Neural cell adhesion molecule in normal, denervated, and myopathic human muscle

The neural cell adhesion molecule (N-CAM) is a cell-surface glycoprotein that may mediate some intercellular adhesive interactions in the nervous system. In adult rat muscle, N-CAM is concentrated near neuromuscular junctions and on satellite cells, but is nearly undetectable in nonsynaptic portions of myofibers. However, N-CAM is abundant throughout myofibers in denervated and regenerating muscles. Using affinity-purified antibodies to N-CAM, we were able to demonstrate a similar distribution and regulation of N-CAM in human muscle. Myofiber N-CAM was not detectable immunohistochemically in any of 10 normal biopsies or in 4 biopsies that were abnormal but showed no evidence of fiber denervation or regeneration. N-CAM was present, however, at end plates, nerves, and satellite cells in normal human muscle. In contrast, myofiber N-CAM was detected in 16 of 16 patients with histological evidence of denervation and in 10 of 10 patients who had myopathy with degenerating/regenerating myofibers. In addition, 2 of 2 histologically nondiagnostic biopsies from patients with amyotrophic lateral sclerosis contained N-CAM-positive myofibers. Immunoblot analysis also detected N-CAM in denervated and myopathic, but not normal, human muscle. These results suggest that N-CAM may play a role in muscle reinnervation or regeneration and that N-CAM immunohistochemistry may complement conventional techniques in the diagnosis of neuromuscular disease.     

Accurate reinnervation of motor end plates after disruption of sheath cells and muscle fibers

After injury, regenerating motor axons grow back to form neuromuscular junctions at the original synaptic sites on muscle fibers. The pathways they grow along consist of basement membrane, Schwann cells, and perineurium that remained after degeneration of the original axons. All the factors necessary for directing axons to the original synaptic sites persist in muscles even after disruption of myofibers. The aim of the present experiments was to determine whether structural integrity of nerve sheath cells is required for precise reinnervation in the presence and absence of muscle fiber targets. The region of innervation of the cutaneous pectoris muscle of the frog was briefly frozen to eliminate all living cells from neuromuscular junctions, intramuscular nerve bundles, and from a 1-3-mm length of the nerve trunk. Only extracellular matrices persisted within the frozen region of muscle and nerve. These consisted of the basement membrane sheaths of myofibers, of Schwann cells, and of perineurial cells and the small fragments of disrupted cells that were bound to them. In some preparations new muscle fibers developed within the basement membrane sheaths. Regenerating axons grew through the naked basement membrane sheaths of original Schwann cells, formed numerous branches, and contacted the myofibers precisely at the original synaptic sites. By 5 weeks 75% of the original synaptic sites became reinnervated; the terminals were indistinguishable from those at normal neuromuscular junctions. In contrast, preparations in which all muscle fibers were prevented from regenerating far fewer synaptic sites became reinnervated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association of IGF-I and IGF-II with myofiber regeneration in vivo

This study examined expression of insulinlike growth factor (IGF) in the myofibers and nonmyofibrillar structures of murine soleus muscle following contraction-induced damage. Identifying the cellular sources of this myogenic growth factor could improve muscle rehabilitation strategies. Immunohistochemical analysis of muscle sections indicated that the number of myofibers expressing both IGF-I and IGF-II increased significantly at 4, 7, and 10 days following injury, compared with control. Muscle spindles and vascular tissue expressed only IGF-II, and staining intensity did not change following injury. The number of fibers expressing developmental myosin heavy chain increased significantly at 7 and 10 days postinjury, and these usually coexpressed IGF. No IGF-specific staining of interstitial/inflammatory cells was observed. Therefore, expression of IGF after mechanically induced fiber damage occurs exclusively within regenerating fibers without supplemental delivery of IGF to the tissue by inflammatory cells or changes in constitutive expression of IGF-II in vascular tissue.     

Cell adhesion molecules (CAMs) in adrenal medulla in situ and in vitro: enhancement of chromaffin cell L1/Ng-CAM expression by NGF

We have studied the expression of the cell adhesion molecules (CAMs) L1/Ng-CAM, N-CAM, J1/tenascin, and myelin-associated glycoprotein and their common carbohydrate L2/HNK-1 epitope in normal rat adrenal gland sections as well as in adrenal medulla cell culture with and without NGF stimulation. In situ L1/Ng-CAM was observed on the surface of some but not all chromaffin cell clusters, including their closely associated extracellular matrix (ECM). N-CAM immunoreactivity was present on all chromaffin cells and ECM. The ECM of whole medullas also expressed J1/tenascin molecules. In long-term cultures, nerve growth factor (NGF) stimulation enhanced L1/Ng-CAM, N-CAM, and Thy 1.1 immunolabeling on chromaffin cells and their processes. Process outgrowth was greater from chromaffin cell clusters containing S-100 positive Schwann cells as compared to dispersed single chromaffin cells. When long bundles of chromaffin cell fibers were present, S-100, L1/Ng-CAM, and N-CAM positive Schwann cells were always found and were grouped in distinct clusters in the intervals between the chromaffin cells. In some areas, however, after NGF stimulation some chromaffin cell process development occurred despite an apparent lack of close contact with Schwann cells. NGF-activated chromaffin cells also demonstrated neurofilament- and vimentin-like-immunoreactive filaments within cell bodies and their processes. Chromaffin cells were usually found on a layer of N-CAM and fibronectin positive fibroblasts, and often were associated with laminin-immunoreactive material. These data suggest a possible role of N-CAM and L1/Ng-CAM as well as ECM laminin in process outgrowth from chromaffin cells.     

Human fetal muscle-specific antigen is restricted to regenerating myofibers in diseased adult muscle

A monoclonal antibody, 5.1H11, directed against human fetal muscle and myogenic cells in tissue culture, was used for immunofluorescence analysis of frozen sections of muscle biopsies from 24 patients with different diseases of muscle. The staining pattern was highly specific; detectable levels of the 5.1H11 antigen were restricted to regenerating myofibers as assessed by comparison with serial sections stained with hematoxylineosin. There was no appreciable staining of intrafusal, normal adult extra fusal, denervated, degenerating, or necrotic muscle fibers. Thus, the 5.1H11 antibody allows unambiguous identification of regenerating myofibers in biopsy specimens.     

Extracellular matrix molecules and their possible roles in the regeneration of frog nervous system

Recent biochemical and histochemical analyses explored different components of the extracellular matrix (ECM) in the nervous system, and either permissive or non-permissive roles in neuronal development and regeneration were suggested. The aim of this study was to detect the distribution pattern of a few of these molecules in the nervous system of intact frogs and during nerve regeneration. The hyaluronan (HA) and tenascin C reactions were negative in the peripheral nerves, but appeared in their entry zones. In the CNS, different populations of neurons were surrounded with HA and tenascin C-positive material, forming a perineuronal net (PN). The phosphacan reaction was weakly positive in the PNS, and a moderate intensity was detected in the entry zone and in the PN. Laminin and fibronectin immunoreactivity was strong in the PNS, but laminin could not be detected in the CNS. In animals with cut and regenerating vestibulocochlear nerve, the distribution of the ECM molecules in the CNS and PNS characteristically changed from that of the normal pattern. Our results showed a non-homogenous distribution of ECM components in the frog nervous system that could be associated with their different roles in physiological and pathological processes.     

Expression of extracellular matrix components in a highly infiltrative in vivo glioma model

This work demonstrates the expression of extracellular matrix (ECM) components in a highly infiltrative brain tumor model developed by simple inoculation of spheroids from five human glioma biopsy tissues directly into the brains of immunodeficient rats. Non-invasive tumors derived from one glioblastoma biopsy specimen and two glioma cell lines (D-54MG and U-251MG) were also included in this study. The extent of tumor cell infiltration was studied using a pan-human monoclonal anti-vimentin antibody. The cellular origin for several of these ECM components was identified using human-specific monoclonal antibodies and polyclonal antibodies detecting epitopes from both species. Immunostaining revealed a diffuse parenchymal staining of glioma-produced tenascin, whereas vitronectin was produced mainly by the invading glioma cells. ECM components such as laminin, fibronectin and collagen type IV were most probably produced by the host and were mainly associated with the blood vessels in the tumors. However, some parenchymal staining with regional variations was observed. The expression pattern of these components was different in cell lines tumors as compared to the biopsy specimen tumors. The alpha3 and beta1 integrin subunits were mainly observed in areas of tumor cell invasion in the invasive tumors. In conclusion, the observed staining patterns clarify the cellular origin and indicate the possible biological function of tenascin, vitronectin, laminin, fibronectin and collagen type IV in these highly invasive malignant tumors of glial origin.     

Mammalian target of rapamycin complex 1 is involved in differentiation of regenerating myofibers in vivo

This work was undertaken to provide further insight into the role of mammalian target of rapamycin complex 1 (mTORC1) in skeletal muscle regeneration, focusing on myofiber size recovery. Rats were treated or not with rapamycin, an mTORC1 inhibitor. Soleus muscles were then subjected to cryolesion and analyzed 1, 10, and 21 days later. A decrease in soleus myofiber cross-section area on post-cryolesion days 10 and 21 was accentuated by rapamycin, which was also effective in reducing protein synthesis in these freeze-injured muscles. The incidence of proliferating satellite cells during regeneration was unaltered by rapamycin, although immunolabeling for neonatal myosin heavy chain (MHC) was weaker in cryolesion+rapamycin muscles than in cryolesion-only muscles. In addition, the decline in tetanic contraction of freeze-injured muscles was accentuated by rapamycin. This study indicates that mTORC1 plays a key role in the recovery of muscle mass and the differentiation of regenerating myofibers, independently of necrosis and satellite cell proliferation mechanisms.     

Transplanted myoblasts can migrate several millimeters to fuse with damaged myofibers in nonhuman primate skeletal muscle

A major restriction of the intramuscular transplantation of myoblasts is that the grafted cells fuse mostly with myofibers along the injection trajectories. This has been attributed to a "lack of migration ability" of the grafted myoblasts. It has been assumed that grafted myoblasts remain motionless in the sites of delivery and fuse only with myofibers with which they come into contact. In the present study, we analyzed this phenomenon in 17 cynomolgus monkeys. We found that intramuscularly injected myoblasts within 1 hour after their injection are mainly located in the perimysium and not distributed along the injection trajectories. This suggested that the grafted myoblasts later migrate from the perimysium to fuse with myofibers that are damaged by the injections. Therefore, we analyzed whether β-galactosidase-labeled myoblasts injected subcutaneously over skeletal muscles migrate in needle-damaged and nondamaged muscle regions. We observed that grafted myoblasts migrated up to 1cm in depth from the muscle surface into the muscles, although they seemingly fused mainly with damaged myofibers. Our findings suggest that myoblast transplantation is not necessarily restricted bya "lack of migration ability" of the grafted cells but by the fact that myoblasts fuse with regenerating myofibers and not with undamaged myofibers.     

A variant of Fukuyama congenital muscular dystrophy in a non-Japanese child

We report a case of Fukuyama congenital muscular dystrophy with inflammatory infiltrate on muscle biopsy in an American girl of non-Japanese ancestry. The child was hypotonic, had decreased muscle strength in all extremities, and poor head control. Her mental and motor development were delayed. She developed generalized seizures at 19 months of age. Her muscle enzymes were abnormal; cranial computed tomography demonstrated hypoplasia of the cerebellum. Electromyogram was normal. Deltoid muscle biopsy documented scattered basophilic regenerating myofibers and focal atrophic fibers with focal increases of endomysial connective tissue, small endomysial foci of inflammatory cells, and occasional perimysial, perivenular lymphocytic infiltrates. Prednisone therapy produced some decrease in serum muscle enzyme levels.     

Regulation of class I MHC expression in skeletal muscle: deleterious effect of aberrant expression on myogenesis

Aberrant expression of class I major histocompatibility complex (MHC) occurs on myofibers in inflammatory myopathies. The mechanisms responsible for such expression are unknown. Here we show that class I MHC expression is developmentally regulated during muscle regeneration with significant levels only in myoblasts. Injection of gamma-IFN plasmid leads to muscle inflammation, induction of class I MHC in regenerating, but not mature myofibers, and attenuation of regeneration. Gene transfer of class I MHC in vivo and in vitro also attenuates muscle differentiation. Thus, cytokines may contribute to the pathogenesis of inflammatory myopathies by upregulating class I MHC and leading to deleterious effects on muscle repair.     

The synaptic CT carbohydrate modulates binding and expression of extracellular matrix proteins in skeletal muscle: Partial dependence on utrophin

The CT carbohydrate, Neu5Ac/Neu5Gcα2,3[GalNAcβ1,4]Galβ1,4GlcNAcβ-, is specifically expressed at the neuromuscular junction in skeletal myofibers of adult vertebrates. When Galgt2, the glycosyltransferase that creates the synaptic β1,4GalNAc portion of this glycan, is overexpressed in extrasynaptic regions of the myofiber membrane, α dystroglycan becomes glycosylated with the CT carbohydrate and this coincides with the ectopic expression of synaptic dystroglycan-binding proteins, including laminin α4, laminin α5, and utrophin. Here we show that both synaptic and extrasynaptic forms of laminin and agrin have increased binding to the CT carbohydrate compared to sialyl-N-acetyllactosamine, its extrasynaptically expressed precursor. Muscle laminins also show increased binding to CT-glycosylated muscle α dystroglycan relative to its non-CT-containing glycoforms. Overexpression of Galgt2 in transgenic mouse skeletal muscle increased the mRNA expression of extracellular matrix (ECM) genes, including agrin and laminin α5, as well as utrophin, integrin α7, and neuregulin. Increased expression of ECM proteins in Galgt2 transgenic skeletal muscles was partially dependent on utrophin, but utrophin was not required for Galgt2-induced changes in muscle growth or neuromuscular development. These experiments demonstrate that overexpression of a synaptic carbohydrate can increase both ECM binding to α dystroglycan and ECM expression in skeletal muscle, and they suggest a mechanism by which Galgt2 overexpression may inhibit muscular dystrophy and affect neuromuscular development.     

Dystrophin and a dystrophin-related protein in intrafusal muscle fibers,and neuromuscular and myotendinous junctions

Summary To determine whether or not and how dystrophin exists in neuromuscular junctions (NMJs) and myotendinous junctions (MTJs), we studied the mid-belly and peripheral portions of control and mdx muscles, immunohistochemically and immunoelectrophoretically, using six kinds of polyclonal antibodies, and an antibody against a dystrophin-related protein (DRP). In controls these regions and the polar region of intrafusal muscle fibers showed a rather clearer immunohistochemical dystrophin reaction than those of extrafusal muscle fibers with all antibodies used. In the muscles of mdx mice NMJs only showed a positive dystrophin reaction with the c-terminal antibody, that is, no reaction with the other five antibodies, and MTJs in mdx showed a positive reaction with the c-terminal antibody and a faint to negative reaction with the other five antibodies. In biopsied human muscles NMJs and MTJs also showed a clear reaction with all ten antibodies, i.e., six polyclonal and four monoclonal ones. Although an immunohistochemical DRP reaction was clearly seen at NMJs, only a faint or no reaction was seen on MTJs and on intrafusal muscle fibers in both mouse and human materials. Western blot analysis of control mouse muscle for dystrophin showed a clearer band for the peripheral portion, which contains many MTJs, than for the mid-belly portion. These data suggest that dystrophin really exists on MTJs, and that dystrophin and DRP exist on NMJs in mouse and human muscles.     

Effect of denervation on regenerating muscle plasma membrane integrity: freeze-fracture and dystrophin immunostaining analyses

Summary We investigated time sequentially, the densities of intramembranous assembly orthogonal arrays of regenerating rat extensor digitorum longus muscle after bupivacaine-induced muscle injury. There was no evidence of orthogonal arrays at the early stage, but the densities of orthogonal arrays increased with the maturation of the innervated regenerating myofibers. In contrast, the orthogonal arrays were scarcely observed at any time point examined in denervated regenerating muscles. Therefore, the neural factor may have an important effect on the appearance of orthogonal arrays. Moreover, we studied the immunostainability of Duchenne muscular dystrophy gene product dystrophin in the regenerating muscle at the same time points. Positive immunostaining was observed in both innervated and denervated regenerating myofibers even from the early stage of regeneration. On the basis of these data, the relationship between orthogonal array and dystrophin are discussed.Supported in part by grants from the Ministry of Education and from the National Center for Nervous, Mental and Muscular Disorders of the Ministry of Health and Welfare (62A-2-20), Japan     

Expression of superfast myosin in aneural regenerates of cat jaw muscle

We investigated whether innervation is necessary for the expression of superfast myosin in regenerating cat jaw-closing muscle. Strips of jaw muscle were permitted to regenerate bilaterally in the beds of a fast limb muscle with innervation on one side being prevented surgically. Immunocytochemical analyses using anti-myosin heavy chain antibodies were done at various times postoperatively, the latest being after 78 days. We found little difference between innervated and uninnervated regenerates up to 27 days postoperatively. All regenerating myotubes expressed fetal myosin. In addition, some myotubes stained for superfast or slow myosin, while others stained for both superfast and slow myosins. Subsequently, uninnervated myotubes became atrophic but continued to express fetal, slow, and superfast myosins while innervated myofibers suppressed fetal and slow myosin expression. These results are consistent with the notion that satellite cells of jaw-closing muscles are committed to express superfast myosin during myogenesis, and that the expression of this program is independent of innervation.     

Human melanoma/NG2 chondroitin sulfate proteoglycan is expressed in the sarcolemma of postnatal human skeletal myofibers. Abnormal expression in merosin-negative and Duchenne muscular dystrophies

NG2 is the rat homologue of the human melanoma chondroitin sulfate proteoglycan (MCSP) preferentially expressed in dividing progenitor cells of the glial and mesenchymal lineage but downregulated after differentiation. It has recently been demonstrated that MCSP/NG2 expression is not restricted to mitotic or malignant cells. We show that MCSP/NG2 expression is detectable in the sarcolemma, and in the neuromuscular junction of human postnatal skeletal muscle, and it gradually reduces with advancing age. In human and murine myogenic cell lines, we found no clear differences in MCSP/NG2 expression between myoblasts and myotubes. Reduced levels of the core protein were found in merosin-negative congenital muscular dystrophy (MDC1A). Duchenne muscular dystrophy patients muscles exhibited an overexpression of the MCSP/NG2 core protein. In gamma-sarcoglycanopathy and calpainopathy, MCSP/NG2 upregulation was restricted to regenerating myofibers. We demonstrate that MCSP/NG2 is expressed in differentiated myofibers, and appears to have a role in the pathogenesis of MDC1A and severe dystrophinopathies.     

Increased laminin a expression in regenerating myofibers neuromuscular disorders

Laminin is a basement membrane (BM) glycoprotein composed of three of five subunits, the A, M, B1, B2, and the S chain. Four forms of laminin, A-B1-B2, A-S-B2, M-B1-B2, and M-S-B2, have been identified. Laminin is implicated in various biological processes such as cell adhesion and differentiation. We studied immunohistochemically the expression of the four laminin subunits A, M, B1, B2 as well as of neural cell adhesion molecule (N-CAM, CD56), a marker of regenerating myofibers, in various neuromuscular disorders. In normal muscle, the predominant subunits of myofiber laminin were M, B1, and B2. The A chain was only faintly expressed in myofiber BM. In inflammatory myopathies and dystrophinopathies myofiber laminin A expression was greatly increased. An average of 80% and 63% of laminin A–positive myofibers in inflammatory myopathies and dystrophinopathies, respectively, were additionally CD56 positive. Laminin A and CD56 expression in denervating diseases and mitochondrial myopathies were negligible. Expression of M, B1, and B2 subunits did not seem to be altered in the diseased conditions examined above. The data suggest that laminin A is upregulated in inflammatory myopathies and dystrophinopathies and, most markedly in regenerating myofibers. © 1995 John Wiley & Sons, Inc.     

Tenascin-C expression in dystrophin-related muscular dystrophy

The mdx mouse has a mutated dystrophin gene and is used as a model for the study of Duchenne muscular dystrophy (DMD). We investigated whether regenerating mdx skeletal muscle contains the extracellular matrix protein tenascin-C (TN-C), which is expressed in wound healing and nerve regeneration. Prior to the initiation of muscle degeneration, both normal and mdx mice displayed similar weak staining for TN-C in skeletal muscle, but by 3 weeks of age the mice differed substantially. TN-C was undetectable in normal muscle except at the myotendinous junction, while in dystrophic muscle, TN-C was prominent in degenerating/regenerating areas but absent from undegenerated muscle. With increasing age, TN-C staining declined around stable regenerated mdx myofibers. TN-C was also observed in muscle from dogs with muscular dystrophy and in human boys with DMD. Therefore, in dystrophic muscle, TN-C expression may be stimulated by the degenerative process and remain upregulated unless the tissue undergoes successful regeneration. © 1996 John Wiley & Sons, Inc.     

The multifunctional role of IGF-1 in peripheral nerve regeneration

The elements of peripheral nerve regeneration comprise a complex combination of nerve growth, muscle satellite cells proliferation and differentiation and vessel growth. There is also increasing evidence that growth factors may act at multiple levels in the regenerative response. One such factor affecting multiple cell processes is insulin-like growth factor (IGF-1). As a neurotrophic factor IGF-1 is known to promote nerve elongation and branching. As a myogenic factor, IGF-1 promotes satellite cell proliferation, differentiation and muscle hypertrophy. As an angiogenic factor, IGF-1 is known to promote angiogenesis in regenerating skeletal muscle by activating VEGF and VEGF receptors. Additionally, recent studies show that IGF-1 may also promote the activation of muscle stem cells during the regenerative process. This review will outline the pathways by which IGF-1 affects multiple layers of the regenerative response and how these pathways converge to promote the regeneration of nerves.