Effects of antibodies to neural cell adhesion molecule (N-CAM) on the differentiation of neuromuscular contacts between ciliary ganglion neurons and myotubes in vitro

Previous experiments have suggested that the neural cell adhesion molecule (N-CAM) may have a role in initial nerve-muscle adhesion. To determine whether N-CAM might be involved in synaptic differentiation, we grew ciliary ganglion neurons and embryonic myotubes together in the presence and absence of monovalent antibodies to N-CAM. In normal cultures, undifferentiated neurites contact myotubes, and the nerve at some of these neurite-myotube contacts acquires concentrations of synaptic vesicle antigens. Most of these vesicle antigen-positive contacts become associated with patches of acetylcholine receptor (AChR) on the surface of the underlying myotube. Contacts without concentrations of vesicle antigens do not become associated with AChR patches. In the presence of antibodies to N-CAM, adhesion between neuronal somata and myotubes was reduced, but neurites contacted myotubes with near-normal frequency. The subsequent differentiation of nerve and muscle at these contacts, as assayed by the localization of vesicle antigens and AChR, proceeded normally in the presence of anti-N-CAM antibodies. The results suggest that N-CAM-mediated adhesion between neurite and myotube is not required for synaptic differentiation.     

Neuromuscular transmission to identified primary and secondary myotubes: a reevaluation of polyneuronal innervation patterns in rat embryos.

The early morphogenesis of rat skeletal muscle is a biphasic process involving two sequentially generated populations of myotubes. A small population of primary myotubes appears early and is followed by a much larger population of secondary myotubes which appear progressively over a number of days. All previously published electrophysiological studies of developing muscle have failed to appreciate the relevance of biphasic myotube production. Here we reevaluate the status of early motor innervation, taking into account the wide range of sizes and levels of maturity within the two myotube populations. Evoked end-plate potentials (EPPs) were recorded from fibers of E17-20 rat sternocostalis muscles. Impaled fibers were then marked by ejection of HRP from the recording pipet, enabling ultrastructural identification of fibers from which recordings had been made. The average number of synaptic inputs per fiber increased to a peak at E19, and the rate of rise of the EPPs increased with age. The majority of impaled fibers (76%) were subsequently found to be primary myotubes, even at ages when secondary myotubes formed the majority of fibers in the muscle. Electrophysiological studies during early stages of secondary myotube development therefore sample largely from the more mature primary fibers and probably give the wrong impression of the extent and degree of polyneuronal innervation and of synaptic rearrangement within the muscle. In addition, the results show that very young secondary myotubes are distinguished by EPPs of longer latency, slower rate of rise, and smaller size than those of other types of myotubes. These results suggest that young secondary myotubes are predominantly activated by EPPs which originate in adjoining primary myotubes and propagate electronically to the secondary myotube. We propose a new model of early synaptic rearrangement which accommodates the biphasic nature of muscle development. We also suggest that secondary myotubes do not require direct neural input for the initiation of their development.     

Development of basal lamina in synaptic and extrasynaptic portions of embryonic rat muscle

Each vertebrate skeletal muscle fiber is ensheathed by a basal lamina (BL) which passes through the synaptic cleft of the neuromuscular junction. In the adult, the synaptic portion of the BL is both functionally and chemically specialized. We have used an immunofluorescence method to compare the development of synaptic and extrasynaptic portions of BL in embryonic rat intercostal muscles. Immunohistochemical staining of adult muscle fibers with monoclonal and serum antibodies defines "synaptic" antigens (including acetylcholinesterase) that are concentrated in synaptic BL, "extrasynaptic" antigens that are concentrated in extrasynaptic regions, and "shared" antigens (including collagen IV, fibronectin, laminin, and a heparan sulfate proteoglycan) that are present in both synaptic and extrasynaptic BL ( Sanes and Chiu , 1983). Synapses appear on newly formed myotubes on embryonic Day 14 (E14; birth is on E22 ). Patches of BL that contain shared and extrasynaptic antigens are present on myotube surfaces by E15, and BL forms a continuous sheath by E17. Shared antigens are present at but not confined to synaptic areas by E15. Two synaptic antigens appear in synaptic areas a day later, and are not detectable extrasynaptically . At least one extrasynaptic antigen is present at immature synapses, and lost or masked by E19 . Thus synaptic BL is not assembled as a unit; rather, components are added, lost, or modified as synaptogenesis proceeds.     

Distribution of N-CAM in synaptic and extrasynaptic portions of developing and adult skeletal muscle

Previous studies of denervated and cultured muscle have shown that the expression of the neural cell adhesion molecule (N-CAM) in muscle is regulated by the muscle's state of innervation and that N-CAM might mediate some developmentally important nerve-muscle interactions. As a first step in learning whether N-CAM might regulate or be regulated by nerve-muscle interactions during normal development, we have used light and electron microscopic immunohistochemical methods to study its distribution in embryonic, perinatal, and adult rat muscle. In embryonic muscle, N-CAM is uniformly present on the surface of myotubes and in intramuscular nerves; N-CAM is also present on myoblasts, both in vivo and in cultures of embryonic muscle. N-CAM is lost from the nerves as myelination proceeds, and from myotubes as they mature. The loss of N-CAM from extrasynaptic portions of the myotube is a complex process, comprising a rapid rearrangement as secondary myotubes form, a phase of decline late in embryogenesis, a transient reappearance perinatally, and a more gradual disappearance during the first two postnatal weeks. Throughout embryonic and perinatal life, N-CAM is present at similar levels in synaptic and extrasynaptic regions of the myotube surface. However, N-CAM becomes concentrated in synaptic regions postnatally: it is present in postsynaptic and perisynaptic areas of the muscle fiber, both on the surface and intracellularly (in T-tubules), but undetectable in portions of muscle fibers distant from synapses. In addition, N-CAM is present on the surfaces of motor nerve terminals and of Schwann cells that cap nerve terminals, but absent from myelinated portions of motor axons and from myelinating Schwann cells. Thus, in the adult, N-CAM is present in synaptic but not extrasynaptic portions of all three cell types that comprise the neuromuscular junction. The times and places at which N-CAM appears are consistent with its playing several distinct roles in myogenesis, synaptogenesis, and synaptic maintenance, including alignment of secondary along primary myotubes, early interactions of axons with myotubes, and adhesion of Schwann cells to nerve terminals.     

A reevaluation of the role of innervation in primary and secondary myogenesis in developing chick muscle

The neural dependence of primary and secondary myogenesis and its relation to fiber-type differentiation was immunocytochemically investigated in chicken limb muscles. In a previous study, we demonstrated that a novel combination of slow myosin and fast Ca2(+)-ATPase antibodies differentially stained mutually exclusive populations of myotubes, which in the slow region of the iliofibularis allowed us to visualize primary and secondary myotubes and to quantify their development. When these antibodies were used to stain myotubes in muscles that were either chronically paralyzed by d-tubocurarine or denervated, we were surprised to observe by both LM and EM analysis that secondary myotubes formed in both cases, in contrast to the widely held tenet that nerve activity is necessary for secondary myogenesis. Also, an unexpected decrease in the number of primary myotubes occurred before the onset of secondary myotube formation. Although the total quantity of myotubes formed was drastically reduced by curare treatment or denervation, the ratio of fast to slow myotubes increased normally between st 34 and 39 1/2. Paralysis by curare did produce a striking increase in the size of individual myotube clusters, indicating that blocking nerve activity either increases adhesion between myotubes or prevents a normal decrease in adhesion during development which may be necessary for myofiber separation from clusters. Our findings indicate that both slow primary and fast secondary myotube populations are composed of nerve-dependent and independent individuals and that the relative quantities of fast and slow myotubes are regulated independent of innervation.     

Agrin-induced reorganization of extracellular matrix components on cultured myotubes: relationship to AChR aggregation

Agrin, an extracellular matrix-associated protein extracted from synapse-rich tissues, induces the accumulation of acetylcholine receptors (AChRs) and other synaptic components into discrete patches on cultured myotubes. The appearance of agrin-like molecules at neuromuscular junctions suggests that it may direct synaptic organization in vivo. In the present study we examined the role of extracellular matrix components in agrin-induced differentiation. We used immunohistochemical techniques to visualize the spatial and temporal distribution of laminin, a heparan sulfate proteoglycan (HSPG), fibronectin, and type IV collagen on cultured chick myotubes during agrin-induced aggregation of AChRs. Myotubes displayed significant amounts of laminin and HSPG, lesser amounts of type IV collagen, and little, if any, fibronectin. Agrin treatment caused cell surface laminin and HSPG to patch, while collagen and fibronectin distributions were generally unaffected. Many of the agrin-induced laminin and HSPG patches colocalized with AChR patches, raising the possibility of a causal relationship between matrix patching and AChR accumulations. However, patching of AChRs (complete within a few hours) preceded that of laminin or HSPG (not complete until 15-20 h), making it unlikely that matrix accumulations initiate AChR patching at agrin-induced sites. Conversely, when AChR patching was blocked by treatment with anti-AChR antibody mAb 35, agrin was still able to effect patching of laminin and HSPG. Taken together, these findings suggest that agrin-induced accumulations of AChR and laminin/HSPG are not mechanistically linked.     

Plasminogen activators and inhibitors in the neuromuscular system: III. The serpin protease nexin I is synthesized by muscle and localized at neuromuscular synapses

Recent studies suggest that the nature of events leading to the formation, maintenance, and elimination of synapses may be regulated by cascade-type, locally expressed proteases and protease inhibitors acting on adhesive extracellular matrix components. We have identified a molecule in conditioned medium of murine skeletal muscle cells that in molecular weight, target protease inhibition, heparin-binding and cross-reactivity with authenic antisera is similar to the human serine proteinase inhibitor, protease nexin I. Protease nexin I is a 43-50 kDa glycoprotein of the serpin superfamily (arg-serpin class). Purified anti-protease nexin I antibody (anti-47 kDa) stains adult mouse skeletal muscle in discrete foci that precisely superimpose on synaptic neuromuscular junctions. Protease nexin I appears in patches on surfaces of cultured mouse skeletal myotubes, but not on myoblasts. These patches co-localize with acetylcholine receptor clusters and acetylcholinesterase staining during cellular maturation in culture. Evidence that protease nexin I is a synaptic, extracellular antigen is particularly intriguing since it has been shown to be identical, in structure and activity, with a factor released by glial cells, called glia-derived nexin that stimulates mouse neuroblastoma cell neurite outgrowth and inhibits granule cell migration. Protease nexin I inhibits both tumor cell and myoblast plasminogen activator-mediated destruction of extracellular matrix. Thus, such observations as presented in this report provide further evidence for involvement of cascade proteolytic systems, and their post-translational regulation by specific serpins, in the remodeling that occurs in synapse formation and elimination.     

Neutrophils injure cultured skeletal myotubes

The purpose of the study was to test the hypothesis that neutrophils can injure cultured skeletal myotubes. Human myotubes were grown and then cultured with human blood neutrophils. Myotube injury was quantitatively and qualitatively determined using a cytotoxicity (51Cr) assay and electron microscopy, respectively. For the 51Cr assay, neutrophils, under non-in vitro-stimulated and N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated conditions, were cultured with myotubes at effector-to-target cell (E:T) ratios of 10, 30, and 50 for 6 h. Statistical analyses revealed that myotube injury was proportional to the E:T ratio and was greater in FMLP-stimulated conditions relative to non-in vitro-stimulated conditions. Transmission electron microscopy, using lanthanum as an extracellular tracer, revealed in cocultures a diffuse appearance of lanthanum in the cytoplasm of myotubes and a localized appearance within cytoplasmic vacuoles of myotubes. These observations and their absence in control cultures (myotubes only) suggest that neutrophils caused membrane rupture and increased myotube endocytosis, respectively. Myotube membrane blebs were prevalent in scanning and transmission electron micrographs of cultures consisting of neutrophils and myotubes (E:T ratio of 5) and were absent in control cultures. These data support the hypothesis that neutrophils can injure skeletal myotubes in vitro and may indicate that neutrophils exacerbate muscle injury and/or delay muscle regeneration in vivo.     

Antibodies that bind specifically to synaptic sites on muscle fiber basal lamina

Basal lamina (BL) ensheathes each skeletal muscle fiber and passes through the synaptic cleft at the neuromuscular junction. Synaptic portions of the BL are known to play important roles in the formation, function, and maintenance of the neuromuscular junction. Here we demonstrate molecular differences between synaptic and extrasynaptic BL. We obtained antisera to immunogens that might be derived from or share determinants with muscle fiber BL, and used immunohistochemical techniques to study the binding of antibodies to rat skeletal muscle. Four antisera contained antibodies that distinguished synaptic from extrasynaptic portions of the muscle fiber's surface. They were anti- anterior lens capsule, anti-acetylcholinesterase, anti-lens capsule collagen, and anti-muscle basement membrane collagen; the last two sera were selective only after antibodies binding to extrasynaptic areas had been removed by adsorption with connective tissue from endplate-free regions of muscle. Synaptic antigens revealed by each of the four sera were present on the external cell surface and persisted after removal of nerve terminal. Schwann cell, and postsynaptic plasma membrane. Thus, the antigens are contained in or connected to BL of the synaptic cleft. Details of staining patterns, differential susceptibility of antigens to proteolysis, and adsorption experiments showed that the antibodies define at least three different determinants that are present in synaptic but not extrasynaptic BL.     

Myotube Formation on Micro-patterned Glass: Intracellular Organization and Protein Distribution in C2C12 Skeletal Muscle Cells

Proliferation and fusion of myoblasts are needed for the generation and repair of multinucleated skeletal muscle fibers in vivo. Studies of myocyte differentiation, cell fusion, and muscle repair are limited by an appropriate in vitro muscle cell culture system. We developed a novel cell culture technique [two-dimensional muscle syncytia (2DMS) technique] that results in formation of myotubes, organized in parallel much like the arrangement in muscle tissue. This technique is based on UV lithography–produced micro-patterned glass on which conventionally cultured C2C12 myoblasts proliferate, align, and fuse to neatly arranged contractile myotubes in parallel arrays. Combining this technique with fluorescent microscopy, we observed alignment of actin filament bundles and a perinuclear distribution of glucose transporter 4 after myotube formation. Newly formed myotubes contained adjacently located MyoD-positive and MyoD-negative nuclei, suggesting fusion of MyoD-positive and MyoD-negative cells. In comparison, the closely related myogenic factor Myf5 did not exhibit this pattern of distribution. Furthermore, cytoplasmic patches of MyoD colocalized with bundles of filamentous actin near myotube nuclei. At later stages of differentiation, all nuclei in the myotubes were MyoD negative. The 2DMS system is thus a useful tool for studies on muscle alignment, differentiation, fusion, and subcellular protein localization. (J Histochem Cytochem 56:881–892, 2008)     

Cell surface acetylcholinesterase molecules on multinucleated myotubes are clustered over the nucleus of origin

Multinucleated skeletal muscle fibers are compartmentalized with respect to the expression and organization of several intracellular and cell surface proteins including acetylcholinesterase (AChE). Mosaic muscle fibers formed from homozygous myoblasts expressing two allelic variants of AChE preferentially translate and assemble the polypeptides in the vicinity of the nucleus encoding the mRNA (Rotundo, R. L. 1990. J. Cell Biol. 110:715-719). To determine whether the locally synthesized AChE molecules are targeted to specific regions of the myotube surface, primary quail myoblasts were mixed with mononucleated cells of the mouse muscle C2/C12 cell line and allowed to fuse, forming heterospecific mosaic myotubes. Cell surface enzyme was localized by immunofluorescence using an avian AChE-specific monoclonal antibody. HOECHST 33342 was used to distinguish between quail and mouse nuclei in myotubes. Over 80% of the quail nuclei exhibited clusters of cell surface AChE in mosaic quail-mouse myotubes, whereas only 4% of the mouse nuclei had adjacent quail AChE-positive regions of membrane, all of which were located next to a quail nucleus. In contrast, membrane proteins such as Na+/K+ ATPase, which are not restricted to specific regions of the myotube surface, are free to diffuse over the entire length of the fiber. These studies indicate that the AChE molecules expressed in multinucleated muscle fibers are preferentially transported and localized to regions of surface membrane overlying the nucleus of origin. This targeting could play an important role in establishing and maintaining specialized cell surface domains such as the neuromuscular and myotendinous junctions.     

THE FINE STRUCTURE OF MOTOR ENDPLATE MORPHOGENESIS

The fine structure of the developing neuromuscular junction of rat intercostal muscle has been studied from 16 days in utero to 10 days postpartum. At 16 days, neuromuscular relations consist of close membrane apposition between clusters of axons and groups of myotubes. Focal electron-opaque membrane specializations more intimately connect axon and myotube membranes to each other. What relation these focal contacts bear to future motor endplates is undetermined. The presence of a group of axons lying within a depression in a myotube wall and local thickening of myotube membranes with some overlying basal lamina indicates primitive motor endplate differentiation. At 18 days, large myotubes surrounded by new generations of small muscle cells occur in groups. Clusters of terminal axon sprouts mutually innervate large myotubes and adjacent small muscle cells within the groups. Nerve is separated from muscle plasma membranes by synaptic gaps partially filled by basal lamina. The plasma membranes of large myotubes, where innervated, simulate postsynaptic membranes. At birth, intercostal muscle is composed of separate myofibers. Soleplate nuclei arise coincident with the peripheral migration of myofiber nuclei. A possible source of soleplate nuclei from lateral fusion of small cells' neighboring areas of innervation is suspected but not proven. Adjacent large and small myofibers are mutually innervated by terminal axon networks contained within single Schwann cells. Primary and secondary synaptic clefts are rudimentary. By 10 days, some differentiating motor endplates simulate endplates of mature muscle. Processes of Schwann cells cover primary synaptic clefts. Axon sprouts lie within the primary clefts and are separated from each other. Specific neural control over individual myofibers may occur after neural processes are segregated in this manner.     

Tissue specific O-linked glycosylation of the neural cell adhesion molecule (N-CAM)

We have shown previously that the predominant N-CAM isoform in skeletal muscle myotubes contains as a result of alternative splicing a novel domain (MSD1) in its extracellular region. Here we show that this region represents a site for O-linked carbohydrate attachment. The lipid tailed N-CAM in myotubes was found to bind peanut lectin while the transmembrane isoform from myoblasts lacking MSD1 did not. In addition, N-CAM from a variety of neural sources failed to bind the lectin. Analysis of 3T3 fibroblasts transfected with various N-CAM cDNAs, showed that peanut lectin binding was correlated specifically with the expression of the MSD1 region. The oligosaccharides isolated from a purified preparation of myotube N-CAM were shown to contain an O-linked oligosaccharide whose core structure was a sialylated version of Gal beta 1----3GalNac which is the structure recognized specifically by peanut lectin. These data provide the first evidence for the expression of O-linked carbohydrate on any N-CAM isoform and more specifically target this oligosaccharide to the MSD1 region of myotube N-CAM.     

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.     

Mannose receptor regulates myoblast motility and muscle growth

Myoblast fusion is critical for the formation, growth, and maintenance of skeletal muscle. The initial formation of nascent myotubes requires myoblast-myoblast fusion, but further growth involves myoblast-myotube fusion. We demonstrate that the mannose receptor (MR), a type I transmembrane protein, is required for myoblast-myotube fusion. Mannose receptor (MR)-null myotubes were small in size and contained a decreased myonuclear number both in vitro and in vivo. We hypothesized that this defect may arise from a possible role of MR in cell migration. Time-lapse microscopy revealed that MR-null myoblasts migrated with decreased velocity during myotube growth and were unable to migrate in a directed manner up a chemoattractant gradient. Furthermore, collagen uptake was impaired in MR-null myoblasts, suggesting a role in extracellular matrix remodeling during cell motility. These data identify a novel function for MR during skeletal muscle growth and suggest that myoblast motility may be a key aspect of regulating myotube growth.     

Relationship of muscle growth in vitro to sodium pump activity and transmembrane potential

Serum stimulates embryonic avian skeletal muscle growth in vitro and the growth-related processes of amino acid transport and protein synthesis. Serum also stimulates myotube Na pump activity (measured as ouabain-sensitive rubidium-86 uptake) for at least 2 h after serum addition. Serum-stimulated growth depends on this Na pump activity since ouabain added at the same time as serum totally inhibits the growth responses. The relationship of myotube growth, Na pump activity, and transmembrane potential was studied to determine whether serum-stimulated Na pump activation and growth are coupled by long-term membrane hyperpolarization. When myotube amino acid transport and protein synthesis are prestimulated by serum, ouabain was found to have little inhibitory effect, indicating that the already stimulated growth-related processes are not tightly coupled to continued Na pump activity. Serum-stimulated protein synthesis is tightly coupled to Na pump activity, but only during the first 5-10 min after serum addition. When myotube transmembrane potentials were measured using the lipophilic cation tetraphenylphosphonium, serum at concentrations that stimulate myotube growth and Na pump activity was found to have little effect on the cell's transmembrane potential. Furthermore, partial depolarization of the myotubes with 12- to 55-mM extracellular potassium does not prevent serum stimulation of myotube growth. Monensin was found to hyperpolarize the myotubes, but causes myotube atrophy. These results indicate that although Na pump activity is associated with initiation of serum-stimulated myotube growth, continued Na pump activity is not essential, and there is little relationship between myotube growth and the myotube's transmembrane potential.     

Developmental regulation of laminin accumulation in the extracellular matrix of a mouse muscle cell line

We have investigated the synthesis, accumulation, and secretion of laminin, an extracellular matrix glycoprotein, during differentiation of the C2 mouse skeletal muscle cell line in culture. Myoblasts actively synthesized laminin, as measured by incorporation of [35S]methionine and by a dot-immunobinding assay. In myoblast cultures laminin accumulated in an intracellular compartment and could be extracted with a physiological salt solution containing the detergent Triton X-100. After the culture medium was replaced to promote differentiation of myoblasts to myotubes, laminin synthesis was increased, and laminin began to accumulate in the medium in soluble form. During differentiation, laminin also accumulated in an insoluble cell-associated fraction that required guanidinium chloride for extraction. Indirect immunofluorescence and immunobinding assays showed that myotubes but not myoblasts contained laminin on their external surface. The time course of increase in surface laminin paralleled that of the accumulation of insoluble laminin. These results suggest that the insoluble fraction represents laminin bound to the extracellular matrix at the cell surface. Our experiments demonstrate, contrary to previous observations, that myotube cultures synthesize and accumulate laminin, and further, that the differentiation of proliferating myoblasts to multinucleated myotubes is accompanied by increased laminin synthesis, by secretion of laminin into the medium, and by the deposition of laminin into an extracellular matrix on the myotube surface.