Myogenic skeletal muscle satellite cells communicate by tunnelling nanotubes

Quiescent satellite cells sit on the surface of the muscle fibres under the basal lamina and are activated by a variety of stimuli to disengage, divide and differentiate into myoblasts that can regenerate or repair muscle fibres. Satellite cells adopt their parent's fibre type and must have some means of communication with the parent fibre. The mechanisms behind this communication are not known. We show here that satellite cells form dynamic connections with muscle fibres and other satellite cells by F‐actin based tunnelling nanotubes (TNTs). Our results show that TNTs readily develop between satellite cells and muscle fibres. Once developed, TNTs permit transport of intracellular material, and even cellular organelles such as mitochondria between the muscle fibre and satellite cells. The onset of satellite cell differentiation markers Pax‐7 and MyoD expression was slower in satellite cells cultured in the absence than in the presence of muscle cells. Furthermore physical contact between myofibre and satellite cell progeny is required to maintain subtype identity. Our data establish that TNTs constitute an integral part of myogenic cell communication and that physical cellular interaction control myogenic cell fate determination. J. Cell. Physiol. 223: 376–383, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

The morphology of regeneration of skeletal muscles in the rat

Muscle regeneration was studied by light and electron microscopy in soleus muscles of rat. After segmental crushing the number of fibres increased in some muscles within 30 days, indicating that numerical hyperplasia can take place. Locally applied Ringer solution of 60-70 degrees C caused necrosis of myofibres but left satellite cells and blood supply largely intact. Following phagocytosis, four mechanisms of regeneration were seen. (1) Lost fibres were replaced by clusters of myotubes formed by satellite cells within persisting basal lamina tubes. These clusters displaced the surrounding endomysium and looked like longitudinally 'split' fibres. (2) Viable fibre fragments fused with satellite cells. (3) Satellite cells of surviving fibres proliferated and fused to myotubes localized beneath the basal lamina. (4) Thin new fibres occurred in the interstitium. Their origin remained unknown. After 6 months the mean size of the new myofibres was normal, but the scatter of diameters was increased, central nuclei, fibre 'spliting' and branching, and fibrosis were prominent. Staining for acetylcholinesterase revealed that many fibres were short and not innervated. The similarity with dystrophic muscles in man suggested, that the most prominent histological changes in myopathic muscles may be due to attempts of regeneration.     

Post-larval growth in the lateral white muscle of the eel, Anguilla anguilla

The post-larval growth of lateral white muscle was studied in eels at different stages of post-larval development (glass, yellow and silver eels) by means of histochemical methods for myosin-ATPase (mATPase) activity, immunohistochemistry (for myosin isoforms) and electron microscopy.
Morphological, histo- and immunohistochemical data reveal a uniform appearance of white muscle in glass eels, whereas in following stages the typical mosaic appearance is present. Small-diameter fibres show a more acid-labile mATPase activity than large fibres and react with anti-F, anti-FHC and anti-S sera, but not with anti-SHC serum. In the silver stage, the small fibres tend to decrease in number as the size of the eels increases.
Electron microscopy reveals the presence of satellite cells at every stage: in glass eels there are also 'activated' elements showing scarce myofilaments in their cytoplasm; in yellow eels very small fibres are present, enveloped within the basal lamina of well-differentiated muscle fibres; in silver eels there are no fibres showing signs of immaturity.
Presumably the post-larval development of white muscle involves in juvenile eels a substantial recruitment of fibres from the satellite cell population; later the hyperplasia decreases or ceases and hypertrophy remains the only mechanism for muscle growth.     

The number of satellite cells in slow and fast fibres from human vastus lateralis muscle

The aim of this investigation was to study the distribution of satellite cells in slow (type I fibres) and fast (type II fibres) fibres from human vastus lateralis muscle. This muscle is characterised by a mixed fibre type composition and is considered as the site of choice for biopsies in research work and for clinical diagnosis. Biopsy samples were obtained from five healthy young volunteers and a total of 1,747 type I fibres and 1,760 type II fibres were assessed. Satellite cells and fibre type composition were studied on serial muscle cross-sections stained with specific monoclonal antibodies. From a total of 218 satellite cells, 116 satellite cells were found in contact with type I fibres (53.6±8% of the satellite cells associated to type I fibres) and 102 satellite cells in contact with type II fibres (46.4±8% of the satellite cells associated to type II fibres). There was no significant difference (P=0.4) between the percentages of satellite cells in contact with type I and with type II fibres. Additionally, there was no relationship between the mean number of satellite cells per fibre and the mean cross-sectional area of muscle fibres. In conclusion, our results show that there is no fibre type-specific distribution of satellite cells in a human skeletal muscle with mixed fibre type composition.     

Colonization of the satellite cell niche by skeletal muscle progenitor cells depends on notch signals

Skeletal muscle growth and regeneration rely on myogenic progenitor and satellite cells, the stem cells of postnatal muscle. Elimination of Notch signals during mouse development results in premature differentiation of myogenic progenitors and formation of very small muscle groups. Here we show that this drastic effect is rescued by mutation of the muscle differentiation factor MyoD. However, rescued myogenic progenitors do not assume?a satellite cell position and contribute poorly to myofiber growth. The disrupted homing is due to a deficit in basal lamina assembly around emerging satellite cells and to their impaired adhesion to myofibers. On a molecular level, emerging satellite cells deregulate the expression of basal lamina components and adhesion molecules like integrin α7, collagen XVIIIα1, Megf10, and Mcam. We conclude that Notch signals control homing of satellite cells, stimulating them to?contribute to their own microenvironment and to adhere to myofibers.     

Immunocytochemical localization of taurine in different muscle cell types of the dog and rat

The presence and distribution of the amino acid taurine in different muscle cell types of the dog and rat was examined by immunocytochemical methods. The light microscope study revealed that smooth muscle cells were similarly immunoreactive for taurine, whereas skeletal muscle fibres showed wide differences in taurine immunoreactivity among individual cells. Some skeletal fibres were strongly immunoreactive whereas others did not display immunolabelling. Mononucleated satellite cells, found adjacent to skeletal fibres in a quiescent stage, were also immunostained. Other myoid cells, such as testicular peritubular cells showed a cytoplasmic and a nuclear pool of taurine. By means of electron microscope immunolabelling, the subcellular localization of taurine was studied in vascular and visceral smooth muscle cells. Taurine was present in most subcellular compartments and frequently appeared randomly distributed. Taurine was localized on myofilaments, dense bodies, mitochondria, the plasma membrane and the cell nucleus. Moreover, the labelling density within individual smooth muscle cells was variable and depended on the state of contraction of each single fibre. Contracted cells showed a higher density of gold particles than relaxed cells. Unmyelinated nerve fibres, found adjacent to smooth muscle cells from the muscularis mucosae and the lamina propria of the stomach, were unstained or poorly stained.     

Visualization of satellite cells in living muscle fibres of the frog

Satellite cells were visualized in living muscle fibres of the frog. Single fibres or bundles consisting of a few fibres were isolated after treatment with collagenase, and viewed under the light microscope. Subsequent electron microscopy of identified cells confirmed that they were satellite muscle cells. Under the light microscope, satellite cells appear as fusiform cells, tapering into long fine processes usually orientated parallel to the muscle fibre axis. Horseradish peroxidase injected into the muscle fibre was not transferred to the satellite cells.     

Synapse-specific expression of acetylcholine receptor genes and their products at original synaptic sites in rat soleus muscle fibres regenerating in the absence of innervation.

To test the hypothesis that synaptic basal lamina can induce synapse-specific expression of acetylcholine receptor (AChR) genes, we examined the levels mRNA for the alpha- and epsilon-subunits of the AChR in regenerating rat soleus muscles up to 17 days of regeneration. Following destruction of all muscle fibres and their nuclei by exposure to venom of the Australian tiger snake, new fibres regenerated within the original basal lamina sheaths. Northern blots showed that original mRNA was lost during degeneration. Early in regeneration, both alpha- and epsilon-subunit mRNAs were present throughout the muscle fibres but in situ hybridization showed them to be concentrated primarily at original synaptic sites, even when the nerve was absent during regeneration. A similar concentration was seen in denervated regenerating muscles kept active by electrical stimulation and in muscles frozen 41-44 hours after venom injection to destroy all cells in the synaptic region of the muscle. Acetylcholine-gated ion channels with properties similar to those at normal neuromuscular junctions were concentrated at original synaptic sites on denervated stimulated muscles. Taken together, these findings provide strong evidence that factors that induce the synapse-specific expression of AChR genes are stably bound to synaptic basal lamina.     

Structure and regeneration of the planarian basal lamina: An ultrastructural study

The structure and regeneration of the planarian subepidermal basement membrane or basal lamina have been electron microscopically examined, particularly in relation to the changes of extracellular products at the wounded area. The intact basal lamina consists of three structural elements; namely, an electron-lucent zone, a limiting layer and a microfibrillar layer. Ultrastructural changes during wound healing have suggested that the amorphous material secreted in the interspace between the epidermal cells and blastema contains precursors of the basal lamina. Within the amorphous zone two distinct phases of the basal lamina regeneration are observed: one is a reconstitution of the limiting layer and the other is a polymerization of the microfibrils. The limiting layer arises from areas subjacent to newly developed hemidesmosomes of epidermal cells. The unit microfibrils are formed from an accumulation of the precursors through transitional smaller microfibrils. At the late stage, individual mature microfibrils are regularly lined with the limiting layer and cell membranes of the newly differentiated muscle fibres. On the basis of these observations we suggest that the planarian basal lamina is regenerated by the interaction between epidermal cells and myoblasts.     

The giant neurone system in ophiuroids

Summary The radial nerve cords of members of the class Ophiuroidea consist of two parts, the ectoneural and the hyponeural tissues, which are separated by an acellular basal lamina. The hyponeural tissue is composed entirely of motor fibres. The cell bodies of the hyponeural neurones are arranged in ganglia, one to each segment of the arm, and each containing approximately one hundred cell bodies. Synaptic contact between the two tissues occurs across the basal lamina. Ultrastructural evidence shows that the majority of these synapses operate in the ectoneural to hyponeural direction. Three pairs of nerve bundles, each containing approximately thirty five large motor fibres arise from each ganglion and innervate the intervertebral muscles. The large motor fibres divide into a number of pre-terminal axons in the region in which the motor fibre enters the muscle block. The terminal axons run at right-angles across the muscle fibres and neuromuscular junctions are found at the points of contact between the two; each terminal axon makes contact with a large number of muscle fibres. The hyponeural axons also pass through the juxtaligamental tissue before they reach the muscle blocks and there is some evidence of synaptic contact with the juxtaligamental cells. The juxtaligamental tissue is thought to be associated with changes in the structural properties of the collagenous ligaments of the arm during arm autotomy (Wilkie 1979). Degeneration studies confirmed the layout of the hyponeural motor axons.     

Concomitant increases in myonuclear and satellite cell content in female trapezius muscle following strength training

A skeletal muscle fibre maintains its cytoplasmic volume by means of hundreds of myonuclei distributed along its entire length. Therefore it is hypothesised that changes in fibre size would involve modifications in myonuclear number. In this study, we have examined whether 10 weeks of strength training can induce changes in the number of myonuclei and satellite cells in female trapezius muscles. Biopsies were taken pre- and posttraining from the upper part of the descending trapezius muscle of nine subjects. Muscle samples were analysed for fibre area and myonuclear and satellite cell number using immunohistochemistry. There was a 36% increase in the cross-sectional area of muscle fibres. The hypertrophy of muscle fibres was accompanied by an approximately 70% increase in myonuclear number and a 46% increase in the number of satellite cells. Myonuclei number was positively correlated to satellite cell number indicating that a muscle with an increased concentration of myonuclei will contain a correspondingly higher number of satellite cells. The acquisition of additional myonuclei appears to be required to support the enlargement of multinucleated muscle cells following 10 weeks of strength training. Increased satellite cell content suggests that mitotic divisions of satellite cells produced daughter cells that became satellite cells. Accepted: 30 November 1999     

Skeletal muscle progenitor cells and the role of Pax genes

Satellite cells, which lie under the basal lamina of muscle fibres, are marked by the expression of Pax7, and in many muscles of Pax3 also. A pure population of satellite cells, isolated from a Pax3(GFP/+) mouse line by flow cytometry, contribute very efficiently to skeletal muscle regeneration and also self-renew, thus demonstrating their role as muscle stem cells. Pax3/7 regulates the entry of these cells into the myogenic programme via the activation of the myogenic determination gene, MyoD. Pax7 is also essential for the survival of satellite cells. This dual role underlines the importance of ensuring that a tissue stem cell that has lost its myogenic instruction should not be left to run amok, with the potential risk of tissue deregulation and cancer. A somite-derived population of Pax3/Pax7 positive cells is responsible for muscle growth during development and gives rise to the satellite cells of postnatal muscles. In the absence of both Pax3 and Pax7, these cells die or assume other cell fates. Pax3/7 lies genetically upstream of both MyoD and Myf5, which determine the skeletal muscle fate of these cells. To cite this article: M. Buckingham, C. R. Biologies 330 (2007).     

Cellular adaptation of the trapezius muscle in strength-trained athletes

 The aim of this study was to elucidate the cellular events that occur in the trapezius muscle following several years of strength training. In muscle biopsies from ten elite power lifters (PL) and six control subjects (C), several parameters were studied: cross-sectional area of muscle fibres, myosin heavy chain composition (MHC) and capillary supply [capillaries around fibres (CAF) and CAF/fibre area]. A method was also developed for counting the number of myonuclei and satellite cell nuclei. The proportion of fibres expressing MHC IIA, the cross-sectional area of each fibre type and the number of myonuclei, satellite cells and fibres expressing markers for early myogenesis were significantly higher in PL than in C (P<0.05). A significant correlation between the myonuclear number and the cross-sectional area was observed. Since myonuclei in mature muscle fibres are not able to divide, we suggest that the incorporation of satellite cell nuclei into muscle fibres resulted in the maintenance of a constant nuclear to cytoplasmic ratio. The presence of small diameter fibres expressing markers for early myogenesis indicates the formation of new muscle fibres. Accepted: 17 November 1998     

Satellite cells in movement disorders of various etiologies

The ultrastructural analysis of the muscle biopsy samples from patients with different neuro-muscular diseases revealed; both degeneration and regeneration of the muscle fibres. The number of satellite cells was increased due to their division and myonuclear segregation. Activated satellite cells were converted into myoblasts which probably could replace the injured fragments of the muscle fibres or form the myotubes. The process of muscle fibre regeneration is restricted not only by the damage of the muscle itself, but by the dystrophic process affecting satellite cells.     

Functional morphology of the myosatellitocytes in ontogenesis of higher vertebrates and man

By means of electron microscopic radioautography of RNA and DNA synthesis, morphometry of satellite cells, transmissive electron microscopy applying photometric scanning of the image, a successive development and maturation of myosatellites has been studied in ontogenesis of hens, rats and the man. The initial muscle cells for simplasts are promyoblasts. At the stage of myotubes and young muscle fibres, structural and functional heterogeneity of the satellite cells in the myotubes takes place. It is possible to distinguish satellite promyoblasts (the cells which are at the state of proliferative rest, interact with simplasts, intensively synthesize RNA, temporarily do not fuse with simplasts but have certain metabolic connection with them). Subsequently, promyocytes form myosatellitocytes of the I type, they are constantly found in small amount in the muscle fibre composition during the whole course of the animal's ontogenesis. According to the morphological classification, promyoblasts and promyocytes belong to myosatellitocytes of the II type. When studying myosatellitogenesis in the animals, no other sources of cell production have been found.     

Frequency of M-cadherin-stained satellite cells declines in human muscles during aging.

To answer the question of whether the satellite cell pool in human muscle is reduced during aging, we detected satellite cells in 30- microm-thick transverse sections under the confocal microscope by binding of M-cadherin antibody. The basal lamina was detected with laminin. Nuclei were stained with bisbenzimide or propidium iodide. Satellite cells were counted by applying the disector method and unbiased sampling design. To determine if there are age-related differences in muscle fiber types, morphometric characteristics of muscle fibers were examined on thin sections stained for myofibrillar ATPase. Autopsy samples of vastus lateralis muscle from six young (28.7 +/- 2.3 years) and six old (70.8 +/- 1.3 years) persons who had suffered sudden death were analyzed. Numbers of satellite cells per fiber length (Nsc/Lfib) and number of satellite cells per total number of nuclei (satellite cell nuclei + myonuclei) (Nsc/Nnucl) were significantly lower in the old group (p < 0.05). We demonstrate the importance of proper sampling and counting in estimation of sparsely distributed structures such as satellite cells. Our results support the hypothesis that the satellite cell fraction declines during aging.     

The satellite cell in male and female, developing and adult mouse muscle: distinct stem cells for growth and regeneration

Satellite cells are myogenic cells found between the basal lamina and the sarcolemma of the muscle fibre. Satellite cells are the source of new myofibres; as such, satellite cell transplantation holds promise as a treatment for muscular dystrophies. We have investigated age and sex differences between mouse satellite cells in vitro and assessed the importance of these factors as mediators of donor cell engraftment in an in vivo model of satellite cell transplantation. We found that satellite cell numbers are increased in growing compared to adult and in male compared to female adult mice. We saw no difference in the expression of the myogenic regulatory factors between male and female mice, but distinct profiles were observed according to developmental stage. We show that, in contrast to adult mice, the majority of satellite cells from two week old mice are proliferating to facilitate myofibre growth; however a small proportion of these cells are quiescent and not contributing to this growth programme. Despite observed changes in satellite cell populations, there is no difference in engraftment efficiency either between satellite cells derived from adult or pre-weaned donor mice, male or female donor cells, or between male and female host muscle environments. We suggest there exist two distinct satellite cell populations: one for muscle growth and maintenance and one for muscle regeneration.     

Maturation of myocardial capillaries in the fetal and neonatal rat: an ultrastructural study with a morphometric analysis of the vesicle populations

The ultrastructural morphology of the cellular and extracellular components of the developing myocardial capillary wall--from the 16-day-gestation fetus of the rat to the 21-day neonate--was examined. A morphometric analysis of plasmalemmal vesicles and of coated vesicles and pits of capillary endothelial cells was performed during the same developmental period. As the lateral extensions of the capillary endothelial cells change from irregular to regular in their thickness during development, there is an increase in the number of plasmalemmal vesicles and a progression from clusters of plasmalemmal vesicles to a uniform distribution in the endothelial cell. The ratio of vesicles which are open to the luminal front, which are "free" in the cytoplasm, or which are open to the abluminal front of the endothelial cell was consistent throughout development. The numerical density of plasmalemmal vesicles demonstrates a gradual and significant increase. In contrast, the numbers of coated vesicles and pits are variable within a very narrow range, and no pattern of increase or decrease is discernible during development. Similarly, there is no change in interendothelial cell junctions, which consist of occluding and primitive adhesive junctional types, during development. The lamina densa of the basal lamina gradually develops from discontinuous, patchy densities along the abluminal surface of the endothelial cells to a continuous and distinct layer by 21 days gestation. The presence of the proteoglycan species in the developing basal lamina was assessed with the cationic dye ruthenium red (RR), and the appearance of RR-marked proteoglycans was found to parallel the appearance of lamina densa material. found to parallel the appearance of lamina densa material. The RR sites appear discontinuously in patches; and later, the RR sites appear in a continuous and regular planar lattice in the lamina rara interna and externa at 21 days gestation. A complete array of RR-stainable anionic sites outside a continuous lamina densa near birth indicates that the basal laminae of developing capillaries in the heart are morphologically, and in part biochemically, mature by the end of the first neonatal week. Our results show that the endothelial cells and the subtending basal lamina of myocardial capillaries gradually mature morphologically during the final days of gestation and the first neonatal week.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mode of formation of the flight muscles in a butterfly Pieris brassicae

The formation and development of the dorsal longitudinal flight muscles of the butterfly Pieris brassicae L have been studied by electron and light microscopy. These imaginal muscles arise from two symmetrical pairs of mesothoracic larval muscles, which are morphologically indistinguishable from the other wall muscles at the beginning of the 5th larval instar. However, 2 days before the end of this instar an accumulation of myoblasts is observed at the median region of these muscle fibres. The muscle fibres are penetrated by the myoblasts and broken into fragments. Progressive dedifferentiation of the larval fibrillar material in each of the muscle fragments is observed during the first days of the pupal development. The myoblasts within the basal lamina of the original larval muscle fibres remain associated with the muscle fragments. Myoblasts then fuse with the larval muscle fragments, which simultaneously fuse with each other. This results in the formation of rudimentary imaginal muscle fibres. The development of these fibres, particularly myofibrillogenesis, is studied until the emergence of the imago.