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.     

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.     

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.

     

Morphology of perimysial and endomysial connective tissue in skeletal muscle

The fibrous components of the endomysium and perimysium of various muscles from four animal species were examined by scanning electron microscopy. All muscles were qualitatively similar. Endomysium consisted of a dense feltwork of collagen fibrils completely covering individual muscle fibre surfaces. Perimysium consisted of three fibrous components, (1) coarse, crimped fibres laid down in a well-ordered criss-cross pattern, (2) a loose feltwork of non-crimped fibrils, (3) fine noncrimped bundles of fibrils with no directional organization. The perimysium showed gradation from dense sheets of collagen down to the most delicate of sheets found on every muscle fibre surface overlying the endomysium.     

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)     

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     

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.     

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.     

The fine structure of myotendinous and myo-epithelial junctions in the guinea pig tongue (author's transl)]

The insertion of muscle fibers in the subepithelial connective tissue layer of the guinea pig tongue was studied light and electron microscopically. Fibers of the tractus verticalis approach the epithelium penetrating the lamina propria, both the reticular and papillar layer. Terminating muscle fibers split up and form branching finger-like cytoplasmic processes. The myotendinous junctions of such terminal processes fine structurally correspond to myotendinous junctions generally observed in skeletal or smooth muscles. The entire brush-like formation, however, is more far-reaching and highly differentiated. Filament bundles (spine-like profiles) originate from the plasmalemma and extend to the lamina densa of the basal lamina, especially in those regions where actin filaments are attached to the plasmalemma. Microfibrils (10 to 12 nm diameter) reach the lamina densa of the basal lamina. They form bundles which are continuous with fibrotubular strands of elaunin fibers and elastic fiber microfibrils. Furthermore, microfibrils are interwoven with collagen fibrils.     

Micromechanical modeling of the epimysium of the skeletal muscles

A micromechanical model has been developed to investigate the mechanical properties of the epimysium. In the present model, the collagen fibers in the epimysium are embedded randomly in the ground substance. Two parallel wavy collagen fibers and the surrounding ground substance are used as the repeat unit (unit cell), and the epimysium is considered as an aggregate of unit cells. Each unit cell is distributed in the epimysium with some different angle to the muscle fiber direction. The model allows the progressive straightening of the collagen fiber as well as the effects of fiber reorientation. The predictions of the model compare favorably against experiment. The effects of the collagen fiber volume fraction, collagen fiber waviness at the rest length and the mechanical properties of the collagen fibers and the ground substance are analyzed. This model allows the analysis of mechanical behavior of most soft tissues if appropriate experimental data are available.     

Diversity of extracellular matrix morphology in vertebrate skeletal muscle

Existing data suggest the extracellular matrix (ECM) of vertebrate skeletal muscle consists of several morphologically distinct layers: an endomysium, perimysium, and epimysium surrounding muscle fibers, fascicles, and whole muscles, respectively. These ECM layers are hypothesized to serve important functional roles within muscle, influencing passive mechanics, providing avenues for force transmission, and influencing dynamic shape changes during contraction. The morphology of the skeletal muscle ECM is well described in mammals and birds; however, ECM morphology in other vertebrate groups including amphibians, fish, and reptiles remains largely unexamined. It remains unclear whether a multilayered ECM is a common feature of vertebrate skeletal muscle, and whether functional roles attributed to the ECM should be considered in mechanical analyses of non-mammalian and non-avian muscle. To explore the prevalence of a multilayered ECM, we used a cell maceration and scanning electron microscopy technique to visualize the organization of ECM collagen in muscle from six vertebrates: bullfrogs (Lithobates catesbeianus), turkeys (Meleagris gallopavo), alligators (Alligator mississippiensis), cane toads (Rhinella marina), laboratory mice (Mus musculus), and carp (Cyprinus carpio). All muscles studied contained a collagen-reinforced ECM with multiple morphologically distinct layers. An endomysium surrounding muscle fibers was apparent in all samples. A perimysium surrounding groups of muscle fibers was apparent in all but carp epaxial muscle; a muscle anatomically, functionally, and phylogenetically distinct from the others studied. An epimysium was apparent in all samples taken at the muscle periphery. These findings show that a multilayered ECM is a common feature of vertebrate muscle and suggest that a functionally relevant ECM should be considered in mechanical models of vertebrate muscle generally. It remains unclear whether cross-species variations in ECM architecture are the result of phylogenetic, anatomical, or functional differences, but understanding the influence of such variation on muscle mechanics may prove a fruitful area for future research.     

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.     

Attachment of Salmonella spp. to chicken muscle surfaces.

Immersion of chicken muscle fascia in water or physiological saline caused collagen associated with the connective tissue to expand and form a dense network of fibers on the surface. Similar changes were noted for muscle perimysium. Two test strains of Salmonella spp. attached to the collagen fibers only when muscle was immersed for extended times in water. Bacteria did not attach to the fascia or perimysium of muscle that was transiently immersed in suspensions. The presence of sodium chloride in the suspension media prevented firm attachment, whereas saline rinses removed many attached cells.     

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.     

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.     

Immunohistochemical analysis of the distribution of type VI collagen in the lingual mucosa of rats during the morphogenesis of filiform papillae

Iwasaki, S., Yoshizawa, H. and Aoyagi, H. 2012. Immunohistochemical analysis of the distribution of type VI collagen in the lingual mucosa of rats during the morphogenesis of filiform papillae. —Acta Zoologica (Stockholm) 93 : 80–87. We examined the distribution after immunostaining of immunofluorescence of type VI collagen, differential interference contrast (DIC) images, and images obtained using confocal laser‐scanning microscopy in transmission mode, after toluidine blue staining, during morphogenesis of the filiform papillae, keratinization of the lingual epithelium and myogenesis in the rat tongue on semi‐ultrathin sections of epoxy resin‐embedded samples. Immunoreactivity specific for type VI collagen was dispersed over a relatively wide range of connective tissue in the mesenchyme of fetuses on day 15 after conception (E15), at which time the lingual epithelium was composed of one or two layers of cuboidal cells and the lingual muscle was barely recognizable. Slight immunoreactivity specific for type VI collagen was scattered within the lamina propria in fetuses on E17 and on E19, and immunoreactivity was relatively distinct on the connective tissue around the lingual muscle during myogenesis. In fetuses on E19, the epithelium was already stratified squamous. At postnatal stages from P0 to P14, keratinization of the lingual epithelium advanced gradually as morphogenesis of the filiform papillae proceeded during postnatal development. In newborns on P0, myogenesis of the tongue was almost completed. The intensity of immunoreactivity specific for type VI collagen at postnatal stages was mainly restricted on the endomysium and perimysium around the lingual muscle, while scant immunoreactivity was evident in the connective tissue in the lamina propria. Immunoreactivity around the fully mature lingual muscle on P7 and P14 was weaker than that on E19 and P0. Thus, type VI collagen appeared in the connective tissue that surrounded the lingual muscles such as the endomysium and perimysium, in parallel with changes in extracellular components during myogenesis of the tongue.     

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).     

Strain-induced reorientation of an intramuscular connective tissue network: implications for passive muscle elasticity

The most abundant intramuscular connective tissue component, the perimysium, of bovine M. sternomandibularis muscle was shown to be a crossed-ply arrangement of crimped collagen fibres which reorientate and decrimp on changing muscle fibre sarcomere length. Reorientation of perimysial strands was observed by light microscopy and identification of these strands as collagen fibres was confirmed by high-angle X-ray diffraction. Mean collagen fibre direction with respect to the muscle fibres ranged from approximately 80 degrees at sarcomere length = 1.1 micron to approximately 20 degrees at 3.9 microns. This behaviour was well described by a model of a crimped planar network surrounding a muscle fibre bundle of constant volume but varying length. Modelling of the mechanical properties of the perimysium at different sarcomere lengths produced a load-sarcomere length curve which was in good agreement with the passive elastic properties of the muscle, especially at long sarcomere lengths. It is concluded that the role of the perimysial collagen network is to prevent over-stretching of the muscle fibre bundles.