Study of muscle regeneration using in vitro 2D 1H spectroscopy.

The in vivo spectrum of regenerating muscles shows a specific cross-correlation signal assigned to the (n-3) fatty acyl chain, which peaks during the myoblast fusion phase. In order to identify the origin of this signal and to take all the lipid metabolites into account, we investigated the degeneration-regeneration process by 1H 2D NMR of lipid muscle extracts. We observed an increase in the total amount of lipids during the regeneration process, although the lipid profile did not show any drastic change during this process. The changes in the NMR signal observed in vivo and, in particular, the appearance of the specific (n-3) fatty acyl chain signal appears to arise from mobile lipid compartments located in fusing cells.

The NO way to increase muscular utrophin expression?

Duchenne muscular dystrophy (DMD), a severe X-linked recessive disorder that results in progressive muscle degeneration, is due to a lack of dystrophin, a membrane cytoskeletal protein. An approach to the search for a treatment is to compensate for dystrophin loss by utrophin, another cytoskeletal protein. During development, in normal as in dystrophic embryos, utrophin is found at the membrane surface of immature skeletal fibres and is progressively replaced by dystrophin. Thus, it is possible to consider utrophin as a 'foetal homologue' of dystrophin. In a previous work, we studied the effect of L-arginine, the substrate of nitric oxide synthetase (NOS), on utrophin expression at the muscle membrane. Using a novel antibody, we confirm here that the immunocytochemical staining was indeed due to an increase in utrophin at the sarcolemma. The result is observed not only on mdx (an animal model of DMD) myotubes in culture but also in mdx mice treated with L-arginine. In addition, we show here the utrophin increase in muscle extracts of mdx mice treated with L-arginine, after electrophoretic separation and western-blotting using this novel antibody, and thus extending the electrophoretic results previously obtained on myotube cultures to muscles of treated mice.

Regulated expression of the proteoglycan SPOCK in the neuromuscular system.

SPOCK is prevalent in developing synaptic fields of the central nervous system (Charbonnier et al., 2000. Mech. Dev. 90, 317-321). The expression of SPOCK during neuromuscular junction (NMJ) formation was compared to agrin and acetylcholine receptor (AChR) distribution. SPOCK is detected within the myogenic masses during the early steps of embryonic development, and distributed in the cytoplasm of myotubes before coclustering with AChRs. In the adult, SPOCK is present in axons and is highly expressed by Schwann cells. SPOCK altered expression pattern after nerve lesioning, or cholinergic transmission blockade, strongly indicate that its cellular distribution at the NMJ depends on innervation.

Nitric oxide and l-arginine cause an accumulation of utrophin at the sarcolemma: a possible compensation for dystrophin loss in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD), a severe X-linked recessive disorder which results in progressive muscle degeneration, is due to a lack of dystrophin, a membrane cytoskeletal protein. An approach to treatment is to compensate for dystrophin loss with utrophin, another cytoskeletal protein with over 80% homology with dystrophin. Utrophin is expressed, at the neuromuscular junction, in normal and DMD muscles and there is evidence that it may perform the same cellular functions as dystrophin. So, the identification of molecules or drugs that could up-regulate utrophin is a very important goal for therapy. We show that in adult normal and mdx mice (an animal model of Duchenne myopathy) treated with l-arginine, the substrate of nitric oxide synthase (NOS), a pool of utrophin localized at the membrane appeared and increased, respectively. In normal and mdx myotubes in culture, l-arginine, nitric oxide (NO), or hydroxyurea increased utrophin levels and enhanced its membrane localization. This effect did not occur with d-arginine, showing the involvement of NOS in this process. The NO-induced increase in utrophin was prevented by oxadiazolo-quinoxalin-1-one, an inhibitor of a soluble guanylate cyclase implicated in NO effects. These results open the way to a potential treatment for Duchenne and Becker dystrophies.

Establishment of a human thymic myoid cell line. Phenotypic and functional characteristics.

The subset of myoid cells is a normal component of the thymic stroma. To characterize these cells, we immortalized stromal cells from human thymus by using a plasmid vector encoding the SV40 T oncogene. Among the eight cell lines obtained, one had myoid characteristics including desmin and troponin antigens. This new line was designated MITC (myoid immortalized thymic cells). These cells expressed both the fetal and adult forms of muscle acetylcholine receptor (AChR) at the mRNA level, as well as the myogenic transcription factor MyoD1. alpha-Subunit AChR protein expression was detected by flow cytometry and the AChR was functional in patch-clamp studies. In addition, AChR expression was down-modulated by myasthenia gravis sera or by monoclonal antibody anti-AChR on MITC line similarly to TE671 rhabdomyosarcoma cells, making the MITC line an interesting tool for AChR antigenic modulation experiments. Finally, the MITC line expressed LFA-3, produced several cytokines able to act on T cells, and protected total thymocytes from spontaneous apoptosis in vitro. These results are compatible with a role of thymic myoid cells in some steps of thymocyte development. Therefore MITC line appears to be a useful tool to investigate the physiological role of thymic myoid cells.

Characteristics of skeletal muscle in mdx mutant mice.

We review the extensive research conducted on the mdx mouse since 1987, when demonstration of the absence of dystrophin in mdx muscle led to X-chromosome-linked muscular dystrophy (mdx) being considered as a homolog of Duchenne muscular dystrophy. Certain results are contradictory. We consider most aspects of mdx skeletal muscle: (i) the distribution and roles of dystrophin, utrophin, and associated proteins; (ii) morphological characteristics of the skeletal muscle and hypotheses put forward to explain the regeneration characteristic of the mdx mouse; (iii) special features of the diaphragm; (iv) changes in basic fibroblast growth factor, ion flux, innervation, cytoskeleton, adhesive proteins, mastocytes, and metabolism; and (v) different lines of therapeutic research.

The Schwann cell at the neuromuscular junction.

Synapses obtained in vitro in a system of co-culture of muscle cells and neurons are of embryonic type. We prepared a monoclonal antibody (6.17) which recognizes a molecule synthesized by Schwann cells and used it to show that the main characteristics of maturity (decrease in number of synapses, appearance of junctional folds, and suppression of butyrylcholinesterase expression) are under the control of Schwann cells. In addition, Schwann cells have the capacity to aggregate the acetylcholine receptors in myotube cultures.

Accumulation of acetylcholine receptors is a necessary condition for normal accumulation of acetylcholinesterase during in vitro neuromuscular synaptogenesis.

To study a step of the very complex processes of the formation of the neuromuscular junction (NMJ), we have analysed the clustering of acetylcholine receptors (AChR) and acetylcholinesterase (AChE) in myotubes cultured in various conditions. On the surface of rat myotubes cultured in the presence of spinal cord cells from embryonic rat, numerous AChE clusters appeared. Such clusters are always co-localized with AChR clusters, but the reverse is not true: the number of AChR clusters largely exceeds that of AChE clusters. Very few AChE clusters formed when such co-cultures were treated with monoclonal antibodies (mAbs) against the main immunogenic region (MIR) of the AChR, which provoke internalization and degradation of the AChRs of the muscular membrane. The total levels of AChE and proportions of molecular forms were unaffected. We also used non-innervated myotubes in which addition of agrin, a protein normally synthesized by motoneurons, transported to nerve terminals and inserted into the synaptic basal lamina, induces the formation of small clusters of AChE. When added to rat myotubes devoid of membrane AChR, agrin-induced AChE clusters did not form. Finally, we analysed the capacity of the variant of the C2 mouse muscle cell line deficient in AChR (1R-) to form clusters of AChE in co-cultures with spinal cord cells from rat: no formation of AChE clusters could be observed. In all these different systems of cultures, the conditions which prevented clustering of AChR (anti-AChR antibodies, deficiency of the variant C2 cell line) also suppressed AChE clustering. We concluded that clustering of AChR is a prerequisite for clustering of AChE, so that NMJ formation implies the sequential accumulation of these two components.

Schwann cells modify expression of acetylcholinesterase and butyrylcholinesterase at rat neuromuscular junctions.

Using a monoclonal antibody (6.17) directed against a Schwann antigen, we have shown that Schwann cells synthesize a molecule implicated in a change of expression of synaptic cholinesterases, AChE and BChE, during muscle differentiation. In vitro, during synaptogenesis, the two enzymes are first present at developing synapses, and addition of Schwann cells to muscle-neuron co-cultures induces a disappearance of BChE, leaving only AChE activity as in the adult neuromuscular junction. This effect is inhibited by the 6.17 antibody. Thus, a molecule produced by Schwann cells is involved in the maturation of the neuromuscular synapse, in addition to the neuronal factors (CGRP, ARIA/heregulin, agrin), which are known to control the synthesis, maturation and accumulation of acetylcholine receptors and other synaptic components. In addition, in vivo, in the newborn rat, butyrylcholinesterase and acetylcholinesterase activities are initially present in equal amounts in the neural zone, but butyrylcholinesterase levels diminish sharply between 7 and 15 days after birth, the stage at which the synaptic Schwann cell membrane becomes juxtaposed with the muscle membrane.

Inhibition of proliferation in 8-week-old mdx mouse muscle fibroblasts in vitro.

Our purpose is to understand why mdx muscle does not show the progressive degeneration observed in human Duchenne muscular dystrophy (DMD) muscle. In the mouse, the regenerative process compensates for the necrosis of the muscle fibers, particularly during the acute phase of the disease (5-9 weeks). In DMD muscle, there is a gradual failure of the regenerative process and the muscle fibers are replaced by connective and fatty tissue. We propose that distinct properties of mdx and DMD muscle fibroblasts could be one of the reasons for the differences between the mdx and DMD phenotypes. We found that fibroblasts taken from human DMD and control muscle had similar in vitro proliferative capacities. The proliferation rate of mouse muscle fibroblasts decreased during the acute phase of the disease, and inhibition was complete in fibroblasts from 8-week-old mdx mice. Moreover, the medium conditioned by these cells inhibited fibroblast proliferation. The effect was specific for fibroblasts, since this conditioned medium stimulated myoblast proliferation, as did control fibroblast-conditioned medium. These results suggest that 8-week-old mdx mouse muscle fibroblasts produce an inhibitor of their own proliferation and a growth factor specific for myoblasts in vitro. If these factors are secreted in vivo, the growth inhibitory factory may stop fibroblast proliferation whereas the mitogenic activity could stimulate satellite cell proliferation, thus favouring muscle regeneration.

Effect of sera from myasthenia gravis patients and of alpha-bungarotoxin on acetylcholinesterase during in vitro neuromuscular synaptogenesis.

Myasthenia gravis (MG) is mediated by circulating antibodies directed against acetylcholine receptor (AChR) but the antibody titre is poorly correlated with the clinical severity of the disease. We analysed acetylcholinesterase (AChE) activity, molecular forms and distribution during in vitro synaptogenesis, in the presence of sera from MG patient. We observed that the formation of AChE patches is inhibited in proportion to the anti-AChR antibody titre, whatever the clinical severity of the disease. The total activity and the proportion of the different molecular forms were unchanged suggesting that AChE level and distribution are controlled by independent mechanisms. To clarify the relationship between the mechanisms of AChE concentration during synaptogenesis and AChR concentration, we compared the effect of MG sera (receptors are internalised and degraded) and of the acetylcholine antagonist alpha-bungarotoxin (non-functional receptors are still present in the muscular membrane). In the presence of alpha-bungarotoxin, the number of AChR clusters, and AChE activity and concentration were equivalent to control values. The comparison of the results obtained with antibodies and alpha-bungarotoxin suggests that the presence and/or concentration of AChR is a necessary condition for normal concentration of AChE during synaptogenesis.

The effect of tetanus toxin on in vitro synaptogenesis.

Cultures of spinal cord neurons and cocultures of rat embryo neurons and muscle cells have been studied in the presence of tetanus toxin (TT) at a concentration of 40 micrograms/ml of medium. TT strongly stimulated neurite outgrowth, notably branching from the cell bodies. In addition it induced a marked, overall increase in acetylcholine receptor (AChR), but inhibited focalisation of AChR and acetylcholinesterase (AChE) at the synaptic sites. TT seems to act on neurite emergence, on the neuronal factor(s) controlling AChE and AChR concentrations, and on the factor(s) modulating degradation and/or synthesis of AChR.

Acetylcholinesterase in cocultures of rat myotubes and spinal cord neurons: effects of collagenase and cis-hydroxyproline on molecular forms, intra- and extracellular distribution, and formation of patches at neuromuscular contacts.

Cultures of rat myotubes from 18-day-old embryos produce both globular (G) and asymmetric (A) forms of acetylcholinesterase (AChE; EC 3.1.1.7), mostly G1, G4, and A12 and a small proportion of A8. We show that all forms are partly intracellular and partly exposed to the extracellular medium; the A forms and their intra- and extracellular distribution are not modified when myotubes are grown in the presence of spinal cord neurons. In these cocultures, however, AChE patches may be detected immunohistochemically at sites of neuromuscular contacts. These patches represent a very minor proportion of AChE activity. We found that collagenase removes AChE patches but not the acetylcholine receptor clusters with which they coincide. This digestion specifically decreases the level of the A12 form. cis-Hydroxyproline, an inhibitor of collagen synthesis, reduces the level of G1 and blocks the synthesis of A forms.

In vitro evidence for a neurite growth-promoting activity in Trembler mouse serum.

Basal lamina components, such as heparan sulfate proteoglycan (HSPG) and laminin play an important role in neuritic outgrowth for CNS and PNS neurons in culture. The mutant mouse 'Trembler' is characterized by hypomyelinization and production of an excess of basal lamina layers around Schwann cells in peripheral nerves. In order to analyse whether or not the serum of the mutant animals contains neurite outgrowth-promoting factors, we cultured rat spinal cord neurons in the presence of Trembler serum. Under these conditions, the outgrowth of neurites was increased approx. 2 times as compared to control serum. Trembler serum induces neuritic outgrowth characterized both by an increase in number of primary neurites emerging from the nerve cell body as well as by an increase in peripheral branching of neurites. To characterize the factors implicated in this increase we added antibodies directed against HSPG or laminin to the mutant serum. As a result, the increase in neuritic outgrowth was reduced or abolished in both cases. Trembler effect on neurite growth disappeared when the number of the non-neuronal cells was reduced, suggesting that the mutant serum did not act directly on neurons but by the intermediary action of non-neuronal cells.

Effect of myasthenic patient sera on the number and distribution of acetylcholine receptors in muscle and nerve-muscle cultures from rat. Correlations with clinical state.

We studied the functional activities (FA) of sera obtained from 83 myasthenic patients on rat muscle cultures. Using the same sets of cultures, two parameters were evaluated after exposure to sera: residual fraction (RF) of acetylcholine receptors (AChR) coupled to 125I-labelled alpha-bungarotoxin (alpha Bgt) (81 sera) and the number of rhodamine labelled clusters (56 sera). Two types of culture were assayed: muscle alone and nerve-muscle cocultures (12 cases). In all combinations (fluorescence, radiolabelling, muscle alone and nerve-muscle cocultures), we found a significant correlation between FA and antibody (Ab) titre, and no correlation between FA and clinical severity: only sera with a high or intermediate Ab titre were effective, whatever the clinical severity of disease. With active sera, AChR loss was about 50% whereas the disappearance of AChR clusters was quite complete, which suggests AChR redistribution induced by MG sera.

[Is muscular acetylcholinesterase activity correlated with the intracellular concentration of free calcium?]. / L'activité de l'acétylcholinestérase musculaire est-elle corrélée à la concentration du Ca2+ intracellulaire libre?

The role of the calcium ion Ca2+ as an agent of intracellular control in a variety of physiological processes is well established. In vertebrate skeletal muscle fibers, Ca2+ is involved in muscle contraction, modulation of membrane permeability and regulation of metabolic activity. Recently it was suggested that ion fluxes through membranes regulate the level of two cholinergic macromolecules, the acetylcholine receptor and the A12 form of acetylcholinesterase (AChE), the presumed synaptic form of the enzyme. Muscle cells paralysed by veratridine, which maintains the Na+ channel in the open state, showed an increase in total AChE and in the levels of the A12 form. The effect of veratridine on AChE was blocked in the presence of agents that block Ca2+ permeability suggesting that Ca2+ is involved in this effect. To understand whether the level of muscle AChE is related in some way to the level of free intracellular Ca2+, we analysed the variations of Ca2+ levels in rat muscle cells treated by agents which modify the ionic permeabilities. This level was determined by spectrofluorimetry using the fluorescent Ca2+ indicator: Quin 2. However no correlation between these parameters was observed in our experimental conditions.

Primary muscle cells cultivated in medium conditioned by spinal cord cells show changes in messenger RNA as detected by translation in ovo accompanied by synthesis of extracellular matrix components.

Primary muscle cell cultures were prepared from rat embryos and maintained in normal (unconditioned) and spinal cord (conditioned) media. In order to relate translational regulation to the morphological changes associated with each culture condition, mRNA was isolated from both culture variants and subjected to in ovo translation. Xenopus oocytes were injected with mRNA and their incubation media checked for the presence of newly formed proteins coded by the mRNAs and secreted into the medium. Electrophoretograms indicated mRNA-dependent synthesis of several proteins in the molecular mass range of 30-260 kD. To further characterize these proteins, antisera directed against several extracellular matrix proteins were used for immunoprecipitation: antigens recognized by anti-heparan-sulfate-proteoglycan and anti-fibronectin were enhanced in conditioned cells, whereas laminin was found to be reduced.

Presynaptic or postsynaptic origin of acetylcholinesterase at neuromuscular junctions? An immunological study in heterologous nerve-muscle cultures.

Numerous studies have shown that the acetylcholine receptor (AChR) is inserted in the plasma membrane of the muscle fiber, and that it is focalized at the site of neuromuscular junctions, as an effect of neural influence. In contrast, acetylcholinesterase (AChE) may be presynaptic or anchored in the basal lamina, as well as postsynaptic at neuromuscular junctions. We investigated the origin of the junctional enzyme, particularly the collagen-tailed asymmetric A12 forms, by studying the AChE contents of heterologous rat and chicken neuromuscular cocultures by immunohistochemical and biochemical methods. We found that the overall content of AChE, in the neuromuscular cocultures, including the A12 form, was essentially identical to the sum of the contents of separate myotube and motoneuron cultures. The sedimentation coefficients of the rat and chicken asymmetric forms are sufficiently different to clearly differentiate these enzymes in sucrose gradients: 16 S for rat, 20 S for chicken A12 AChE. Sedimentation analyses of AChE in cocultures thus showed that the A12 form was of muscular origin. In the case of aneural cultures of myotubes, histochemical staining of AChE activity or immunohistochemical staining with specific antibodies showed only very scarce, faint concentrations of enzyme. Some patches of acetylcholine receptor (AChR) were, however, visible in these cultures. Neuromuscular contacts are readily established in cocultures of myotubes with embryonic motoneurons from spinal cords. In the presence of motoneurons, the myotubes presented a larger number of AChR patches. The most remarkable feature of neuromuscular cocultures was the presence of numerous intense AChE patches which always coincided with AChR clusters. By specifically staining nerve terminals with tetanus toxin, we could show an excellent correlation between neuromuscular contacts and the presence of AChE-AChR patches. We found that the AChE patches in heterologous cocultures could be stained exclusively by the anti-myotube AChE antiserum. The focalized enzyme is therefore exclusively, or very predominantly, provided by the myotube.

Simultaneous demonstration of 3 synaptic markers: nerve endings, localization of AChE and AChR clusters at neuromuscular junctions of the rat in vitro.

Our triple labelling method allows the AChE and AChR distributions and the nerve endings to be stained in the same preparation. This method which provides a detailed visualization of the synaptic contacts and is capable of revealing a weak AChE activity is particularly valuable in studies of synaptogenesis.

Infection of cultured rat myotubes and neurons from the spinal cord by rabies virus.

Rabies virus multiplication was investigated in cultured primary rat myotubes and neurons. The susceptibility of these two cell types to fixed rabies challenge virus strain (CVS) was monitored by fluorescence and virus titration. Differentiated rat myotubes were susceptible to rabies virus infection, and showed an increasing accumulation of viral material from day one to day four. However, these cells did not release infective viral particles, nor did they accumulate infectious virions in the cytoplasm. In contrast, infected neurons released large amounts of infectious particles. Electron microscopy observation of infected myotubes showed minor alterations and the presence of typical viral inclusions in the cytoplasm without mature virions assembling viral membranes. Competition binding experiments show that alpha-bungarotoxin inhibits rabies virus infection from 10(-5) to 10(-7) M, whereas lower toxin concentrations failed to have any effect. These data do not confirm the hypothesis of a fixed rabies virus amplification step at the site of the viral entry. On the other hand, the high susceptibility of peripheral neurons to rabies virus infection is an argument for the direct uptake of virions by these cells. The restrictive viral multiplication in the myotubes is an alternative explanation for the local persistence of rabies virus at the site of inoculation.