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.     

Observations on the Fine Structure of the Turtle Atrium

The general fine structure of the atrial musculature of the turtle heart is described, including; the nature of the sarcolemma; the cross-banded structure of the myofibrils; the character of the sarcoplasm, and the form and disposition of its organelles. An abundant granular component of the sarcoplasm in this species is tentatively identified as a particulate form of glycogen. The myocardium is composed of individual cells joined end to end at primitive intercalated discs, and side to side at sites of cohesion that resemble the desmosomes of epithelia. Transitional forms are found between desmosomes and intercalated discs. Both consist of a thickened area of the cell membrane with an accumulation of dense material in the subjacent cytoplasm. This dense amorphous component is often continuous with the Z substance of the myofibrils and may be of the same composition. The observations reported reemphasize the basic similarity between desmosomes and terminal bars of epithelia and intercalated discs of cardiac muscle. Numerous unmyelinated nerves are found beneath the endocardium. Some of these occupy recesses in the surface of Schwann cells; others are naked axons. No specialized nerve endings are found. Axons passing near the sarcolemma contain synaptic vesicles, and it is believed that this degree of proximity is sufficient to constitute a functioning myoneural junction.     

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.     

In vitro attachment of skeletal muscle fibers to a collagen gel duplicates the structure of the myotendinous junction

The myotendinous junction (MTJ) and its associated cells and connective tissue are important structures involved in transmission of contractile force from skeletal muscle to tendon. A model culture system was developed to investigate the formation of the MTJ and its attachment to collagen fibers. Skeletal muscle cells were cultured in a well modeled from two layers of a native gel of type I collagen. Muscle cells cultured in this manner formed attachments to the collagen gel and developed into highly contractile multinucleated muscle fibers with the development of extensive terminal invaginations of the sarcolemma. In addition, the subsarcolemma at the ends of muscle fibers showed areas of increased electron density which corresponded well with the termini of myofibrils. The results indicate that the development of sarcolemmal invaginations at the end of a muscle fiber probably occurs intrinsically during muscle development in vivo. The direct association of collagen fibers with the basal lamina at the end of muscle fibers was only occasionally observed in culture, suggesting that other fibrils or proteins may also be involved in the attachment of collagen fibers to the basal lamina of muscle fibers at the MTJ.     

Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts

The regulation of collagen fibril, bundle, and lamella formation by the corneal fibroblasts, as well as the organization of these elements into an orthogonal stroma, was studied by transmission electron microscopy and high voltage electron microscopy. Transmission and high voltage electron microscopy of chick embryo corneas each demonstrated a series of unique extracellular compartments. Collagen fibrillogenesis occurred within small surface recesses. These small recesses usually contained between 5 and 12 collagen fibrils with typically mature diameters and constant intrafibrillar spacing. The lateral fusion of the recesses resulted in larger recesses and consequent formation of prominent cell surface foldings. Within these surface foldings, bundles that contained 50-100 collagen fibrils were formed. The surface foldings continued to fuse and the cell surface retracted, forming large surface-associated compartments in which bundles coalesced to form lamellae. High voltage electron microscopy of 0.5 micron sections cut parallel to the corneal surface revealed that the corneal fibroblasts and their processes had two major axes at approximately right angles to one another. The surface compartments involved in the production of the corneal stroma were aligned along the fibroblast axes and the orthogonality of the cell was in register with that of the extracellular matrix. In this manner, corneal fibroblasts formed collagen fibrils, bundles, and lamellae within a controlled environment and thereby determined the architecture of the corneal stroma by the configuration of the cell and its associated compartments.     

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.     

Extracellular compartments in tendon morphogenesis: collagen fibril, bundle, and macroaggregate formation

The formation of collagen fibrils, fibril bundles, and tissue-specific collagen macroaggregates by chick embryo tendon fibroblasts was studied using conventional and high voltage electron microscopy. During chick tendon morphogenesis, there are at least three extracellular compartments responsible for three levels of matrix organization: collagen fibrils, bundles, and collagen macroaggregates. Our observations indicate that the initial extracellular events in collagen fibrillogenesis occur within narrow cytoplasmic recesses, presumably under close cellular regulation. Collagen fibrils are formed within these deep, narrow recesses, which are continuous with the extracellular space. Where these narrow recesses fuse with the cell surface, it becomes highly convoluted with folds and processes that envelope forming fibril bundles. The bundles laterally associate and coalesce, forming aggregates within a third cell-defined extracellular compartment. Our interpretation is that this third compartment forms as cell processes retract and cytoplasm is withdrawn between bundles. These studies define a hierarchical organization within the tendon, extending from fibril assembly to fascicle formation. Correlation of different levels of extracellular compartmentalization with tissue architecture provides insight into the cellular controls involved in collagen fibril and higher order assembly and a better understanding of how collagen fibrils are collected into structural groups, positioned, and woven into functional tissue-specific collagen macroaggregates.     

Three-dimensional ultrastructure of human tendons.

The three-dimensional ultrastructure of human tendons has been studied. Epitenon and peritenon consist of a dense network of longitudinal, oblique and transversal collagen fibrils crossing the tendon fibres. The internal structure of tendon fibres is also complex. The collagen fibrils are oriented not only longitudinally but also transversely and horizontally. The longitudinal fibrils do not run only parallel but also cross each other forming spirals (plaits). These fibril bundles are bound together by a three-dimensional collagen fibril network of endotenon. In the myotendinous junction the surface of the muscle cells form processes. A network of tendineal collagen fibrils fills the recesses between the muscle cell processes penetrating the basement membrane of these processes. This complex ultrastructure of human tendons most likely offers a good buffer system against longitudinal, transversal, horizontal as well as rotational forces during movement and activity.     

Morphology and histochemistry of the myotendineal junction of the rat calf muscles. Histochemical, immunohistochemical and electron-microscopic study.

The macromolecular composition and morphometry of the myotendineal junction (MTJ) of slow-twitch (type 1) and fast-twitch (type 2) muscle fibers were studied in gastrocnemius-soleus-Achilles unit of the rat. Proteoglycans and glycosaminoglycans, type III collagen, fibronectin and laminin could be detected at the myotendineal junction. Due to the membrane folding finger-like processes were seen at the MTJ. The processes of type 1 fibers were greater in size. However, due to the subdivisions the processes of type 2 muscle fibers had a significantly greater surface length per muscle cell diameter than type 1 fibers. The myotendineal endings of both fiber types had a characteristic basal lamina, which was about three times thicker than in the longitudinal site of the same muscle cells. The basal lamina of type 1 fibers at the MTJ was significantly thicker than that of type 2 fibers.     

Association between mitochondria and gap junctions in mammalian myocardial cells

In ventricular myocardial cells of mouse, guinea-pig, dog, and monkey, mitochondria frequently form close associations with gap junctions, the two structures being separated by a space of 20 nm or less. Similar appositions are found in both the mature atria and the developing myocardium of the mouse. The gap junctions assume a variety of configurations with respect to the apposed mitochondria. These include profiles in which the gap junctions conform closely to the contours of mitochondria, as well as profiles in which finger-like sarcolemmal evaginations, composed entirely of gap junctions, extend longitudinally or transversely into an adjoining cell to envelop mitochondria. In mouse ventricular wall, over 40% of the length of gap junctions is juxtaposed to mitochondria, and strands of connecting material are often present in the interspace between the two structures. In addition, in freeze-fracture replicas, portions of mitochondria are found attached to areas of myocardial sarcolemma that contain gap-junctional particles. Since mitochondria are known to sequester Ca²⁺ ion, it is possible that the close association between mitochondria and gap junction may function to buffer the intracellular Ca²⁺ concentration near the gap junctions, and thereby regulate the ionic permeability of the gap junctions.     

Macromolecular composition of the sarcolemma and endomysium in the rat

The macromolecular composition of sarcolemma and endomysium was studied by classical staining methods for glycosaminoglycans and using immunological techniques for proteins. Both proteoglycans and glycosaminoglycans (heparan sulphate, dermatan sulphate, chondroitin sulphate) could be detected in the sarcolemma. Type IV and type V collagen and laminin were found exclusively in the sarcolemma and endomysium. Type I and type III collagen as well as fibronectin were detected both in the endomysium and perimysium.     

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.     

Specific attachment of collagen to cardiac myocytes: In vivo and in vitro

The formation and attachment of collagen to the sarcolemma of cardiac myocytes were examined in vivo in neonatal rats and hamsters and in vitro in cultures of neonatal rat myocytes. Scanning, transmission, and high-voltage electron microscopy were used to show that the collagen struts attach to specific sites on the sarcolemma just lateral to the Z band of neonatal animals. In vitro, collagen preferentially attaches to distal end of myocytes at a site where internal stress fibers also attach to the sarcolemma. Formation of the collagen struts appeared to be a multistep process involving several components of the extracellular matrix. The role of the collagen struts is involved in the distribution of force generated by muscle contraction.     

Partial purification of an intercalated disc-containing cardiac plasma membrane fraction

The sarcolemma of cardiac muscle cells contains a specialised junctional region, the intercalated disc which includes three types of intercellular junction, the macula and fascia adherens and the nexus or gap junction. To facilitate the isolation of these junctions a procedure for the partial purification from mouse hearts of a subcellular fraction containing the intercalated disc region of the sarcolemma was developed. This involved investigating methods of tissue disruption that preserve the integrity of the intercalated disc and minimise myofibrillar entrapment of organelles. Examination of the distribution of marker enzymes showed that relative to the homogenate the intercalated disc fraction prepared by sucrose density centrifugation was only enriched 1.5- to 3-fold in 5'-nucleotidase and (Na+ + K+)-ATPase activities, whereas mitochondrial and sarcoplasmic reticulum marker enzymes were low. The properties of the intercalated disc-containing fraction were compared with the vesicular sarcolemmal fractions devoid of junctional complexes prepared by other methods.     

Fine structure of the receptors at the myotendinous junction of human extraocular muscles

The myotendinous junction of the human extraocular muscles was studied by electron microscopy. Some peculiar receptorial structures have been found in the majority of the samples examined. These structures are very small and consist of 1) the terminal portion of one muscle fibre, 2) the tendon into which it inserts and 3), within the tendon, a rich nerve arborization, whose branches are always very close to the muscle component. Only one discontinuous layer, made up of flat cells, which lack a basal lamina and often show pinocytotic vesicles, encapsules every musculo-tendinous complex. The tendinous component consists of amorphous ground substance of different electron density, of collagen and elastic fibres and is divided in compartments by ramified cells, which make an inner capsular-like covering to the nerve fibres. Three types of afferent nerve endings can be identified. One type is usually more frequent than the others, possesses a large number of neurotubules and neurofilaments and few mitochondria and is always surrounded by a Schwann cell which forms finger-like processes penetrating into the axoplasm. The second type is only partially enveloped by the Schwann cell. The axoplasm is devoid of neurotubules and contains few neurofilaments, several mitochondria and groups of small clear vesicles placed in the areas uncovered by the glial sheath. The third one is completely surrounded by the Schwann cell, but is devoid of neurotubules and neurofilaments and full of mitochondria. These morphological features correspond well with the probable role of these receptorial structures, which is to ensure very exact and precise ocular movements.     

Histologic study of the pyelo-ureteral junction

The ureter structure was analyzed under light microscope on serial sections in newborn children affected by obstruction of the pyelo-ureteric junction. In the obstructive segments, preceded by ureter portions dilated and provided with close-packed layers of smooth muscle layers, the tunica mucosa was lacking epithelial cover, its lamina propria was thickened, being built by conspicuous bundles of collagen fibers, and the tunica muscularis showed scarce and disrupted groups of muscle cells invaded by connective tissue. Numerous mastocytes were seen in the mucosa and muscularis tunicae. The results suggest that the breaking of the epithelium may be a primary pathogenetic event followed by passage of urine in the subjacent tissues in turn responsible for a diffuse connective reaction, and, therefore for a final fibrosis of the ureter wall. The role of the mastocytes in the etiopathogenesis of the pyelo-ureteric junction obstruction was also discussed.     

Confocal laser microscopy of dystrophin localization in guinea pig skeletal muscle fibers

A confocal laser microscope was used to analyze the localization pattern of dystrophin along the sarcolemma in guinea pig skeletal muscle fibers. Hind leg muscles of the normal animals were freshly dissected and frozen for cryostat sections, which were then stained with a monoclonal antidystrophin antibody. In confocal laser microscopy, immunofluorescence staining in relatively thick sections could be sharply imaged in thin optical sections. When longitudinal and transverse sections of muscle fibers were examined, the immunostaining of dystrophin was seen as linearly aligned fluorescent dots or intermittent lines along the sarcolemma. In longitudinally cut muscle fibers, many fluorescent dots, but not all, corresponded to the sarcomere pattern, especially the I band. Sections cut tangential to the sarcolemma also showed a lattice-like pattern of longitudinal and transverse striations of fluorescent dots. Double staining for dystrophin and vinculin showed that the two proteins were not exactly colocalized. The end portions of muscle fibers were much more intensely stained with antidystrophin antibody than the central portions, following the contour of elaborate surface specializations at the myo-tendon junction. The staining pattern at the myo-tendon junction was also discontinuous. These confocal microscopic observations suggest that dystrophin may be localized in a nonuniform, discontinuous pattern along the sarcolemma and in some relationship with the underlying myofibrils.     

Arrangement of smooth muscle cells and intramuscular septa in the taenia coli

Summary Bands of electron-dense material beneath the cell membrane of smooth muscle cells of the guinea-pig taenia coli provide attachment to thin myofilaments and to intermediate (10 nm) filaments; about 50% of the cell membrane is occupied by dense bands in muscle cells transversely sectioned at the level of their nucleus, and between 50 and 100% in smaller cell profiles nearer the cell's ends. In addition to the known cell-to-cell junctions (intermediate contacts), more complex apparatuses anchor muscle cells together, either end-to-end or end-to-side or side-to-side. They consist of elaborate folds, invaginations and protrusions accompanied by large amounts of basal lamina material. In the end-to-end anchoring apparatuses numerous finger-like and laminar processes from the two cells interdigitate. Other muscle cells have a star-shaped profile in the last few microns of their length, or show longitudinal invaginations occupied by a thickened basal lamina and occasionally by collagen fibrils. The septa of connective tissue extend only for a few hundred microns along the length of the taenia. In taeniae fixed in condition of mild stretch the muscle cells form an angle of about 5° with the septa. In muscles fixed during isotonic contraction the angle increases to about 20–22°, and in longitudinal sections the muscle cells appear arranged in a herring-bone pattern. The collagen concentration in the taenia coli is 4–6 times greater that in skeletal and cardiac muscles. These various structures are discussed in terms of their possible role in the mechanism of force transmission.I thank Mr. S.J. Sarsfield and Miss E.M. Franke for expert technical assistance, and Dr. Adam Yamey for much help in the experiments on collagen content. This work is supported by grants from the Medical Research Council     

The accessory papillary muscle with inferior J-waves - peculiarity or hidden danger?

A 24-year-old woman admitted with mild chest distress associated with activity without chest complaint for twenty days. Two orifices were visible at the level of the mitral valve with a transthoracic short-axis view of the two-dimensional and three-dimensional echocardiography. The left ventricle was mildly dilatated and the left ventricular wall was thickened, especially at the apex and anterolateral wall, and appeared sponge-like. There were numerous, excessively prominent trabeculations associated with intertrabecular recesses. Although the coexistence of NVM and DOMV could be a coincidence, we believe that both defects were probably caused by a developmental arrest of the left ventricular myocardium in the present case.     

Topographical difference of cytoskeletal organization in smooth muscle cells of rat duodenum revealed by quick-freezing and deep-etching method

The sarcolemmal domain of rat duodenal smooth muscle cells includes caveolae and associated cytoskeletal or filamentous elements. We have used the quick-freezing, deep-etching method to examine the three dimensional relationships between these components. Replica membranes for separated strips of rat duodenal muscle layers were routinely prepared after extraction soluble proteins from cytoplasm and extracellular matrix. As results, 1) cytoskeletal elements in smooth muscle cells consisted mainly of striated thin filaments; 2) thin filaments were connected with some plasma membranes through filaments associated with the sarcolemma, which formed fine network structures beneath the sarcolemma; 3) many bridging structures between the filaments associated with the sarcolemma and the extracellular matrix were frequently detected in the plasma membrane; and 4) compact filaments associated with the sarcolemma almost disappeared near the caveolae, and only thin filaments were anchored to their neck parts. The special arrangement of the cytoskeletal components, which is probably necessary for the intestinal motility, characterizes the topographical difference of the smooth muscle sarcolemma.