates the effects of oxidative stress due to exhaustive training on uncoupling protein 2 (UCP2 and Bcl-2/Bax in rat skeletal muscles. A total of 18 Sprague-Dawley female rats were randomly divided into three groups: the control group (CON, the trained control group (TC, and the exhaustive trained group (ET. Malondialdehyde (MDA, superoxide dismutase (SOD, xanthine oxidase (XOD, ATPase, UCP2, and Bcl-2/Bax ratio in red gastrocnemius muscles were measured. Exhaustive training induced ROS increase in red gastrocnemius muscles, which led to a decrease in the cell antiapoptotic ability (Bcl-2/Bax ratio. An increase in UCP2 expression can reduce ROS production and affect mitochondrial energy production. Thus, oxidative stress plays a significant role in overtraining.

Exercise-induced increase in interstitial bradykinin and adenosine concentrations in skeletal muscle and peritendinous tissue in humans

DEFF Research Database (Denmark)

Langberg, H; Bjørn, C; Boushel, Robert Christopher

2002-01-01

been established. Microdialysis (molecular mass cut-off 5 kDa) was performed simultaneously in calf muscle and peritendinous Achilles tissue at rest and during 10 min periods of incremental (0.75 W, 2 W, 3.5 W and 4.75 W) dynamic plantar flexion exercise in 10 healthy individuals (mean age 27 years...... increased both in muscle (from 0.48 +/- 0.07 micromol l(-1) to 1.59 +/- 0.35 micromol l(-1); P increases the interstitial concentrations......Bradykinin is known to cause vasodilatation in resistance vessels and may, together with adenosine, be an important regulator of tissue blood flow during exercise. Whether tissue concentrations of bradykinin change with exercise in skeletal muscle and tendon-related connective tissue has not yet...

The skeletal consequences of thyrotoxicosis.

Science.gov (United States)

Nicholls, Jonathan J; Brassill, Mary Jane; Williams, Graham R; Bassett, J H Duncan

2012-06-01

Euthyroid status is essential for normal skeletal development and the maintenance of adult bone structure and strength. Established thyrotoxicosis has long been recognised as a cause of high bone turnover osteoporosis and fracture but more recent studies have suggested that subclinical hyperthyroidism and long-term suppressive doses of thyroxine (T4) may also result in decreased bone mineral density (BMD) and an increased risk of fragility fracture, particularly in postmenopausal women. Furthermore, large population studies of euthyroid individuals have demonstrated that a hypothalamic-pituitary-thyroid axis set point at the upper end of the normal reference range is associated with reduced BMD and increased fracture susceptibility. Despite these findings, the cellular and molecular mechanisms of thyroid hormone action in bone remain controversial and incompletely understood. In this review, we discuss the role of thyroid hormones in bone and the skeletal consequences of hyperthyroidism.

Tribbles 3 Mediates Endoplasmic Reticulum Stress-Induced Insulin Resistance in Skeletal Muscle

Science.gov (United States)

Koh, Ho-Jin; Toyoda, Taro; Didesch, Michelle M.; Lee, Min-Young; Sleeman, Mark W.; Kulkarni, Rohit N.; Musi, Nicolas; Hirshman, Michael F.; Goodyear, Laurie J.

2013-01-01

Endoplasmic Reticulum (ER) stress has been linked to insulin resistance in multiple tissues but the role of ER stress in skeletal muscle has not been explored. ER stress has also been reported to increase tribbles 3 (TRB3) expression in multiple cell lines. Here, we report that high fat feeding in mice, and obesity and type 2 diabetes in humans significantly increases TRB3 and ER stress markers in skeletal muscle. Overexpression of TRB3 in C2C12 myotubes and mouse tibialis anterior muscles significantly impairs insulin signaling. Incubation of C2C12 cells and mouse skeletal muscle with ER stressors thapsigargin and tunicamycin increases TRB3 and impairs insulin signaling and glucose uptake, effects reversed in cells overexpressing RNAi for TRB3 and in muscles from TRB3 knockout mice. Furthermore, TRB3 knockout mice are protected from high fat diet-induced insulin resistance in skeletal muscle. These data demonstrate that TRB3 mediates ER stress-induced insulin resistance in skeletal muscle. PMID:23695665

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

Science.gov (United States)

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

2014-01-01

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

Proteomics of Skeletal Muscle

DEFF Research Database (Denmark)

Deshmukh, Atul

2016-01-01

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

PGC-1alpha and PGC-1beta have both similar and distinct effects on myofiber switching toward an oxidative phenotype

DEFF Research Database (Denmark)

Mortensen, Ole Hartvig; Frandsen, Lis; Schjerling, Peter

2006-01-01

Peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -1beta (PGC-1alpha and PGC-1beta) were overexpressed by adenovirus-mediated gene transfer in cultures of primary rat skeletal muscle cells derived from neonatal myoblasts. Effects on muscle fiber type transition and metabolism...... an increase in the mRNA expression of TRB3, a negative regulator of insulin signaling. These results show that both PGC-1alpha and PGC-1beta are involved in the regulation of skeletal muscle fiber transition and metabolism and that they have both overlapping and differing effects....... were studied from days 5 to 22 of culture. PGC-1alpha and PGC-1beta overexpression caused a three- to fourfold increase in mRNA level, a doubling of enzymatic activity of citrate synthase, a slight increase in short-chain acyl-CoA dehydrogenase mRNA, a doubling of the mRNA level, and a 30-50% increase...

In utero undernutrition programs skeletal and cardiac muscle metabolism

Directory of Open Access Journals (Sweden)

Brittany eBeauchamp

2016-01-01

Full Text Available In utero undernutrition is associated with increased risk for insulin resistance, obesity, and cardiovascular disease during adult life. A common phenotype associated with low birth weight is reduced skeletal muscle mass. Given the central role of skeletal muscle in whole body metabolism, alterations in its mass as well as its metabolic characteristics may contribute to disease risk. This review highlights the metabolic alterations in cardiac and skeletal muscle associated with in utero undernutrition and low birth weight. These tissues have high metabolic demands and are known to be sites of major metabolic dysfunction in obesity, type 2 diabetes, and cardiovascular disease. Recent research demonstrates that mitochondrial energetics are decreased in skeletal and cardiac muscles of adult offspring from undernourished mothers. These effects apparently lead to the development of a thrifty phenotype, which may represent overall a compensatory mechanism programmed in utero to handle times of limited nutrient availability. However, in an environment characterized by food abundance, the effects are maladaptive and increase adulthood risks of metabolic disease.

Skeletal surveys in multiple myeloma

International Nuclear Information System (INIS)

Sebes, J.I.; Niell, H.B.; Palmieri, G.M.A.; Reidy, T.J.

1986-01-01

Thirty-three patients with multiple myeloma were studied with serial skeletal surveys, serum immunoglobulin levels, and postabsorptive urinary hydroxyproline (Spot-HYPRO) determinations. Twenty receiving chemotherapy were also followed with skeletal surveys in order to evaluate bone response to treatment. A close association was found between skeletal findings and changes in immunoglubulin levels with positive correlation in 71% of the patients. A similar association was found between skeletal disease and Spot-HYPRO level changes in 65%. Five of 12 patients (42%) with partial or complete clinical response to chemotherapy, demonstrated improvement in the appearance of skeletal lesions. Positive correlation between the roentgenographic changes and clinical markers of myeloma as well as therapeutic response, indicates that skeletal surveys are useful and effective in monitoring patients with multiple myeloma. (orig.)

Intrauterine growth retardation increases the susceptibility of pigs to high-fat diet-induced mitochondrial dysfunction in skeletal muscle.

Directory of Open Access Journals (Sweden)

Jingbo Liu

Full Text Available It has been recognized that there is a relationship between prenatal growth restriction and the development of metabolic-related diseases in later life, a process involved in mitochondrial dysfunction. In addition, intrauterine growth retardation (IUGR increases the susceptibility of offspring to high-fat (HF diet-induced metabolic syndrome. Recent findings suggested that HF feeding decreased mitochondrial oxidative capacity and impaired mitochondrial function in skeletal muscle. Therefore, we hypothesized that the long-term consequences of IUGR on mitochondrial biogenesis and function make the offspring more susceptible to HF diet-induced mitochondrial dysfunction. Normal birth weight (NBW, and IUGR pigs were allotted to control or HF diet in a completely randomized design, individually. After 4 weeks of feeding, growth performance and molecular pathways related to mitochondrial function were determined. The results showed that IUGR decreased growth performance and plasma insulin concentrations. In offspring fed a HF diet, IUGR was associated with enhanced plasma leptin levels, increased concentrations of triglyceride and malondialdehyde (MDA, and reduced glycogen and ATP contents in skeletal muscle. High fat diet-fed IUGR offspring exhibited decreased activities of lactate dehydrogenase (LDH and glucose-6-phosphate dehydrogenase (G6PD. These alterations in metabolic traits of IUGR pigs were accompanied by impaired mitochondrial respiration function, reduced mitochondrial DNA (mtDNA contents, and down-regulated mRNA expression levels of genes responsible for mitochondrial biogenesis and function. In conclusion, our results suggest that IUGR make the offspring more susceptible to HF diet-induced mitochondrial dysfunction.

Intrauterine Growth Retardation Increases the Susceptibility of Pigs to High-Fat Diet-Induced Mitochondrial Dysfunction in Skeletal Muscle

Science.gov (United States)

Liu, Jingbo; Chen, Daiwen; Yao, Ying; Yu, Bing; Mao, Xiangbing; He, Jun; Huang, Zhiqing; Zheng, Ping

2012-01-01

It has been recognized that there is a relationship between prenatal growth restriction and the development of metabolic-related diseases in later life, a process involved in mitochondrial dysfunction. In addition, intrauterine growth retardation (IUGR) increases the susceptibility of offspring to high-fat (HF) diet-induced metabolic syndrome. Recent findings suggested that HF feeding decreased mitochondrial oxidative capacity and impaired mitochondrial function in skeletal muscle. Therefore, we hypothesized that the long-term consequences of IUGR on mitochondrial biogenesis and function make the offspring more susceptible to HF diet-induced mitochondrial dysfunction. Normal birth weight (NBW), and IUGR pigs were allotted to control or HF diet in a completely randomized design, individually. After 4 weeks of feeding, growth performance and molecular pathways related to mitochondrial function were determined. The results showed that IUGR decreased growth performance and plasma insulin concentrations. In offspring fed a HF diet, IUGR was associated with enhanced plasma leptin levels, increased concentrations of triglyceride and malondialdehyde (MDA), and reduced glycogen and ATP contents in skeletal muscle. High fat diet-fed IUGR offspring exhibited decreased activities of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PD). These alterations in metabolic traits of IUGR pigs were accompanied by impaired mitochondrial respiration function, reduced mitochondrial DNA (mtDNA) contents, and down-regulated mRNA expression levels of genes responsible for mitochondrial biogenesis and function. In conclusion, our results suggest that IUGR make the offspring more susceptible to HF diet-induced mitochondrial dysfunction. PMID:22523560

Increased autophagy and apoptosis contribute to muscle atrophy in a myotonic dystrophy type 1 Drosophila model

Directory of Open Access Journals (Sweden)

Ariadna Bargiela

2015-07-01

Full Text Available Muscle mass wasting is one of the most debilitating symptoms of myotonic dystrophy type 1 (DM1 disease, ultimately leading to immobility, respiratory defects, dysarthria, dysphagia and death in advanced stages of the disease. In order to study the molecular mechanisms leading to the degenerative loss of adult muscle tissue in DM1, we generated an inducible Drosophila model of expanded CTG trinucleotide repeat toxicity that resembles an adult-onset form of the disease. Heat-shock induced expression of 480 CUG repeats in adult flies resulted in a reduction in the area of the indirect flight muscles. In these model flies, reduction of muscle area was concomitant with increased apoptosis and autophagy. Inhibition of apoptosis or autophagy mediated by the overexpression of DIAP1, mTOR (also known as Tor or muscleblind, or by RNA interference (RNAi-mediated silencing of autophagy regulatory genes, achieved a rescue of the muscle-loss phenotype. In fact, mTOR overexpression rescued muscle size to a size comparable to that in control flies. These results were validated in skeletal muscle biopsies from DM1 patients in which we found downregulated autophagy and apoptosis repressor genes, and also in DM1 myoblasts where we found increased autophagy. These findings provide new insights into the signaling pathways involved in DM1 disease pathogenesis.

LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration

Science.gov (United States)

Wang, Lijun; Zhao, Yu; Bao, Xichen; Zhu, Xihua; Kwok, Yvonne Ka-yin; Sun, Kun; Chen, Xiaona; Huang, Yongheng; Jauch, Ralf; Esteban, Miguel A; Sun, Hao; Wang, Huating

2015-01-01

Emerging studies document the roles of long non-coding RNAs (LncRNAs) in regulating gene expression at chromatin level but relatively less is known how they regulate DNA methylation. Here we identify an lncRNA, Dum (developmental pluripotency-associated 2 (Dppa2) Upstream binding Muscle lncRNA) in skeletal myoblast cells. The expression of Dum is dynamically regulated during myogenesis in vitro and in vivo. It is also transcriptionally induced by MyoD binding upon myoblast differentiation. Functional analyses show that it promotes myoblast differentiation and damage-induced muscle regeneration. Mechanistically, Dum was found to silence its neighboring gene, Dppa2, in cis through recruiting Dnmt1, Dnmt3a and Dnmt3b. Furthermore, intrachromosomal looping between Dum locus and Dppa2 promoter is necessary for Dum/Dppa2 interaction. Collectively, we have identified a novel lncRNA that interacts with Dnmts to regulate myogenesis. PMID:25686699

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.

Skeletal metastases from hepatoma: frequency, distribution, and radiographic features

International Nuclear Information System (INIS)

Kuhlman, J.E.; Fishman, E.K.; Leichner, P.K.; Magid, D.; Order, S.E.; Siegelman, S.S.

1986-01-01

Over the past 6 years, the authors evaluated 300 patients with hepatoma as part of phase 1 and 2 treatment protocol trials. Analysis of the available clinical data and radiographic studies revealed 22 patients (7.3%) with skeletal metastases demonstrated by radiography, computed tomography (CT), and/or nuclear scintigraphy. The plain film appearance of skeletal metastases from hepatoma was osteolytic in all cases. CT scanning best demonstrated the expansile, destructive nature of these metastases, which were often associated with large, bulky soft-tissue masses. Skeletal metastases from hepatomas demonstrated increased radiotracer uptake on standard bone scans and were gallium avid, similar to the hepatoma itself. In addition, they could be targeted therapeutically with I-131 antiferritin immunoglobulin. The most frequent sites of skeletal metastases were the ribs, spine, femur, pelvis, and humerus. An initial symptom in ten patients was skeletal pain corresponding to the osseous metastases. In five patients, pathologic fractures of the proximal femur or humerus developed and required total hip replacement or open-reduction internal fixation. Patients with long-standing cirrhosis or known hepatocellular carcinoma who also have skeletal symptoms should be evaluated for possible osseous metastases

Whole-body MRI in comparison to skeletal scintigraphy for detection of skeletal metastases in patients with solid tumors

International Nuclear Information System (INIS)

Ghanem, N.; Altehoefer, C.; Winterer, J.; Schaefer, O.; Bley, T.A.; Langer, M.; Kelly, T.; Moser, E.

2004-01-01

The aim of this study was to compare the diagnostic efficacy of whole-body magnetic resonance imaging (WB-MRI) as a new and rapid examination technique with skeletal scintigraphy for detection of skeletal metastases from solid tumors. In 129 patients with solid malignant tumors, WB-MRI was performed for individual comparison with skeletal scintigraphy. Examinations were performed with the innovative AngioSURF trademark rolling table with integrated phased array surface coil and coronary TIRM sequences for different body regions. The results for WB-MRI and skeletal scintigraphy were concordant in 81% of the cases, whereby both procedures excluded skeletal metastases in 43%. WB-MRI and skeletal scintigraphy demonstrated skeletal metastases in 38% of the cases, whereby WB-MRI provided more comprehensive findings in 45%. In 12% of the cases, skeletal scintigraphy was superior to WB-MRI and in 19% the findings were discordant, whereby WB-MRI detected skeletal metastases in 15 cases which had not been found on skeletal scintigraphy. In nine cases, skeletal scintigraphy was positive when the WB-MRI was negative. In 60% of the cases, WB-MRI evidenced tumor-associated findings. WB-MRI represents a promising new staging technique for detection of skeletal metastases, which is more sensitive in many cases than skeletal scintigraphy in detecting and assessing the extent of skeletal metastases - and tumor-associated findings that are relevant for treatment strategy. (orig.) [de

Alternate-Day High-Fat Diet Induces an Increase in Mitochondrial Enzyme Activities and Protein Content in Rat Skeletal Muscle.

Science.gov (United States)

Li, Xi; Higashida, Kazuhiko; Kawamura, Takuji; Higuchi, Mitsuru

2016-04-06

Long-term high-fat diet increases muscle mitochondrial enzyme activity and endurance performance. However, excessive calorie intake causes intra-abdominal fat accumulation and metabolic syndrome. The purpose of this study was to investigate the effect of an alternating day high-fat diet on muscle mitochondrial enzyme activities, protein content, and intra-abdominal fat mass in rats. Male Wistar rats were given a standard chow diet (CON), high-fat diet (HFD), or alternate-day high-fat diet (ALT) for 4 weeks. Rats in the ALT group were fed a high-fat diet and standard chow every other day for 4 weeks. After the dietary intervention, mitochondrial enzyme activities and protein content in skeletal muscle were measured. Although body weight did not differ among groups, the epididymal fat mass in the HFD group was higher than those of the CON and ALT groups. Citrate synthase and beta-hydroxyacyl CoA dehydrogenase activities in the plantaris muscle of rats in HFD and ALT were significantly higher than that in CON rats, whereas there was no difference between HFD and ALT groups. No significant difference was observed in muscle glycogen concentration or glucose transporter-4 protein content among the three groups. These results suggest that an alternate-day high-fat diet induces increases in mitochondrial enzyme activities and protein content in rat skeletal muscle without intra-abdominal fat accumulation.

Noradrenaline spillover during exercise in active versus resting skeletal muscle in man

DEFF Research Database (Denmark)

Savard, G; Strange, S; Kiens, Bente

1987-01-01

Increases in plasma noradrenaline (NA) concentration occur during moderate to heavy exercise in man. This study was undertaken to examine the spillover of NA from both resting and contracting skeletal muscle during exercise. Six male subjects performed one-legged knee-extension so that all...... in the exercising leg than in the resting leg both during 50% and 100% leg exercise. These results suggest that contracting skeletal muscle may contribute to a larger extent than resting skeletal muscle to increasing the level of plasma NA during exercise. Contractile activity may influence the NA spillover from...

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

Directory of Open Access Journals (Sweden)

Yukino Hatazawa

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

Selection, processing and clinical application of muscle-skeletal tissue

International Nuclear Information System (INIS)

Luna Z, D.; Reyes F, M.L.; Lavalley E, C.; Castaneda J, G.

2007-01-01

Due to the increase in the average of the world population's life, people die each time to more age, this makes that the tissues of support of the human body, as those muscle-skeletal tissues, when increasing the individual's age go weakening, this in turn leads to the increment of the illnesses like the osteoporosis and the arthritis, that undoubtedly gives as a result more injure of the muscle-skeletal tissues joined a greater number of traffic accidents where particularly these tissues are affected, for that the demand of tissues muscle-skeletal for transplant every day will be bigger. The production of these tissues in the Bank of Radio sterilized Tissues, besides helping people to improve its quality of life saved foreign currencies because most of the muscle-skeletal tissues transplanted in Mexico are of import. The use of the irradiation to sterilize tissues for transplant has shown to be one of the best techniques with that purpose for what the International Atomic Energy Agency believes a Technical cooperation program to establish banks of tissues using the nuclear energy, helping mainly to countries in development. In this work the stages that follows the bank of radio sterilized tissues of the National Institute of Nuclear Research for the cadaverous donor's of muscle-skeletal tissue selection are described, as well as the processing and the clinical application of these tissues. (Author)

Extra-osseous uterine pathophysiology demonstrated on skeletal scintigraphy

International Nuclear Information System (INIS)

Mansberg, R.; Lewis, G.

1999-01-01

Full text: Skeletal scintigraphy is a sensitive procedure for evaluating disease and trauma involving the skeleton. Extra-skeletal pathophysiology is also often demonstrated. This may include uptake by tumours, soft tissue calcification and infection as well as renal pathology. Skeletal scintigraphy is often performed to evaluate hip and back pain and extra-osseous uterine pathophysiology can be demonstrated in both the early and late phases of the study as in the following cases. Three women underwent skeletal scintigraphy for the investigation of low back pain in two patients and post-partum hip pain in one. A large vascular uterus with deviation of the bladder was demonstrated in the post-partum patient. Increased pelvic vascularity and bladder deviation in the second patient was shown by ultrasound to correspond to a left-sided fibroid with associated adenomyosis. In the third case, right-sided pelvic vascularity and left bladder deviation were shown on ultrasound to be due to an anteverted, anteflexed uterus tilted to the right. These cases illustrate the importance of documenting extra-osseous findings on skeletal scintigraphy and the benefits of correlation with anatomical imaging

Genes Whose Gain or Loss-Of-Function Increases Skeletal Muscle Mass in Mice: A Systematic Literature Review

Directory of Open Access Journals (Sweden)

Sander A. J. Verbrugge

2018-05-01

Full Text Available Skeletal muscle mass differs greatly in mice and humans and this is partially inherited. To identify muscle hypertrophy candidate genes we conducted a systematic review to identify genes whose experimental loss or gain-of-function results in significant skeletal muscle hypertrophy in mice. We found 47 genes that meet our search criteria and cause muscle hypertrophy after gene manipulation. They are from high to small effect size: Ski, Fst, Acvr2b, Akt1, Mstn, Klf10, Rheb, Igf1, Pappa, Ppard, Ikbkb, Fstl3, Atgr1a, Ucn3, Mcu, Junb, Ncor1, Gprasp1, Grb10, Mmp9, Dgkz, Ppargc1a (specifically the Ppargc1a4 isoform, Smad4, Ltbp4, Bmpr1a, Crtc2, Xiap, Dgat1, Thra, Adrb2, Asb15, Cast, Eif2b5, Bdkrb2, Tpt1, Nr3c1, Nr4a1, Gnas, Pld1, Crym, Camkk1, Yap1, Inhba, Tp53inp2, Inhbb, Nol3, Esr1. Knock out, knock down, overexpression or a higher activity of these genes causes overall muscle hypertrophy as measured by an increased muscle weight or cross sectional area. The mean effect sizes range from 5 to 345% depending on the manipulated gene as well as the muscle size variable and muscle investigated. Bioinformatical analyses reveal that Asb15, Klf10, Tpt1 are most highly expressed hypertrophy genes in human skeletal muscle when compared to other tissues. Many of the muscle hypertrophy-regulating genes are involved in transcription and ubiquitination. Especially genes belonging to three signaling pathways are able to induce hypertrophy: (a Igf1-Akt-mTOR pathway, (b myostatin-Smad signaling, and (c the angiotensin-bradykinin signaling pathway. The expression of several muscle hypertrophy-inducing genes and the phosphorylation of their protein products changes after human resistance and high intensity exercise, in maximally stimulated mouse muscle or in overloaded mouse plantaris.

Lyophilized skeletal imaging composition

International Nuclear Information System (INIS)

Vanduzee, B.F.

1983-01-01

This invention encompasses a process for producing a dry-powder skeletal imaging kit. An aqueous solution of a diphosphonate, a stannous reductant, and, optionally, a stabilizer is prepared. The solution is adjusted to a pH within the range 4.2 to 4.8 and the pH-adjusted solution is then lyophilized. The adjustment of pH, within a particular range, during the process of manufacturing lyophilized diphosphonate containing skeletal imaging kits yields a kit which produces a technetium skeletal imaging agent with superior imaging properties. This improved performance is manifested through faster blood clearance and higher skeletal uptake of the technetium imaging agent

Cardiac troponin T and fast skeletal muscle denervation in ageing.

Science.gov (United States)

Xu, Zherong; Feng, Xin; Dong, Juan; Wang, Zhong-Min; Lee, Jingyun; Furdui, Cristina; Files, Daniel Clark; Beavers, Kristen M; Kritchevsky, Stephen; Milligan, Carolanne; Jin, Jian-Ping; Delbono, Osvaldo; Zhang, Tan

2017-10-01

Ageing skeletal muscle undergoes chronic denervation, and the neuromuscular junction (NMJ), the key structure that connects motor neuron nerves with muscle cells, shows increased defects with ageing. Previous studies in various species have shown that with ageing, type II fast-twitch skeletal muscle fibres show more atrophy and NMJ deterioration than type I slow-twitch fibres. However, how this process is regulated is largely unknown. A better understanding of the mechanisms regulating skeletal muscle fibre-type specific denervation at the NMJ could be critical to identifying novel treatments for sarcopenia. Cardiac troponin T (cTnT), the heart muscle-specific isoform of TnT, is a key component of the mechanisms of muscle contraction. It is expressed in skeletal muscle during early development, after acute sciatic nerve denervation, in various neuromuscular diseases and possibly in ageing muscle. Yet the subcellular localization and function of cTnT in skeletal muscle is largely unknown. Studies were carried out on isolated skeletal muscles from mice, vervet monkeys, and humans. Immunoblotting, immunoprecipitation, and mass spectrometry were used to analyse protein expression, real-time reverse transcription polymerase chain reaction was used to measure gene expression, immunofluorescence staining was performed for subcellular distribution assay of proteins, and electromyographic recording was used to analyse neurotransmission at the NMJ. Levels of cTnT expression in skeletal muscle increased with ageing in mice. In addition, cTnT was highly enriched at the NMJ region-but mainly in the fast-twitch, not the slow-twitch, muscle of old mice. We further found that the protein kinase A (PKA) RIα subunit was largely removed from, while PKA RIIα and RIIβ are enriched at, the NMJ-again, preferentially in fast-twitch but not slow-twitch muscle in old mice. Knocking down cTnT in fast skeletal muscle of old mice: (i) increased PKA RIα and reduced PKA RIIα at the NMJ; (ii

Wnt/β-catenin signaling changes C2C12 myoblast proliferation and differentiation by inducing Id3 expression

International Nuclear Information System (INIS)

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

2012-01-01

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

Intermittent pneumatic compression of legs increases microcirculation in distant skeletal muscle.

Science.gov (United States)

Liu, K; Chen, L E; Seaber, A V; Johnson, G W; Urbaniak, J R

1999-01-01

Intermittent pneumatic compression has been established as a method of clinically preventing deep vein thrombosis, but the mechanism has not been documented. This study observed the effects of intermittent pneumatic compression of legs on the microcirculation of distant skeletal muscle. The cremaster muscles of 80 male rats were exposed, a specially designed intermittent pneumatic-compression device was applied to both legs for 60 minutes, and the microcirculation of the muscles was assessed by measurement of the vessel diameter in three categories (10-20, 21-40, and 41-70 microm) for 120 minutes. The results showed significant vasodilation in arterial and venous vessels during the application of intermittent pneumatic compression, which disappeared after termination of the compression. The vasodilation reached a maximum 30 minutes after initiation of the compression and could be completely blocked by an inhibitor of nitric oxide synthase, NG-monomethyl-L-arginine (10 micromol/min). A 120-minute infusion of NG-monomethyl-L-arginine, beginning coincident with 60 minutes of intermittent pneumatic compression, resulted in a significant decrease in arterial diameter that remained at almost the same level after termination of the compression. The magnitude of the decrease in diameter in the group treated with intermittent pneumatic compression and NG-monomethyl-L-arginine was comparable with that in the group treated with NG-monomethyl-L-arginine alone. The results imply that the production of nitric oxide is involved in the positive influence of intermittent pneumatic compression on circulation. It is postulated that the rapid increase in venous velocity induced by intermittent pneumatic compression produces strong shear stress on the vascular endothelium, which stimulates an increased release of nitric oxide and thereby causes systemic vasodilation.

Deep bite malocclusion: exploration of the skeletal and dental factors

International Nuclear Information System (INIS)

Bhateja, N.K.; Fida, M.; Shaikh, A.

2016-01-01

Correction of deep bite is crucial for maintenance of dental hard and soft tissue structures and for prevention of temporomandibular joint disorders. Exploration of underlying skeletal and dental factors is essential for efficient and individualized treatment planning. To date etiological factors of dental and skeletal deep bite have not been explored in Pakistani orthodontic patients. The objectives of this study were to explore frequencies of dental and skeletal etiological factors in deep bite patients and to determine correlations amongst dental and skeletal etiological factors of deep bite. Methods: The study included a total of 113 subjects (males=35; females=78) with no craniofacial syndromes or prior orthodontic treatment. Pre-treatment orthodontic records were used to evaluate various dental and skeletal parameters. Descriptive statistics of each parameter were calculated. The various study parameters were correlated using Pearson's Correlation. Results: Deep curve of Spee was most frequently seen factor of dental deep bite (72.6%), followed by increased coronal length of upper incisors (28.3%), retroclined upper incisors (17.7%), retroclined lower incisors (8%) and increased coronal length of lower incisors (5.3%). Decreased gonial angle was most commonly found factor of skeletal deep bite (43.4%), followed by decreased mandibular plane angle (27.4%) and maxillary plane's clockwise rotation (26.5%). Frankfort mandibular plane angle and gonial angle showed a strong positive correlation (r=0.66, p=0.000). Conclusions: Reduced gonial angle is most frequently seen skeletal factor, signifying the importance of angulation and growth of ramus in development of deep bite. Deep curve of Spee is most frequently seen dental etiological component in deep bite subjects, hence signifying the importance of intruding the lower anterior teeth. (author)

Maintenance of DNA repair capacity in differentiating rat muscle cells in vitro

International Nuclear Information System (INIS)

Koval, T.M.; Kaufman, S.J.

1981-01-01

Unscheduled DNA synthesis was measured at several times during the differentiation of cultured rat skeletal muscle cells in response to exposures to 254 nm UV light. There was no change in the amount of repair DNA synthesis as the cells fuse and differentiate from postmitotic prefusion myoblasts to multinucleated contracting myotubes. (author)

Fine-structural distribution of MMP-2 and MMP-9 activities in the rat skeletal muscle upon training: a study by high-resolution in situ zymography.

Science.gov (United States)

Yeghiazaryan, Marine; Żybura-Broda, Katarzyna; Cabaj, Anna; Włodarczyk, Jakub; Sławińska, Urszula; Rylski, Marcin; Wilczyński, Grzegorz M

2012-07-01

Matrix metalloproteinases (MMPs) are key regulators of extracellular matrix remodeling, but have also important intracellular targets. The purpose of this study was to examine the activity and subcellular localization of the gelatinases MMP-2 and MMP-9 in skeletal muscle of control and physically trained rats. In control hind limb muscle, the activity of the gelatinases was barely detectable. In contrast, after 5 days of intense exercise, in Soleus (Sol), but not Extensor digitorum longus (EDL) muscle, significant upregulation of gelatinolytic activity in myofibers was observed mainly in the nuclei, as assessed by high resolution in situ zymography. The nuclei of quiescent satellite cells did not contain the activity. Within the myonuclei, the gelatinolytic activity colocalized with an activated RNA Polymerase II. Also in Sol, but not in EDL, there were few foci of mononuclear cells with strongly positive cytoplasm, associated with apparent necrotic myofibers. These cells were identified as activated satellite cells/myoblasts. No extracellular gelatinase activity was observed. Gel zymography combined with subcellular fractionation revealed training-related upregulation of active MMP-2 in the nuclear fraction, and increase of active MMP-9 in the cytoplasmic fraction of Sol. Using RT-PCR, selective increase in MMP-9 mRNA was observed. We conclude that training activates nuclear MMP-2, and increases expression and activity of cytoplasmic MMP-9 in Sol, but not in EDL. Our results suggest that the gelatinases are involved in muscle adaptation to training, and that MMP-2 may play a novel role in myonuclear functions.

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