The cesium-induced delay in myoblast membrane fusion is accompanied by changes in isolated membrane lipids

We have recently demonstrated that cesium ions delay the sharp decrease in both membrane conductivity and membrane permittivity of chick embryo myoblasts seen at fusion (Santini, M.T., Bonincontro, A., Cametti, C. and Indovina, P.L. (1988) Biochim. Biophys. Acta 945, 56-64). Analysis of the conductivity dispersion data (obtained in the radiowave frequency range) indicated that cesium delays fusion by about 30 h. We suggested that cesium is affecting both active ionic transport by blocking potassium channels as well as interfering with membrane lipid and/or protein charges. In the present study, we have investigated both the possible role of membrane lipids in myoblast fusion and the possible effects of cesium on these lipids. Our data indicate that lipid changes do occur in the isolated myoblast plasma membrane of controls during myogenic differentiation especially prior to fusion and that in cesium cultures these variations do not occur. These variations are in accordance with current membrane fusion theory. Specifically, there is a decrease in bilayer-stabilizing lipids (phosphatidylcholine) and an increase in bilayer-destabilizing ones (phosphatidylethanolamine and phosphatidic acid) and cholesterol during the fusion process. In addition, although slight, during fusion there appears to be a decrease in phosphatidylinositol which is believed to be involved in the inositol phosphate second messenger system. In cesium cultures, in which fusion is greatly delayed, the same lipid changes do not take place and those that are observed seem to reflect the fusion delay.     

Changes in mitochondrial activity during avian myoblast differentiation: Influence of triiodothyronine or v-erbA expression

Numerous data suggest that mitochondrial activity is involved in the regulation of cell growth and differentiation. Therefore, we have studied the changes in mitochondrial activity in avian myoblast cultures (QM7 line) undergoing differentiation or in BrdU-treated, differentiation-deficient cells. As we have previously shown that triiodothyronine and v-erb A expression stimulate myogenic differentiation, we have also observed their influence upon mitochondrial activity. Comparison of control and BrdU-treated myoblasts indicated that precocious differentiation events were associated with a stimulation of citrate synthase and cytochrome oxidase activities. They also induced a transient decrease in mitochondrial membrane potential assessed by rhodamine 123 uptake. In control myoblasts, a general stimulation of mitochondrial activity was recorded at cell confluence, prior to terminal differentiation. These events did not occur in BrdU-treated myoblasts, thus indicating that they were tightly linked to myoblast commitment. Whereas no significant triiodothyronine influence could be detected upon mitochondrial activity, we observed that v-erb A expression significantly depresses the mitochondrial membrane potential in control myoblasts. This action was not observed in BrdU-treated myoblasts, thus suggesting that it involves an indirect pathway linked to differentiation. Moreover, the oncoprotein abrogated the decrease in E2-PDH subunit level observed at cell confluence. These data underline that changes in mitochondrial activity occurred prior to myoblast terminal differentiation and could be involved in the processes regulating myogenesis. In addition, they provide the first evidence that the v-erb A oncoprotein influences mitochondrial activity. © 1996 Wiley-Liss, Inc.     

Quantitation of changes in cell surface determinants during skeletal muscle cell differentiation using monospecific antibody

The differentiation of skeletal muscle is characterized by recognition, alignment, and subsequent fusion of myoblast cells at their surfaces to form large, multinucleated myotubes. Monoclonal antibodies were used to investigate anti-genie changes in the cell surface membrane specific for various stages of myogenesis. Chick embryonic skeletal muscle cells were cultured in vitro to the desired stage of differentiation and then injected into BALB/c mice. Spleen cells from the immunized mice were hybridized with NS-1 or P3 8653 mouse myeloma cells. Hybrid cell clones were selected in HAT medium and screened using an indirect radioimmunoassay for the production of monoclonal antibodies specific to myogenic cell surfaces. Target cells for the radioimmunoassay included three stages of myogenesis (myoblasts, midfusion myoblasts, and myotubes) and chick lung cells as a control for polymorphic antigens. Sixty-one clones were obtained which produced antibodies specific for myogenic cells. Thirty-five of these clones were generated from mice immunized with midfusion myoblast stages of myogenesis and 26 were obtained from mice immunized with the later myotube stage of myogenesis. Quantitative measurements by RIA of myogenic determinants per cell surface area on each target cell type revealed that most of the determinants decrease during myogenesis when midfusion myoblasts are used as the immunogen. When myotube stages are used as the immunogen, more determinants increase with cell differentiation. Therefore, the most common pattern of determinant change is for them to be present at all stages of myogenesis but to vary quantitively through development. There are determinants unique to each stage of myogenesis and marked quantitative differences within a cell stage for each determinant.     

Phorbol myristate acetate (PMA) fails to prevent the appearance of markers associated with muscle and liver differentiation in culture

The tumour promoter PMA has been shown to both prevent and induce differentiation of a variety of cell types in culture. The reason for its paradoxical effects is not known. However, it is clear that PMA alters the cell membrane and therefore it is possible that PMA may only be effective in instances where differentiation is accompanied by changes to the cell membrane e.g. myoblast fusion during myogenesis. In this study, its effects on myoblast fusion as well as the appearance of the muscle specific isoenzyme of creatine phosphokinase (M-CPK) which is not fusion dependent is examined. It is shown that M-CPK accumulates in myogenic cultures exposed to PMA although fusion is prevented. PMA is also tested in foetal rat hepatocytes which differentiate and acquire the enzyme tyrosine aminotransferase during culture. There is no evidence which suggests that this change is membrane dependent. The tumour promoter does not prevent the accumulation of tyrosine aminotransferase in cultured foetal rat hepatocytes.     

Insulin receptor substrate protein 53kDa (IRSp53) is a negative regulator of myogenic differentiation

Fusion of mononucleated myoblasts to generate multinucleated myotubes is a critical step in skeletal muscle development. Filopodia, the actin cytoskeleton based membrane protrusions, have been observed early during myoblast fusion, indicating that they could play a direct role in myogenic differentiation. The control of filopodia formation in myoblasts remains poorly understood. Here we show that the expression of IRSp53 (Insulin Receptor Substrate protein 53kDa), a known regulator of filopodia formation, is down-regulated during differentiation of both mouse primary myoblasts and a mouse myoblast cell line C2C12. Over-expression of IRSp53 in C2C12 cells led to induction of filopodia and decrease in cell adhesion, concomitantly with inhibition of myogenic differentiation. In contrast, knocking down the IRSp53 expression in C2C12 cells led to a small but significant increase in myotube development. The decreased cell adhesion of C2C12 cells over-expressing IRSp53 is correlated with a reduction in the number of vinculin patches in these cells. Mutations in the conserved IMD domain (IRSp53 and MIM (missing in metastasis) homology domain) or SH3 domain of IRSp53 abolished the ability of this protein to inhibit myogenic differentiation and reduce cell adhesion. Over-expression of the IMD domain alone was sufficient to decrease the cell-extracellular matrix adhesion and to inhibit myogenesis in a manner dependent on its function in membrane shaping. Based on our data, we propose that IRSp53 is a negative regulator of myogenic differentiation which correlates with the observed down regulation of IRSp53 expression during myoblast differentiation to myotubes.     

Effects of cesium on in vitro myoblast differentiation: An electron microscopic study

Summary This paper describes the microscopic evidence supporting a cesium-induced delay in the fusion of chick embryo myoblast membranes during in vitro myogenic differentiation. We have recently demonstrated that the sharp decrease in the conductivity and permittivity of the membranes of these myogenic cells at the time of fusion is delayed 30 h by the addition of cesium to the culture medium (Santini et al., Biochim. Biophys. Acta 945:56–64; 1988). We report here that this delay in fusion is substantiated by direct microscopic observation and that cesium also induces ultrastructural changes in the myoblast cells themselves. Possible mechanisms by which cesium may cause both the delay in fusion as well as the ultrastructural changes observed are discussed. This investigation was partially supported by an Italian Consiglio Nazionale delle Ricerche grant 85.00.304.02 (to P. L. I.).     

Regulation of Membrane Transport Sites for Amino Acids in Myogenic Cells

Abstract. Myoblasts from 12-day chick embryos in cell culture transport the nonmetabolizable amino acid α-aminoisobutyric acid (AIB) two to three-fold more rapidly than multinucleated myotubes which form from them. This decrease in transport is due to a relative decrease in the number of transport sites per unit area of cell surface suggesting a compositional change in the plasma membrane during myogenesis. In studies reported here, AIB transport was monitored throughout myogenesis and correlated with other aspects of differentiation. During myogenesis the number of amino acid transport sites remains constant per myotube nucleus. As myogenesis proceeds, there is a marked increase in cellular protein and cell surface without a commensurate increase in amino acid transport sites. The net consequence of the surface area change is fewer amino acid transport sites per unit area of myotube membrane surface. The decrease in membrane transport sites for AIB per unit area of membrane is not a result of length of time in culture per se, medium depletion, or cell density, but is a result of differentiation, since blocking myoblast fusion by deprivation of calcium delays the decrease in AIB transport sites per unit cell surface area while reversal of the calcium deprivation block is accompanied by a rapid decrease in the number of AIB transport sites per unit cell surface area. Thus, the decrease in AIB transport sites is an aspect of differentiation which accompanies the marked elaboration of surface membrane during myogenesis.     

Regulation of membrane transport sites for amino acids in myogenic cells. A differentiation dependent phenomenon

Myoblasts from 12-day chick embryos in cell culture transport the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB) two to three-fold more rapidly than multinucleated myotubes which form from them. This decrease in transport is due to a relative decrease in the number of transport sites per unit area of cell surface suggesting a compositional change in the plasma membrane during myogenesis. In studies reported here, AIB transport was monitored throughout myogenesis and correlated with other aspects of differentiation. During myogenesis the number of amino acid transport sites remains constant per myotube nucleus. As myogenesis proceeds, there is a marked increase in cellular protein and cell surface without a commensurate increase in amino acid transport sites. The net consequence of the surface area change is fewer amino acid transport sites per unit area of myotube membrane surface. The decrease in membrane transport sites for AIB per unit area of membrane is not a result of length of time in culture per se, medium depletion, or cell density, but is a result of differentiation, since blocking myoblast fusion by deprivation of calcium delays the decrease in AIB transport sites per unit cell surface area while reversal of the calcium deprivation block is accompanied by a rapid decrease in the number of AIB transport sites per unit cell surface area. Thus, the decrease in AIB transport sites is an aspect of differentiation which accompanies the marked elaboration of surface membrane during myogenesis.     

Regulation of hexose transport in rat myoblasts during growth and differentiation

We report here the effects of growth conditions and myogenic differentiation on rat myoblast hexose transport activities. We have previously shown that in undifferentiated myoblasts the preferred substrates for the high (HAHT)- and low (LAHT)-affinity hexose transport systems are 2-deoxyglucose (2-DG) and 3-O-methyl-D-glucose (3-OMG), respectively. The present study shows that at cell density higher than 4.4 x 10(4) cells/cm2, the activities of both transport processes decrease with increasing cell densities of the undifferentiated myoblasts. Since the transport affinities are not altered, the observed decrease is compatible with the notion that the number of functional hexose transporters may be decreased in the plasma membrane. Myogenic differentiation is found to alter the 2-DG, but not the 3-OMG, transport affinity. The Km values of 2-DG uptake are elevated upon the onset of fusion and are directly proportional to the extent of fusion. This relationship between myogenesis and hexose transport is further explored by using cultures impaired in myogenesis. Treatment of cells with 5-bromo-2'-deoxyuridine abolishes not only myogenesis but also the myogenesis-induced change in 2-DG transport affinity. Similarly, alteration in 2-DG transport affinity cannot be observed in a myogenesis-defective mutant, D1. However, under myogenesis-permissive condition, the myogenesis of this mutant is also accompanied by changes in its 2-DG transport affinity. The myotube 2-DG transport system also differs from its myoblast counterpart in its response to sulfhydryl reagents and in its turnover rate. It may be surmised from the above observations that myogenesis results in the alteration of the turnover rate or in the modification of the 2-DG transport system. Although glucose starvation has no effect on myogenesis, it is found to alter the substrate specificity and transport capacity of HAHT. In conclusion, the present study shows that hexose transport in rat myoblasts is very sensitive to the growth conditions and the stages of differentiation of the cultures. This may explain why different hexose transport properties have been observed with myoblasts grown under different conditions.     

Localized and transient changes in plasma membrane fluidity during in vitro myoblast fusion: an 1H-NMR study.

High resolution proton NMR was used to study the cell surface molecular events which take place during in vitro myoblast differentiation and fusion. The CH3 and (CH2)n spectral signals were followed throughout in vitro myogenic development. The results show that although both the T1 and T2 relaxation times of the CH3 and (CH2)n groups are sensitive to the fusion process, T1 is the most sensitive. Both T1 of CH3 and (CH2)n increased before fusion indicating a higher degree of molecular motion and then returned to their original values. These results demonstrate how mobile lipid domains observed with proton NMR can be used to study the changes taking place during myoblast differentiation, particularly myoblast membrane fusion.     

Membrane dynamics of differentiating cultured embryonic chick skeletal muscle cells by fluorescence microscopy techniques

Changes in membrane fluidity during myogenesis have been studied by fluorescence microscopy of individual cells growing in monolayer cultures of embryonic chick skeletal muscle cells. Membrane fluidity was determined by the techniques of fluorescence photobleaching recovery (FDR), with the use of a lipidsoluble carbocyanine dye, and by fluorescence depolarization (FD), with perylene used as the lipid probe. The fluidity of myoblast plasma membranes, as determined from FPR measurements in membrane areas above nuclei, increased during the period of myoblast fusion and then returned to its initial level. The membrane fluidity of fibroblasts, also found in these primary cultures, remained constant. The fluidity in specific regions along the length of the myoblast membrane was studied by FD, and it was observed that the extended arms of the myoblast have the highest fluidity on the cell and that the tips at the ends of the arms had the lowest fluidity. However, since the perylene probe used in the FD experiments appeared to label cytoplasmic components, changes in fluidity measured with this probe reflect changes in membrane fluidity as well as in cytoplasmic fluidity. The relative change in each of these compartments cannot yet be ascertained. Tips have specialized surface structures, filopodia and lamellipodia, which may be accompanied by a more immobile membrane as well as a more rigid cytoplasm. Rounded cells, which may also have a more convoluted surface structure, show a lower apparent membrane fluidity than extended cells.     

Requirement for G protein activity at a specific time during embryonic chick myogenesis

Signaling between embryonic myoblasts involves prostaglandin metabolism, the activation of a membrane receptor and changes in polyphosphatidyl inositol metabolism. Many of these membrane-localized events occur between 33 to 35 h of differentiation, concomitant with a dramatic change in membrane organization, in myoblast aggregates in culture. Since many receptors affect inositol phosphate metabolism by activating a GTP-binding protein (G protein), we asked if there was evidence for such a protein in myogenic signaling. We show that during the period of differentiation in culture when prostaglandin is needed to bind to a transient receptor, a pertussis toxin-sensitive but cholera toxin-insensitive G protein must act. If this activation is blocked, the characteristic change in myoblast cell adhesion and subsequent membrane fusion do not occur. We suggest that a G protein couples the activated prostaglandin receptor and the change in polyphosphatidyl inositol metabolism and that this membrane transduction step is necessary for subsequent membrane differentiation events during myogenesis.     

Evidence for the involvement of cathepsin B in skeletal myoblast differentiation

Our previous studies suggest that the cysteine protease cathepsin B (catB) is involved in skeletal myoblast differentiation (myogenesis). To test this hypothesis, we examined the effect of trapping one of the two catB alleles on the ability of C2C12 cells to differentiate. During differentiation, catB gene-trapped C2C12 mouse myoblasts (RT-27) demonstrated a similar pattern of intracellular catB activity and protein expression compared to that observed in control C2C12 myoblasts and myoblasts trapped in a gene other than catB. However, compared to control myoblast cell lines, levels of catB activity and protein at each stage of RT-27 differentiation were reduced. The reductions in levels of catB were associated with reductions in several myogenic phenotypes including reduced levels of creatine phosphokinase activity and myosin heavy chain protein, two late biochemical markers of myogenesis, and reduced myotube size and extent of myotube formation over time. Comparable reductions were not observed for myogenin protein, an early biochemical marker of myogenesis, or in myokinase activity and catB related cathepsin L-type activity, two non-specific proteins. Finally, both control and catB gene-trapped myoblasts secreted active catB at pH 7.0. However levels of active pericellular/secreted catB were 50% lower in catB gene-trapped myoblasts. Collectively, these results support a functional link between catB expression and skeletal myogenesis and suggest a role for active pericellular/secreted catB in myoblast fusion.     

TIEG1 negatively controls the myoblast pool indispensable for fusion during myogenic differentiation of C2C12 cells

The transforming growth factor (TGF)-β inducible early gene (TIEG)-1 is implicated in the control of cell proliferation, differentiation, and apoptosis in some cell types. Since TIEG1 functioning may be associated with TGF-β, a suppressor of myogenesis, TIEG1 is also likely to be involved in myogenesis. Therefore, we investigated the function of TIEG1 during myogenic differentiation in vitro using the murine myoblasts cell line, C2C12. TIEG1 expression increased during differentiation of C2C12 cells. Constitutive expression of TIEG1 reduced survival and decreased myotube formation. Conversely, knocking down TIEG1 expression increased the number of viable cells during differentiation, and accelerated myoblast fusion into multinucleated myotubes. However, expression of the myogenic differentiation marker, myogenin, remained unaffected by TIEG1 knockdown. The mechanism underlying these events was investigated by focusing on the regulation of myoblast numbers after induction of differentiation. The knockdown of TIEG1 led to changes in cell cycle status and inhibition of apoptosis during the initial stages of differentiation. Microarray and real-time PCR analyses showed that the regulators of cell cycle progression were highly expressed in TIEG1 knockdown cells. Therefore, TIEG1 is a negative regulator of the myoblast pool that causes inhibition of myotube formation during myogenic differentiation.     

Phospholipase D1 facilitates second-phase myoblast fusion and skeletal muscle regeneration

Myoblast differentiation and fusion is a well-orchestrated multistep process that is essential for skeletal muscle development and regeneration. Phospholipase D1 (PLD1) has been implicated in the initiation of myoblast differentiation in vitro. However, whether PLD1 plays additional roles in myoblast fusion and exerts a function in myogenesis in vivo remains unknown. Here we show that PLD1 expression is up-regulated in myogenic cells during muscle regeneration after cardiotoxin injury and that genetic ablation of PLD1 results in delayed myofiber regeneration. Myoblasts derived from PLD1-null mice or treated with PLD1-specific inhibitor are unable to form mature myotubes, indicating defects in second-phase myoblast fusion. Concomitantly, the PLD1 product phosphatidic acid is transiently detected on the plasma membrane of differentiating myocytes, and its production is inhibited by PLD1 knockdown. Exogenous lysophosphatidylcholine, a key membrane lipid for fusion pore formation, partially rescues fusion defect resulting from PLD1 inhibition. Thus these studies demonstrate a role for PLD1 in myoblast fusion during myogenesis in which PLD1 facilitates the fusion of mononuclear myocytes with nascent myotubes.     

Changes in intramembranous particle topography and concanavalin A receptor mobility associated with myoblast differentiation.

These studies have examined the distribution of plasma membrane intramembranous particles (PMP) visualized by freeze fracture and concanavalin A receptors seen by ultrastructural cytochemistry of differentiated and undifferentiated L6 myoblasts. Undifferentiated mononucleated cells have a clustered distribution of PMP on the majority of the fracture faces. Associated with cell differentiation and cell fusion a more uniform distribution of PMP is observed. Changes also occur with myoblast differentiation in the topography and dynamics of receptors bound to concanavalin A. If undifferentiated or differentiated cells are fixed with glutaraldehyde and then reacted with con-A a uniform distribution of con-A is seen on the cell surfaces. In contrast to this if unfixed live cells are reacted at 37 degrees C with con-A a profound redistribution occurs on differentiated cells (greater than 99% showing redistribution) while receptors remain in a uniform array on undifferentiated cells (approximately 95% uniform distribution). In addition to the membrane binding, con-A is observed to bind to an extracellular filamentous matrix seen in high density undifferentiated cultures which then appears to be degraded with differentiation and myoblast fusion. These studies show that a number of membrane changes, both structural and dynamic occur with myoblast differentiation.     

α-Cyclodextrin enhances myoblast fusion and muscle differentiation by the release of IL-4

Muscle fibers are formed during embryonic development by the fusion of mononucleated myoblasts. The spatial structure and molecular composition of the sarcolemma are crucial for the myoblast recognition and fusion steps. Cyclodextrins are a group of substances that have the ability to solubilize lipids through the formation of molecular inclusion complexes. Previously, we have shown that methyl-β-cyclodextrin (MbCD) enhances muscle differentiation. Here, we analyzed the effects of α-cyclodextrin (aCD) during myogenesis. Myogenic cultures treated with aCD showed an increase in myoblast fusion and in the expression of myogenin, sarcomeric tropomyosin and desmin. aCD-conditioned media accelerates myogenesis in a similar way as aCD does, and increased levels of IL-4 were found in aCD-conditioned media. aCD-induced effects on myogenesis were inhibited by an anti-IL4 antibody. These results show that α-cyclodextrin induces myogenic differentiation by the release of IL-4.     

Changes in Intramembranous Particle Topography and Concanavalin A Receptor Mobility Associated with Myoblast Differentiation

These studies have examined the distribution of plasma membrane intramembranous particles (PMP) visualized by freeze fracture and concanavalin A receptors seen by ultrastructural cytochemistry of differentiated and undifferentiated L6 myoblasts. Undifferentiated mononucleated cells have a clustered distribution of PMP on the majority of the fracture faces. Associated with cell differentiation and cell fusion a more uniform distribution of PMP is observed. Changes also occur with myoblast differentiation in the topography and dynamics of receptors bound to concanavalin A. If undifferentiated or differentiated cells are fixed with glutaraldehyde and then reacted with con-A a uniform distribution of con-A is seen on the cell surfaces. In contrast to this if unfixed live cells are reacted at 37° C with con-A a profound redistribution occurs on differentiated cells (greater than 99% showing redistribution) while receptors remain in a uniform array on undifferentiated cells (approximately 95% uniform distribution). In addition to the membrane binding, con-A is observed to bind to an extracellular filamentous matrix seen in high density undifferentiated cultures which then appears to be degraded with differentiation and myoblast fusion. These studies show that a number of membrane changes, both structural and dynamic occur with myoblast differentiation.     

Expression of a developmentally regulated antigen on the surface of skeletal and cardiac muscle cells

H36 is a species-specific, cell-surface antigen on differentiating newborn rat skeletal myoblasts and myogenic lines. This membrane antigen has been defined by a monoclonal antibody raised by the fusion of SP 2/0-Ag14 myeloma cells with spleen cells from mice immunized with myotubes derived from the myogenic E63 line. H36 antigen, isolated by immunoaffinity chromatography, is comprised of two polypeptides with apparent molecular weights of 98,000 and 117,000. Fluorescence photometry and radioimmunoassays have been used to follow quantitative and topographic changes in the H36 determinant during myogenesis. H36 is present at a basal level on replicating myoblasts; it increases on prefusion myoblasts and persists on myotubes. At or near the time of prefusion, it becomes concentrated between adjacent aligned myoblasts and localized on membrane "blebs". H36 is present on both skeletal and cardiac cells but absent from a variety of cells that include fibroblasts, neuronal cells, and smooth muscle. There are approximately 4 x 10(5) determinants per myoblast, and the Ka of the antibody is 3.8 x 10(8) liters/mol. The distributions of H36 on the top and attached surfaces of myoblasts and myotubes are distinct, which suggests localized specialization of these surfaces. H36 is an integral membrane component and upon cross-linking, it associates with the detergent-insoluble cytoskeletal framework. Inhibition of myogenesis by 5-bromodeoxyuridine or by calcium deprivation prevents the developmentally associated changes in the expression of H36. H36 is also absent or markedly reduced on the fu- and Ama102 developmentally defective mutant myoblast lines. We conclude that H36 is a muscle-specific, developmentally regulated cell-surface antigen that may have a role in myoblast differentiation and that can be used to determine the embryonic lineages of skeletal and cardiac muscle.