Replication of animal viruses in differentiating muscle cells: vaccinia and herpes simplex virus type 1.

Cells cultured from the breast muscles of 11 to 12-day-old chick embryos were infected in the undifferentiated mitotic myoblast stage or in the terminally differentiated non-mitotic myotube stage with one of two DNA viruses, vaccinia and herpes simplex virus type 1 (HSV-1). DNA synthesis was measured and production ov virus-specific DNA detected in cells infected as myoblasts or myotubes by isotope labelling, autoradiographic and buoyant density centrifugation techniques. Furthermore, fully fused myotubes resemble myoblasts in their ability to support productive infection by these DNA viruses although DNA replication and nuclear division have ceased in myotubes and only minimum levels of host-cell DNA polymerase activity are present.     

人类胚胎来源成肌细胞的培养和鉴定

背景：人类胚胎骨骼肌含有成肌细胞,其在体外条件下的培养及在体外能否融合形成肌管,以及表达的相应的标志物,目前尚不明确。 目的：验证源于人类胚胎骨骼肌的成肌细胞在体外条件下的培养条件,能否在体外融合形成肌管,能否表达神经细胞的标志物。 方法：采用组织块培养法对人类胚胎肌肉来源的成肌细胞进行原代培养,以免疫细胞化学染色检测培养的细胞肌肉细胞标志物desmin、myogenin、平滑肌肌动蛋白、myosin和神经细胞标志物β-tubulin Ⅲ、nestin、neurofilament 200 (NF200)、胶质纤维酸性蛋白的表达。 结果与结论：从人类胚胎肌肉组织中成功培养出成肌细胞,表达成肌细胞的标志物desmin和myogenin,同时也表达神经元特异性烯醇化酶、nestin和NF200,细胞能够在体外融合形成含有多个细胞核的肌管,融合的肌管可以表达NF200、β-tubulin Ⅲ和胶质纤维酸性蛋白等神经细胞的标志物。结果证实,人类胚胎肌肉来源的成肌细胞能够同时表达神经细胞和肌肉细胞的标志物,培养的成肌细胞和肌管细胞表达神经元特异性烯醇化酶、β-tubulin Ⅲ、nestin、NF 200和胶质纤维酸性蛋白。说明这几种神经细胞标志物不能用于肌肉来源的细胞向神经细胞跨分化的鉴定研究。     

Control of myotube contraction using electrical pulse stimulation for bio-actuator

The contractility of tissue-engineered muscle on the application of electrical signals is required for the development of bio-actuators and for muscle tissue regeneration. Investigations have already reported on the contraction of myotubes differentiated from myoblasts and the construction of tissue-engineered skeletal muscle using electrical pulses. However, the relationship between myotube contraction and electrical pulses has not been quantitatively evaluated. We quantitatively investigated the effect of electrical pulse frequency on the excitability of myotubes and developed bio-actuators made of tissue-engineered skeletal muscle. C2C12 cells were seeded on a collagen-coated dish and in collagen gel and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and antibiotics. When the cells reached confluence or after 2 days in culture, the medium was shifted to DMEM containing 7% horse serum to allow them to differentiate to C2C12 myotubes. We electrically stimulated the myotubes and tissue-engineered skeletal muscle, and contractions were observed under a microscope. The myotubes contracted synchronously with electrical pulses between 0.5 and 5 Hz and unfused tetanus was generated at 10 Hz. The contractile performance of tissue-engineered skeletal muscle made of collagen gel and C2C12 was similar to that of the myotubes. Both the rheobase and chronaxie of the myotubes were lowest when the electric field was applied parallel to the myotube axis, and the values were 8.33 ± 2.78 mA and 1.19 ± 0.38 ms, respectively. The motion of C2C12 myotube contraction depended on the pulse frequency and showed anisotropy in the electric field. These results suggest that a tissue-engineered bio-actuator may be controlled using electrical signals.     

A delay in the appearance of myosin in dystrophic chicken embryos

Growth patterns during myogenesis were observed in vitro using normal and dystrophic New Hampshire Red chick embryos. The pectoral muscles from 6 d old embryos were removed, dissociated and grown in culture. Smaller muscle straps, both in size and number, as well as pseudostraps were seen in cultures from the myopathic embryos. There was a 1 1/2 d delay in the appearance of thick myosin-like myofilaments as well as large polyribosomes actively synthesizing myosin in the dystrophic muscle cells. Myosin may be a necessary constituent during myogenesis for the occurrence of normal fusion and thus myotubes.     

The relationship of nerve to myoblasts and newly-formed secondary myotubes in the fourth lumbrical muscle of the rat foetus

Summary The formation of normal numbers of skeletal muscle fibres depends on functional innervation of the muscle before and during the period of secondary myotube formation, but little has been known about the physical relationship between nerve terminals and the myoblasts and secondary myotubes over the critical period. This paper reports the results of a serial-section electron microscopic study of the IVth lumbrical muscle of the rat hindlimb, studied on embryonic day 20 (E20), a time when all secondary myotubes are less than 24 h old, and new ones are rapidly forming. Most myoblasts lying within the endplate region of the muscle received some direct neural contact; in almost all cases, the contact originated from an extension of a differentiated nerve terminal present at the endplate of an adjacent primary myotube. At six of 15 neural contact sites on myoblasts, primitive synaptic specialization was present. The newly-formed secondary myotubes were also directly, although sparsely, innervated in nine of ten instances. One secondary myotube was never seen to be innervated, despite extensive serial tracing. Nerve terminals passing to secondary myotubes were also principally derived from the innervation zone of the earlier-formed primary myotubes. Primary myotubes were profusely innervated by multiple axons. The results suggest that most nerve terminals are initially accommodated on the primary generation of myotubes, but progressively transfer to pre-fusion myoblasts or to secondary myotubes as these appear. In general, very young secondary myotubes are innervated by only a single axon, rather than being polyneuronally innervated. The existence of some secondary myotubes which lack any direct innervation suggests that intimate nerve contact may not be obligatory for formation of new secondary myotubes.     

The effect of methylprednisolone on intracellular calcium of normal and dystrophic human skeletal muscle cells.

Clinical trials have shown that a glucocorticoid, the methyiprednisolone (PDN), has a beneficial effect on muscle strength and function in Duchenne muscular dystrophy (DMD) patients. The aim of this study was to test if the effect of PDN could be mediated via a possible action on intracellular calcium. The intracellular calcium activity, at rest and during calcium mobilizing drug superfusion protocols was recorded in normal and dystrophic human cocultured muscle cells. PDN (10 microM) pretreatment induced an elevation of the resting calcium concentration of 51, 34 and 38% in proliferating normal myoblasts, DMD myoblasts and DMD myotubes, respectively, while normal myotubes resting [Ca2+]i was not altered.     

Generation of a monoclonal antibody reactive to prefusion myocytes

We established a novel monoclonal antibody, Yaksa that is specific to a subpopulation of myogenic cells. The Yaksa antigen is not expressed on the surface of growing myoblasts but only on a subpopulation of myogenin-positive myocytes. When Yaksa antigen-positive mononucleated cells were freshly prepared from a murine myogenic cell by a cell sorter, they fused with each other and formed multinucleated myotubes shortly after replating while Yaksa antigen-negative cells scarcely generated myotubes. These results suggest that Yaksa could segregate fusion-competent, mononucleated cells from fusion–incompetent cells during muscle differentiation. The Yaksa antigen was also expressed in developing muscle and regenerating muscle in vivo and it was localized at sites of cell–cell contact between mono-nucleated muscle cells and between mono-nucleated muscle cells and myotubes. Thus, Yaksa that marks prefusion myocytes before myotube formation can be a useful tool to elucidate the cellular and molecular mechanisms of myogenic cell fusion.     

Human mesenchymal stem cells ectopically expressing full-length dystrophin can complement Duchenne muscular dystrophy myotubes by cell fusion

Duchenne muscular dystrophy (DMD) is the most prevalent inheritable muscle disease. It is caused by mutations in the approximately 2.5-megabase dystrophin (Dys) encoding gene. Therapeutic attempts at DMD have relied on injection of allogeneic Dys-positive myoblasts. The immune rejection of these cells and their limited availability have prompted the search for alternative therapies and sources of myogenic cells. Stem cell-based gene therapy aims to restore tissue function by the transplantation of gene-corrected autologous cells. It depends on (i) the capacity of stem cells to participate in tissue regeneration and (ii) the efficient genetic correction of defective autologous stem cells. We explored the potential of bone marrow-derived human mesenchymal stem cells (hMSCs) genetically modified with the full-length Dys-coding sequence to engage in myogenesis. By tagging hMSCs with enhanced green fluorescent protein (EGFP) or the membrane dye PKH26, we demonstrated that they could participate in myotube formation when cultured together with differentiating human myoblasts. Experiments performed with EGFP-marked hMSCs and DsRed-labeled DMD myoblasts revealed that the EGFP-positive DMD myotubes were also DsRed-positive indicating that hMSCs participate in human myogenesis through cellular fusion. Finally, we showed that hMSCs transduced with a tropism-modified high-capacity hybrid viral vector encoding full-length Dys could complement the genetic defect of DMD myotubes.     

Morphometric analysis of the developing, murine aneural soleus muscle.

The pattern of organogenesis of the aneural soleus muscle of the 129ReJ mouse [rendered aneural by laser ablation of the lumbosacral spinal cord at 14 days in utero (during the period of primary myotube formation, but prior to the formation of secondary myotubes)] was evaluated quantitatively with spaced, serial ultrathin sections and computer-assisted morphometric analysis. Aneural muscles from 16- and 18-day gestation and newborn mice were analyzed to determine age-related changes in a number of parameters including: muscles' maximal girths, numbers of myotubes, myotube diameter distributions, and cluster frequency. Data were compared with a similar study of the organogenesis of the normal soleus muscle (Ontell et al: Am J Anat 181:279-288, 1988). Basic patterns of morphogenesis of the soleus muscle were unchanged by spinal cord ablation, and differences in development between the aneural and innervated muscles were quantitative rather than qualitative. At birth, the aneural muscle contained approximately 76% of the myotubes found in the innervated muscle (approximately 840 myotubes in the innervated muscle and approximately 640 in the aneural muscle). Evidence is presented consistent with the hypothesis that primary myotube formation is reduced by approximately 32% in the aneural muscles and that while extensive secondary myotube formation occurs (approximately 78% of the myotube present at birth in these muscles are secondary myotubes), there is a significant reduction in the number of secondary myotubes in aneural muscles. It is suggested that the reduced numbers of secondary myotubes may be related to the reduction in the number of primary myotubes, which are known to act as scaffolds for secondary myotube formation. The time course of secondary myotube formation and of cluster formation and cluster dispersal and the number of cells per cluster are similar in age-matched, innervated and aneural muscles. The absence of innervation has little effect on myotube growth until birth, when comparison of the myotube diameter distributions reveals a slight alteration in myotube diameter distributions of aneural as compared with innervated muscles.     

Culture of skeletal muscle cells in unprecedented proximity to a gold surface

Culturing of skeletal muscle cells on conductive surfaces is required to develop electronic device-muscle junctions for tissue engineering and medical applications. We characterized from a molecular and morphological point of view myogenic cells cultured on gold and on cysteamine-coated gold, as compared to the standard plastic for cell culture. Our results show that cell proliferation and survival are comparable between cells grown on either of the gold surface or plastic. The majority of the cells cultured on gold surfaces retain the ability to respond to differentiation cues, as shown by nuclear translocation of myogenin. Following terminal differentiation, the myotubes cultured on cysteamine-coated gold resemble myotube cultures obtained on plastic for the size and orientation of the myotube bundles retaining most of myosin expression; on the contrary, the myotube cultures on gold show a clumped morphology, likely due to repulsive cell-substratum interaction resulting in aberrant differentiation. On the basis of the aforementioned evidences, the culture of muscle cells on cysteamine-coated gold represents an advance with respect to previously reported substrata. The cysteamine self-assembled monolayer coating is a simple approach to accomplish cultures of myotubes in unprecedented tight proximity to conductive surfaces.     

Differentiation of myoblasts in serum-free media: effects of modified media are cell line-specific

Myoblast cell lines are grown and differentiated readily in cell culture. Two cell lines typically used for investigating the growth and differentiation of muscle are the mouse cell line C2C12 and the rat cell line L6. The differentiation of these cells in vitro requires a switch from a serum-rich medium to a less rich medium after the cells have reached confluence. Since the components present in serum are not well characterized, the use of a better defined medium for these studies was investigated. C2C12 and L6 myoblasts were differentiated in both serum-containing and serum-free media. The differentiation state of these cultures was then tested both microscopically and biochemically. Cultures were checked for myotube formation, the activity of creatine phosphokinase and the presence of sarcomeric actin. In C2C12 cells, the extent of differentiation was greater in the serum-free than in the serum-containing system. In both media types, the C2C12 cells produced sarcomeric actin, showing the presence of sarcomere structure in the myotubes. In L6 cells, however, myotubes were readily formed in medium containing 2% horse serum, but not in the serum-free system. In addition, the ability of C2C12 cells to differentiate on substrates coated with extracellular matrix proteins was shown to be media-dependent. The presence of extracellular matrix proteins did not enable L6 cells to form myotubes when cultured in serum-free media. Primary cultures of chick myoblasts were able to differentiate in both media tested, with Dulbecco's modified Eagle medium containing horse serum being a more efficient medium for cell fusion. This study shows a divergence in muscle cell line responses in three cell lines, two of which are typically used as 'model systems' for understanding muscle growth and development.     

Lactate transport during differentiation of skeletal muscle cells: evidence for a specific carrier in L6 myotubes

El abida , K., Duvallet , A., Thieulart , L., Rieu , M. & Beaudry , M. 1992. Lactate transport during differentiation of skeletal muscle cells: evidence for a specific carrier in L6 myotubes. Acta Physiol Scand 144 , 469471. Received 12 June 1 991 , accepted 8 October 1991. ISSN 0001–6772. Laboratoire de physiologie des adaptations, CIIU Cochin, 24 rue du Faubourg Saint Jacques, 75104 Paris, France. Cellular uptake of lactate involves a carrier in numerous types of mammalian cells. In cultured rat L6 myoblasts, lactate diffuses freely. We show that during myogenesis a carrier appears at the myotube stage. The degree of cellular differentiation was determined by microscopic observation and by measurement of a marker of biochemical differentiation: the increase in specific activity of creatine kinases.     

Influence of anabolic agents on protein synthesis and degradation in muscle cells grown in culture

Muscle cell culture (L6) studies were conducted to determine whether anabolic agents have a direct effect on the muscle cell. The effects of zeranol, testosterone propionate, estradiol benzoate, progesterone, dexamethasone and anabolic agent-dexamethasone combinations on protein synthesis and degradation were measured. Myoblast and myotube cultures were pretreated with 1 microM compounds for 12, 24 and 48 h before a 6-h synthesis or degradation measuring period. Protein synthesis was determined as cpm of [3H] leucine incorporated per mg cell protein. Protein degradation was measured by a pulse-chase procedure using [3H] leucine and expressed as the percentage labeled protein degraded in 6 h. Progesterone slightly increased (P less than .05) protein synthesis in myoblast cultures. Testosterone propionate had no effect on synthesis. Protein synthesis was decreased by estradiol benzoate (P less than .01) in myotube cultures. Protein degradation was not altered appreciably by anabolic agents. Protein synthesis was initially inhibited in myotubes (P less than .05) by dexamethasone, but increased (P less than .01) in myoblasts and myotubes in the extended incubation time. Dexamethasone also consistently increased protein degradation, but this required several hours to be expressed. Anabolic agents did not interfere with dexamethasone-induced increases in protein synthesis and degradation. The magnitude of response and sensitivity were similar for both the myoblast and the more fully differentiated myotube for all compounds tested. These results indicate that anabolic agents at the 1 microM level do not have a direct anabolic effect on muscle or alter glucocorticoid-induced catabolic response in muscle.     

VAMP2 Marks Quiescent Satellite Cells and Myotubes,but not Activated Myoblasts

We examined the expression and intracellular localization of vesicle-associated membrane protein 2 (VAMP2) during the differentiation of skeletal muscle cells by immunofluorescence microscopy. In isolated single myofibers, VAMP2 was expressed in quiescent satellite cells, downregulated in proliferating myoblastic cells, and re-expressed with differentiation. In the myoblastic cell line C2C12, VAMP2 was expressed at a low level in the proliferating stage, and then increased after differentiation into myotubes. Based on these results, we propose that VAMP2 can be used as a molecular marker for both quiescent satellite cells and myotubes, but not for proliferating myoblasts. We also found the partial colocalization of VAMP2 with transferrin- or Rab11-labeled vesicles in myotubes, suggesting a role of VAMP2 in the trafficking of recycling endosomes.     

An ultrastructural study of the dufferentiation of skeletal muscle in the bovine fetus

Summary The differentiation of skeletal muscle was studied by electron microscopy in bovine fetuses from 47 days gestation to neonatal calves 3 days of age.Initally, the muscle was composed of clusters of myotubes with mononucleated myoblasts between them. In 2-month-old fetuses these myoblasts became apposed to the differentiating muscle cells and were enclosed within the rudimentary basal lamina of the myotubes. At this stage the clusters of myotubes consisted of central, larger diameter, more differentiated myotubes and also the mononucleated satellite cells. The differentiated myotubes separated from the clusters accompanied by satellite cells which continued proliferating and fused together to form new generations of satellite myotubes. In this manner new clusters of myotubes were formed. By 4–5 months some of the separating myotubes began to form individual myotubes and independent myofibers were prominent in fetuses of 5–8 months of age.The myofibers in the 8-month-old fetuses showed diversification into fiber types by differences in the thickness of the Z-line, the prominence of the sarcotubular system, the amount of glycogen and lipid droplets and also the number of mitochondria.     

Characteristics of the Localization of Connexin 43 in Satellite Cells during Skeletal Muscle Regeneration In Vivo

For myogenesis, new myotubes are formed by the fusion of differentiated myoblasts. In the sequence of events for myotube formation, intercellular communication through gap junctions composed of connexin 43 (Cx43) plays critical roles in regulating the alignment and fusion of myoblasts in advances of myotube formation in vitro. On the other hand, the relationship between the expression patterns of Cx43 and the process of myotube formation in satellite cells during muscle regeneration in vivo remains poorly understood. The present study investigated the relationship between Cx43 and satellite cells in muscle regeneration in vivo. The expression of Cx43 was detected in skeletal muscles on day 1 post-muscle injury, but not in control muscles. Interestingly, the expression of Cx43 was not localized on the inside of the basement membrane of myofibers in the regenerating muscles. Moreover, although the clusters of differentiated satellite cells, which represent a more advanced stage of myotube formation, were observed on the inside of the basement membrane of myofibers in regenerating muscles, the expression of Cx43 was not localized in the clusters of these satellite cells. Therefore, in the present study, it was suggested that Cx43 may not directly contribute to muscle regeneration via satellite cells.     

Protein turnover and growth of L8 muscle cultures in serum from rats fed clenbuterol

Beta-adrenergic agonists stimulate muscle growth in chronically treated animals. The response may be primary (receptor binding directly stimulates anabolism) or secondary (circulating levels of hormones or other factors regulate muscle mass). Although B-receptors are functional in L8 muscle cells in culture, a direct effect of B-agonists on protein turnover in culture has not been demonstrated. To determine whether the stimulation of muscle growth is a secondary effect, we assessed the anabolic activity of sera from both clenbuterol-treated rats and normal rats in L8 cultured muscle cells. Rats were fed either normal diet or diet containing ten parts per million clenbuterol for seven days. Blood was collected from the inferior vena cava of rats under metofane anaesthesia and serum prepared. The anabolic activities of serum were measured in cultures of either myoblasts (still dividing) or fused myotubes (non-dividing) to distinguish mitogenic action from alteration in rates of protein turnover. Sera from both normal and clenbuterol-treated rats gave the same stimulation of protein synthesis in myotube cultures. Sera from both groups of rats contained factors which inhibited protein degradation, stimulated amino isobutyric acid (AIB) and thymidine uptake (myoblasts only). These processes were not augmented by sera from rats treated with clenbuterol. Protein accumulation was equivalent after 48 hours of exposure to either test sera. In conclusion, adult rat serum contained anabolic factors but there was no evidence for an increase in activity in serum from clenbuterol-treated rats.