The effect of immobilization on myotendinous junction: an ultrastructural, histochemical and immunohistochemical study.

The effect of immobilization on the myotendinous junction of the calf muscles in the rat was studied histochemically, immunohistochemically and morphometrically with a transmission electron microscope. After 3 weeks of immobilization, the contact area between the muscle cells and tendineal collagen fibres was reduced by almost 50% in both type I (slow-twitch) and type II (fast-twitch) muscle fibres. The terminal finger-like processes of the muscle cells became shallow and cylindrical or were completely atrophied. Their basal membranes were slightly thickened. Histochemically, the most remarkable alteration in the myotendinous junction was the marked decrease in the sulphate containing glycosaminoglycans. In the basal lamina of the muscle fibres, the glycosaminoglycan and proteoglycan content was also reduced. Immunohistochemical analyses revealed that the amount of type III collagen was markedly increased on the myotendinous interface, but the amount and distribution of type I collagen was not affected by immobilization. These findings suggest that immobilization causes degenerative changes at the myotendinous junction, which, in turn, most likely decrease its tensile strength and may predispose it to rupture during activity.     

The effect of immobilization on myotendinous junction: an ultrastructural,histochemical and immunohistochemical study

Kannus , P., Jozsa , L., Kvist , M., Lehto , M. & Järvinen , M. 1992. The effect of immobilization on myotendinous junction: an ultrastructural, histochemical and immunohistochemical study. Acta Physiol Scand 144 , 387–394. Received 28 April 1 991 , accepted 13 October 1991. ISSN 0001–6772. Tampere Research Station of Sports Medicine, UKK-Institute, and Department of Surgery, Tampere University Central Hospital, Tampere, Finland; Department of Morphology, National Institute of Traumatology, Budapest, Hungary; and Sports Medical Research Unit, Paavo Nurmi Center, University of Turku, Turku, Finland. The effect of immobilization on the myotendinous junction of the calf muscles in the rat was studied histochemically, immunohistochemically and morphometrically with a transmission electron microscope. After 3 weeks of immobilization, the contact area between the muscle cells and tendineal collagen fibres was reduced by almost 50% in both type I (slow-twitch) and type II (fast-twitch) muscle fibres. The terminal finger-like processes of the muscle cells became shallow and cylindrical or were completely atrophied. Their basal membranes were slightly thickened. Histochemically, the most remarkable alteration in the myotendinous junction was the marked decrease in the sulphate containing glyco-saminoglycans. In the basal lamina of the muscle fibres, the glycosaminoglycan and proteoglycan content was also reduced. Immunohistochemical analyses revealed that the amount of type III collagen was markedly increased on the myotendinous interface, but the amount and distribution of type I collagen was not affected by immobilization. These findings suggest that immobilization causes degenerative changes at the myotendinous junction, which, in turn, most likely decrease its tensile strength and may predispose it to rupture during activity.     

Fine structure of the myotendinous junction and “terminal coupling” in the skeletal muscle of the lamprey,Lampetra japonica

The myotendinous junction in the skeletal muscle of adult lamprey Lampetra japonica was studied with an electron microscope. Numerous finger-like sarcolemmal invaginations were present at the ends of muscle fibers to form the myotendinous junction. Parietal fibers of each muscle unit showed more closely distributed sarcolemmal invaginations than central fibers. Features of the myotendinous junction. Parietal fibers of each muscle unit showed more closely distributed sarcolemmal invaginations than central fibers. Features of the myotendinous junction generally conform to the accounts in the literature. The sarcolemmal invagination was covered on its sarcoplasmic aspect by the connecting filament layer and the dense amorphous attachment layer, and on the extracellular aspect by the intermediary layer and the external lamina with collagen fibrils arising from the myosepta. Sarcolemmal invaginations were sometimes seen to consist of a pair of sarcolemmas of adjacent muscle fibers within a muscle unit, which is characteristic to the myotendinous junction of lamprey. It is noteworthy that the connecting filament layer is much thinner than that, e.g., in the tadpole tail muscles (Nakao, '74). Furthermore, it is much thicker in the parietal fibers than in the central fibers. The sarcolemma of the terminal segment of the invagination frequently showed specific coupling with cisterns of the sarcoplasmic reticulum (terminal coupling). The external lamina is partially or completely deficient in the terminal segment of sarcolemmal invaginations which form terminal couplings so that collagen fibrils contained in the invagination appear to be in direct contact with the sarcolemma; however, definite relationships of collagen fibrils with the sarcolemma and the external lamina in the terminal segment of invagination still remain obscure. This type of coupling is considered to play a role in the coupling of excitation to contraction of muscle fibers as triads and diads.     

Fine structure of the myotendinous junction and "terminal coupling" in the skeletal muscle of the lamprey, Lampetra japonica.

The myotendinous junction in the skeletal muscle of adult lamprey Lampetra japonica was studied with an electron microscope. Numerous finger-like sarcolemmal invaginations were present at the ends of muscle fibers to form the myotendinous junction. Parietal fibers of each muscle unit showed more closely distributed sarcolemmal invaginations than central fibers. Features of the myotendinous junction generally conform to the accounts in the literature. The sarcolemmal invagination was covered on its sarcoplasmic aspect by the connecting filament layer and the dense amorphous attachment layer, and on the extracellular aspect by the intermediary layer and the external lamina with collagen fibrils arising from the myosepta. Sarcolemmal invaginations were sometimes seen to consist of a pair of sarcolemmas of adjacent muscle fibers within a muscle unit, which is characteristic to the myotendinous junction of lamprey. It is noteworthy that the connecting filament layer is much thinner than that, e. g., in the tadpole tail muscles (Nakao, '74). Furthermore, it is much thicker in the parietal fibers than in the central fibers. The sarcolemma of the terminal segment of the invagination frequently showed specific coupling with cisterns of the sarcoplasmic reticulum (terminal coupling). The external lamina is partially or completely deficient in the terminal segment of sarcolemmal invaginations which form terminal couplings so that collagen fibrils contained in the invagination appear to be in direct contact with the sarcolemma; however, definite relationships of collagen fibrils with the sarcolemma and the external lamina in the terminal segment of invagination still remain obscure. This type of coupling is considered to play a role in the coupling of excitation to contraction of muscle fibers as triads and diads.     

Type VI collagen in mouse masseter tendon,from osseous attachment to myotendinous junction

Background and Methods: The association of masseter tendon type VI collagen with other extracellular matrix (ECM) components was examined from osseous attachment to myotendinous junction by immunohistochemistry and transmission electron microscopy with ATP treatment and enzyme digestion. Results: In the tendon proper, fibrocytes extended their processes among bundles of striated collagen fibrils and associated with adjacent cells through amorphous materials, thus forming a three-dimensional network. The amorphous or filamentous material was observed around the fibrocyte cell body and along the cell processes, where the localization of type VI collagen was confirmed by immunohistochemistry using anti-type VI collagen antibody. After treatment with 20 mM adenosine 5′-triphosphate (ATP), 100 nm periodic fibrils, an aggregated form of type VI collagen, were formed in the place where amorphous or filamentous material was present before the treatment. In myotendinous junction, the ATP-aggregated periodic fibrils were observed to associate with the external lamina of the muscle cells as well as among junctional tendon collagen fibrils. In the tendonbone boundary, ATP-aggregated periodic fibrils were observed around fibrocartilage-like cells in the uncalcifying area but not in the calcification front. Prolonged ATP treatment or hyaluronidase predigestion caused the formation of type VI collagen periodic fibrils in the area near the calcified matrix. Conclusions: The distribution of type VI collagen in mouse masseter tendon is different in different anatomical position. This may reflect the different functional demand for this collagen. © 1995 Wiley-Liss, Inc.     

Divalent cation-dependent adhesion at the myotendinous junction: ultrastructure and mechanics of failure

Summary Junctional microfibrils, which span the lamina lucida of the vertebrate myotendinous junction, are thought to function in force transmission at the junction. This hypothesis has been tested by disrupting junctional microfibrils through elimination of extracellular divalent cations, and determining the effects of this treatment on the ultrastructure and mechanics of whole frog skeletal muscles passively stretched to failure. Muscles incubated in divalent cation-free solution failed exclusively in the lamina lucida of the myotendinous junction, while control muscles all failed within the muscle fibres, several millimetres away from the junction. Failure sites from divalent cation-free muscles incubated with antibodies against collagen type IV, laminin, and tenascin showed no labelling of the avulsed ends of the muscle fibres, indicating that remnants of junctional microfibrils observed on the cell surface are not composed of any of these extracellular proteins. All three proteins were present on the tendon side of the failure site, confirming that the lamina densa remains attached to the tendon. Breaking stress for control muscles was 3.47×10⁵ N m^-2, and for divalent cation-free muscles, 1.84×10⁵ N m^-2, or approximately half the control value. Breaking strain averaged 1.17 for divalent cation-free muscles and 1.39 for controls, although the difference was not significant. We conclude that junctional microfibrils are components of a divalent cation-dependent adhesion mechanism at the myotendinous junction. In addition, ultrastructural analysis of divalent cation-free fibres stretched just short of failure suggests that a second, divalent cation-independent mechanism persists along the non-junctional cell surface, and can transmit substantial passive tension from myofibrils laterally to the extracellular matrix, bypassing the failed myotendinous junction.     

Tendon and myo-tendinous junction in an overloaded skeletal muscle of the rat

Summary Overloading of rat plantaris muscles was produced by aseptic ablation of the synergists. The morphological changes occurring after 1 or 2 weeks were investigated at the light and electron microscopical level in the distal tendon of the plantaris and at the myotendinous junction. Sham-operated rats were prepared as controls. In the tendon, quiescent fibrocytes were replaced by activated fibroblasts displaying a vesicular nucleus with prominent nucleoli and an outstanding increase in cytomembranes, particularly the rough endoplasmic reticulum and the Golgi complex. The plasmalemma of the fibroblasts was modified by the presence of caveolae and the surbsurface cytoplasm contained many membrane-bound vacuoles. In the tendon, the collagen bundles were disrupted, resulting in the formation of empty longitudinally oriented spaces; in these spaces, as in the pericapillary areas, no inflammatory cells were observed. At the myotendinous junction, fibroblast activation was consistently observed in both control and overloaded specimens. At this level, the sarcolemma of the finger-like projections of muscle fibres presented many caveolae close to clusters of large subsurface vacuoles. These observations indicate that, at the beginning of the compensatory hypertrophy, the adaptative changes to overloading include a non-inflammatory reaction of the tendon characterized by enhanced collagen synthesis and intensive membrane renewal and recycling. From the mechanical point of view this reaction can impair the tendon resistance to stretch. At the myotendinous junction the increased membrane turnover of the sarcolemma and the fibroblast activation can be considered permanent phenomena consequent to the increased stress exerted upon the interface connecting the contractile apparatus to the stroma.     

Dystrophin is required for normal thin filament-membrane associations at myotendinous junctions.

Dystrophin, the deficient gene product in Duchenne muscular dystrophy, is located subjacent to the muscle cell membrane at myotendinous junctions, as well as along the entire muscle cell. Myotendinous junctions are sites at which thin filaments normally are linked to one another and to the cell membrane, by both lateral and end-on associations between the thin filaments and membrane. The cell membrane at these sites in normal muscle is folded extensively. Dystrophic junctions display normal contacts between the ends of thin filaments and subsarcolemmal densities. However dystrophic junctions are deficient in lateral associations between thin filaments and the membrane and display less membrane folding than controls. These structural defects would result in stress concentrations at sites of thin filament attachment to the membrane, which can cause membrane tearing during muscle activation, especially in large-diameter and mature muscle cells. This deficiency in dystrophic myotendinous junction structure may contribute to our understanding of previously unaccountable aspects of the etiology of Duchenne muscular dystrophy.     

In the human,true myocutaneous junctions of skeletal muscle fibers are limited to the face

Within the panniculus carnosus-associated skeletal muscles in the human, the palmaris brevis and the platysma showed myotendinous/myofascial junctions with clear distance to the corium and the specific connection collagen type XXII. The orbicularis oris muscle, in contrast, contained bundles of striated muscle fibers reaching the corium at two distinct levels: the predominant inner ending was connected to the elastic network of the inner corium and the outer ending was within the more superficial collagen network. At both locations, the striated muscle fibers showed brush-like cytoplasmic protrusions connecting a network which was not oriented toward the muscle fibers. Collagen type XXII was not present.     

Ultrastructural comparison of slack and stretched myotendinous junctions,based on a three-dimensional model of the connecting domain

Summary The vertebrate myotendinous junction contains junctional microfibrils, located in the lamina lucida of the basement membrane. The junctional microfibrils are thought to transmit muscular force across the junctional lamina lucida, also called the connecting domain. If true, deformation of the terminal muscle cell processes and connecting domain during force transmission would be detected as a change in spacing and/or orientation of the junctional microfibrils. This study compared connecting domain morphology in frog semitendinosus muscles fixed in two extremes of resting tension, to elucidate the mechanical properties of the myotendinous junction. An initial study of connecting domain ultrastructure revealed that junctional microfibrils are punctate or spinelike in shape, and that they are distributed in a linear, helically-oriented array on the muscle cell surface. The rows in the surface lattice are 10–15 nm in thickness, have a centre-to-centre distance between rows of approximately 24 nm, and are oriented at approximately 41^o with respect of the long axis of the muscle fibre. Comparison of slack and highly stretched myotendinous junctions shows no significant changes in spacing or orientation of either individual junctional microfibrils or rows in the helical surface lattice. Thus, both the connecting domain and terminal cell processes at the myotendinous junction are essentially inextensible under the loading conditions used in this study.     

Graded arrangement of collagen fibrils in the equine superficial digital flexor tendon

By using ultramorphological and biochemical methods, we analyzed the regional differences between the three parts of the equine superficial digital flexor tendon (SDFT), namely, the myotendinous junction (MTJ), middle metacarpal (mM), and osteotendinous junction (OTJ). Cross-sectional images showed unique distributions of collagen fibrils of varying diameters in each region. Small collagen fibrils (diameter <100 nm) were distributed predominantly in the MTJ region, and the OTJ region was relatively rich in large collagen fibrils (diameter >200 nm). In the mM region, the collagen fibrils were intermediately distributed between the MTJ and OTJ. The results indicate a graded arrangement of collagen fibrils in the tendon. Type V collagen was detected preferentially in the MTJ region. Since type V collagen is believed to be one of the collagens regulating collagen fibril formation, its possible functionality in the MTJ region in terms of fibril formation and fibril arrangement in the tendon has been discussed here.     

Graded Arrangement of Collagen Fibrils in the Equine Superficial Digital Flexor Tendon

By using ultramorphological and biochemical methods, we analyzed the regional differences between the three parts of the equine superficial digital flexor tendon (SDFT), namely, the myotendinous junction (MTJ), middle metacarpal (mM), and osteotendinous junction (OTJ). Cross-sectional images showed unique distributions of collagen fibrils of varying diameters in each region. Small collagen fibrils (diameter <100 nm) were distributed predominantly in the MTJ region, and the OTJ region was relatively rich in large collagen fibrils (diameter >200 nm). In the mM region, the collagen fibrils were intermediately distributed between the MTJ and OTJ. The results indicate a graded arrangement of collagen fibrils in the tendon. Type V collagen was detected preferentially in the MTJ region. Since type V collagen is believed to be one of the collagens regulating collagen fibril formation, its possible functionality in the MTJ region in terms of fibril formation and fibril arrangement in the tendon has been discussed here.     

Gender differences in the passive stiffness of the human gastrocnemius muscle during stretch

The aim of this study was to determine whether muscle stiffness measured in vivo was different between males and females. Distal displacement of the gastrocnemius medialis myotendinous junction was measured directly using ultrasonography during passive dorsiflexion in eight males and eight females (age range 19–28 years). Plantarflexion torque and myotendinous junction displacement were measured at 5° intervals, where 0° was with the foot at right angles to the tibia. Stiffness of the gastrocnemius medialis muscle was calculated between 0° and 25° of dorsiflexion, and defined as passive plantarflexion torque/distal displacement of the myotendinous junction (N m cm⁻¹). Relative muscle stiffness was also calculated as distal displacement relative to resting muscle length, and as passive torque relative to plantarflexion maximal voluntary contraction torque. No significant gender difference was observed in passive dorsiflexion torque, or in passive torque/maximal voluntary torque throughout the range of motion. Distal displacement of the gastrocnemius myotendinous junction was 26% more in females than in males (P < 0.05). Myotendinous junction displacement was 5.0 ± 1.4% of resting gastrocnemius medialis length in females, and 3.9 ± 0.6% in males. Over 25° of passive dorsiflexion, gastrocnemius medialis muscle stiffness was greater in males than in females by 44% (P < 0.05). In conclusion, based on the in vivo assessment of myotendinous junction displacement, passive gastrocnemius medialis muscle stiffness is greater in males than in females.     

Development of myotendinous‐like junctions that anchor cardiac valves requires fibromodulin and lumican

Results : FMOD deficient mice and double deficient FMOD; LUM mice exhibited anomalies in regions where cardiac valve tissue interdigitates with adjacent muscle for support. Ectopic connective and/or myocardial tissue(s) was associated with the more severe cardiac valve anomalies in FMOD; LUM deficient mice. At postnatal day 0 (P0) there was an increase in the mesenchymal cell number in the regions where valve cusps anchor in FMOD; LUM deficient mice compared to WT. The cardiac valve anomalies correlated with the highest levels of FMOD expression in the heart and also where myotendinous junctions (MTJ) components biglycan, collagen type I alpha 1, and collagen type VI, are also localized. 相似文献   

NUMBER AND MORPHOLOGY OF MECHANORECEPTORS IN THE MYOTENDINOUS JUNCTION OF PARALYSED HUMAN MUSCLE

The mechanoreceptor system of the myotendinous junction (MTJ) of human palmaris longus muscle obtained at autopsy was studied histologically from six patients with flaccid paralysis (complete acute tetraplegia 4–6 weeks before the autopsy, due to a spinal cord injury), eight patients with spastic paralysis (chronic hemiplegia due to cerebral stroke) and ten neurologically normal controls. Four types of nerve endings, Ruffini and Pacini corpuscles, Golgi tendon organs, and free nerve endings, could be identified in the MTJs of the controls. In the MTJs of the patients with flaccid and spastic paralysis, the free nerve endings were not present and the mechanoreceptors that were found were few in number, degenerated, fibrotic, and atrophic. These mechanoreceptors had lost their connection with the muscle fibres and tendon bundles and were frequently located within pathological accumulations of fatty tissue in the myotendinous region. The number and distribution of mechanoreceptors in the MTJ were almost identical in patients with flaccid and spastic paralysis.     

The fine structure of innervated myotendinous cylinders in extraocular muscles of rhesus monkeys

Summary Many of the myelinated nerve fibres of the distal myotendinous region of rectus muscles terminate on muscle fibre tips. The terminal expansions contain aggregated, small clear vesicles and mitochondria. Neuromuscular clefts at the contacts measure 20–40 nm and are uninterrupted by a basal lamina; the sarcoplasm opposite the contacts is unmodified. Some terminals invaginate the muscle fibre tips and others contact the sides of processes formed by splitting of the tips. The muscle fibre termination, its tendon and the nerve fibre branches are encapsulated to form an end-organ averaging 125 m in length and described as a myotendinous cylinder.Approximately 350 innervated myotendinous cylinders were estimated to be present in the horizontal recti with rather fewer in the vertical rectus muscles. Many of them occur shortly before the main myotendinous junction. All muscle fibres contributing to myotendinous cylinders were identified as the compact, felderstruktur, multi-innervated variety with directly apposed myofibrils that are known to be non-twitch fibres. All felderstruktur fibre terminations examined were encapsulated but 19% of them were not innervated.The nerve terminals of myotendinous cylinders are similar to those described by Dogiel (1906) as palisade endings and it is argued that they meet the morphological criteria of sensory neuromuscular endings. Their disposition suggests a capacity to monitor felderstruktur muscle fibre contraction.     

Basophilic bodies of skeletal muscle in hypothyroidism: enzyme histochemical and ultrastructural studies

Basophilic bodies of skeletal muscles from two patients with hypothyroidism were examined by enzyme histochemistry and ultrastructural study of ultrathin sections stained with periodic-acid-thiocarbohydrazide-silver proteinate for polysaccharides. Some additional characterizations of basophilic bodies were observed: basophilic bodies were found exclusively in type 1 fiber; basophilic bodies were devoid of myofibrillary adenosine triphosphatase, oxidative enzymes, and phosphorylase; and both fibrillary and granular components of basophilic bodies stained strongly for polysaccharides. The polysaccharide nature of basophilic bodies is in keeping with the previous suggestion that the formation of basophilic bodies in hypothyroid patients is related to an impairment of carbohydrate metabolism. Their selective involvement of type 1 fiber and preferential occurrence at the myotendinous junction remain obscure.     

Organization of the myotendinous junction is dependent on the presence of alpha7beta1 integrin

The laminin receptor alpha7beta1 is enriched at the myotendinous junctions, and mice with a targeted inactivation of the alpha7 gene develop a form of muscular dystrophy that primarily affects this structure. By ultrastructural analysis of alpha7-deficient mice, in comparison with wild-type and mdx mice, we attempted to elucidate the role of alpha7 integrin for the integrity and function of the myotendinous junctions. Ultrastructurally, myotendinous junctions of alpha7-deficient myofibers lose their interdigitations and the myofilaments retract from the sarcolemmal membrane, whereas the lateral side of the myofibers remains morphologically normal. The basement membrane at the myotendinous junctions in alpha7 -/- mice is significantly broadened, and immunogold-histochemistry has demonstrated that the laminin alpha2 chain is not localized here but, instead, in the matrix of the neighboring tendon. In contrast, mdx mice have normal myotendinous junctions, with a matrix protein pattern also found in wild-type mice, however the lateral sides of the myofibers are severely damaged. These results suggest that the alpha7beta1 integrin is a major receptor connecting the muscle cell to the tendon and helps to organize the myotendinous junction, whereas the dystrophin-glycoprotein complex is necessary for the lateral integrity of the muscle cell.     

Lack of cytosolic and transmembrane domains of type XIII collagen results in progressive myopathy

Type XIII collagen is a type II transmembrane protein found at many sites of cell adhesion in tissues. Homologous recombination was used to generate a transgenic mouse line (Col13a1(N/N)) that expresses N-terminally altered type XIII collagen molecules lacking the short cytosolic and transmembrane domains but retaining the large collagenous ectodomain. The mutant molecules were correctly transported to focal adhesions in cultured fibroblasts derived from the Col13a1(N/N) mice, but the cells showed decreased adhesion when plated on type IV collagen. These mice were viable and fertile, and in immunofluorescence stainings the mutant protein was located in adhesive tissue structures in the same manner as normal alpha1(XIII) chains. In immunoelectron microscopy of wild-type mice type XIII collagen was detected at the plasma membrane of skeletal muscle cells whereas in the mutant mice the protein was located in the adjacent extracellular matrix. Affected skeletal muscles showed abnormal myofibers with a fuzzy plasma membrane-basement membrane interphase along the muscle fiber and at the myotendinous junctions, disorganized myofilaments, and streaming of z-disks. The findings were progressive and the phenotype was aggravated by exercise. Thus type XIII collagen seems to participate in the linkage between muscle fiber and basement membrane, a function impaired by lack of the cytosolic and transmembrane domains.     

Molecular alterations in the perijunctional region of frog skeletal muscle fibres following denervation

Summary The anatomical distribution of a frog skeletal muscle antigen was studied using immunofluorescence microscopy and a monoclonal antibody 3B6 that was produced against denervated skeletal muscle. In innervated muscles, the monoclonal antibody 3B6 stain was associated with the inner surface of the muscle plasma membrane at the endplate and myotendinous junction. After denervation, the monoclonal antibody 3B6 stain extended from the endplate laterally around the perimeter of muscle fibres and longitudinally well beyond the endplate for a total length of 600–1000 m. The monoclonal antibody 3B6 stain thus forms a cylindrical structure centred on the endplate. This observation shows that denervation produces a non-homogeneous molecular change in skeletal muscle fibres: an antigen that is present in high concentrations at innervated endplates appears in restricted perijunctional regions of denervated muscle fibres. It further suggests that perijunctional regions of denervated muscle fibres differ from the remaining non-endplate regions in molecular composition and possibly also in function.