Prostaglandins and myoblast fusion

Physiological concentrations of prostaglandin E₁ (10^?7 and 10^?10M) provoke a discrete burst of cell fusion in cultures of primary chick myoblasts, 5 hr after their addition but well before the start of fusion, under control conditions. Two inhibitors of prostaglandin synthesis, aspirin (2-acetoxybenzoic acid) and indomethacin (1-[p-chlorobenzoyl]-5-methoxy-2-methylindole-3-acetic acid), have been used to examine the possibility of prostaglandin production by the undifferentiated myoblasts. Both inhibitors produce a marked inhibition of cell fusion which is possible to reverse by the further addition of 10^?5M prostaglandin E_↑. The findings provide evidence of prostaglandin synthesis in the cultures and suggest that prostaglandin E_↑ is required for the generation of a transient increase in intracellular cyclic AMP which brings about the cellular changes necessary for fusion to occur.     

Cholesterol availability modulates myoblast fusion

The requirement of cholesterol for myoblast fusion has been linked to the primary step in the fusion process, calcium-dependent aggregation (recognition). Inhibition of cholesterol synthesis with 25- hydroxycholesterol or compactin in the absence of exogenous lipid dramatically inhibits calcium-mediated aggregation and concomitant fusion within several hours. Restimulating cholesterol synthesis or supplying exogenous cholesterol rapidly restores aggregation activity. Over this time period, however, the sterol:phospholipid ratio is unaltered, suggesting a local rather than a general membrane cholesterol requirement for the expression of aggregation activity. The aggregation response to a change in sterol availability occurs on a shorter time scale than that required to inhibit the synthesis of the protein(s) with aggregation activity; thus, the cholesterol-requiring step is posttranslational. We suggest that the assembly or maintenance of the aggregation activity depends on a continued local supply of cholesterol.     

Normal myoblast fusion requires myoferlin

Muscle growth occurs during embryonic development and continues in adult life as regeneration. During embryonic muscle growth and regeneration in mature muscle, singly nucleated myoblasts fuse to each other to form myotubes. In muscle growth, singly nucleated myoblasts can also fuse to existing large, syncytial myofibers as a mechanism of increasing muscle mass without increasing myofiber number. Myoblast fusion requires the alignment and fusion of two apposed lipid bilayers. The repair of muscle plasma membrane disruptions also relies on the fusion of two apposed lipid bilayers. The protein dysferlin, the product of the Limb Girdle Muscular Dystrophy type 2 locus, has been shown to be necessary for efficient, calcium-sensitive, membrane resealing. We now show that the related protein myoferlin is highly expressed in myoblasts undergoing fusion, and is expressed at the site of myoblasts fusing to myotubes. Like dysferlin, we found that myoferlin binds phospholipids in a calcium-sensitive manner that requires the first C2A domain. We generated mice with a null allele of myoferlin. Myoferlin null myoblasts undergo initial fusion events, but they form large myotubes less efficiently in vitro, consistent with a defect in a later stage of myogenesis. In vivo, myoferlin null mice have smaller muscles than controls do, and myoferlin null muscle lacks large diameter myofibers. Additionally, myoferlin null muscle does not regenerate as well as wild-type muscle does, and instead displays a dystrophic phenotype. These data support a role for myoferlin in the maturation of myotubes and the formation of large myotubes that arise from the fusion of myoblasts to multinucleate myotubes.     

Differential inhibition of myoblast fusion.

The aggregation and fusion of myoblasts in the presence of either metabolic inhibitors or alterations in the incubation medium or under conditions which result in structural changes in the cells was studied using previously described assays for the intercellular interactions of myoblasts in suspension [Knudsen, K. A., and Horwitz, A. F. (1977). Develop. Biol.58, 328]. These perturbations inhibit myoblast fusion differently. For example, energy poisons, prior trypsin or glutaraldehyde treatment, and inhibitors of protein or cholesterol synthesis all inhibit the Ca²⁺-mediated myoblast aggregation. In contrast, whereas myoblasts aggregate in the presence of 20 mM Mg²⁺, these aggregates are dispersed, even after 1–2 hr, with EDTA or trypsin. Furthermore, enriching the fatty acyl chains in elaidate or prior incubation of the myoblasts in the presence of cytochalasin B or colchicine results in aggregates which, after 1–2 hr, are dispersed by trypsin but not by EDTA. Aggregates of unaltered, control myoblasts, on the other hand, begin to show resistance to dispersion by trypsin after these times. These observations support the suggestion that multinucleate cell formation results from a sequence of events. The influence of these perturbations on cellular aggregation also provides some initial, tentative insight into the molecular mechanism of myoblast fusion. Recognition (calcium-mediated aggregate formation) appears to be mediated by a protein(s) that is turning over during the period of fusion competence, while membrane union (formation of aggregates resistant to dispersion by trypsin) most likely involves the direct participation of membrane lipid.     

Inhibition of myoblast fusion by bromoconduritol

It has recently been reported that the glucosidase I inhibitor, N-methyl-1-deoxynojirimycin (MDJN), inhibits myoblast fusion whereas the mannosidase inhibitor, 1-deoxymannojirimycin (ManDJN), has no effect on fusion. We now report that bromoconduritol, which is an active-site-directed covalent inhibitor of glucosidase II, also inhibits fusion at concentrations that have no effect on the plating efficiency or growth of rat L6 myoblasts. Significant inhibition of fusion was obtained at concentrations as low as 50 micrograms of bromoconduritol/mL, whereas inhibition of cell growth did not occur until concentrations of 250 micrograms/mL were reached. Rat L6 myoblasts were grown in the presence and absence of processing inhibitors and were surface labelled with 125I. Analysis of the iodinated proteins by two-dimensional gel electrophoresis demonstrated that a number of high-molecular-weight proteins (greater than 90,000) detected at the surface of control cells were absent from the surface of cells treated with MDJN or bromoconduritol. It is suggested that MDJN and bromoconduritol prevent the translocation of these proteins to the cell surface. The high-molecular-weight proteins detected at the surface of control cells were also detectable in ManDJN-treated cells, indicating that inhibition of N-linked complex oligosaccharide formation does not affect the translocation of these proteins to the myoblast cell surface.     

The soluble metalloendoprotease required in myoblast fusion remains intracellular

The fusion of myoblasts to myotubes requires an endogenous soluble metalloendoprotease. To determine whether this protease is released by fusing myoblasts, or stays within the cell, we examined the effects of membrane-impermeant and a membrane-permeant metalloendoprotease inhibitors. Membrane-permeant 1,10-phenanthroline, and membrane-impermeant bathophenanthroline disulfonic acid both inhibited soluble metalloendoprotease activity in homogenized myoblasts with equal potency. However, while 1,10-phenanthroline inhibited fusion, bathophenanthroline disulfonic acid had no effect. In addition, metalloendoprotease activity could not be detected in the media of fusing myoblasts, but was in the cells. These observations support the conclusion that the soluble metalloendoprotease required in fusion remains within the myoblast.     

GRAF1 promotes ferlin-dependent myoblast fusion

Myoblast fusion (a critical process by which muscles grow) occurs in a multi-step fashion that requires actin and membrane remodeling; but important questions remain regarding the spatial/temporal regulation of and interrelationship between these processes. We recently reported that the Rho-GAP, GRAF1, was particularly abundant in muscles undergoing fusion to form multinucleated fibers and that enforced expression of GRAF1 in cultured myoblasts induced robust fusion by a process that required GAP-dependent actin remodeling and BAR domain-dependent membrane sculpting. Herein we developed a novel line of GRAF1-deficient mice to explore a role for this protein in the formation/maturation of myotubes in vivo. Post-natal muscles from GRAF1-depleted mice exhibited a significant and persistent reduction in cross-sectional area, impaired regenerative capacity and a significant decrease in force production indicative of lack of efficient myoblast fusion. A significant fusion defect was recapitulated in isolated myoblasts depleted of GRAF1 or its closely related family member GRAF2. Mechanistically, we show that GRAF1 and 2 facilitate myoblast fusion, at least in part, by promoting vesicle-mediated translocation of fusogenic ferlin proteins to the plasma membrane.     

Rat myoblast fusion requires metalloendoprotease activity

The calcium-dependent fusion of cultured rat myoblasts to multinucleate myotubes appears to require the activity of a neutral metalloendoprotease at the time of fusion. Metalloendoprotease inhibitors and synthetic dipeptide substrates prevent myoblast fusion when added to fusion-competent myoblasts with the addition of calcium. Metalloendoprotease activity has been identified and partially characterized in myoblast membranes with a fluorogenic protease substrate, and is inhibited by the same compounds that prevent fusion.     

Membrane events involved in myoblast fusion

Myoblast fusion has been studied in cultures of chick embryonic muscle utilizing ultrastructural techniques. The multinucleated muscle cells (myotubes) are generated by the fusion of two plasma membranes from adjacent cells, apparently by forming a single bilayer that is particle-free in freeze-fracture replicas. This single bilayer subsequently collapses, and cytoplasmic continuity is established between the cells. The fusion between the two plasma membranes appears to take place primarily within particle-free domains (probably phospholipid enriched), and cytoplasmic unilamellar, particle-free vesicles are occasionally associated with these regions. These vesicles structurally resemble phospholipid vesicles (liposomes). They are present in normal myoblasts, but they are absent in certain fusion-arrested myoblast popluations, such as those treated with either 5-bromo-deoxyuridine (BUdR), cycloheximide (CHX), or pospholipase C (PLC). The unilamellar, particle-free vesicles are present in close proximity to the plasma membranes, and physical contact is observed frequently between the vesicle membrane and the plasma membrane. The regions of vesicle membrane-plasma membrane interaction are characteristically free of intramembrane particles. A model for myoblast fusion is presented that is based onan interpretation of these observations. This model suggests that the cytoplasmic vesicles initiate the generation of particle-depleted membrane domains, both being essential components in the fusion process.     

Muscle development: molecules of myoblast fusion

The fusion of myoblasts to make multinucleate muscle fibres is central to muscle development. Recent work on Drosophila has identified two members of the immunoglobulin superfamily that have key roles in controlling the specificity of myoblast fusion.     

Inhibition of myoblast fusion by lysosomotropic amines

Fusion of cultured chick embryo myoblasts was inhibited by treatment with several lysosomotropic amines. The concentrations required for half-maximal inhibition of fusion were approximately 2 μM for chloroquine, 30 μM for tributylamine, 3.2 mM for ammonium chloride, and 3.3 mM for methylamine. All the amines inhibited fusion appreciably at concentrations lower than those that reduced cell density. Both the rate and extent of fusion were affected by the amines, which had to be present for about 20 hr before the usual onset of fusion. Inhibition of fusion was reversible by transfer of inhibited cells to fresh medium. The amines did not cause accumulation of a nondialyzable inhibitor in the culture medium. Levels of creatine kinase increased by eight-fold or more between 18 and 65 hr in cultures treated with tributylamine or chloroquine, although this increase was not as pronounced as in control cultures. The increased creatine kinase activity in amine-treated cultures was due mainly to the BB and MB isozymes, with relatively little increase in the MM isozyme.     

Loss of hyaluronate-dependent coat during myoblast fusion

Cultured myoblasts were found to exhibit extensive, Streptomyces hyaluronidase-sensitive pericellular coats as revealed by exclusion of particles (fixed red blood cells). These coats are not discernible subsequent to fusion of the myoblasts to form myotubes. The myoblasts contained 2.5 times more hyaluronate attached to their cell surface than myotubes when the data was expressed per unit of protein, but no change in hyaluronate was evident on a per DNA basis. Hyaluronidase activities in the cultures were equivalent when expressed per unit of protein. We conclude that, although the myotubes accumulate larger amounts of protein than myoblasts, there is no compensatory increase in hyaluronate.     

3D analysis of founder cell and fusion competent myoblast arrangements outlines a new model of myoblast fusion

Formation of the Drosophila larval body wall muscles requires the specification, coordinated cellular behaviors and fusion of two cell types: Founder Cells (FCs) that control the identity of the individual muscle and Fusion Competent Myoblasts (FCMs) that provide mass. These two cell types come together to control the final size, shape and attachment of individual muscles. However, the spatial arrangement of these cells over time, the sequence of fusion events and the contribution of these cellular relationships to the fusion process have not been addressed. We analyzed the three-dimensional arrangements of FCs and FCMs over the course of myoblast fusion and assayed whether these issues impact the process of myoblast fusion. We examined the timing of the fusion process by analyzing the fusion profile of individual muscles in wild type and fusion mutants. We showed that there are two temporal phases of myoblast fusion in wild type embryos. Limited fusion events occur during the first 3 h of fusion, while the majority of fusion events occur in the remaining 2.5 h. Altogether, our data have led us to propose a new model of myoblast fusion where the frequency of myoblast fusion events may be influenced by the spatial arrangements of FCs and FCMs.     

Fibronectin-independent myoblast fusion in suspension cultures.

Myoblasts cultivated in suspension in serum-free medium were used to examine whether fibronectin influences myoblast fusion. No effect on cell fusion was observed when the medium was supplemented with antibodies against fibronectin (at a concentration effective in inhibiting the myoblast attachment to gelatinized dishes mediated by 1 % horse serum). Purified horse serum fibronectin (70 μg/ml) also had no effect. The assay did, however, detect both inhibition of fusion in low-calcium medium and stimulation of fusion with added embryo extract, horse serum, and fibronectin-depleted horse serum. Thus, although fibronectin may influence cell motility or other processes necessary for fusion in monolayer cultures, it does not affect the fusion process itself.     

A role for lipid in myoblast fusion

Alteration of the membrane fatty acyl composition modulates the fusion of myoblasts into multinucleate myotubes. The rate of fusion after addition of calcium to 50–52 hour cultures of chick pectoral myoblasts is markedly inhibited in cells possessing acyl chains enriched in elaidate and is enhanced in those enriched in oleate. The modulations appear to occur after the cells have recognized one another and adhered strongly but before the membranes have united. These observations lead to a hypothesis for membrane union (fusion) in which the lipids participate directly perhaps by a mechanism analogous to that proposed for the fusion of lipid vesicles.     

Inhibition of myoblast fusion by tunicamycin and pantomycin

The formation of myotubes by a continuous rat myoblast line, L6, can be inhibited by non-toxic concentrations of tunicamycin and pantomycin. The effect of tunicamycin, an inhibitor of UDP-N-acetyl-glucosamine: dolichol phosphate N-acetylglucosaminyltransferase, could be reversed by N-acetylglucosamine but not by mannose, glucose or UDP-N-acetylglucosamine.