Histological features of endomysium,perimysium and epimysium in rat lateral pterygoid muscle

The distribution of the endomysium, perimysium, and epimysium and their constituent connective tissue fiber types in the mature rat lateral pterygoid muscle was examined with the light microscope. The endomysium and perimysium were relatively thin and consisted mainly of reticular fibers. The epimysium was thicker than the intramuscular sheaths and consisted of both collagen and reticular fibers; however, the thickness and constituent connective tissue fiber types of these sheaths varied regionally. Near the articular capsule and disc, the endomysium, perimysium, and epimysium were all thicker than in other regions of the muscle and consisted of collagen, reticular, and elastic fibers. The perimysium bound the bundles of muscle fibers together and frequently included blood vessels and nerves. As the superior head of the pterygoid muscle approached its insertion, sheaths of perimysium divided this head into smaller and smaller bundles of muscle fibers. In the inferior head, some of the perimysial sheaths and part of the epimysium were aponeurotic, and many muscle fibers attached to them. There were few such aponeurotic regions in the superior head. © 1996 Wiley-Liss, Inc.     

Immunohistochemical localization of genetically distinct types of collagen in muscle of kuruma prawn Penaeus japonicus

• 1.1. The location of genetically distinct types of collagen in muscular tissue of the kuruma prawn was examined using immunohistochemical techniques.
• 2.2. Collagen was distributed not only in muscle connective tissues, which were classified into three forms, epimysium, perimysium and endomysium, but also in subcuticular membrane, which was mainly composed of two layers, hypodermis and subcuticular connective tissue.
• 3.3. The α1(Pr) component existed in all connective tissues in the kuruma prawn muscle. Type AR-II collagen was distributed in all the connective tissues except for the hypodermis, while the α2(Pr) component existed in the thin connective tissues, the perimysium and endomysium, and in the hypodermis.

     

The dynamics of compartmentalization of embryonic muscle by extracellular matrix molecules

In order to delineate the role of proteoglycans in muscle development, the immunohistological localization of glycosaminoglycans and proteoglycan core proteins was studied in embryonic chick leg at Hamburger-Hamilton stages (St.) 36, 39, 43, and 46, and at 2 weeks posthatching. A specific anatomical landmark was chosen (the junction between the pars pelvica and the pars accessoria of the flexor cruris lateralis muscle) in order to ensure the study of anatomically equivalent sites. Frozen cross sections were immunostained with monoclonal antibodies to chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, and keratan sulfate glycosaminoglycans; to the core proteins of muscle/mesenchymal chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, and basement membrane heparan sulfate proteoglycan; and to laminin and tenascin. Extracellular matrix zones corresponding to the endomysium, perimysium, epimysium, basement membrane, and myotendinous junction each show characteristic immunostaining patterns from St. 36 to St. 46 and have unique matrix compositions by St. 46. In some cases, there is a sequential or coordinate expression of epitopes, first in the epimysium, then the perimysium, and last in the endomysium. Dermatan sulfate proteoglycan is detected in the epimysium at St. 36, in the perimysium at St. 39 (there is no perimysium structure at St. 36), and is not detected in the endomysium until St. 43. A putative mesenchymal proteoglycan core protein (reactive to the monoclonal antibody MY-174) is detected at St. 39 in both epimysium and perimysium, but is not detected in the endomysium until St. 43. Keratan sulfate antibody immunostains epimysium at St. 39 and perimysium at St. 46, but is never detected in the endomysium. Some epitopes are expressed independently in each of the extracellular matrix zones: antibody to tenascin stains only a subset of the epimysium, at the myotendinous junction; and heparan sulfate proteoglycan and laminin are detected only in the endomysium. Between St. 36 and St. 39, the muscle/MY-174-reactive proteoglycan core protein staining decreases in intensity in the endomysium and becomes positive in the epimysium and perimysium. An inverse relationship is found between (1) the disappearance of muscle/MY-174-reactive proteoglycan core protein staining at the surface of myotubes from St. 36 to St. 39 and (2) the infiltration of laminin and heparan sulfate proteoglycan staining encompassing groups of myotubes (St. 36) to circumferential staining of all myotubes (St. 39).(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of hyaluronan in various muscular tissues

Summary The histochemical distribution of hyaluronan (hyaluronic acid, HYA) was analysed in various types of muscles in the rat by use of a hyaluronan-binding protein (HABP) and the avidin-biotin/peroxidase complex staining procedure. Microwave-aided fixation was used to retain the extracellular location of the glycosaminoglycan. In skeletal muscles, HYA was detected in the connective tissue sheath surrounding the muscles (epimysium), in the septa subdividing the muscle fibre bundles (perimysium) and in the connective tissue surrounding each muscle fibre (endomysium). HYA was heterogeneously distributed in all striated muscles. In skeletal muscles with small fibre dimensions (e.g., the lateral rectus muscle of the eye and the middle ear muscles), HYA was predominantly accumulated around the individual muscle fibres. Perivascular and perineural connective tissue formations were distinctly HYA-positive. In cardiac muscles, HYA was randomly distributed around the branching and interconnecting muscle fibres. In comparison, smooth muscle tissue was devoid of HYA.     

Myogenesis and histogenesis of skeletal muscle on flexible membranes in vitro

Summary Primary muscle cell cultures consisting of single myocytes and fibroblasts are grown on flexible, optically clear biomembranes. Muscle cell growth, fusion and terminal differentiation are normal. A most effective membrane for these cultures is commercially available Saran Wrap. Muscle cultures on Saran will, once differentiated, contract vigorously and will deform the Saran which is pinned to a Sylgard base. At first, the muscle forms a two-dimensional network which ultimately detaches from the Saran membrane allowing an undergrowth of fibroblasts so that these connective tissue cells completely surround groups of muscle fibers. A three-dimensional network is thus formed, held in place through durable adhesions to stainless steel pins. This three-dimensional, highly contractile network is seen to consist of all three connective tissue compartments seenin vivo, the endomysium, perimysium and epimysium. Finally, this muscle shows advanced levels of maturation in that neonatal and adult isoforms of myosin heavy chain are detected together with high levels of myosin fast light chain 3. Antibody 2E9 to neonatal myosin heavy chain was obtained from Dr. Everett Bandman. MF 20 which reacts with all myosin heavy chain isoforms including the embryonic isoform and MF 14 which reacts specifically with adult myosin heavy chain were obtained from Drs. Bader and Fischman. Antibody to myosin fast light chain 3 was obtained from Dr. Susan Lowey. Antibody to fibronectin was obtained from Dr. Douglas Fambrough. This work was supported by grants to R. C. S. from the Muscular Dystrophy Association and from NIH. Editor's Statement The paper represents a novel and interesting approach to the co-culture of myotubes with fibroblasts which allows three dimensional development of endomysium, perimysium and epimysium and expression of adult-type muscle proteins. Such organogenic development is not normally seen in vitro. The technique should prove useful in elucidating development aspects of muscle cells and their relationship with connective support.     

Characterization of muscle epimysium, perimysium and endomysium collagens

In the past it has been proven difficult to separate and characterize collagen from muscle because of its relative paucity in this tissue. The present report presents a comprehensive methodology, combining methods previously described by McCollester [(1962) Biochim. Biophys. Acta 57, 427-437] and Laurent, Cockerill, McAnulty & Hastings [(1981) Anal. Biochem. 113, 301-312], in which the three major tracts of muscle connective tissue, the epimysium, perimysium and endomysium, may be prepared and separated from the bulk of muscle protein. Connective tissue thus prepared may be washed with salt and treated with pepsin to liberate soluble native collagen, or can be washed with sodium dodecyl sulphate to produce a very clean insoluble collagenous product. This latter type of preparation may be used for quantification of the ratio of the major genetic forms of collagen or for measurement of reducible cross-link content to give reproducible results. It was shown that both the epimysium and perimysium contain type I collagen as the major component and type III collagen as a minor component; perimysium also contained traces of type V collagen. The endomysium, the sheaths of individual muscle fibres, was shown to contain both type I and type III collagen as major components. Type V collagen was also present in small amounts, and type IV collagen, the collagenous component of basement membranes, was purified from endomysial preparations. This is the first biochemical demonstration of the presence of type IV collagen in muscle endomysium. The preparation was shown to be very similar to other type IV collagens from other basement membranes on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and was indistinguishable from EHS sarcoma collagen and placenta type IV collagen in the electron microscope after rotary shadowing.     

不同脊椎动物骨骼肌的解剖结构与动物进化程度的关系

目的:依据发育重演律的理论,比较进化程度不同的脊椎动物骨骼肌是否存在结构层次的差异。方法:选取进化程度不同的脊椎动物,如哺乳动物、鸟类、两栖动物及鱼类,选择各类有代表性并容易取材的动物,通过苏木精伊红染色(HE染色)的方法对健康的昆明白小鼠、家兔、家鸽、牛蛙、鲫鱼背部及腿部肌肉横切面进行观察。结果:昆明白小鼠、家兔、家鸽、牛蛙、鲫鱼的骨骼肌都有相类似的层次结构,即每块骨骼肌由数个肌束构成,骨骼肌外被肌外膜,肌束由肌束膜包绕,每个肌束又由众多肌纤维构成,肌纤维由肌内膜包绕。骨骼肌的层次结构与动物的进化程度和实验取材部位无关。结论:表明进化程度不同的脊椎动物骨骼肌的进化程度相近。表明骨骼肌的3层结构并非在脊椎动物阶段进化完成的。     

Chick myotendinous antigen. I. A monoclonal antibody as a marker for tendon and muscle morphogenesis

Extracellular matrix components are likely to be involved in the interaction of muscle with nonmuscle cells during morphogenesis and in adult skeletal muscle. With the aim of identifying relevant molecules, we generated monoclonal antibodies that react with the endomysium, i.e., the extracellular matrix on the surface of single muscle fibers. Antibody M1, which is described here, specifically labeled the endomysium of chick anterior latissimus dorsi muscle (but neither the perimysium nor, with the exception of blood vessels and perineurium, the epimysium ). Endomysium labeling was restricted to proximal and distal portions of muscle fibers near their insertion points to tendon, but absent from medial regions of the muscle. Myotendinous junctions and tendon fascicles were intensely labeled by M1 antibody. In chick embryos, " myotendinous antigen" (as we tentatively call the epitope recognized by M1 antibody) appeared first in the perichondrium of vertebrae and limb cartilage elements, from where it gradually extended to the premuscle masses. Around day 6, tendon primordia were clearly labeled. The other structures labeled by M1 antibody in chick embryos were developing smooth muscle tissues, especially aorta, gizzard, and lung buds. In general, tissues labeled with M1 antibody appeared to be a subset of the ones accumulating fibronectin. In cell cultures, M1 antibody binds to fuzzy, fibrillar material on the substrate and cell surfaces of living fibroblast and myogenic cells, which confirms an extracellular location of the antigenic site. The appearance of myotendinous antigen during limb morphogenesis and its distribution in adult muscle and tendon are compatible with the idea that it might be involved in attaching muscle fibers to tendon fascicles. Its biochemical characterization is described in the accompanying paper ( Chiquet , M., and D. Fambrough , 1984, J. Cell Biol. 98:1937-1946).     

Analysis of fibronectin expression during human muscle differentiation

Fibronectin expression during human muscle differentiation was investigated by determining its distribution in foetal, normal adult and dystrophic muscle and in foetal, normal adult and dystrophic muscle cultures during myogenesis. Muscle sections and muscle cultures were studied by indirect immunofluorescence staining using polyclonal and monoclonal anti-human antibodies. Mass and clonal muscle cultures were prepared from foetal, adult and dystrophic muscle tissue. Immunofluorescence staining detected fibronectin on the epimysium, perimysium and endomysium of transverse sections of normal adult muscle, while sarcoplasm was devoid of this glycoprotein. In foetal muscle, some fibers showed a prominent ring of fibronectin. In mass and clonal cultures, myoblasts were found to synthesize and accumulate fibronectin while myotubes did not. No difference in fibronectin distribution was observed between Duchenne Muscular Dystrophy (DMD) and control myotubes. An enzyme-linked immunoassay (ELISA), performed on homogenated muscle, sonicated fibroblasts and muscle cells, showed a high fibronectin level in fibroblasts when compared with the other samples tested.     

Differential and restricted expression of novel collagen VI chains in mouse

Recently, three novel collagen VI chains, α4, α5 and α6, were identified. These are thought to substitute for the collagen VI α3 chain, probably forming α1α2α4, α1α2α5 or α1α2α6 heterotrimers. The expression pattern of the novel chains is so far largely unknown. In the present study, we compared the tissue distribution of the novel collagen VI chains in mouse with that of the α3 chain by immunohistochemistry, immunoelectron microscopy and immunoblots. In contrast to the widely expressed α3 chain, the novel chains show a highly differential, restricted and often complementary expression. The α4 chain is strongly expressed in the intestinal smooth muscle, surrounding the follicles in ovary, and in testis. The α5 chain is present in perimysium and at the neuromuscular junctions in skeletal muscle, in skin, in the kidney glomerulus, in the interfollicular stroma in ovary and in the tunica albuginea of testis. The α6 chain is most abundant in the endomysium and perimysium of skeletal muscle and in myocard. Immunoelectron microscopy of skeletal muscle localized the α6 chain to the reticular lamina of muscle fibers. The highly differential and restricted expression points to the possibility of tissue-specific roles of the novel chains in collagen VI assembly and function.     

Biological resorption of fibers from chitosan in endomysium and perimysium of muscular tissue

The resorption of fibers from chitosan implanted into emdomysium and perimysium of the rat’s broadest muscle of the back is comparatively studied in vivo by the scanning electron microscopy and histologic analysis methods. It is shown that the mechanism and rate of resorption of the fibers from chitosan depend on the fiber localization in the muscular tissue. Implantation of chitosan fibers into endomysium, where they have been in direct contact with muscle fibers, results in 14 days in the formation of transverse cracks, fiber fragmentation, and their partial resorption. Complete resorption of fibers in endomysium is observed in 30 days. Fibers implanted into perimysium maintain integrity in 7 days of the experiment, and a fibrous tissue is formed around the fibers. There is no destruction of chitosan fibers in 45 days of the exposition. The biocompatibility of the chitosan fibers is confirmed by the effective adhesion and proliferation mesenchyme stem cells on their surface.     

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.     

Age-related changes in the mechanical properties of the epimysium in skeletal muscles of rats

Skeletal muscle is composed of muscle fibers and an extracellular matrix (ECM). The collagen fiber network of the ECM is a major contributor to the passive force of skeletal muscles at high strain. We investigated the effect of aging on the biomechanical and structural properties of epimysium of the tibialis anterior muscles (TBA) of rats to understand the mechanisms responsible for the age-related changes. The biomechanical properties were tested directly in vitro by uniaxial extension of epimysium. The presence of age-related changes in the arrangement and size of the collagen fibrils in the epimysium was examined by scanning electron microscopy (SEM). A mathematical model was subsequently developed based on the structure-function relationships that predicted the compliance of the epimysium. Biomechanically, the epimysium from old rats was much stiffer than that of the young rats. No differences were found in the ultrastructure and thickness of the epimysium or size of the collagen fibrils between young and old rats. The changes in the arrangement and size of the collagen fibrils do not appear to be the principal cause of the increased stiffness of the epimysium from the old rats. Other changes in the structural composition of the epimysium from old rats likely has a strong effect on the increased stiffness. The age-related increase in the stiffness of the epimysium could play an important role in the impaired lateral force transmission in the muscles of the elderly.     

Developmental expression of extracellular matrix components in intramuscular connective tissue of bovine semitendinosus muscle

 We have investigated the expression patterns of extracellular matrix components in intramuscular connective tissue during the development of bovine semitendinosus muscle by means of indirect immunofluorescence techniques. Types I, III, V, and VI collagen and fibronectin were located in the endomysium and the perimysium. Type IV collagen, laminin, and heparan sulfate proteoglycans (PGs) were exclusively located in the endomysium, and dermatan sulfate PGs existed only in the perimysium. The localization of these components in the intramuscular connective tissue of semitendinosus muscle remained unchanged throughout prenatal and postnatal growth of cattle, suggesting that they are essential for forming and maintaining structures of the endomysium and perimysium in bovine semitendinosus muscle. On the other hand, decorin was undetectable in the endomysium of neonates, although other matrix components were already expressed. It was expressed slightly in the endomysium of 2-month-old calves, and clearly detectable in the endomysium of cattle more than 6 months old. Chondroitin sulfate PGs were barely detectable in the perimysium of fetuses and neonatal calves, and progressively appeared during postnatal development of the muscle. It seems likely that these PGs are closely related to the postnatal development of the endomysium and perimysium. Accepted: 30 October 1996     

Increase in decorin and biglycan in Duchenne Muscular Dystrophy: role of fibroblasts as cell source of these proteoglycans in the disease

Fibrosis is a common pathological feature observed in muscles of patients with Duchenne muscular dystrophy (DMD). Biglycan and decorin are small chondroitin/dermatan sulfate proteoglycans in the muscle extracellular matrix (ECM) that belong to the family of structurally related proteoglycans called small leucine-rich repeat proteins. Decorin is considered an anti-fibrotic agent, preventing the process by blocking TGF-beta activity. There is no information about their expression in DMD patients. We found an increased amount of both proteoglycans in the ECM of skeletal muscle biopsies obtained from DMD patients. Both biglycan and decorin were augmented in the perimysium of muscle tissue, but only decorin increased in the endomysium as seen by immunohistochemical analyses. Fibroblasts were isolated from explants obtained from muscle of DMD patients and the incorporation of radioactive sulfate showed an increased synthesis of both decorin and biglycan in cultured fibroblasts compared to controls. The size of decorin and biglycan synthesized by DMD and control fibroblasts seems to be similar in size and anion charge. These findings show that decorin and biglycan are increased in DMD skeletal muscle and suggest that fibroblasts would be, at least, one source for these proteoglycans likely playing a role in the muscle response to dystrophic cell damage.     

Morphology of connective tissue in skeletal muscle

The arrangement and distribution of connective tissue in six different skeletal muscles and smooth muscle was examined by scanning electron microscopy. The endomysial arrangement of collagen was similar in all types of muscle and consisted of three components: (1) myocyte-myocyte connectives; (2) myocyte-capillary connectives; and (3) a weave network of collagen intimately associated with the basal laminae of the myocytes. The perimysium of the different muscles was qualitatively similar but quantitatively dissimilar. The perimysium consisted of large tendon-like bundles of interwoven collagen which connected with the dense weave collagen that surrounded groups of muscles. The arrangement of the collagen in the perimysium and endomysium would explain differences in the mechanical properties of the different muscle. The contribution of the connective tissue to mechanical properties of muscle is discussed.     

Finite element analysis of mechanics of lateral transmission of force in single muscle fiber

Most of the myofibers in long muscles of vertebrates terminate within fascicles without reaching either end of the tendon, thus force generated in myofibers has to be transmitted laterally through the extracellular matrix (ECM) to adjacent fibers; which is defined as the lateral transmission of force in skeletal muscles. The goal of this study was to determine the mechanisms of lateral transmission of force between the myofiber and ECM. In this study, a 2D finite element model of single muscle fiber was developed to study the effects of mechanical properties of the endomysium and the tapered ends of myofiber on lateral transmission of force. Results showed that most of the force generated is transmitted near the end of the myofiber through shear to the endomysium, and the force transmitted to the end of the model increases with increased stiffness of ECM. This study also demonstrated that the tapered angle of the myofiber ends can reduce the stress concentration near the myofiber end while laterally transmitting force efficiently.     

Immunohistochemical evidence for the existence of rat cytosolic sialidase in rat skeletal muscles

 Histochemical evidence is required to demonstrate the presence of biochemically defined cytosolic sialidase. To meet this requirement, we examined the immunohistochemical localization of the enzyme in rat skeletal muscles. Sections of chemically fixed tissues were incubated with a polyclonal antibody raised against a synthetic peptide which corresponded to a part of the enzyme protein. After incubation with the primary antibody, cryosections for fluorescence microscopy and resin sections for electron microscopy were incubated with a fluorochrome- and colloidal gold-labeled secondary antibody, respectively. Immunofluorescence was diffusely distributed in the muscle fibers and was also found in the perimysium and blood vessels. Many immunogold particles were scattered over the sarcoplasm, myofibrils, nucleoplasm, and matrix of mitochondria. The immunogold particles were also found in the equivalent compartments of axons, Schwann cells, and cells of endomysium and blood vessels. The specificity of the primary antibody was elucidated by immunoblotting and an immunoprecipitation test. These findings clearly indicate that this type of sialidase is essentially located in the cytosolic compartment. Consequently, the name, cytosolic sialidase, will be appropriate for this enzyme. Additionally it is indicated that this enzyme is also present in cells other than skeletal muscle fibers. Accepted: 29 January 1997