Some observations on the fine structure of the myotendinous junction in myotomal muscles of the tadpole tail

Summary Myotendinous junctions in the myotomal tail muscles of the tadpole of Rana rugosa were examined by electron microscopy. At the site of the myotendinous junction, the sarcolemma is covered on its sarcoplasmic aspect by the connecting filament layer and the attachment layer, and on the extracellular aspect by the intermediary layer and the external lamina, with associated collagen fibrils. The intermediary layer consists of filamentous structures which closely resemble microfibrils (Hanak and Böck, 1971), spine-like or thread-like profiles (Korneliussen, 1973) and intermediary layer (Nakao, 1975a, b) in the myotendinous junctions of other vertebrate skeletal muscles.Particularly interesting is the fact that all the coverings and linings of the sarcolemma, including the external lamina, are completely absent in the terminal segment of the finger-like sarcolemmal invagination characteristic of the myotendinous junction. Furthermore, special types of coupling between a sac of sarcoplasmic reticulum and a part of the sarcolemmal invagination are frequently observed. These couplings always occur along the region of the sarcolemma where the external lamina is absent. The couplings show features similar to those of the triad, such as SR feet , scalloped SR membranes and granular content of the SR sac, suggesting that they are analogous and functionally similar to the triad and other equivalent structures.     

The myotendinous junction of the smooth feather muscles (mm. pennati)

Summary Smooth feather muscles (mm. pennati) consist of bundles of smooth muscle cells which are attached to the feather follicles by short elastic tendons. In addition, some muscle bundles are interrupted by elastic tendons. The elastic tendon is composed of longitudinally arranged elastic fibers which branch and wavy bundles of collagen fibrils. Smooth muscle cells of the muscle bundles are attached to each other by desmosome-like junctions and by fusion of the basal laminae. The cytoplasm of the muscle cells is characterized by conspicuous thick filaments and abundant thin and intermediate filaments. These are attached to band-like dense patches (dense bands) at the plasma membrane which are particularly broad at the tapering end of the muscle cell. The contact surface between smooth muscle cells and their elastic tendon is considerably increased (i) by deep finger-like invaginations and indentations located at the tapering muscle end, and (ii) by branching of the coarse elastic fibers into slender processes, which are attached to the richly folded surface of the muscle cell endings by peripheral microfibrils. This intimate interlocking closely resembles the myotendinous junctions in skeletal muscle. In addition to fibroblasts and fibrocytes, the myotendinous junction of the young growing chicks contains numerous so-called myofibroblasts, which are suggested to represent smooth muscle cells differentiating into fibroblasts of the developing tendon.Dedicated to Professor Dr. Helmut Leonhardt on the occasion of his 60th birthdaySupported by a grant from the Deutsche Forschungsgemeinschaft (Dr. 91/1)     

Strains at the myotendinous junction predicted by a micromechanical model

The goal of this work was to create a finite element micromechanical model of the myotendinous junction (MTJ) to examine how the structure and mechanics of the MTJ affect the local micro-scale strains experienced by muscle fibers. We validated the model through comparisons with histological longitudinal sections of muscles fixed in slack and stretched positions. The model predicted deformations of the A-bands within the fiber near the MTJ that were similar to those measured from the histological sections. We then used the model to predict the dependence of local fiber strains on activation and the mechanical properties of the endomysium. The model predicted that peak micro-scale strains increase with activation and as the compliance of the endomysium decreases. Analysis of the models revealed that, in passive stretch, local fiber strains are governed by the difference of the mechanical properties between the fibers and the endomysium. In active stretch, strain distributions are governed by the difference in cross-sectional area along the length of the tapered region of the fiber near the MTJ. The endomysium provides passive resistance that balances the active forces and prevents the tapered region of the fiber from undergoing excessive strain. These model predictions lead to the following hypotheses: (i) the increased likelihood of injury during active lengthening of muscle fibers may be due to the increase in peak strain with activation and (ii) endomysium may play a role in protecting fibers from injury by reducing the strains within the fiber at the MTJ.     

Fine structure of the myotendinous junction of lathyritic rat muscle with special reference to connectin, a muscle elastic protein

The fine structure of the myotendinous junction of the skeletal muscle of lathyritic rats caused by β-aminopropionitrile was investigated. In the junction there are finger-like processes of muscle fibers, in which thin filaments were extended from the last Z lines of myofibrils and attached to the sarcolemma of the processes. By the heavy meromyosin decoration technique, these thin filaments were identified as actin filaments. In the lathyritic muscle, the thin filaments were markedly fewer in number and distributed sparsely in the sarcoplasm.The content of connectin, an elastic protein, which is localized in myofibrils and also in sarcolemma was significantly decreased in the lathyritic muscle. A possible relationship between the changes in the fine structure of the myotendinous junction and in the connectin contents is discussed.     

Myosin mRNA accumulation and myofibrillogenesis at the myotendinous junction of stretched muscle fibers

Myofiber growth and myofibril assembly at the myotendinous junction (MTJ) of stretch-hypertrophied rabbit skeletal muscle was studied by in situ hybridization, immunofluorescence, and electron microscopy. In situ hybridization identified higher levels of myosin heavy chain (MHC) mRNA at the MTJ of fibers stretched for 4 d. Electron microscopy at the MTJ of these lengthening fibers revealed a large cytoplasmic space devoid of myofibrils, but containing polysomes, sarcoplasmic reticulum and T-membranes, mitochondria, Golgi complexes, and nascent filament assemblies. Tallies from electron micrographs indicate that myofibril assembly in stretched fibers followed a set sequence of events. (a) In stretched fiber ends almost the entire sarcolemmal membrane was electron dense but only a portion had attached myofibrils. Vinculin, detected by immunofluorescence, was greatly increased at the MTJ membrane of stretched muscles. (b) Thin filaments were anchored to the sarcolemma at the electron dense sites. (c) Thick filaments associated with these thin filaments in an unregistered manner. (d) Z-bodies splice into thin filaments and subsequently thin and thick filaments fall into sarcomeric register. Thus, the MTJ is a site of mRNA accumulation which sets up regional protein synthesis and myofibril assembly. Stretched muscles also lengthen by the addition of myotubes at their ends. After 6 d of stretch these myotubes make up the majority of fibers at the muscle ends. Essentially all these myotubes repeat the developmental program of primary myotubes and express slow MHC. MHC mRNA distribution in myotubes is disorganized as is the distribution of their myofibrils.     

Location of the integrin complex and extracellular matrix molecules at the chicken myotendinous junction

Summary The distribution of several extracellular matrix macromolecules was investigated at the myotendinous junction of adult chicken gastrocnemius muscle. Localization using monoclonal antibodies specific for 3 basal lamina components (type IV collagen, laminin, and a basement membrane form of heparan sulfate proteoglycan) showed strong fluorescent staining of the myotendinous junction for heparan sulfate proteoglycan and laminin, but not for type IV collagen. In addition, a strong fluorescent stain was observed at the myotendinous junction using a monoclonal antibody against the subunit of the chicken integrin complex (antibody JG 22). Neither fibronectin nor tenascin were concentrated at the myotendinous junction, but instead were present in a fibrillar staining pattern throughout the connective tissue which was closely associated with the myotendinous junction. Tenascin also gave bright fluorescent staining of tendon, but no detectable staining of the perimysium or endomysium. Type I collagen was observed throughout the tendon and in the perimysium, but only faintly in the endomysium. In contrast, type III collagen was present brightly in the endomysium and in the perimysium, but could not be detected in the tendon except when associated with blood vessels and in the epitendineum, which stained intensely. Type VI collagen was found throughout the tendon and in all connective tissue partitions of skeletal muscle. The results indicate that one or more molecules of the integrin family may play an important role in the attachment of muscle to the tendon. This interaction does not appear to involve extensive binding to fibronectin or tenascin, but may involve laminin and heparan sulfate proteoglycan.     

Muscle mechanosensitive receptors close to the myotendinous junction of the Achilles tendon elicit a pressor reflex.

Feedback regulation by activation of mechanosensitive afferents in the exercising muscle causes the cardiovascular and sympathetic nerve responses, which follow tension development and are almost identical between static contraction and passive stretch. The precise location of the mechanoreceptors contributing to the exercise pressor reflex, however, remained unknown. To test the hypothesis that the mechanoreceptors will be located around the myotendinous junction to monitor a change in muscle tension than a change in muscle length, we examined the reflex cardiovascular responses to passive stretch of the triceps surae muscle in anesthetized rats with three interventions; systemic injection of gadolinium, cutting the Achilles tendon, and local injection of lidocaine into the myotendinous junction. Gadolinium (42 micromol/kg iv) blunted the increases in heart rate and mean arterial blood pressure during passive stretch by 36 and 22-26%, respectively, suggesting that the reflex cardiovascular responses were evoked by stimulation of muscle mechanosensitive receptors. The cardiovascular responses to passive stretch were not different between the cut Achilles tendon and the intact tendon in the same rats, suggesting that any mechanoreceptors, terminated in the more distal part of the tendon, did not contribute to the reflex cardiovascular responses. Lidocaine (volume, 0.04-0.1 ml) injected into the myotendinous junction blunted the stretch-induced increases in heart rate and mean arterial blood pressure by 37-49 and 27-34%, respectively. We conclude that the muscle mechanosensitive receptors evoking the reflex cardiovascular responses at least partly locate at or close to the myotendinous junction of the Achilles tendon.     

Organization and fine structure of extraocular muscles in Carassius and Rana

Summary 1. Two types of muscle fibers, red (-slow) and white (-twitch), have been described in the extraocular muscles of Carassius and Rana, respectively. 2. Red and white muscle fibers occupy a definite position in particular eye muscles and occur in almost constant numerical relation. 3. The red fibers in the fish extraocular muscles are supposedly slow. The position of the triads is at the level of the A/I junction, whereas that of the white muscle fibers is at the Z line level. 4. In the frog the extraocular muscles consist of two types of muscle fibers, which have morphological features of slow and fast fibers, respectively, the triads being localized at the Z line level.This work has been supported by the Polish Academy of Sciences.Authors express their thanks to Doc. Dr. J. Kawiak for help in densitometrography.     

Fine structure of the retino-optic nerve junction in the chicken

The aim of this study is to investigate a fine structure of the retino-optic nerve junction in the chicken. We especially focused on the myelin sheaths and astrocytes in the intraocular optic nerve (ION) and its adjoining parts. A part of the axons of retinal nerve fiber layer (NFL) were myelinated. Ganglion cell axons were ensheathed by loose myelin in the NFL and by a compact one in the ION and optic nerve (ON). Myelin structure changed from loose type to a compact one within the very narrow NFL-ION junction. Loose myelin forming cells are dark type of oligodendrocytes in the retina. From the most peripheral ON to the choroidal part of ION, astrocytes contained abundant microtubules. The optic nerve around the lamina cribrosa is exposed to mechanical force during eye movement. It is suggested that these microtubules may perform the cytoskeletal function. Astrocytes in the retinal part of ION had longer processes filled with abundant gliofilaments. They may provide the mechanical support for the ganglion cell axons, which are exposed directly to intraocular pressure. Although astrocytes in the retinal level of ION extended their processes into the retina, their soma was never found in the retina.     

Fine structure of the gap junction in the tunicate heart

Summary The plasma membranes of the tunicate heart exhibit an abundance of macular gap junctions distributed widely over the membrane surface. A study of these junctions by the freeze-etch technique was undertaken in an effort to elucidate the fine structure of this important membrane modification in a primitive heart. In cross or near-cross fractured junctions the junctional particles in contiguous membranes appear to be paired in register and to meet in the midline. In favorable face views, the junctional particles are seen to be disposed in hexagonal array. The individual particles display a distinct rosette-like substructure consistent with a six-membered ring of globular protein molecules clustered around a central channel. Similar junctional-type particles can be found in nonjunctional areas of membrane suggesting that the transport mechanism which they may represent is not restricted to the gap junction.Career Investigator of the American Heart AssociationWe wish to thank Dr. J.B. Jillett for use of the facilities of the Portobello Marine Biological Station; Mr. W.S. Bertaud, Physics and Engineering Laboratory, D.S.I.R., Lower Hutt, who kindly supervised the preparation of some of the freeze-etch replicas; Dr. R.H. Millar of the Dunstaffnage Marine Research Laboratory, Oban, Argyll, Scotland, who identified the tunicate used in the present (and previous) study; Prof. W.D. Trotter who made facilities in the Department of Anatomy, University of Otago Medical School, Dunedin, available to one of us (V.L.); and Mrs. S.M. O'Kane for excellent technical assistance. Generous support from the American Heart Association (to V.L.) and from the Medical Research Council of New Zealand (to D.G.R.) is gratefully acknowledged     

Dynamic formation of microenvironments at the myotendinous junction correlates with muscle fiber morphogenesis in zebrafish

Muscle development involves the specification and morphogenesis of muscle fibers that attach to tendons. After attachment, muscles and tendons then function as an integrated unit to transduce force to the skeletal system and stabilize joints. The attachment site is the myotendinous junction, or MTJ, and is the primary site of force transmission. We find that attachment of fast-twitch myofibers to the MTJ correlates with the formation of novel microenvironments within the MTJ. The expression or activation of two proteins involved in anchoring the intracellular cytoskeleton to the extracellular matrix, Focal adhesion kinase (Fak) and beta-dystroglycan is up-regulated. Conversely, the extracellular matrix protein Fibronectin (Fn) is down-regulated. This degradation of Fn as fast-twitch fibers attach to the MTJ results in Fn protein defining a novel microenvironment within the MTJ adjacent to slow-twitch, but not fast-twitch, muscle. Interestingly, however, Fak, laminin, Fn and beta-dystroglycan concentrate at the MTJ in mutants that do not have slow-twitch fibers. Taken together, these data elucidate novel and dynamic microenvironments within the MTJ and indicate that MTJ morphogenesis is spatially and temporally complex.     

Fine structure of the alary muscles of the American cockroach

The alary muscles of the cockroach, Periplaneta americana, are striated with an A-band of 3·0 to 3·5 μm long. Each muscle fibre was 10 to 12 μm in diameter and Z-lines appeared as small discrete units staggered throughout the sarcoplasm. Mitochondria were conspicuously located near the Z-line areas and were absent from the middle portion of the sarcomere. A transverse membrane system was present which formed dyad structures with a relatively sparse sarcoplasmic reticulum. Cockroach alary muscles were innervated by axons containing electron-dense granules of near 100 nm in diameter. These are thought to be typical of ‘neurosecretory’ axons based on their ultrastructural appearance.     

Development of the zebrafish myoseptum with emphasis on the myotendinous junction

Zebrafish myosepta connect two adjacent muscle cells and transmit muscular forces to axial structures during swimming via the myotendinous junction (MTJ). The MTJ establishes transmembrane linkages system consisting of extracellular matrix molecules (ECM) surrounding the basement membrane, cytoskeletal elements anchored to sarcolema, and all intermediate proteins that link ECM to actin filaments. Using a series of zebrafish specimens aged between 24 h post-fertilization and 2 years old, the present paper describes at the transmission electron microscope level the development of extracellular and intracellular elements of the MTJ. The transverse myoseptum development starts during the segmentation period by deposition of sparse and loosely organized collagen fibrils. During the hatching period, a link between actin filaments and sarcolemma is established. The basal lamina underlining sarcolemma is well differentiated. Later, collagen fibrils display an orthogonal orientation and fibroblast-like cells invade the myoseptal stroma. A dense network of collagen fibrils is progressively formed that both anchor myoseptal fibroblasts and sarcolemmal basement membrane. The differentiation of a functional MTJ is achieved when sarcolemma interacts with both cytoskeletal filaments and extracellular components. This solid structural link between contractile apparatus and ECM leads to sarcolemma deformations resulting in the formation of regular invaginations, and allows force transmission during muscle contraction. This paper presents the first ultrastructural atlas of the zebrafish MTJ development, which represents an useful tool to analyse the mechanisms of the myotendinous system formation and their disruption in muscle disorders.     

A protein homologous to the Torpedo postsynaptic 58K protein is present at the myotendinous junction

The 58K protein is a peripheral membrane protein enriched in the acetylcholine receptor (AChR)-rich postsynaptic membrane of Torpedo electric organ. Because of its coexistence with AChRs in the postsynaptic membrane in both electrocytes and skeletal muscle, it is thought to be involved in the formation and maintenance of AChR clusters. Using an mAb against the 58K protein of Torpedo electric organ, we have identified a single protein band in SDS-PAGE analysis of Xenopus myotomal muscle with an apparent molecular mass of 48 kD. With this antibody, the distribution of this protein was examined in the myotomal muscle fibers with immunofluorescence techniques. We found that the 48K protein is concentrated at the myotendinous junctions (MTJs) of these muscle fibers. The MTJ is also enriched in talin and vinculin. By double labeling muscle fibers with antibodies against talin and the 48K protein, these two proteins were found to colocalize at the membrane invaginations of the MTJ. In cultured myotomal muscle cells, the 48K protein and talin are also colocalized at sites of membrane-myofibril interaction. The 48K protein is, however, not found at focal adhesion sites in nonmuscle cells, which are enriched in talin. These data suggest that the 48K protein is specifically involved in the interaction of myofibrillar actin filaments with the plasma membrane at the MTJ. In addition to the MTJ localization, 48K protein is also present at AChR clusters both in vivo and in vitro. Thus, this protein is shared by both the MTJ and the neuromuscular junction.     

Structural remodeling of unweighted soleus myotendinous junction in monkey

This study describes the morphology of the soleus myotendinous junction (MTJ) in the Rhesus monkey. Ultrastructural observations revealed a structural complexity that probably reflects functional adaptations. We also studied ultrastructural modifications of the MTJ in response to 14 days of hypokinesia and microgravity (Bion 11 mission). The reduced limb mobility of the animals, placed in a safety seat aboard the satellite, induced a sarcolemmal remodeling that was enhanced by the microgravity conditions. Signs of MTJ remodeling such as alterations of contractile apparatus and myofilament-anchoring structures, T-tubule dilation, and autophagic vacuoles could be ascribed to the microgravity.     

Fine structure of myomyous junctions in the mouse skeletal muscles

The reaction product of acetylcholinesterase (AChE) activity is known to be specifically localized at a neuromuscular junction and a muscle-tendon junction of the striated skeletal muscles. In addition to the two junctions, we recently found some linear precipitates due to AChE activity running transversely across a fibre of the semitendinosus, rectus abdominis, gastrocnemius, tibialis anterior and diaphragm muscles in mice. Under an electron microscope, the linear precipitates were seen at the extracellular side of the muscle fibre endings. Most of the endings contacted each other to form a junction, which has been called the 'myomyous junction (M-Mj)'. The patterns of the M-Mj were grouped into three types: (1) a junction in which all contacts were firm, without any connective tissue, and invaginated deeply; (2) the ones in which numerous collagen fibres were visible in the space between the two separate opposing muscle fibres; (3) an intermediate type between (1) and (2), i.e. a junction with partial contacts. The muscle fibre ending forming M-Mj was constructed of finger-like processes like that of a muscle-tendon junction. However, the processes of a M-Mj adhered so closely to each other that no collagen fibrils could penetrate into their folds.