Cultures Of Embryonic Muscle Cells Research Articles

Acidic Compartment Size, Positioning, and Function during Myogenesis and Their Modulation by the Wnt/Beta-Catenin Pathway.

Lysosomes and acidic compartments are involved in breaking down of macromolecules, membrane recycling, and regulation of signaling pathways. Here, we analyzed the role of acidic compartments during muscle differentiation and the involvement of the Wnt/beta-catenin pathway in lysosomal function during myogenesis. Acridine orange was used to localize and quantify acidic cellular compartments in primary cultures of embryonic muscle cells from Gallus gallus. Our results show an increase in acidic compartment size and area, as well as changes in their positioning during the initial steps of myogenesis. The inhibition of lysosomal function by either the chloroquine Lys05 or the downregulation of LAMP-2 with siRNA impaired chick myogenesis, by inhibiting myoblast fusion. Two activators of the Wnt/beta-catenin pathway, BIO and Wnt3a, were able to rescue the inhibitory effects of Lys05 in myogenesis. These results suggest a new role for the Wnt/beta-catenin pathway in the regulation of acidic compartment size, positioning, and function in muscle cells.

Cloning and expression of three K + channel cDNAs from Xenopus muscle

Embryonic Xenopus muscle cells grown in culture express voltage-gated K + currents with inactivating and non-inactivating kinetics. Here we report the cloning of three K + channel cDNAs, designated XKv1.2′, XKv1.4 and XKv1.10, from muscle which may underlie these currents. XKv1.2′ cDNA appears to be an allelic variant of the XKv1.2 previously cloned from Xenopus. The second cDNA encodes a homologue of Kv1.4 that has not been previously cloned from Xenopus. The predicted XKv1.4 protein shows 73% overall similarity to mouse and chick Kv1.4, but shows significant divergence in the region corresponding to the chain of the inactivating ‘ball and chain’ domain. The third K + channel cDNA isolated from Xenopus muscle is a novel Kv1 isoform designated XKv1.10. The predicted protein shares about 70% similarity with other members of the Kv1 subfamily, and about 40% with members of the Kv2, Kv3 and Kv4 subfamilies. XKv1.4 mRNA appears as early as stage 10.5 in whole embryos and is prominent in muscle throughout development from stage 14 to adult. XKv1.2′ mRNA is detected by stage 11.5 in whole embryos, but remains at low levels in embryonic skeletal muscle (stages 14 and 21), and is absent from adult muscle. XKv1.10 mRNA is first detected at stage 21 in whole embryos, and is present in muscle from this stage onwards. All three transcripts are present in spinal cord at stage 21. The results support the notion that channels encoded by XKv1.4 contribute to the inactivating K + current observed in embryonic muscle cells in culture.

Antibodies against cysteine string proteins inhibit evoked neurotransmitter release at Xenopus neuromuscular junctions.

Cysteine string proteins (CSPs) are evolutionarily conserved proteins that are associated with synaptic vesicles and other regulated secretory organelles. To investigate the role of CSPs in vertebrate neuromuscular transmission, we introduced anti-CSP antibodies into the cell bodies of Xenopus spinal motor neurons that form synapses with embryonic muscle cells in culture. These antibodies produced a rapid (within 3-6 min), and in most cases complete, inhibition of stimulus-dependent neurotransmitter secretion. However, spontaneous neurotransmitter release was stable (both in frequency and amplitude) throughout the period of antibody exposure. Several control experiments validated the specificity of the anti-CSP antibody effects. First, the anti-CSP antibody actions were not mimicked either by antibodies against another synaptic vesicle protein SV2, or by nonspecific immunoglobins. Second, heat treatment of the anti-CSP antibodies eliminated their effect on evoked secretion. Third, immunoblot experiments showed that the anti-CSP and anti-SV2 antibodies were highly selective for their respective antigens in these Xenopus cultures. We conclude from these results that CSPs are vital constituents of the pathway for regulated neurotransmitter release in vertebrates. Moreover, the selective inhibition of evoked, but not spontaneous transmitter release by anti-CSP antibodies indicates that there is a fundamental difference in the machinery that mediates these secretory processes.

Expression of lactate dehydrogenase, myosin heavy chain and myogenic regulatory factor genes in rabbit embryonic muscle cell cultures.

The expression of myogenic regulatory factors (MRFs), lactate dehydrogenase (LDH) and myosin heavy chains (MyHC), as markers of myogenesis, metabolism and contractility respectively, were investigated during differentiation of rabbit embryonic muscle cells in primary culture. Myf5, MyoD and myogenin mRNAs were abundantly expressed at day 1 of culture. The expression of Myf5 and MyoD mRNA transcripts decreased sharply as myoblasts fused and differentiated into myotubes, whilst myogenin mRNA was maintained throughout the duration of the culture. In contrast, MRF4 mRNA was weakly expressed on day 1 of culture, its expression increased slightly as myoblasts fused and reached a maximum level in 7-day-old cultures containing striated myofibres. The specific activity of LDH increased linearly during myoblast proliferation and fusion. In 7-day-old cultures, LDH-M mRNA (dominant in glycolytic muscles) and LDH-H mRNA (predominant in perinatal and oxidative muscles) represented 38% and 62% of total LDH mRNA respectively. At this stage, immunocytochemical staining with perinatal and adult-type MyHC antibodies showed that embryonic and perinatal MyHC isoforms were expressed in all myotubes, while few of them were stained by type I MyHC antibody. However, none of them expressed adult type II MyHC. The latter results were further supported by RT-PCR analysis of adult-type MyHC mRNA which showed that only the type I MyHC mRNA transcript was expressed. These data were in agreement with those reported in vivo on perinatal rabbit muscles. They differed from those obtained on cultured satellite cells isolated from adult rabbit fast-twitch or slow-twitch muscles which did not express embryonic MyHC, and instead expressed fast- or slow-type MyHC according to their muscle origin. Taken together, these results further suggest that myogenic mononucleated cells express different properties in vitro according to their developmental origin as well as properties related to those of the muscles from which they were isolated.

106 Interaction of voltage-gated k + channel subunits

Embryonic Xenopus muscle cells grown in culture express voltage-gated K+ currents with inactivating and non-inactivating kinetics. Here we report the cloning of three K+ channel cDNAs, designated XKv1.2′, XKv1.4 and XKv1.10, from muscle which may underlie these currents. XKv1.2′ cDNA appears to be an allelic variant of the XKv1.2 previously cloned from Xenopus. The second cDNA encodes a homologue of Kv1.4 that has not been previously cloned from Xenopus. The predicted XKv1.4 protein shows 73% overall similarity to mouse and chick Kv1.4, but shows significant divergence in the region corresponding to the chain of the inactivating ‘ball and chain’ domain. The third K+ channel cDNA isolated from Xenopus muscle is a novel Kv1 isoform designated XKv1.10. The predicted protein shares about 70% similarity with other members of the Kv1 subfamily, and about 40% with members of the Kv2, Kv3 and Kv4 subfamilies. XKv1.4 mRNA appears as early as stage 10.5 in whole embryos and is prominent in muscle throughout development from stage 14 to adult. XKv1.2′ mRNA is detected by stage 11.5 in whole embryos, but remains at low levels in embryonic skeletal muscle (stages 14 and 21), and is absent from adult muscle. XKv1.10 mRNA is first detected at stage 21 in whole embryos, and is present in muscle from this stage onwards. All three transcripts are present in spinal cord at stage 21. The results support the notion that channels encoded by XKv1.4 contribute to the inactivating K+ current observed in embryonic muscle cells in culture.

The effects of β-d-xynoside on the synthesis of proteolgycans by skeletal muscle: Lack of effect on decorin and differential polymerization of core protein-bound and xyloside-linked chondroitin sulfate

Developing skeletal muscle cells, as both myoblasts and myotubules, synthesize a distinctive large chondroitin sulfate proteoglycan. To probe the role of this proteoglycan in myogenesis, chick embryonic muscle cells in culture were treated with beta-D-xyloside, a compound which interferes with proteoglycan synthesis by acting as an artificial acceptor for glycosaminoglycan synthesis and thereby competing with the proteoglycan core protein. Analysis of the proteoglycans indicates that with increasing concentrations of beta-D-xyloside, synthesis of the chondroitin sulfate proteoglycan is inhibited, with concomitant massive synthesis of xyloside-linked chondroitin sulfate glycosaminoglycans. Xyloside does not appear to inhibit glycosaminoglycan attachment onto the small heparan sulfate and dermatan sulfate proteoglycans which are synthesized in the muscle cultures, even though, because of the mechanism of action of beta-xyloside, these proteoglycans should be affected. At submaximal concentrations of beta-xyloside, there is synthesis of both large chondroitin sulfate proteoglycans and xyloside-linked chondroitin sulfate. The xyloside-linked chondroitin sulfate chains have the same sulfation pattern as the core protein-bound skeletal muscle chondroitin sulfate (90% 6-sulfated isomer), but are much smaller (24,000 vs. 65,000 in molecular weight). The discrepancy in size but identify of sulfation indicates that, although sulfation takes place normally on either the core protein or the xyloside acceptor, termination of glycosylation occurs earlier for xyloside-initiated chondroitin sulfate. In spite of these dramatic effects on chondroitin sulfate proteoglycan synthesis, beta-xyloside elicits no observable effects on in vitro myogenesis. This suggests that the function served by the large chondroitin sulfate proteoglycan is not required in the more simplified environment of the muscle cultures.

Ca 2+/calmodulin-dependent phosphorylation of the 100-kDa protein in chick embryonic muscle cells in culture

The pattern of protein phosphorylation was found to change in differentiating chick embryonic myoblasts in culture. The extent of phosphorylation of 42-, 50-, and 100-kDa proteins increased while that of a 63-kDa protein declined in extracts of myoblasts that had been cultured for increasing periods. Of these, the increase in phosphorylation of the 100-kDa protein occurred most dramatically in extracts of myoblasts in an early stage of differentiation and was specifically inhibited by trifluoperazine (TFP) and other calmodulin (CaM) antagonists including chlorpromazine and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7). Treatment of increasing concentrations of TFP to culture medium also decreased the phosphorylation state of the 100-kDa protein and the degree of myoblast fusion in parallel. In addition, levels of both the kinase activity and the 100-kDa protein but not of CaM appeared to rise in the cells cultured for longer periods. These results suggest that (1) a Ca 2+/CaM-dependent protein kinase is responsible for phosphorylation of the 100-kDa protein, (2) the TFP-mediated myoblast fusion block may be associated with the inhibitory effect of the drug against the kinase activity, and (3) the increase in phosphorylation state of the 100-kDa protein during myogenic differentiation is due to the rise in levels of the kinase and its substrate.

Purothionin from wheat endosperm reversibly blocks myogenic differentiation of chick embryonic muscle cells in culture

Purothionin from wheat endosperm is a cysteine-rich, basic polypeptide of about 5000 Da, which modifies membrane permeability of cultured mammalian cells. This peptide was found to block fusion of chick embryonic muscle cells in culture but allows proliferation and alignment. A purothionin concentration of 6 μg/ml (1.2 μ M) was necessary for the complete prevention of myotube formation. Under similar conditions, incorporation of [ 35S]methionine occurred normally but the synthesis of muscle-specific proteins including creatine kinase and acetylcholine receptor was strongly inhibited. In addition, purothionin blocked the uptake of 86Rb +, immediately after its addition to the cultured myoblasts. No such effects were found with the purothionin chemically modified with acetic or succinic anhydride. Thus, the basic residues in purothionin appear to be associated with the inhibition of myogenic differentiation. These results suggest that purothionin exerts its regulatory effect on the transition from proliferative to differentiative myoblasts by interfering with membrane permeability or intercellular contact and recognition, which are necessary for the initiation of muscle differentiation.

Expression of nuclear lamin A and muscle-specific proteins in differentiating muscle cells in ovo and in vitro.

Primary cultures and tissue samples of chicken embryonic muscle were immunologically probed for the expression of muscle-specific proteins, such as myosin heavy chain and the tropomyosins, as well as for the nuclear lamina protein, lamin A. As determined by quantitative immunoblotting, the expression of lamin A and the muscle-specific proteins were at low levels or absent in predifferentiation myoblasts both in vitro and in ovo. During differentiation, an increase of lamin A expression preceded the induction to high levels of expression of muscle-specific proteins. Immunofluorescence staining of chicken embryonic muscle cells in culture also indicates an accumulation of lamin A before the induction of muscle-specific proteins expression. Furthermore, the accumulation of lamin A reached a plateau before the muscle-specific proteins during muscle development. In two dimensional NEPHGE gel analysis of immunoprecipitated lamin A, no detectable change in the ratio of the acidic/basic isoelectric variants of lamin A was observed during myogenesis. A potential role for lamin A in the mechanisms which underlie the differential and coordinate expression of muscle-specific genes is proposed.

A monoclonal antibody against the skeletal muscle enzyme, creatine kinase

Creatine kinase (CK) is often used as an index of differentiation in cultures of embryonic muscle cells [l] and its presence in serum is also used to monitor muscle damage in disease [2,3]. Immunological assays of CK can be made specific for any one of the CK isoenzymes and polyclonal rabbit antisera have been used for the study of muscle-specific CK (MM-CK) accumulation and distribution in differentiating myoblast cultures [4,5]. A monoclonal antibody (McAb) for these assays would have the added advantages of providing an unlimited supply of antibody with defined properties which can be selected for during McAb preparation. review), McAb CK-JOE does appear to be suitable for measuring MM-CK levels in muscle cell cultures.

The control of glucose 1,6-bisphosphate by developmental state and hormonal stimulation in cultured muscle tissue.

1. The concentration of glucose 1,6-bisphosphate, a potent regulator of muscle glucose metabolism, was examined in embryonic muscle cells in culture. 2. The concentration in fused myotubes was twice that in unfused myoblasts. 3. The effect of various hormones and agonists on the glucose 1,6-bisphosphate concentration in both pre- and post-fusion muscle cells was examined. In pre-fusion cells no effect of adrenaline or cyclic AMP was observed, but stimulation by vasopressin, adrenaline + propranolol, ionophore A23187 and dibutyryl cyclic GMP significantly decreased glucose 1,6-bisphosphate. In post-fusion cells similar effects were observed, except that stimulation by adrenaline and by dibutyryl cyclic AMP significantly increased metabolite concentration. 4. All effects increased with time (over a 1 h period), except for that of vasopressin, which was transient. 5. The changes in glucose 1,6-bisphosphate concentration were accompanied by changes in the fructose 1,6-bisphosphate/fructose 6-phosphate ratio, implying an effect on phosphofructokinase activity.

Function of the Carbohydrate Moieties of Glycoproteins

AbstractTo determine the function of the carbohydrate moiety of glycoproteins, we have used tunicamycin, an analog of N‐acetylglucosamine, to inhibit the glycosylation of N‐glycosidically linked glycoproteins. First, we examined the effect of this drug on the intracellular processing, export and biological activity of fibronectin–the major cell surface glycoprotein of chick embryo fibroblasts. Chick fibroblasts treated with tunicamycin produced only nonglycosylated fibronectin and the export or secretion of the carbohydrate‐free protein species was not totally impaired. We did observe that there was a substantial decrease in the absolute amount of nonglycosylated fibronectin on the cell surface and in the culture medium. This decrease was shown to be due to increased proteolytic degradation of the nonglycosylated protein species.To examine the biological activity of nonglycosylated fibronectin, we compared the activities of the glycosylated and nonglycosylated forms of this protein utilizing in vitro assay procedures. We have shown that isolated, nonglycosylated fibronectin retained the biological properties characteristic of the glycosylated protein; they are: 1) promotion of cell‐cell and cell‐substratum adhesion, 2) restoration of normal behavior and phenotype to transformed cells, and 3) promotion of cell binding to collagen. The isolated, nonglycosylated protein was shown to be more sensitive to degradation by proteolytic enzymes, in agreement with the data obtained “in vivo.”The requirement of glycosylation for the export of acetylcholine receptor was also examined. We found that treatment of embryonic muscle cells in culture with tunicamycin did not inhibit the export of this protein to the cell surface. As with fibronectin, there was a substantial decrease in the amount of receptor present on the cell surface, due to enhanced proteolysis of the nonglycosylated protein. The simultaneous treatment of cells with the protease inhibitor leupeptin diminished the rate of degradation of the nonglycosylated receptor and restored the expression of receptor on the cell surface.Finally, the requirement for N‐glycosidically linked glycoproteins during differentiation of embryonic myoblasts into multinucleated, functional muscle fibers was also investigated. Tunicamycin blocked the expression of glycoproteins on the cell surface and strongly inhibited fusion when added to cultures of differentiating muscle cells prior to fusion. The inhibition of fusion was partially prevented when tunicamycin was administered in the presence of protease inhibitors such as leupeptin and pepstatin. Both glycosylation and fusion were completely restored to normal after removal of tunicamycin from the medium. These studies provide strong support for the idea that myoblast fusion is partially mediated by surface glycoproteins with asparagine‐linked oligosaccharides. However, the requirement for the carbohydrate portion of the glycoprotein appears to be indirect in that it acts to stabilize the protein moiety against proteolytic degradation.To elucidate the mechanism responsible for the enhancement of proteolysis of cell surface glycoproteins following treatment with tunicamycin, we investigated the effect of tunicamycin on the intracellular processing of proteolytic enzymes. Treatment of chick embryo fibroblasts with tunicamycin resulted in more than a 10‐fold increase in the amount of protease activity released into the culture medium. The enzyme activity has been tentatively identified as cathepsin B based on substrate specificity, pH optimum and inhibition with leupeptin.These results as well as extensive work by other investigators [see references [1–11] for recent reviews] suggest that the carbohydrate moiety of surface glycoproteins is not required for their synthesis, secretion or biological function, but instead helps to protect the protein against proteolytic degradation. In contrast, in agreement with the results of Neufeld et al [12–24] and Sly et al [15, 16], the carbohydrate moiety of lysosomal enzymes is required for their intracellular retention.

Interaction of the cytoskeletal framework with acetylcholine receptor on th surface of embryonic muscle cells in culture.

To monitor the interaction of cell surface acetylcholine (AcCho) receptors with the cytoskeleton, cultured muscle cells were labeled with radioactive or fluorescent alpha-bungarotoxin and extracted with Triton X-100, using conditions that preserve internal structure. A significant population of the AcCho receptors is retained on the skeletal framework remaining after detergent extraction. The skeleton organization responsible for restricting AcCho receptors to a patched region may also result in their retention after detergent extraction.

Membrane lipid heterogeneity associated with acetylcholine receptor particle aggregates in Xenopus embryonic muscle cells.

Filipin, digitonin, and saponin react with membrane cholesterol to produce unique membrane alterations (sterol-specific complexes) that are easily discernible in freeze-fracture replicas. We have treated both noninnervated and innervated Xenopus embryonic muscle cells in culture with these agents. Freeze-fracture of these treated muscle cells showed that most areas of the muscle plasma membrane contain sterol-specific complexes (19- to 40-nm protuberances and dimples with filipin, a scalloped appearance with digitonin, or an irregular, rough appearance with saponin). However, these complexes were virtually absent from membrane areas of junctional and nonjunctional aggregates of acetylcholine receptor particles. This result suggests that the membrane matrix of these aggregates is low in cholesterol and that this membrane lipid heterogeneity may be linked to the mechanisms involved in their formation and stabilization on muscle cells in culture.

Inhibition of fusion of embryonic muscle cells in culture by tunicamycin is prevented by leupeptin.

The carbohydrate requirement for alignment and fusion of embryonic quail muscle cells has been examined in tissue culture by use of tunicamycin (TM). The mononucleated, spindle-shaped proliferating myoblasts were treated with TM at various times before fusion and differentiation into multinucleated muscle fibers capable of spontaneous contraction. Tm blocked protein glycosylation and expression of glycoproteins on the cell surface, and strongly inhibited fusion when added to cultures of differentiating muscle cells before the fusion "burst," but had no apparent effect on cell alignment. The inhibition of fusion was partially prevented when TM was administered in the presence of protease inhibitors such as leupeptin and pepstatin, but the inhibition of glycosylation was not prevented. Both glycosylation and fusion were completely restored to normal by the removal of the antibiotic from the medium. These studies provide strong support for the idea that myoblast fusion is partially mediated by glycoproteins with asparagine-linked oligosaccharides. However, the requirement for the carbohydrate portion of the glycoprotein appears to be indirect in that it acts to stabilize the protein moiety against proteolytic degradation. Our findings do not rule out the possibility that oligosaccharide units of surface glycolipids have some role in myoblast fusion.

Carbohydrate requirement for expression and stability of acetylcholine receptor on the surface of embryonic muscle cells in culture.

We have investigated the significance of protein glycosylation for metabolism of acetylcholine receptors (AcChoR) in primary cultures of embryonic chicken muscle cells. Tunicamycin, a specific inhibitor of the glycosylation of asparagine residues on glycoproteins, decreased AcChoR accumulation and accelerated its degradation. In contrast, there was no evidence that tunicamycin treatment affected AcChoR biosynthesis, intracellular transport, or incorporation into surface membranes. Leupeptin, an inhibitor of intracellular proteases, markedly increased accumulation of AcChoR on the external surface of muscle cells treated with tunicamycin. Our findings indicate that impairment of protein glycosylation prevents accumulation of AcChoR by increasing its susceptibility to degradation by cellular proteases.

Extraction with triton X-100 of active polysomes from monolayer cultures of embryonic muscle cells

1. A new procedure has been developed for the extraction of undegraded polysomes from monolayer cultures of developing chick embryo muscle. The cells were lysed with 0.5 % Triton X-100, the cell debris removed by centrifugation, and the supernatant containing the polysomes was analyzed on sucrose gradients. 2. Polysomes extracted in this way, as well as charging enzymes and tRNA's, are not denatured and are capable of synthesizing protein in vitro. 3. The large myosin synthesizing polysomes which have been observed in extracts of uncultured developing muscle tissue have been observed in extracts of cultured muscle. Cultured muscle also contains a lower proportion of monomers than usually seen in polysome preparations from other sources.