Microinjection of calpastatin inhibits fusion in myoblasts

Rat satellite cells (RSC) were microinjected with purified calpastatin or m-calpain, and myoblasts from a C2C12 mouse line were microinjected with purified calpastatin. Microinjection with calpastatin completely prevented fusion of myoblasts from both sources, whereas microinjection with m-calpain significantly increased the rate of fusion of cultured RSC; 44% of the nuclei of RSC cultures were in multinucleated myotubes within 48 h after microinjection with m-calpain plus labeled dextran, whereas only 15% of the nuclei were in multinucleated myotubes after microinjection with dextran alone. Western analyses indicated that neither RSC nor C2C12 myoblasts contained detectable amounts of mu-calpain before fusion. The levels of calpastatin in C2C12 myoblasts increased as cells passed from the proliferative stage to the onset of fusion, and these levels increased substantially in both the C2C12 and the RSC cells as they progressed to the late or postfusion stage. Both RSC and C2C12 myoblasts contained an 80-kDa polypeptide that was labeled with an anti-m-calpain antibody in Western blots. The results are consistent with a role of the calpain system (m-calpain in these myoblast lines) in remodeling of the cytoskeletal/plasma membrane interactions during cell fusion.     

Effects of retinol,fatty acids and glycerol monooleate on the fusion of chick embryo myoblasts in vitro

Cell fusion of embryonic chick myoblasts has been studied in the presence of fat-soluble agents that induce erythrocytes to fuse. Retinol inhibited myoblast fusion but the cells recovered their ability to fuse within 48 h of removal of the retinol from the medium. Myristic acid, oleic acid, glycerol monooleate, linolenic acid and arachidonic acid similarly prevented fusion in myogenic cultures. By contrast, linoleic acid moderately enhanced the fusion of chick skeletal myoblasts. In addition, stearic acid, which does not fuse erythrocytes, inhibited myoblast fusion whereas the saturated, non-fusogenic fatty acid, arachidic acid, was without effect.     

Influence of Concanavalin A, wheat germ agglutinin, and soybean agglutinin on the fusion of myoblasts in vitro

Although muscle cell fusion was shown to be an energy-requiring process, release of myoblasts from an EGTA fusion block could be accomplished with Earle's balanced salt solution (containing 1.8 mM Ca++) free of glucose or any other energy-produced metabolite. The effect of concanavalin A, abrin, and the lectins from wheat germ, soybean, and Lens culinaris on myoblast fusion was examined with synchronized myoblast cultures upon release from fusion block. At a concentration of 15 mug/ml, these lectins were found to inhibit the fusion process to the extent of 62%, 41%, 32%, 8%, and 19%, respectively. Concanavalin A inhibition could be prevented by alpha-methyl-D-mannoside. The inhibitory effect of all the lectins except abrin could be reversed by changing to the normal, serum-containing medium. The number of binding sites was 3.4 X 10(7), 6.1 X 10(7), and 1.7 X 10(6), respectively. Although myoblasts were found to have about twice as many binding sites for wheat germ agglutinin as for concanavalin A, concanavalin A was determined to be twice as effective as wheat germ agglutinin as an inhibitor of myoblast fusion. These findngs raise the possibility that specific cell surface glycoproteins may be an important factor in this process.     

Oxygen enhances fusion of cultured chick embryo myoblasts

Fusion of mononucleate myoblasts to form multinucleated myotubes increases when skeletal muscle cells are grown in progressively higher oxygen concentrations (5%, 20%, and 40% oxygen). At four days of growth fusion of myoblasts (as expressed by the percent of all muscle nuclei that are located in myotubes) is 57 ± 2% in 5% oxygen, 68 ± 1% in 20% oxygen, and 78 ± 2% in 40% oxygen (P<0.001). However, at a concentration of 40%, oxygen depresses the rate of cell division and thereby affects the number of myoblasts available for fusion. Thus, oxygen concentration significantly modifies growth of skeletal muscle in vitro. Its net effect on myotube formation results from the interaction of its separate effects to enhance cell fusion and to depress cell proliferation.     

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.     

A rapid assay for fusion of embryonic chick myoblasts

A rapid and sensitive assay for measuring myoblast fusion in suspension cultures of embryonic chick pectoral myoblasts is described. Fusion-competent cells are generated by growth in suspension using a low calcium medium. Fusion-promoting levels of calcium are added, and the suspensions incubated for 1–6 h. The cells are then trypsinized to disperse cellular aggregates and sized in a Coulter particle counter. This assay minimizes many of the artifacts inherent in measurements of fusion in monolayer cultures, and is designed for the rapid screening of agents for their effects on fusion.     

Leptin promotes proliferation and inhibits differentiation in porcine skeletal myoblasts

Leptin, a major regulator of body weight, was recently suggested to play a role in myoblasts. We conducted an experiment to determine whether leptin can influence the proliferation and differentiation of porcine skeletal myoblasts. Myoblasts occurred in non-leptin and leptin forms in various concentrations for various periods of cell states. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and flow cytometry assays demonstrated that leptin significantly promoted myoblast proliferation and increased cell accumulation in the S + G2/M phase, in a dose-dependent manner. Furthermore, in morphologic experiments, the formation of myotubes and the myogenic index was markedly reduced by leptin. In addition, biochemical analysis showed that leptin decreased creatine kinase (CK) activity and the amount of myogenin and myosin heavy chain (MyHC) protein. Taking all this together, our study indicated that exogenous leptin promoted proliferation but inhibited differentiation in porcine skeletal myoblasts, suggesting that leptin might be an important mediator in the regulation of the growth and development of muscle cells.     

Studies on the effect of ketoconazole on the fusion of L6 myoblasts

Summary The effect of ketoconazole on the fusion of L6 myoblasts was studied. Ketoconazole was a potent inhibitor of myoblast fusion at concentrations as low as 0.1 M, but fusion was restored when the inhibitor was removed. The inhibitor resulted in decreased binding of conA and WGA to cell surface oligosaccharides showing that it was inhibiting N-linked cell surface glycoproteins. Inhibition of fusion by ketoconazole was accompanied by reduced creatine phosphokinase activities showing that it is affecting biochemical differentiation. Incorporation of labelled mannose from GDP-mannose into lipid-sugar and lipid-oligosaccharide complexes involved in the synthesis of N-linked oligosaccharides was also inhibited by ketoconazole, but the inhibition was reversed by addition of exogenous dolichol phosphate to the incorporation mixture. The main conclusion from these studies was that ketoconazole inhibited fusion of L6 myoblasts by affecting the synthesis of dolichol-phosphate required for the synthesis of lipid-oligosaccharides needed for the synthesis of fusogenic cell surface N-linked glycoproteins.Abbreviations HMG-CoA 3-hydroxy-3-methylglutaryl Coenzyme A - Dol-P Dolichol Phosphate - Man Mannose - GlcNAc N-acetylglucosamine - Glc Glucose - conA concanavalin A - WGA Wheat Germ Agglutinin - CPK Creatine Phosphokinase     

Prostaglandin binding activity and myoblast fusion in aggregates of avian myoblasts

Myoblast aggregates provide a system for studying cell interactions which have several advantages over standard, stationary cultures. In gyrotory rotation, aggregate size can be controlled and is independent of cell migration. In muscle aggregates, fibroblasts are excluded, yet myoblast differentiation and fusion occur in a highly synchronous fashion. Specific PG binding occurs in chick or quail myoblast aggregates: in chick the peak of binding is at 35-36 hr. Aggregation is complete 16 hr before PG binding activity appears. This suggests either that gyrotory aggregation is not identical to myoblast recognition, or that PG binding activity occurs subsequent to myoblast recognition. Myoblast aggregates begin to release PG before 18 hr. The amount detected remains constant until binding begins at 34 hr when PG binding to the aggregates begins. Thus, both the release of PG and PG receptor activity are characteristics of the myoblasts and release of prostaglandin precedes appearance of the binding activity. As a first step in identifying the PG receptor and determining its appearance on the myoblast cell surface, we have prepared antisera against myoblast surfaces which blocks receptor-ligand interaction and have absorbed it against both peripheral and intrinsic membrane fractions. The results indicate that the PG receptor is a myoblast peripheral membrane macromolecule.     

A role for acetylcholine receptors in the fusion of chick myoblasts

The role of acetylcholine receptors in the control of chick myoblast fusion in culture has been explored. Spontaneous fusion of myoblasts was inhibited by the nicotinic acetylcholine receptor antagonists alpha-bungarotoxin, Naja naja toxin and monoclonal antibody mcAb 5.5. The muscarinic antagonists QNB and n-methyl scopolamine were without effect. Atropine had no effect below 1 microM, where it blocks muscarinic receptors; at higher concentrations, when it blocks nicotinic receptors also, atropine inhibited myoblast fusion. The inhibitions imposed by acetylcholine receptor antagonists lasted for approximately 12 h; fusion stimulated by other endogenous substances then took over. The inhibition was limited to myoblast fusion. The increases in cell number, DNA content, the level of creatine phosphokinase activity (both total and muscle-specific isozyme) and the appearance of heavy chain myosin, which accompany muscle differentiation, followed a normal time course. Pre-fusion myoblasts, fusing myoblasts, and young myotubes specifically bound labeled alpha-bungarotoxin, indicating the presence of acetylcholine receptors. The nicotinic acetylcholine receptor agonist, carbachol, induced uptake of [14C]Guanidinium through the acetylcholine receptor. Myoblasts, aligned myoblasts and young myotubes expressed the synthetic enzyme Choline acetyltransferase and stained positively with antibodies against acetylcholine. The appearance of ChAT activity in myogenic cultures was prevented by treatment with BUDR; nonmyogenic cells in the cultures expressed ChAT at a level which was too low to account for the activity in myogenic cultures. We conclude that activation of the nicotinic acetylcholine receptor is part of the mechanism controlling spontaneous myoblast fusion and that myoblasts synthesize an endogenous, fusion-inducing agent that activates the nicotinic ACh receptor.     

Okadaic acid blocks membrane fusion of chick embryonic myoblasts in culture

Okadaic acid was found to block membrane fusion of chick embryonic myoblasts in culture. It also induced morphological change of the cells from bipolar to spherical shape. These effects were dose-dependent, and could be reversed upon removal of the drug from the culture medium. It showed, however, no effect on the induction of muscle specific proteins including tropomyosin and creatine kinase. When okadaic acid was treated to the cell lysates, the phosphorylation state of many proteins significantly increased. These results suggest that the inhibition of myoblast fusion by okadaic acid may be mediated by the increase in the phosphorylation of certain, unknown protein(s) that regulate the fusion process.     

Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes

Cell membrane consists of various lipids such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Among them, PS is a molecular marker of apoptosis, because it is located to the inner leaflet of plasma membrane generally but it is moved to the outer leaflet during programmed cell death. The process of apoptosis has been implicated in the fusion of muscle progenitor cells, myoblasts, into myotubes. However, it remained unclear whether PS regulates muscle cell differentiation directly. In this paper, localization of PS to the outer leaflet of plasma membrane in proliferating primary myoblasts and during fusion of these myoblasts into myotubes is validated using Annexin V. Moreover, we show the presence of PS clusters at the cell–cell contact points, suggesting the importance of membrane ruffling and PS exposure for the myogenic cell fusion. Confirming this conclusion, experimentally constructed PS, but not PC liposomes dramatically enhance the formation of myotubes from myoblasts, thus demonstrating a direct positive effect of PS on the muscle cell fusion. In contrast, myoblasts exposed to PC liposomes produce long myotubes with low numbers of myonuclei. Moreover, pharmacological masking of PS on the myoblast surface inhibits fusion of these cells into myotubes in a dose-dependent manner.     

Effect of fibroblast growth factor on the division and fusion of bovine myoblasts

The effect of fibroblast growth factor (FGF) on the rate of proliferation and fusion of bovine myoblast has been examined. Addition to the cultures of 0.1 mug-1 mug/ml of FGF stimulates the rate of proliferation and delays the fusion of primary cultures of bovine myoblasts cultured in 10% serum. Final cell densities reached in the presence of 0.1 mug/ml of FGF were fivefold higher than in controls; with 1 mug/ml, they were 10-fold higher. Increases in cell density were paralleled by increases in acetylcholine receptor sites as measured by the binding of 125I-alpha-bungarotoxin. Both fusion and the appearance of acetylcholine receptor sites were delayed in the presence of FGF. Growth hormone, insulin and testosterone, which have been reported to be mitogenic for rat and chick embryo myoblasts, did not have significant effects on DNA synthesis in bovine myoblasts when compared to the FGF. Conversely, FGF did not stimulate the proliferation of chick embryo myoblasts, indicating that it is not active in all vertebrate species.     

Viral transformation of rat myoblasts: effects on fusion and surface properties.

Lines of rat myoblasts infected by avian sarcoma viruses have been isolated, cloned, and used to study the effects of viral transformation on myogenic differentiation and the surface changes associated with differentiation. The lines transformed by sarcoma viruses failed to fuse into myotubes and did not show the increase in myosin synthesis normally associated with fusion. The parental nontransformed line showed, subsequent to fusion, a surface alteration detectable by external labeling methods. This alteration, an increase in the level of an external protein of MW > 200 × 10³, is similar to that observed in fibroblasts arrested in the G₁ phase of the cell cycle. This protein was absent or greatly reduced on the surfaces of the myoblast lines that had been transformed by sarcoma viruses. Therefore, viral transformation causes loss of several properties normally associated with arrest of myoblasts in G₁.