Expression of the Extracellular Fatty Acid Binding Protein (Ex-FABP) during Muscle Fiber Formationin Vivoandin Vitro

We report that Ex-FABP, an extracellular protein belonging to the lipocalin family and involved in the extracellular transport of long-chain fatty acids, is expressed in the forming myotubes bothin vivoandin vitro.The presence of the protein and of the mRNA was observed in newly formed myotubes at early stages of chick embryo development by immunohistochemistry and byin situhybridization. At later stages of development myofibers still expressed both the mRNA and the protein. Ex-FABP expression was observed also in the developing myocardium and the muscular layer of large blood vessels. In agreement with these findings, an initial expression of the mRNA and protein secretion by cultured chicken myoblasts were observed only after the onset of myoblast fusion. Double-immunofluorescence staining of these cultured cells revealed that multinucleate myotubes were stained by antibodies directed against both the Ex-FABP and the sarcomeric myosin, whereas immature myotubes and single myoblasts were not. When added to cultured myoblasts, antibodies against the Ex-FABP induced a strong enhancement of the production of the same protein. In all experiments some cell sufferance and a transient impairment of myotube formation were also observed. The finding that the continuous removal of the Ex-FABP from the culture medium of myoblasts, due to the formation of immune complexes, resulted in an overproduction of the protein suggests a feedback (autocrine) control during myotube differentiation and maturation. We propose that the requirement for increased transport and metabolism of free fatty acid released from the membrane phospholipids and storage lipids, mediated by Ex-FABP, may be essential during differentiation of multinucleated myotubes or that an increased local demand of fatty acids and metabolites may act as a local hormone in tissues differentiating and undergoing morphogenesis.     

Pax7基因在C 2C12细胞内的表达及诱导失活(简报)

配对框(Paired box)首先是在果蝇的分节基因中发现的一段DNA保守序列,编码能与DNA特异结合的一个蛋白质结构域。这些序列在进化中有一定的保守性,在许多种生物基因组内存在,其中包括小鼠和人。至今为止,在小鼠中分离到九个含配对框的Pax基因,按基因发现时序,分别定名为Pax 1至Pax 9。Pax 7是其中的一个成员,它不但含有配对框,还含有八肽结构(Octapeptide)和     

p38激酶在肌肉发生及运动中的作用

p38通路是真核细胞转导细胞外信号到细胞内引起细胞反应的一类重要信号通路,它能够调控多种细胞内分子事件。p38是p38通路发挥生物学作用的末端激酶,它能够通过调控MyoD、Myf-5活性或表达、组蛋白修饰、染色体重塑、某些细胞分化相关mRNA的更替等方式影响肌肉分化进程。运动能够激活p38,p38可能通过NF-κB/IL-6/成肌调节因子(myogenic regulatory factor,MRF)及肌细胞专一增强因子2(muscle-specific enhancement factor2,MEF2)、过氧化物增殖物活化受体γ辅激活因子1α(peroxisome proliferator activated receptor γ coactivator-1α,PGC-1α)、葡萄糖转运蛋白4(glucose transporter 4,GLUT4)在运动介导的肌肉发生、线粒体发生、血糖摄取能力提高等几方面发挥作用。     

Monoclonal Antibodies Which Alter the Morphology of Cultured Chick Myogenic Cells

Two monoclonal antibodies that cause changes in the morphology of cultured myogenic cells are described. Antibody JG9 causes myoblasts to round up and causes myotubes to become thin, cable-like structures with multiple round swellings. Antibody JG22 causes both myoblasts and myotubes to become round refractile cells poorly attached to the substratum. The effects of both antibodies are reversible. Fab fragments of JG22 can cause the morphological change. A tentative identification of the antigen recognized by JG22 is made.     

Primary human muscle satellite cell culture: Variations of cell yield,proliferation and differentiation rates according to age and sex of donors,site of muscle biopsy,and delay before processing

The present study was performed to determine the influence on human satellite cell yield, proliferation, and differentiation rates of: 1) sex and age of donors; 2) site of the muscle biopsy; and 3) delay before processing of the muscle biopsy sample. We used a standardized primary muscle cell culture procedure on 206 normal muscle samples obtained from different muscle groups of patients aged from 20 to 88 years, at time of orthopedic surgery. Sex of donors did not influence muscle culture parameters. In contrast, aging tended to affect muscle cell yield (age group 50–59 years vs 70–79 years, P < 0.08), but not myogenic cell abilities to proliferate and to fuse into myotubes. The anatomic origin of muscle samples used for culture appeared to influence culture parameters. In contrast with other tested muscles, the tensor fasciae muscle gave both a good cell yield (174 ± 25 10³ cells per gram) and homogeneous proliferation and differentiation rates. Storage of the muscle sample at 4°C in transport medium was associated with a very high cell yield when processing was done in early hours after biopsy (277 ± 50 10³ cells/g), a high and stable cell yield when processing was done from day 1 to day 3 after biopsy (185 ± 15 10³ cells/g), and a poor cell yield when processing was done after day 4 (111 ± 13 10³ cells/g). Storage of muscle biopsy samples at 4°C for 1 to 4 days was associated with good proliferation and fusion rates. In conclusion, these data validate a convenient procedure of primary human muscle cell culture, using tensor fasciae muscle biopsy, which is easily done at time of orthopedic surgery, obtained from men and women of all ages (if possible less than 70 years to obtain good cell yield), and allowing of 1–3 days of storage before processing that may compensate uncertainty of the exact time of availability of muscle samples for the scientist.     

p38 MAPK interacts with actin and modulates filament assembly during skeletal muscle differentiation

Skeletal muscle differentiation is marked by enhanced myotube formation and increased cytoskeletal rearrangement. Actin, a cytoskeletal protein is involved in various cellular functions such as glucose transport, intracellular trafficking, cell shape, and coordinated cell movement in response to various extracellular signals. The present study reveals an association between actin and p38 MAPK only in differentiated myotubes, not in proliferating myoblasts. Actin filament disassembly caused by cytochalasinD can be reversed using the potent activator of p38 MAPK, anisomycin. Pretreatment of myotubes with anisomycin partially resisted the effect of cytochalasinD. However, inhibition of p38 MAPK completely abolished the anisomycin-mediated actin remodeling. Data suggests that p38 MAPK interacts with actin and modulates actin filament rearrangement in differentiated L6E9 skeletal muscle cells.     

Molecular control of myogenesis: antagonism between growth and differentiation

Insight into the molecular mechanisms that control establishment of the skeletal muscle phenotype has recently been obtained through cloning of a family of muscle-specific regulatory factors that can activate myogenesis when transfected into non-muscle cells. This family of factors, which includes MyoD, myogenin, myf-5, and MRF4, can bind DNA and transactivate muscle-specific genes in collaboration with ubiquitous cellular factors. Growth factors play an antagonistic role in myogenesis by suppressing the actions of the myogenic regulatory factor family. This review will focus on the regulation and mechanism of action of this family of myogenic regulatory factors and on the central role of peptide growth factors in modulating their expression and biological activities.     

Traf7, a MyoD1 transcriptional target, regulates nuclear factor-κB activity during myogenesis

We have identified the E3 ligase Traf7 as a direct MyoD1 target and show that cell cycle exit-an early event in muscle differentiation-is linked to decreased Traf7 expression. Depletion of Traf7 accelerates myogenesis, in part through downregulation of nuclear factor-κB (NF-κB) activity. We used a proteomic screen to identify NEMO, the NF-κB essential modulator, as a Traf7-interacting protein. Finally, we show that ubiquitylation of NF-κB essential modulator is regulated exclusively by Traf7 activity in myoblasts. Our results suggest a new mechanism by which MyoD1 function is coupled to NF-κB activity through Traf7, regulating the balance between cell cycle progression and differentiation during myogenesis.     

Effects of dietary l-arginine supplementation to gilts during early gestation on foetal survival, growth and myofiber formation

The effects of l-arginine on porcine foetal development and myogenesis were determined. Twenty Swiss Large White gilts were randomly allocated to either the control (C) or l-arginine treatment (A). In addition to the standard gestation diet, A-sows received 26 g l-arginine daily from days 14 to 28 of gestation. At day 75 of pregnancy, sows were sacrificed and the number and weight of foetuses were recorded. From each litter, the lightest, heaviest and the ones with an average foetal weight (FtW) were selected. Primary (P), secondary (S) and total myofiber number as well as S/P ratio were determined in the semitendinosus (ST) and rhomboideus (RH) muscles. In A-sows, the number of viable foetuses (13.0 v. 9.3) and total FtW (4925 v. 3729 g) was greater (P ⩽ 0.04) than in C-sows. Compared to C-sow foetuses, the ST of A-sow foetuses had 7% more (17 699 v. 16 477; P = 0.04) P myofibers and the S/P ratio in both muscles was lower (ST = 20.3 v. 21.5; RH = 24.1 v. 27.1; P ⩽ 0.07). Regardless of the maternal diet, the S myofiber number and the S/P ratio in both muscles were greater (P ⩽ 0.01) in foetuses with a high FtW compared to low FtW. These data suggest that l-arginine supplemented to gilts during early gestation enhanced foetal survival and in the ST positively affected the primary phase of myofiber formation.     

Insulin-like growth factors,hepatocyte growth factor and transforming growth factor-alpha in mouse tongue myogenesis

Many reports have shown that tongue striated muscles have several unique characteristics not found in other skeletal muscles such as limb and trunk. Several peptide growth factors are reported to play important roles in skeletal myogenesis. In this article, the roles of insulin-like growth factors (IGF), hepatocyte growth factor (HGF) and transforming growth factor (TGF)-alpha in mouse tongue myogenesis were studied using an organ culture system of the mandible or tongue obtained from mouse embryos. It was found that IGF-I promotes the differentiation of tongue myoblasts. HGF plays an essential role in the migration and proliferation of tongue myogenic cells, and inhibits the differentiation of tongue myoblasts. TGF-alpha does not play an essential role in the proliferation of tongue myogenic cells, but does promote the early differentiation of tongue myoblasts. The role of IGF-I in the differentiation of tongue myoblasts, and that of HGF in the migration, proliferation and differentiation of tongue myogenic cells appear to be almost identical to their roles in the myogenesis of limb and cultured myogenic cell lines. However, the role of TGF-alpha in the proliferation and differentiation of tongue myogenic cells appears to be different from its role in the myogenesis of limb and cultured myogenic cell lines such as C2 and L6.     

The influence of maternal nutrition on expression of genes responsible for adipogenesis and myogenesis in the bovine fetus

The objective of this study was to determine whether altered maternal energy supply during mid-gestation results in differences in muscle histology or genes regulating fetal adipose and muscle development. In total, 22 Angus cross-bred heifers (BW=527.73±8.3 kg) were assigned randomly to the three dietary treatments providing 146% (HIGH; n=7), 87% (INT; n=7) or 72% (LOW; n=8) of the energy requirements for heifers from day 85 to day 180 of gestation. Fetuses were removed via cesarean section at day 180 of gestation and longissimus muscle (LM) and subcutaneous fat were collected and prepared for analysis of gene expression. Samples from the LM and semitendinosus (ST) were evaluated for muscle fiber diameter, area and number. The right hind limb was dissected and analyzed to determine compositional analysis. Fetal growth and muscle histology characteristics of the LM and ST were similar among treatments. Preadipocyte factor-1 expression was up-regulated in fetal LM (P<0.05) of HIGH fetuses as compared with INT, whereas LOW fetuses showed increased CCAAT/enhancer-binding protein-β (C/EBP-β) expression in LM as compared with INT (P<0.05). Peroxisome proliferator-activated receptor γand C/EBP-α did not differ as a result of dietary treatment in LM or subcutaneous fat samples. There was a tendency for increased expression of fatty acid synthase in LM of LOW fetuses as compared with INT (P<0.10). Myogenin was more highly expressed (P<0.05) in LM of the LOW fetuses, whereas μ-calpain expression was increased in the HIGH treatment compared with INT. A tendency for increased expression of IGF-II was observed for both LOW and HIGH fetuses compared with INT (P<0.10). Expression of stearoyl-CoA desaturase, myoblast determination protein 1, myogenic factor 5, myogenic regulatory factor-4, m-calpain, calpastatin, IGF-I and myostatin was similar between treatments. Collectively, these results suggest that fetal growth characteristics are not affected by the level of maternal nutritional manipulation imposed in this study during mid-gestation. However, differences in expression of fetal genes regulating adipose and muscle tissue growth and development could lead to differences in postnatal composition and warrants further investigation.     

Skeletal myogenesis by human embryonic stem cells

We have examined the myogenic potential of human embryonic stem （hES） cells in a xeno-transplantation animal model. Here we show that precursors differentiated from hES cells can undergo myogenesis in an adult environment and give rise to a range of cell types in the myogenic lineage. This study provides direct evidences that hES cells can regenerate both muscle and satellite cells in vivo and are another promising cell type for treating muscle degenerative disorders in addition to other myogenic cell types.     

Involvement of phosphatidylserine externalization in the down-regulation of c-myb expression in differentiating C2C12 cells

Abstract Analysis of c-myb gene down-regulation in differentiating C212 cells revealed that in proliferating cells, c-myb expression is high and ceases as the proliferation rate decreases. However, a low level of c-myb mRNA was detected in confluent non-proliferating differentiating cells for an extended period of time before it declined to an undetectable level. The time course of c-myb gene silencing in differentiating cells correlated with exposition of phosphatidylserine (PS) on the cell surface. Moreover, the interaction of exposed PS with exogenously added annexin V perturbed PS-mediated cell signaling and transiently up-regulated the declining c-myb expression. We, therefore, suggest that cell surface-exposed PS, which plays a role in the process of myotube formation, is also involved in the down-regulation of c-myb expression.     

Lethal(1)fibrillardysgenesis (I(1)fdg), a mutation affecting muscle development in the embryo of Drosophila melanogaster

The I(1)fdg mutation demonstrates two separate phases of lethality, depending on developmental conditions. At 32–33°C, an embryonic lethality is expressed whereas at lower temperatures a larval-pupal lethality is observed. This larval-pupal lethality characteristically produces noncondensed, curved puparia, and since the contraction of the pupa depends on strong muscular contraction, this phase of lethality implicates some involvement of abnormal musculature. The embryonic expression of I(1)fdg at 32–33°C is the subject of this study. In these embryos, which are alive but immobile (incapable of hatching), the fibrillar organization and fiber morphology of the somatic musculature varies from being apparently normal to being grossly abnormal. While the abnormalities appear as unusual distributions of fiber organelles, abnormal convolutions of the muscle fibers, and disorganizations of fibrillar components, it seems most probable that the underlying defect ultimately responsible resides in some system essential for Z body alignment and sarcomere formation. Accompanying the embryonic lethality, certain abnormalities in midgut development are observed which at present do not appear to be related to the defects observed in the somatic muscle.     

Effects of ascorbate on myogenesis in micromass culture

Summary Micromass cultures from stage 23 and 24 chick wing mesenchyme were grown in serum-containing medium with or without additional ascorbic acid. It was found that ascorbic acid administered as a single pulse or present continuously throughout culture, in concentrations as low as 25 μg/ml, was sufficient to abolish 80% of myogenesis as assessed by immunolocalization using muscle-specific antibodies. This effect was not significantly altered when cultures were maintained in a serum-free medium that promotes myogenesis. In contrast to the above findings, spectrophotometric analysis of accumulated sulphated glycosaminoglycans, an indicator of chondrogenesis, was elevated by ascorbate treatment. Furthermore, a similar level of glycosaminoglycan stimulation was found in ascorbate treated stage 23 distal-tip limb cultures that were essentially free of myogenic cells. We conclude, therefore, that the presence of myoblasts in whole-limb cultures has no appreciable inhibitory effects on chondrogenesis. This work was supported by the Nuffield Foundation, England.     

Myogenic differentiation of mesenchymal stem cells co‐cultured with primary myoblasts

TE (tissue engineering) of skeletal muscle is a promising method to reconstruct loss of muscle tissue. This study evaluates MSCs (mesenchymal stem cells) as new cell source for this application. As a new approach to differentiate the MSCs towards the myogenic lineage, co‐cultivation with primary myoblasts has been developed and the myogenic potential of GFP (green fluorescent protein)‐transduced rat MSC co‐cultured with primary rat myoblasts was assessed by ICC (immunocytochemistry). Myogenic potential of MSC was analysed by ICC, FACS and qPCR (quantitative PCR). MSC—myoblast fusion phenomena leading to hybrid myotubes were evaluated using a novel method to evaluate myotube fusion ratios based on phase contrast and fluorescence microscopy. Furthermore, MSC constitutively expressed the myogenic markers MEF2 (myogenic enhancer factor 2) and α‐sarcomeric actin, and MEF2 expression was up‐regulated upon co‐cultivation with primary myoblasts and the addition of myogenic medium supplements. Significantly higher numbers of MSC nuclei were involved in myotube formations when bFGF (basic fibroblast growth factor) and dexamethasone were added to co‐cultures. In summary, we have determined optimal co‐culture conditions for MSC myogenic differentiation up to myotube formations as a promising step towards applicability of MSC as a cell source for skeletal muscle TE as well as other muscle cell‐based therapies.