Expression of fast and slow isoforms of the Ca2+-ATPase in developing chick skeletal muscle

The expression of fast and slow isoforms of the sarcoplasmic reticulum Ca2+-ATPase was studied in the developing chick embryo and in tissue-cultured myotubes. Monoclonal antibodies specific for each isoform were used as probes of protein expression. Analysis of expression of Ca2+-ATPase isoforms in chick thigh muscles by immunofluorescence microscopy revealed that all muscle fibers expressed both isoforms during their development. Primary generation muscle fibers expressed predominantly the slow isoform. Secondary generation fibers expressed both isoforms at comparable levels. Loss of the "inappropriate" isoforms occurred late in embryonic development. Immunoblot analysis of embryonic thigh muscle proteins indicated that the expression of the slow isoform varied little from embryonic Day 6 (ED6) to ED19, while expression of the fast isoform increased dramatically just prior to ED19. Tissue-cultured myotubes derived from ED12 chick thigh muscle myoblasts, plated at high density, expressed both isoforms of the Ca2+-ATPase at very similar levels. Clonal analysis of myoblasts taken from early (ED6) and late (ED12) chick thigh muscles showed that all muscle colonies expressed both forms, consistent with in vivo results. Fiber-type specific isoforms of the Ca2+-ATPase and myosin heavy chain are not coordinately expressed in developing chick skeletal muscle.     

Developmentally regulated lectin in embryonic chick muscle and a myogenic cell line.

Soluble extracts of embryonic chick pectoral muscle and myoblast clone L6 agglutinated trypsin treated glutaraldehyde fixed rabbit erythrocytes. Agglutination activity was blocked by thiodigalactoside, lactose and related saccharides but not by many other saccharides. Agglutination activity of chick pectoral muscle extracts increased at least one order of magnitude between 8 and 16 days of chick embryo development, as the pectoral muscle differentiated. With L6 myoblasts there was a three-fold increase in activity of the extracts as the myoblasts fused to form multinucleated myotubes.     

Role of muscle fibroblasts in the deposition of type-IV collagen in the basal lamina of myotubes

In cell cultures of quail, chick, or mouse skeletal muscle, both myogenic and fibrogenic cells synthesize and secrete type-IV collagen, a major structural component of the basal lamina. Type-IV collagen, together with laminin, forms characteristic patches and strands on the surface of developing myotubes, marking the onset of basement-membrane formation. The pattern for type-IV collagen and laminin is unique to these proteins and is not paralleled by other matrix proteins, such as fibronectin or type-I or -III collagen. In the present study, we used species-specific antibodies to either mouse or chick type-IV collagen to demonstrate the ability of fibroblast--derived type-IV collagen to incorporate in the basal lamina of myotubes. In combination cultures of embryonic quail skeletal myoblasts and mouse muscle fibroblasts, antibodies specific for mouse type-IV collagen revealed the deposition of type-IV collagen on the surface of quail myotubes in the pattern typical of the beginning of basement-membrane formation. Control cultures consisting of only quail muscle cells containing myoblasts and fibroblasts demonstrated no such reaction with these antibodies. Deposits of mouse type-IV collagen were also observed on the surface of quail myotubes when conditioned medium from mouse muscle fibroblasts was added to quail myoblast cultures. Similarly, in combination cultures of mouse myoblasts and chick muscle fibroblasts, chick type-IV-collagen deposits were identified on the surface of mouse myotubes. These results indicate that type-IV collagen synthesized by muscle fibroblasts may be incorporated into the basal lamina forming on the plasmalemma of myotubes, and may explain ultrastructural studies by Lipton on the contribution of fibroblasts to the formation of basement membranes in skeletal muscle.     

The insulin signal initiating cellular differentiation is preserved by chick embryo myoblasts incubated at 2 degrees C

Insulin pulse treatment, lasting for 1 to 3 min, stimulates differentiation of chick embryo myoblasts to myotubes and myofibers in a serum-free medium. The use of serum-free medium supplemented with 2,2'-thiodiethanol permits terminal differentiation of chick embryo myoblasts to cross-striated, spontaneously contracting myofibers. The experiments carried out showed that incubation of chick embryo myoblasts after the insulin pulse treatment for 10 days at 2 degrees C does not inhibit the progress of their differentiation. The data demonstrate that differentiation can be interrupted at any time by transferring cells to 2 degrees C and resumed without delay after returning to 37 degrees C.     

Centrioles are lost as embryonic myoblasts fuse into myotubes in vitro

Embryonic chick myoblasts possess an extensive network of cytoplasmic microtubules which emanate from a single, perinuclear centrosome containing a microtubule-organizing center (MTOC) and the centrioles. However, after myoblasts fuse into myotubes the centrosome is no longer apparent, and instead long parallel arrays of microtubules are seen. From ultrastructural studies on developing muscle tissue, it has been proposed that centrioles are present in myoblasts but are absent from fused muscle fibers. We have examined this hypothesis in vitro in cultures of chick embryonic muscle cells using sera which specifically label centrioles. Almost all (90-97%) mononucleated cells in these cultures, including myoblasts aligned just prior to fusion, contain a pair of centrioles in close proximity to the nucleus. However, in newly fused multinucleated myotubes as well as in older myotubes that had developed myofibrils, centrioles were rarely found (1-10% positive cells). This study thus provides direct evidence for a loss of centrioles from muscle cells soon after they fuse to form myotubes.     

On the non-equivalence of skeletal muscle satellite cells and embryonic myoblasts

Postnatal satellite cells, isolated from normal or previously denervated skeletal muscles of juvenile quails, were tested as to their capacity to participate in embryonic muscle ontogeny. They were grafted into 2-day chick embryo hosts, in place of a piece of brachial somitic mesoderm. Satellite cell implants were prepared from pellets either of freshly isolated cells or of cells precultured in vitro under proliferative conditions. Myogenic capacity of the implanted cells was attested by their ability to fuse into myotubes when cultured under differentiation conditions. In no case did the implanted satellite cells invade the adjacent wing bud or participate in wing muscle morphogenesis. They did not either give rise to myotubes at the site of implantation, nor did they even survive longer than 3 days in the embryonic environment. These negative results indicate that postnatal satellite cells, unlike embryonic myoblasts, are unable to take part in muscle embryogenesis. Although they derive from the same somitic myogenic cell line as the embryonic myoblasts, they therefore represent a differentiated non-totipotent type of myogenic cell.     

Inhibition of the formation of myotubes in vitro by inhibitors of transglutaminase

The effects of competitive inhibitors of transglutaminase on the formation of myotubes by the fusion of myoblasts in vitro has been investigated. Myotube formation was inhibited when myoblasts from 11-day-old chick embryos were cultured in vitro in the presence of 10 mM histamine or 0.2 mM dansyl cadaverine. The inhibitions observed were reversed when the treated cells were subsequently cultured in normal medium. Glycine methyl ester also inhibited myotube formation but sarcosine methyl ester, which is not a competitive inhibitor of transglutaminase, had little if any inhibitory action. The formation of myotubes was not inhibited by cultivation in normal medium adjusted to pH 8.0-8.1, indicating that the observed effects of histamine and of dansyl cadaverine were not mediated by a lysosomotropic effect. Inhibition of myotube formation in the presence of histamine was accompanied by the production of abnormal multinucleated cells, indicating that myoblast fusion occurred in the treated cultures but that the fused cells failed to elongate into normal myotubes. Transglutaminase activity has been found in cell-free lysates of embryonic chick myoblasts and it is concluded that a transglutaminase enzyme, activated by an increase in the concentration of intracellular Ca2+, plays an important role in stabilising the cytoskeletal network of developing myotubes.     

Stimulation of myogenesis by 2,2'-thiodiethanol in suboptimal tissue culture conditions

It is shown that 2,2'-thiodiethanol, a product of yperite hydrolysis, strongly stimulates differentiation of chick embryo myogenic cells. In its presence myoblasts fused, yielding myotubes with the same efficiency in standard media for chick embryo fibroblast-like cell culture (containing 4% bovine serum and 1% chick serum) as in media specially designed to promote myoblast fusion (containing 10% horse serum and 5% chick serum). What is more, the myofibres formed in the presence of 0.1% 2,2'-thiodiethanol morphologically resembled more closely myofibres formed in vivo than those formed in the presence of horse serum.     

Immunological studies of the embryonic muscle cell surface. Antiserum to the prefusion myoblast

Xenogeneic antisera raised in rabbits have been used to detect compositional changes at the cell surfaces of differentiating embryonic chick skeletal muscle. In this report, we present the serological characterization of antiserum (Anti-M-24) against muscle tissue and developmental stage-specific cell surface antigens of the prefusion myoblast. Cells from primary cultures of 12-d-old embryonic chick hindlimb muscle were injected into rabbits, and the resulting antisera were selectively absorbed to obtain immunological specificity. Cytotoxicity and immunohistochemical assays were used to test this antiserum. Absorption with embryonic or adult chick heart, brain, retina, liver, erythrocytes, or skeletal muscle fibroblasts failed to remove all reactivity of Anti-M-24 for myogenic cells at all stages of development. After absorption with embryonic myotubes, however, Anti-M-24 no longer reacted with differentiated myofibers, but did react with prefusion myoblasts. The myoblast surface antigens detected with Anti-M-24 are components of the muscle cell membrane: (a) these macromolecules are free to diffuse laterally within the myoblast membrane; (b) Anti-M-24, in the presence of complement, induced lysis of the muscle cell membrane; and (c) intact monolayers of viable myoblasts completely absorbed reactivity of Anti-M-24 for myoblasts. These antigens are not loosely adsorbed culture medium components or an artifact of tissue culture because: (a) absorption of Anti-M-24 with homogenized embryonic muscle removed all antibodies to cultured myoblasts; (b) Anti-M-24 reacted with myoblast surfaces in vivo; and (c) absorption of Anti-M-24 with culture media did not affect the titer of this antiserum for myoblasts. We conclude that myogenic cells at all stages of development possess externally exposed antigens which are undetected on other embryonic and adult chick tissues. In addition, myoblasts exhibit surface antigenic determinants that are either masked, absent, or present in very low concentrations on skeletal muscle fibroblasts, embryonic myotubes, or adult myofibers. These antigens are free to diffuse laterally within the myoblast membrane and may be modulated in response to appropriate environmental cues during myodifferentiation.     

A transient increase in amino acid transport modulated by insulin in differentiating muscle cells.

During synchronous differentiation of embryonic chick muscle cells in cultures, the Na-dependent uptake of an amino acid analog, alpha-amino isobutyric acid (AIB) undergoes in abrupt, transient increase. The increase in AIB uptake is concomitant with the rapid fusion of mononucleated myoblasts, and precedes the accumulation of muscle-specific proteins. Subsequently, Na-dependent AIB transport diminishes markedly during postfusional differentiation of myotubes. The rate of AIB uptake is increased by insulin both before and after myoblast fusion. This stimulation by insulin is restricted to the Na-dependent component of total AIB uptake but is apparently not the result of insulin-mediated increase in the trans-membrane Na gradient.     

Electrophysiological properties of the membrane and acetylcholine receptor in developing rat and chick myotubes

Membrane properties of rat and chick myotubes in various stages of development were studied. Resting membrane potentials (Em) increased from -8 to -55 mV in both rat and chick as the myotubes developed from myoblasts to large multinucleated fibers. In the rat myotubes, this increase was not accompanied by significant changes in specific membrane resistivity or changes in Na+ and K+ ion distribution. Nor have we observed a significant electrogenic component to the resting Em of mature rat myotubes under normal circumstances. A progressive increase in the passive permeability of the membrane to K+ relative to Na+ ions has been observed which can account for the changes in Em with development. In contrast to the changes in the ionic selectivity of the membrane, we have found that the ionic selectivity of the ACh receptor of rat and chick myotubes remains constant during the same period of myotube development.     

Slow and fast myosin heavy chain content defines three types of myotubes in early muscle cell cultures

We prepared monoclonal antibodies specific for fast or slow classes of myosin heavy chain isoforms in the chicken and used them to probe myosin expression in cultures of myotubes derived from embryonic chicken myoblasts. Myosin heavy chain expression was assayed by gel electrophoresis and immunoblotting of extracted myosin and by immunostaining of cultures of myotubes. Myotubes that formed from embryonic day 5-6 pectoral myoblasts synthesized both a fast and a slow class of myosin heavy chain, which were electrophoretically and immunologically distinct, but only the fast class of myosin heavy chain was synthesized by myotubes that formed in cultures of embryonic day 8 or older myoblasts. Furthermore, three types of myotubes formed in cultures of embryonic day 5-6 myoblasts: one that contained only a fast myosin heavy chain, a second that contained only a slow myosin heavy chain, and a third that contained both a fast and a slow heavy chain. Myotubes that formed in cultures of embryonic day 8 or older myoblasts, however, were of a single type that synthesized only a fast class of myosin heavy chain. Regardless of whether myoblasts from embryonic day 6 pectoral muscle were cultured alone or mixed with an equal number of myoblasts from embryonic day 12 muscle, the number of myotubes that formed and contained a slow class of myosin was the same. These results demonstrate that the slow class of myosin heavy chain can be synthesized by myotubes formed in cell culture, and that three types of myotubes form in culture from pectoral muscle myoblasts that are isolated early in development, but only one type of myotube forms from older myoblasts; and they suggest that muscle fiber formation probably depends upon different populations of myoblasts that co-exist and remain distinct during myogenesis.     

Prostaglandin binding does not require direct cell-cell contact during chick myogenesis in vitro

Myogenic differentiation in vitro involves at least three events at the cell surface: binding of prostaglandin to the cells, contact-mediated cell-cell recognition, and fusion of the myoblast membranes into myotubes. While the earlier events are thought to be necessary for subsequent fusion, the sequence of events has not been determined. A major impediment to determining the initial event has been the lack of synchrony of cell differentiation in vitro. To overcome this, we cultured chick embryo myoblasts as a suspension of single cells in gyratory rotation in medium without added Ca2+. Under these conditions, myoblasts exhibited characteristic prostaglandin binding at 34 h. Within 30 min, the cells began to aggregate. Because this occurred without change of medium or conditions of rotation, we termed the process autoaggregation. Within 8-10 h. cells within these autoaggregates began to fuse into syncytia. These results suggest that an early cell surface event in embryonic myogenesis is the characteristic binding of prostaglandin to the myoblasts. The results demonstrate that this binding precedes any direct cell-cell contact and suggest that it causes the subsequent change in myoblast cell-cell adhesion.     

Intracellular antigen migration in interspecific myoblast heterokaryons

Intracellular migration of species-specific nuclear antigens was studied in chick-rat heterokaryons. These cells were produced by virus-induced or spontaneous fusion of different chick cells with rat myoblasts or myotubes. Chick erythrocyte nuclei introduced into rat myogenic cells increased in volume and were reactivated to synthesize RNA. As the chick erythrocyte nuclei enlarged, they rapidly accumulated rat nuclear antigens. Rat nucleolar and nucleoplasmic antigens assumed a distribution in the chick nuclei corresponding to that in rat nuclei. In hybrid myotubes formed by the spontaneous fusion of chick myoblasts and rat myoblasts antigen exchange was at a much lower level. Some exchange of both rat and chick nuclear antigens could, however, be detected also in this system. Thus chick nuclear envelope and nucleolar antigens migrated into the rat myoblast nuclei and assumed an intranuclear localization analogous to that in chick nuclei. On the basis of these results it appears that antigenic nuclear macromolecules are constantly exchanged between the rat and chick nuclear compartments and the cytoplasm of the heterokaryon. During the rapid nuclear swelling which occurs when chick erythrocyte nuclei are activated in rat myoblast heterokaryons, the inward migration of rat nuclear antigens into the chick erythrocyte nucleus is more impressive than the migration of chick antigens into the rat nuclei.     

Lack of evidence for the involvement of a β-D-galactosyl-specific lectin in the fusion of chick myoblasts

The role of a β-D-galactosyl-specific lectin, first reported by Teichberg et al., in the fusion of myoblasts in vitro was investigated. The concentration of this lectin in embryonic chick skeletal muscle was found to reach maximal levels at the time of myoblast fusion in vivo. β-D-Galactosyl-β-thiogalactopyranoside and lactose are potent inhibitors of agglutination of trypsinized rabbit erythrocytes caused by the lectin. However, at concentrations of 50 mM these compounds had no effect on either nonsynchronous fusion of myoblasts or on the release of synchronized myoblast cultures from EGTA fusion block. The presence of the agglutinin in the external membranes of chick myoblasts and myotubes could not be demonstrated. It is, therefore, concluded that the involvement of the lectin in the fusion of chick myoblasts remains questionable.     

Isolation of myosin-synthesizing polysomes from cultures of embryonic chicken myoblasts before fusion.

Mononucleated myoblasts and multinucleated myotubes were obtained by culturing embryonic chicken skeletal muscle cells. Comparison of total polysomes isolated from these mononucleated and multinucleated cell cultures by density gradient centrifugation and electron microscopy revealed that mononucleated myoblasts contain polysomes similar to those contained by multinucleated myotubes and large enough to synthesize the 200,000-dalton subunit of myosin. When placed in an in vitro protein-synthesizing assay containing [³H]leucine, total polysomes from both mononucleated and multinucleated myogenic cultures were active in synthesizing polypeptides indistinguishable from myosin heavy chains as detected by measurement of radioactivity in slices through the myosin band on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Fractionation of total polysomes on sucrose density gradients showed that myosin-synthesizing polysomes from mononucleated myoblasts may be slightly smaller than myosin-synthesizing polysomes from myotubes. Multinucleated myotubes contain approximately two times more myosin-synthesizing polysomes per unit of DNA than mononucleated myoblasts, and the proportion of total polysomes constituted by myosin polysomes is only 1.2 times higher in multinucleated myotubes than it is in mononucleated myoblasts. The results of this study suggest that mononucleated myoblasts contain significant amounts of myosin messenger RNA before the burst of myosin synthesis that accompanies muscle differentiation and that a portion of this messenger RNA is associated with ribosomes to form polysomes that will actively translate myosin heavy chains in an in vitro protein-synthesizing assay.     

Appearance of acetylcholine receptors in cultured myoblasts prior to fusion

The development of the acetycholine receptors in chick embryo myoblasts from 11-day old embryos was studied in vitro. Using the purified α-bungarotoxin labeled with radioactive iodide, a high concentration of acetylcholine receptors was found in the prefusing myoblasts; most of these receptors were located in the interior of the myoblasts. However, upon the completion of myoblast fusion, the majority of the acetylcholine receptors appeared on the external cell surface of the myotubes.     

Membrane function in differentiating skeletal muscle cells: I. Kinetic analysis of amino acid transport

Primary cultures of mononucleated myoblasts from 12-day-old chick embryos have a twofold higher rate of α-aminoisobutyric acid (AIB) transport before fusion occurs to form multinucleated myotubes. Several lines of evidence indicate that the uptake of AIB observed in both myoblasts and myotubes is primarily carrier-mediated by a membrane transport system. Increasing the temperature from 24 to 37°C results in a threefold increase in the rate of AIB uptake; both methionine and glycine inhibit AIB uptake by more than 85%; and 2,4-dinitrophenol inhibits AIB uptake by approximately 50%. In addition, the energies of activation (14.5 and 14.0 kcal/mole for myoblasts and myotubes, respectively) are characteristic of carrier-mediated transport. Resolution of AIB uptake into a saturable, carrier-mediated component and a nonsaturable, diffusion component shows that at concentrations of AIB≤1.5 mM over 97% of total AIB uptake is carriermediated in both myoblasts and myotubes. Kinetic analysis of carrier-mediated AIB uptake indicates that myoblasts and myotube membrane carriers have the same affinity for AIB (K_m values = 1.73 and 1.31 mM, respectively). However, the V_max for myoblasts is 23.7 nmole/mg/min while myotubes have a V_max of 12.6 nmole/mg/min. The twofold difference in V_max is shown to be due to a twofold difference in the quantity of membrane transport sites per milligram of protein.     

鸡胚与胎鼠的成纤维细胞条件培养液促进成肌细胞增殖和融合的比较

用培养过鸡胚(来亨鸡)或胎鼠(ICR小鼠)肌组织的成纤维细胞的条件培养液,定量地研究它们对胎鼠或鸡胚的成肌细胞的增殖和融合的影响。所得结果如下:(1) 胎鼠的成纤维细胞条件培养液促进胎鼠或鸡胚成肌细胞增殖,分别为对照组的2.65倍,(P<0.001)或2.35倍,(P<0.01);(2) 鸡胚的成纤维细胞条件培养液促进鸡胚或胎鼠的成肌细胞增殖,分别为对照组的2.66倍,(P<0.01)或2.17倍,(P<0.01);(3) 胎鼠的成纤维细胞条件培养液增加胎鼠或鸡胚的成肌细胞的融合率,分别为对照组的1.9倍或2.6倍;鸡胚的成纤维细胞条件培养液只增加鸡胚成肌细胞的融合率,为对照组的2.1倍,但对胎鼠成肌细胞的融合无明显的影响。 实验结果提示:成纤维细胞条件培养液促进成肌细胞的增殖,两种动物间无明显的差异,但在融合上却有一定的种属特异性。