The ultrastructure of the striated muscle of the tail of Cercaria chackai Nadakal et al., 1969

The electron microscopic study of the tail of Cercaria chackai reveals that it contains four sets of striated muscle bundles located central to the nonstriated circular and longitudinal muscles. The striated muscle consists of longitudinally oriented lamellar myofibres. Each myofibre contains a single "U" shaped myofibril. The banding pattern is analogous to that of vertebrate striated muscle. The sarcolemma is a simple surface membrane. There are no transverse tubular extensions of sarcolemma. The sarcoplasmic reticulum (SR) is very well developed with cisternae, tubules, and vesicles. SR cisternae form dyadic couplings with the sarcolemma. There is a set of flattened tubules of SR origin traversing the myofibril exactly at the Z region. These tubules are unique to the striated muscle of the cercarian tail and may have functional significance. A diagrammatic reconstruction of the myofibre is presented.     

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.     

Dynamics of muscle fibre growth during postnatal mouse development

Background  

Postnatal growth in mouse is rapid, with total skeletal muscle mass increasing several-fold in the first few weeks. Muscle growth can be achieved by either an increase in muscle fibre number or an increase in the size of individual myofibres, or a combination of both. Where myofibre hypertrophy during growth requires the addition of new myonuclei, these are supplied by muscle satellite cells, the resident stem cells of skeletal muscle.     

The isolated muscle fibre as a model of disuse atrophy: characterization using PhAct, a method to quantify f-actin

Research into muscle atrophy and hypertrophy is hampered by limitations of the available experimental models. Interpretation of in vivo experiments is confounded by the complexity of the environment while in vitro models are subject to the marked disparities between cultured myotubes and the mature myofibres of living tissues. Here we develop a method (PhAct) based on ex vivo maintenance of the isolated myofibre as a model of disuse atrophy, using standard microscopy equipment and widely available analysis software, to measure f-actin content per myofibre and per nucleus over two weeks of ex vivo maintenance. We characterize the 35% per week atrophy of the isolated myofibre in terms of early changes in gene expression and investigate the effects on loss of muscle mass of modulatory agents, including Myostatin and Follistatin. By tracing the incorporation of a nucleotide analogue we show that the observed atrophy is not associated with loss or replacement of myonuclei. Such a completely controlled investigation can be conducted with the myofibres of a single muscle. With this novel method we can distinguish those features and mechanisms of atrophy and hypertrophy that are intrinsic to the muscle fibre from those that include activities of other tissues and systemic agents.     

Jaw muscle development as evidence for embryonic repatterning in direct-developing frogs.

The Puerto Rican direct-developing frog Eleutherodactylus coqui (Leptodactylidae) displays a novel mode of jaw muscle development for anuran amphibians. Unlike metamorphosing species, several larval-specific features never form in E. coqui; embryonic muscle primordia initially assume an abbreviated, mid-metamorphic configuration that is soon remodelled to form the adult morphology before hatching. Also lacking are both the distinct population of larval myofibres and the conspicuous, larval-to-adult myofibre turnover that are characteristic of muscle development in metamorphosing species. These modifications are part of a comprehensive alteration in embryonic cranial patterning that has accompanied life history evolution in this highly speciose lineage. Embryonic ''repatterning'' in Eleutherodactylus may reflect underlying developmental mechanisms that mediate the integrated evolution of complex structures. Such mechanisms may also facilitate, in organisms with a primitively complex life cycle, the evolutionary dissociation of embryonic, larval, and adult features.     

Apoptosis in capillary endothelial cells in ageing skeletal muscle

The age‐related loss of skeletal muscle mass and function (sarcopenia) is a consistent hallmark of ageing. Apoptosis plays an important role in muscle atrophy, and the intent of this study was to specify whether apoptosis is restricted to myofibre nuclei (myonuclei) or occurs in satellite cells or stromal cells of extracellular matrix (ECM). Sarcopenia in mouse gastrocnemius muscle was characterized by myofibre atrophy, oxidative type grouping, delocalization of myonuclei and ECM fibrosis. Terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labelling (TUNEL) indicated a sharp rise in apoptosis during ageing. TUNEL coupled with immunostaining for dystrophin, paired box protein‐7 (Pax7) or laminin‐2α, respectively, was used to identify apoptosis in myonuclei, satellite cells and stromal cells. In adult muscle, apoptosis was not detected in myofibres, but was restricted to stromal cells. Moreover, the age‐related rise in apoptotic nuclei was essentially due to stromal cells. Myofibre‐associated apoptosis nevertheless occurred in old muscle, but represented < 20% of the total muscle apoptosis. Specifically, apoptosis in old muscle affected a small proportion (0.8%) of the myonuclei, but a large part (46%) of the Pax7⁺ satellite cells. TUNEL coupled with CD31 immunostaining further attributed stromal apoptosis to capillary endothelial cells. Age‐dependent rise in apoptotic capillary endothelial cells was concomitant with altered levels of key angiogenic regulators, perlecan and a perlecan domain V (endorepellin) proteolytic product. Collectively, our results indicate that sarcopenia is associated with apoptosis of satellite cells and impairment of capillary functions, which is likely to contribute to the decline in muscle mass and functionality during ageing.     

Specific regulation of N-CAM/D2-CAM cell adhesion molecule during skeletal muscle development

The expression of the N-CAM/D2-CAM cell adhesion molecule was studied in skeletal muscle. In cell cultures derived from adult human muscle N-CAM/D2-CAM was found at the cell surface of myoblasts and myotubes but not fibroblasts, showing that N-CAM/D2-CAM is a specific gene product of muscle. Western blots showed that the anti N-CAM/D2-CAM antibody reacted with a single protein band of 180 000 daltons in these cultures that differed in mobility from the broad band of 150 000-200 000 daltons found in brain. N-CAM/D2-CAM is also expressed by muscle at certain stages of development. Human foetal muscle of 10 and 20 weeks gestation showed N-CAM/D2-CAM around developing myofibres while both fast and slow adult muscle fibres did not express N-CAM/D2-CAM, suggesting that the protein is down regulated during myofibre maturation. This was studied further in developing rat muscle where N-CAM/D2-CAM was found on myofibres in the day 1 neonate, but had disappeared by day 9. N-CAM/D2-CAM is, however, re-expressed in human muscle disease where there is muscle regeneration such as in polymyositis, and here is associated with classic regenerating myofibres. N-CAM/D2-CAM expression is temporally regulated and is expressed only at times of synapse formation consistent with the idea that it may be involved in early nerve-muscle interactions.     

Ultrastructure of the Stylet Protractor Muscle in Gyratrix hermaphroditus (Turbellaria,Rhabdocoela)

The subsarcolemmal cisternae of the sarcoplasmic reticulum form peripheral couplings with the sarcolemma. The junctional gap is crossed by periodic densities called junctional processes. Desmosomes provide mechanical coupling between the myofibres. Hemidesmosomes connect the myofibre with a well developed connective tissue sheath.     

Postlarval muscle growth in fish: a DNA flow cytometric and morphometric analysis

The mechanism of postlarval fish myotomal growth was investigated in trout (Salmo gairdneri) by means of morphometric and cytofluorometric analysis. The mechanism by which new fibres are added during postlarval growth (hyperplasia) is not fully understood. In histological cross sections these new fibres have a small diameter which give the muscle a "mosaic" appearance. One hypothesis suggested that they could be derived from the proliferative activity of satellite cells. DNA cytofluorometric analysis of nuclei suspensions obtained from trout white myotomal muscle during different developmental stages (eleutherembyronic; alevin; yearling and adult) showed a consistently low S-cytometric phase during all stage in which myofibres of small diameters were present. The percentage of such small fibres, determined by morphometric analysis, suggested that satellite cells are the proliferative population. In fact, their percentages, as determined by morphometric analysis in histological section, bear a linear relationship with the S-cytometric phase percent nuclei (R = 0.927). Only in adults (67 cm in size) there was a significant decrease in the S-cytometric phase. At this stage, in histological sections, the myotomal muscle no longer had a "mosaic" appearance because of the disappearance of the small fibres. It may, therefore, be supposed that in the cm 67 adult specimens, the proliferative population is entering the G0 phase. It is known, in fact, that muscle growth proceeds only by fibre hypertrophy in trout longer than 70 cm in length (Stickland, 1983).     

Protein 4.1R expression in normal and dystrophic skeletal muscle

4.1R pre-mRNA alternative splicing results in multiple mRNA and protein isoforms that are expressed in virtually all tissues. More specifically, isoforms containing the alternative exon 17a, are exclusively expressed in muscle tissues. In this report, we show that these isoforms are preferentially present in the myoplasm of fast myofibres. 4.1R epitopes are also found at the sarcolemma of both slow and fast myofibres in normal muscle. Interestingly, they are absent from dystrophin-deficient sarcolemma of DMD muscle, and colocalize with partially expressed dystrophin in BMD muscle. We also show that alternative splicing of exons 16 and 17a is regulated during muscle differentiation in an asynchronous fashion, with an early inclusion of exon 16 in forming myotubes, and a late inclusion of exon 17a. Consistently, Western blot analysis led to characterize mainly an approximately 96/98-kDa doublet bearing exons 16-17a-encoding peptide, exclusively occurring in the differentiated muscle.     

Ring bands in fish skeletal muscle: Reorienting the myofibrils and microtubule cytoskeleton within a single cell

Skeletal muscle cells (fibers) contract by shortening their parallel subunits, the myofibrils. Here we show a novel pattern of myofibril orientation in white muscle fibers of large black sea bass, Centropristis striata. Up to 48% of the white fibers in fish >1168 g had peripheral myofibrils undergoing an ～90^o shift in orientation. The resultant ring band wrapped the middle of the muscle fibers and was easily detected with polarized light microscopy. Transmission electron microscopy showed that the reoriented myofibrils shared the cytoplasm with the central longitudinal myofibrils. A microtubule network seen throughout the fibers surrounded nuclei but was mostly parallel to the long‐axis of the myofibrils. In the ring band portion of the fibers the microtubule cytoskeleton also shifted orientation. Sarcolemmal staining with anti‐synapsin was the same in fibers with or without ring bands, suggesting that fibers with ring bands have normal innervation and contractile function. The ring bands appear to be related to body‐mass or age, not fiber size, and also vary along the body, being more frequent at the midpoint of the anteroposterior axis. Similar structures have been reported in different taxa and appear to be associated with hypercontraction of fibers not attached to a rigid structure (bone) or with fibers with unusually weak links between the sarcolemma and cytoskeleton, as in muscular dystrophy. Fish muscle fibers are attached to myosepta, which are flexible and may allow for fibers to hypercontract and thus form ring bands. The consequences of such a ring band pattern might be to restrict the further expansion of the sarcolemma and protect it from further mechanical stress. J. Morphol., 2012. © 2012 Wiley Periodicals, Inc.     

Human exercise-mediated skeletal muscle hypertrophy is an intrinsic process

Muscle cells (fibres) are post-mitotic and thus undergo changes in phenotype by modifying their existing structure. Hypertrophy is a hallmark change that occurs in response to increased loading and can be achieved in humans through repeated bouts of resistance exercise (i.e., training). In resistance exercise, contractions are initiated by neural drive leading to immediate perturbations such as calcium influx, cross-bridge cycling and tension/stress on the cytoskeleton, sarcolemma and extracellular matrix, as well as more delayed cellular events such as the production/release of potential local growth factors (e.g., IGF-1). Resistance exercise can also elevate the systemic concentration of certain hormones (growth hormone, testosterone, IGF-1) that are hypothesized to drive hypertrophy. However, while these hormones are clearly anabolic during childhood and puberty, or when given at supraphysiological exogenous doses, the transient post-exercise elevations in hormone concentration are of little consequence to the either the acute protein synthetic response or to a hypertrophic phenotype after resistance training. Thus, the acute post-exercise increases in systemic hormones are in no way a proxy marker for anabolism since they do not underpin the capacity of the muscle to hypertrophy in any measurable way. In contrast, the acute activation of intrinsically located signalling proteins such as p70^S6K and the acute elevation of muscle protein synthesis are more reflective of the potential to increase in muscle mass with resistance training. Ultimately, local mechanisms are activated by the stress imposed by muscle loading and prime the muscle for protein accretion. Membrane-derived molecules and tension-sensing pathways are two intrinsic mechanisms implicated in upregulating the synthesis and incorporation of muscle proteins into the myofibre in response to mechanical stress derived from loaded contractions.     

Apoptosis of skeletal muscles during development and disease

Cells from multicellular organisms self-destroy when no longer needed or when damaged. They do this by activating genetically controlled machineries that lead to apoptosis. Skeletal muscles in adult animals are fully differentiated syncytial cells. Apoptosis has been described in developing and, recently, in adult skeletal muscle. The cellular and molecular aspects of myoblast and myofibre apoptosis and their role in disease are analysed in this review. Alterations in the pathways that regulate myoblasts proliferation/differentiation lead to induction of apoptosis during myogenesis both in vivo and in vitro. In adult muscle myofibres apoptosis seems to start from segmental areas of myofibres often producing loss of a single myonucleus. The bcl2/bax system is active in muscle when apoptosis occurs. On the other hand conflicting results are reported on the role played by FasL/Fas system. These findings are confirmed by in vitro results on myotubes and on their susceptibility to apoptosis. Though apoptosis has been shown to occur in the skeletal muscle, the role played in diseases and the pattern followed in myogenic cells are far from being clear.     

Physical training is associated with changes in nuclear magnetic resonance and morphometrical parameters of the skeletal muscle in senescent mice

The effect of a three-month training period on T2 relaxation time as well as on myofibre size and type was investigated in the lower limbs of senescent mice. After training, T2 (which is a magnetic resonance imaging parameter known to increase during acute exercise) was significantly higher in trained mice (36.37+/-1.27 vs 37.76+/-2.06 ms, p=0.003, n=8), whereas no change was found in non-trained animals (36.35+/-1.02 vs 36.24+/-1.15 ms, p=0.278, n=8). The percentage of muscle limb area evaluated in vivo on magnetic resonance images before and after the experimental period was unchanged in trained mice (69.84+/-2.50 vs 70.29+/-2.29, p=0.896, n=3) and decreased in non-trained animals (72.98+/-1.68 vs 64.62+/-2.34, p=0.006, n=3). Cross-sectional area of fast and slow myofibres, evaluated on paraffin-embedded samples after immunolabelling for skeletal fast fibre myosin, was lower in non-trained than in trained mice in both gastrocnemius and quadriceps muscle, but no change in slow/fast fibre ratio nor in apoptotic rate was found. These data show that training can prevent sarcopenia in senescent mice by affecting muscle status and inducing myofibre hypertrophy in the absence of significant muscle damage.     

Wnt7a-Fzd7 signalling directly activates the Akt/mTOR anabolic growth pathway in skeletal muscle

Wnt7a signals through its receptor Fzd7 to activate the planar-cell-polarity pathway and drive the symmetric expansion of satellite stem cells resulting in enhanced repair of skeletal muscle. In differentiated myofibres, we observed that Wnt7a binding to Fzd7 directly activates the Akt/mTOR growth pathway, thereby inducing myofibre hypertrophy. Notably, the Fzd7 receptor complex was associated with Gα(s) and PI(3)K and these components were required for Wnt7a to activate the Akt/mTOR growth pathway in myotubes. Wnt7a-Fzd7 activation of this pathway was completely independent of IGF-receptor activation. Together, these experiments demonstrate that Wnt7a-Fzd7 activates distinct pathways at different developmental stages during myogenic lineage progression, and identify a non-canonical anabolic signalling pathway for Wnt7a and its receptor Fzd7 in skeletal muscle.     

Fusion between myogenic cells in Vivo: An ultrastructural study in regenerating murine skeletal muscle

Fusion of myogenic cells in adult murine skeletal muscle regenerating in vivo was examined at the ultrastructural level. Fusion of myoblast to myoblast, myoblast to myotube, and myotube to myotube was observed by 4 to 5 days after injury. Fusion between myogenic cells (myoblasts or myotubes) lacking a definitive glycocalyx or external lamina (basal lamina) occurred at multiple sites. It was defined by zones of cytoplasmic confluence between apposed cells at sites where contiguous segments of the cell membranes were interrupted while their edges had united resulting in linear continuity; vesicles of varying dimensions were frequent in these areas of fusion. Myoblasts were seen invaginating the surface of myofibres and again vesicles were seen in abundance in such regions. Cilia were often observed at this junctional zone suggesting that they might play a role in fusion. In the one example of probable fusion between a myotube and a myofibre, only a single area of cytoplasmic continuity was apparent.     

Integrin on developing and adult skeletal muscle

Avian integrin is a complex of integral membrane glycoproteins that appears to function as a dual receptors for both intracellular cytoskeletal and extracellular matrix components. Antibodies were raised against this complex and used to (1) immunolocalize integrin on cryosections of developing and adult muscle tissue and on developing myotube cultures in vitro and (2) immunoaffinity purify integrin from various fiber-type specific muscles. Integrin localization was compared with that of its putative cytoskeletal-associated and extracellular matrix ligands, talin and vinculin and fibronectin and laminin, respectively. The goal was to identify putative sites of interaction between the muscle sarcolemma and the cytoskeleton and the extracellular matrix and to reveal any differences in the molecular composition at these sites. Integrin's distribution on the sarcolemma of early (Day 12) embryonic limb muscle was random and punctate. On late embryonic (Days 17-19) limb muscle tissue its distribution was generally uniform but with occasional increased densities at specific sites along the sarcolemma. Posthatch (greater than 3 weeks) fast twitch muscle showed a highly regionalized distribution. These regions of integrin concentration coincided with densities of acetylcholine receptors, revealed by TRITC alpha-bungarotoxin labeling, and regions of muscle-tendon interaction, identified by morphological criteria. Tissue culture studies also demonstrated integrin densities at analogous sites in vitro, e.g., acetylcholine receptor clusters and sites at which myofibrils terminate at the sarcolemma. These integrin-rich sites were also shown to be Triton X-100 insoluble and therefore presumably are linked to the cytoskeleton or extracellular matrix. The localization of integrin on developing and adult muscle tissue was compared with that of fibronectin, laminin, vinculin, and talin using double, immunofluorescently labeled cryosections. In general, integrin did not colocalize exclusively with any one of its putative ligands. In the embryo, discrete densities of both talin and vinculin were observed at the myotendinous junction, whereas integrin immunoreactivity was widely distributed on muscle, vasculature, nerve, and connective tissue with no discernible sites of increased density. Laminin was primarily associated with muscle and nerve whereas fibronectin was prominent on connective tissue. On posthatch tissue, the distributions of talin, vinculin, laminin, and fibronectin were similar to those in the embryo, whereas the distribution of integrin was restricted to specific sites. The distribution of integrin was also examined for fiber-type specific differences on adu