Extraocular muscles in the lamprey, Lampetra fluviatilis L. I. Muscle fibres

Six extraocular muscles of the river lamprey, Lampetra fluviatilis L., were studied with the light and electron microscope. On the basis of morphology and histochemistry three types of muscle fibres were distinguished: thin, thick mitochondria-rich and thick multifibrillar fibres. In the thin fibres, 2.8-22.4 microns in diameter, myofibrils are distributed peripherally and show strong ATPase activity. The mitochondria are located paraxially. In the thick mitochondria-rich fibres, 19.4-31.0 microns in diameter, myofibrils are also located peripherally, whereas the central part of the fibre is densely packed with very numerous mitochondria possessing tubular cristae. Thick multifibrillar fibres, with a diameter similar to that of the former type, contain thin myofibrils scattered over the entire cross-section of the fibre. The activity of myofibrillar ATPase is lower in both types of thick fibres than in the thin ones. The tubules of the T system were observed frequently only in the thick multifibrillar fibres. The extraocular muscles of the lamprey are composed of large quantities of muscle fibres. Thin and thick fibres do not form separate layers, but are more or less uniformly distributed throughout the muscle. Many muscle fibres show structural features suggesting their degeneration.     

Abnormalities of the nuclear envelope in porcine muscle affected with congenital myofibrillar hypoplasia.

The nuclear ultrastructure of muscle fibres in congenital myofibrillar hypoplasia of piglets was studied in order to provide more information on the pathological changes present. In the initial stages of the disease, muscle fibres with a small reduction of myofibrils often had an increased number of nuclear pores. In the more advanced stages of the disease, the following nuclear abnormalities were observed in muscle fibres showing an extensive disintegration of myofibrils: 1) large dilations of the perinuclear cisternae, 2) small nuclear protrusions projecting into these dilations and 3) appositions of stray membranes of the disrupted endoplasmic reticulum to the nuclear envelope. The increase in the number of nuclear pores may be significant in the development of pathological changes in congenital myofibrillar hypoplasia which is thought to be a hereditary disease. The observed abnormalities of the nuclear envelope appear to be characteristic for muscle fibres undergoing degeneration.     

Satellite cells in the tail muscles of the urodelan larvae during development

The incidence and ultrastructure of satellite cells in the tail muscles of urodelan larvae were examined during development during which the number of satellite cells is gradually reduced. They are found more frequently in red than in the white fibres in all four stages examined (stage 53, 64, 66+ and juvenile). As development proceeds, intercellular space between satellite cell and muscle fibre is in general gradually extended and is mostly filled with basal lamina. Small muscle cells, satellite fibres, which are situated under the basal lamina of the parent fibre, are morphologically similar to satellite cells but contain a small amount of myofibrils. Three types of satellite fibres are distinguishable on the basis of differences in K2-EDTA-treated ATPase activity, width of Z line, and parent fibre type. Neuromuscular junctions are visible in satellite fibres.     

Muscle development in larvae of a fast growing tropical freshwater fish, the curimatã-pacú

The distribution and ultrastructure of myotomal muscle fibres was studied in larvae and early juveniles of the curimatã-pacú Prochilodus marggravii , a tropical freshwater fish endemic to the São Francisco River system, Brazil. At 26°C, larvae hatched 15 h post-fertilization at a relatively early stage of development with the head still curved around the yolk-sac (head-trunk angle greater than 45°), and prior to pigmentation of the eyes and formation of the jaws, gut and pectoral fins. Although motile the swimming muscles of newly-hatched larvae were largely undifferentiated. The myotomes were made up of a single layer of superficial muscle fibres containing six to eight myofibrils and abundant mitochondria, surrounding an inner core of myoblasts, myotubes and immature muscle fibres. The volume densities of mitochondria and myofibrils in the immature inner muscle fibres of 1-day-old lavae were 14.5 and 6.4% respectively. The body axis straightened within 24 h of hatching and the yolk sac was completely absorbed by 72 h. Larval development was rapid with gill filaments, a muscular stomach, liver and swimbladder present after 7 days. The inner muscle fibres were well differentiated in 7-day-old larvae; the volume density of myofibrils had increased to 63.1% whereas the volume density of mitochondria had decreased to 3.5%. In 14-day-old juveniles the superficial muscle had thickened to a layer two to three fibres thick in the region of the lateral line nerve and capillaries were present in the inner muscle. Muscle growth until 14 days was largely due to the hypertrophy of the fibres present at hatching.     

Three muscle fibre types in the axial muscle of axolotl (Ambystoma mexicanum Shaw) A quantitative lightand electron microscopic study

Morphometric analysis by light microscopy of p-phenylene-diamine stained semithin sections of axolotl tail muscle revealed differences in the cross-sectional area of the fibres and in the number of mitochondria and of lipid inclusions per fibre, and indicated the presence of three distinct types of fibres. The tripartition was found to be statistically highly significant. Representative fibres from each group established by light microscopic morphometry were subjected to an ultrastructural morphometric analysis. The volume content of mitochondria amounted to 9.8% of the fibre volume for red, 4.0% for intermediate and 0.8% for white fibres. The myofibrils composed 60%, 70% and 83% in the same fibres. The volume of the sarcotubular system (t-tubuli and sarcoplasmic reticulum) was 2.5% in red, 4.5% in intermediate and 11.7% in white fibres. The three fibre types also demonstrated differences in myofibrillar cross-striation pattern and number of triads. The reliability of the light microscopic morphometry was tested by correlation with EM montages of the representative fibres.     

Novel thick filament protein of chicken pectoralis muscle: the 86 kd protein. II. Distribution and localization

Antibodies specific for the novel 86 kd protein purified from chicken pectoralis myofibrils stained by indirect immunofluorescence the middle third of each half A-band of isolated myofibrils and myotubes. Pectoralis muscle 86 kd protein, like pectoralis C-protein, displayed a fibre-type specific distribution by being restricted to fast twitch fibres and absent in slow tonic and heart muscle fibres. This was demonstrated by immunoblotting experiments with tissue extracts and by immunofluorescence labelling of cryosections. In primary cell cultures prepared from embryonic chicken breast muscle, 86 kd protein, C-protein and myomesin were all detected in post-mitotic myoblasts where fluorescence was found in a cross-striated pattern along strands of nascent myofibrils. Fluorescence due to the 86 kd protein was restricted to myofibrils within myotubes and no significant labelling of the sarcoplasm was evident. Glycerinated fast twitch muscle fibres, after incubation with antibodies to 86 kd protein, revealed in each half of the A-band nine distinctly labelled stripes, spaced about 43 nm apart. Simultaneous incubation of fibres with antibodies against 86 kd protein and C-protein showed a co-localization of the seven C-protein stripes (stripes 5 to 11), with seven stripes of 86 kd protein. The two additional stripes (stripes 3 and 4) labelled by anti-86 kd antibody continued towards the M-band at the same periodicity from the last C-protein stripe (stripe 5). Thus, partial co-localization of two different thick filament proteins is demonstrated and the identity of transverse stripes at positions 3 and 4 attributed in part to the presence of the new 86 kd protein.     

EXERCISE MYOPATHY: CHANGES IN MYOFIBRILS OF FAST-TWITCH MUSCLE FIBRES

The purpose of the present study was to determine the relationships between the changes of myofibrils in fast-twitch oxidative-glycolytic (type IIA) fibres and fast-twitch glycolytic (type IIB) muscle fibres, protein synthesis and degradation rate in exercise-induced myopathic skeletal muscle. Exhaustive exercise was used to induce myopathy in Wistar rats. Intensity of glycogenolysis in muscle fibres during exercise, protein synthesis rate, degradation rate and structural changes of myofibrils were measured using morphological and biochemical methods. Myofibril cross sectional area (CSA) in type IIA fibres decreased 33% and type IIB fibres 44%. Protein degradation rate increased in both type IIA and IIB fibres, 63% and 69% respectively in comparison with the control group. According to the intensity of glycogenolysis, fast oxidative-glycolytic fibres are recruited more frequently during overtraining. Myofibrils in both types of fast-twitch myopathic muscle fibres are significantly thinner as the result of more intensive protein degradation. Regeneration capacity according to the presence of satellite cells is higher in type IIA fibres than in type IIB fibres in myopathic muscle.     

The so-called tonic muscle fibre type in cyprinid axial muscle: their morphology and response to endurance exercise training

The morphology and the effect of an endurance training programme on tonic muscle fibres were studied in chub Leuciscus cephalus , by means of histochemistry and immunohistochemistry, electronmicroscopy and morphometry/stereology. Location and distribution, SDH- and mATPase-activity, reaction to an anti-tonic myosin antibody and ultrastructural features of the fibre type were investigated. With regard to training conditions, fibre size was not significantly affected. However, an increase in the volume densities of mitochondria, lipid and myofibrils can be observed, suggesting a training influence on the aerobic capacity of the so-called tonic muscle fibres. Based on the quantitative findings, the fine structure and the response to training, similarities with intermediate muscle fibres and the functional role of these so-called tonic muscle fibres are discussed.     

Mechanism of myofibril growth and proliferation in fish muscle.

The mechanisms of myofibril growth proliferation were investigated in the red and white muscles of fish. In both types of muscle the ratio of lattice filament spacings between the Z disk and M line was found to be greater than that required for perfect transformation of a square into a hexagonal lattice. This mismatch was considered to result in the thin filaments being pulled obliquely instead of at right angles to the Z disk. The angle of pull of the thin filaments was measured in longitudinal sections. The splitting process was found to decrease the degree of pull. Splitting was also observed in transverse sections of the peripheral myofibrils. In both red and white fibres these myofibrils were found to commence splitting when they reached a size of approximately 1-2 mum diameter. Evidence from ultrastructural and autoradiographical studies suggested that growth of the myofibrils within the fibres is centrifugal. The outermost myofibrils appear to be the ones which are being built up and which split. The data indicated that in fish muscle a considerable number of filaments may be added to the daughter regions whilst splitting of the myofibril is still continuing.     

The fine structure of pigeon breast muscle

The pectoralis major muscle of the pigeon is composed of two types of muscle fibre. In the Type I fibres, the myofibrils are closely packed and there are few mitochondria. The myofibrils in the Type II fibres are separated by numerous columns of large mitochondria and lipid droplets. The membrane systems of the two types of fibre are similar. The triads occur at the Z-line; the sarcoplasmic reticulum is in the form of large terminal cisternae which are joined by narrow longitudinal tubules to a broad central cisterna. The value of morphological criteria in the classification of muscle fibres is discussed.     

Mucosubstances in the human skeletal muscles.

With histomchemical, and electronmicroscopic-histochemical methods two types of human skeletal muscle fibres were established. The first type of muscle fibres does not contain acidic mucosubstances. The staining reactions and cellulase digestion indicate that, the neutral polysaccharides are cellulose-like substances. The second type of fibres contains only acidic mucosubstances, hyaluronic acid and chondroitine sulphate. The author suggests that the mucosubstances have joint function. These polysaccharides contributed to the jointing both of myofibrils and sarcomers. The polysaccharides can be exhibited in the joint points of contractile elements. In mechanical injury this point became disintegrated.     

Multinucleation during myogenesis of the myotome of Xenopus laevis: a qualitative study

Ultrastructural studies of myogenesis in the myotome of Xenopus laevis reveal that the myotubes developed by stage 33/34 have peripheral myofibrils but are still uninucleate with a single large nucleus. By stage 45, the cytoplasm of the muscle cells is filled with myofibrils and there are many small peripheral nuclei, resulting in multinucleate muscle fibres. With the electron microscope, we have examined myotomes from stages 33/34 to 59 of development and some stages were also investigated by autoradiography. There was no evidence from autoradiographic studies for DNA synthesis in muscle cells, and the increase in the number of myonuclei was accompanied by a decrease in their size. Satellite cells were not seen at the myotube stage but were first seen after the cells had become multinucleate, with many small nuclei close together forming rows. Constrictions were frequently observed in the large single nuclei. It is concluded that division of the myonuclei by amitosis is mainly responsible for the multinucleation that occurs during development of the myotome muscle in Xenopus laevis.     

Histochemical and Ultrastructural Features of the Biceps Brachii of the African Chameleon (Chamaeleo senegalensis)

Abstract Characteristics of reptilian muscle fibres were investigated in the biceps brachii of the African chameleon, Chamaeleo senegalensis. Fibres were classified as slow and fast. These types of fibre were distinguished on the basis of histochemical staining for myofibrillar ATPase (mATPase). Fast fibres stained dark for mATPase while slow fibres stained light. The patterns of innervation of slow and fast fibres were investigated by staining nerve endings for acetylcholinesterase activity. Slow fibres have a pattern of multiple innervation, whereas fast fibres are associated with individual endplates. The organization of the myofibrils and the sarcoplasmic reticulum in slow muscle fibres from the chameleon biceps brachii was compared with that in fast fibres. Slow fibres lacked an M-line and the Z-lines were uneven. They had fibrils that were not clearly separated from each other and the sarcoplasmic reticulum was poorly developed. These features are in sharp contrast to those of fast fibres which had straight Z-lines, clear M-lines and well-developed sarcoplasmic reticulum.     

Temperature and neuromuscular development in the tambaqui

The development of muscle innervation pattern was investigated in larvae of the Amazonian fish, the tambaqui Colossoma macropomum. The time to hatching decreased from 28–29 h at 23.5° C to 11–12 h at 31° C. The larvae hatched after the completion of somitogenesis (38-somite stage) at 23.5° C but only at the 33-somite stage at 28–31° C. Embryos were stained for acetylcholinesterase activity and with an acetylated tubulin antibody in order to visualize neural processes. All muscle fibre types were initially innervated at their myoseptal ends. The development of motor innervation to the trunk muscle was delayed with respect to hatching at higher temperatures. At hatching, muscle fibres were innervated only to somites 16–17 at 28–31° C and somite 23–26 at 23.5–25° C (counting from the head), although the larvae swam vigorously to avoid sinking. In contrast, in newly hatched larvae myofibrils were present right along the trunk at all temperatures in both the superficial and inner muscle fibres. At hatching numerous multi-layered membrane contacts with the ultrastructural characteristics of gap junctions, were found between muscle fibres and at the inter-somite junctions, suggesting the somites were initially electrically coupled. These structures disappeared concomitant with the development of muscle endplates right down the trunk. The larvae started feeding 5 days post-hatch at 28° C. First feeding was associated with a dramatic decrease in the volume density of mitochondria and an increase in the volume density of myofibrils in the inner muscle fibres. The polyneuronal and multi-terminal pattern of innervation characteristic of adult slow-muscle fibres also developed around the time of first feeding.     

Fusion of myocytes with larval muscle remnants during flight muscle development in the Colorado beetle

The transformation of the slow contracting larval m. obliquus lateralis caudalis II during metamorphosis into the asynchronous indirect flight muscle, m. obliquus lateralis dorsalis, in the Colorado beetle, Leptinotarsa decemlineata, was examined by electron microscopy. Particular attention was paid to the fate of the larval muscle fibres, the origin and behaviour of the myoblasts for flight muscle development and the change of the myofibrillar filament lattice of the larva into that of the adult. In the pre-pupal period, the larval muscles dedifferentiate and fragment. At pupation, the muscle fibres consist of cell fragments containing very few myofibrils. The sarcoplasmic reticulum and the transverse tubular system are greatly reduced. The number of myoblasts developed from satellite cells by mitosis increases considerably. They penetrate the muscle fibre and surround the cell fragments. The new fibres of the flight muscle develop from myocytes fused with the larval fragments. The larval basal lamina, surrounding the cell fragments and myoblasts, is present in pupae up to 1 day old. In pupae about 2.5 days old new myofibrils appear that have the adult filament lattice. The insect muscle transformation and the repair of vertebrate muscle after injury show striking resemblances.     

Dedifferentiation and remodeling of motor endplates following experimental localized lesions of muscle fibers

Local anaesthetics, cardiotoxin and mechanical injuries may cause necrosis of muscle fibres while leaving the motor nerve fibres and their terminals intact. With local injuries to mouse muscles carried out by freezing or cutting we made a point of preserving both the nerve terminals and the muscle fibre portions on which these terminals were located. It was thus possible to follow the changes induced at endplates by these lesions. Within two or three days of the freezing or cutting, the muscle fibres underwent very different degrees of regression of the contractile material and T-system. The neuromuscular junctions also underwent changes, principally affecting their postsynaptic portion, in particular the folds of the subneural apparatus. After dedifferentiation of subsynaptic areas, we observed sprouting of the nerve terminal on muscle fibres which survived the amputation of one end and formed actively new myofibrils. This sprouting restored synaptic connections at the original sites, but with new structural features and correlative changes in the distribution of cholinergic receptors and cholinesterases. It is probable that after a phase of involution followed by a phase of recovery, the injured muscle fibres triggered off the nerve terminal sprouting which led to the remodelling of the endplates.     

Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating sites for the assembly of new sarcomeres. How 1.7-2.0 microns nascent sarcomeres can be added distally during elongation while the tips of the myofibrils remain inserted into submembranous adhesion plaques is unknown.     

Cytochemical studies of hydrogen peroxide production in the tadpole tail of Rana japonica during metamorphic climax

Summary The degeneration of tadpole tail tissue was investigated cytochemically by localizing the sites of hydrogen peroxide production. A cerium perhydroxide precipitation method was used. No reaction product was found in resting macrophages and intact muscle fibres during premetamorphosis. In the metamorphosis phase, extensive cerium precipitates were visualized on the outer surface of the plasma membrane of phagocytotic macrophages, fibroblasts, neutrophils, epidermal cells, muscle fibres, notochordal cells, nerve cells and capillary endothelial cells. The reaction products were localized on those parts of the plasma membranes of the macrophages that were in contact with those of adjoining cells. When catalase were added, the amount of deposits decreased. -Tocopherol and indomethacin, but not dexamethasone, significantly inhibited the formation of the reaction products. These findings are taken to indicate that active oxygen is produced on the plasma membrane of activated macrophages and may play a role in the degeneration of the tail tissue.     

Effect of myonuclear number and mitochondrial fusion on Drosophila indirect flight muscle organization and size

Mechanisms involved in establishing the organization and numbers of fibres in a muscle are not completely understood. During Drosophila indirect flight muscle (IFM) formation, muscle growth is achieved by both incorporating hundreds of nuclei, and hypertrophy. As a result, IFMs provide a good model with which to understand the mechanisms that govern overall muscle organization and growth. We present a detailed analysis of the organization of dorsal longitudinal muscles (DLMs), a subset of the IFMs. We show that each DLM is similar to a vertebrate fascicle and consists of multiple muscle fibres. However, increased fascicle size does not necessarily change the number of constituent fibres, but does increase the number of myofibrils packed within the fibres. We also find that altering the number of myoblasts available for fusion changes DLM fascicle size and fibres are loosely packed with myofibrils. Additionally, we show that knock down of genes required for mitochondrial fusion causes a severe reduction in the size of DLM fascicles and fibres. Our results establish the organization levels of DLMs and highlight the importance of the appropriate number of nuclei and mitochondrial fusion in determining the overall organization, growth and size of DLMs.     

Involution of the caudal musculature during metamorphosis in the ascidian,Botryllus schlosseri

Summary The caudal musculature of the free-swimming tadpole of the ascidian, B. schlosseri consists of cylindrical mononucleated cells connected in longitudinal rows flanking the axial notochord. During resorption of the larval tail, which is apparently induced by the contraction of the epidermis, muscle cells are dissociated and pushed into the body cavity where most of them are rapidly engulfed by phagocytes. In the initial stages of tail withdrawal muscle cells display surface alterations due to the disruption of intercellular junctions and disarrangement of myofibrils. Extensive degenerative changes, with shrinkage of mitochondria and disintegration of the contractile material are subsequently observed. Lysosomes and autophagic vacuoles are rarely seen and appear to play a secondary role in the degradation of the muscle cells, which occurs predominantly within the phagocytes. Myofilaments and myofibrils have never been observed within autophagic vacuoles. Clumps of muscle fragments and degenerated phagocytes undergo eventual dissolution in the blood lacunae, concomitantly with the differentiation of the young oozooid.This investigation was supported in part by a grant from the Muscular Dystrophy Associations of America and by CNR contract No. 7100396/04115542 from the Istituto di Biologia del Mare, Venice. We gratefully acknowledge the skillful assistance of Mr. G. Gallian, Mr. M. Fabbri and Mr. G. Tognon. We also thank the staff of the Stazione Idrobiologica at Chioggia for collecting the colonies.