Expression of myofibrillar proteins and parvalbumin isoforms in white muscle of dorada during development

Several polyacrylamide gel electrophoresis techniques were used to study developmental changes in myofibrillar protein composition and parvalbumin distribution in the myotomal muscle of Brycon moorei . Two myosin LC2 chains and two troponin I isoforms were successively detected. Up to four troponin T isoforms were synthesized. Slow red-muscle myofibrils from adult fish showed no common component (except actin) with larval, juvenile or adult fast white-muscle myofibrils. During growth of B. moorei , two classes of parvalbumin isoforms were sequentially expressed: larval PA I, PA IIa, and PA IIb and adult PA III. In adult fish, the content in Tn T-2 isoform decreased from the anterior to the posterior myomeres, in favour of Tn T-1 and Tn T-4. The parvalbumin content also diminished from the rostral to the caudal muscle. The fast rate of transition from larval to adult isoforms appeared to parallel the extremely fast growth of B. moorei . Sequential expression of these isoforms presumably reflected variations in the contractile properties of the muscle fibres, required by changes in physiological demands of the propulsive musculature.     

Myosin heavy chain and parvalbumin expression in swimming and feeding muscles of centrarchid fishes: the molecular basis of the scaling of contractile properties

In centrarchid fishes, such as bluegill (Lepomis macrochirus, Rafinesque) and largemouth bass (Micropterus salmoides, Lacepède), the contractile properties of feeding and swimming muscles show different scaling patterns. While the maximum shortening velocity (V(max)) and rate of relaxation from tetanus of swimming or myotomal muscle slow with growth, the feeding muscle shows distinctive scaling patterns. Cranial epaxial muscle, which is used to elevate the head during feeding strikes, retains fast contractile properties across a range of fish sizes in both species. In bass, the sternohyoideous muscle, which depresses the floor of the mouth during feeding strikes, shows faster contractile properties with growth. The objective of this study was to determine the molecular basis of these different scaling patterns. We examined the expression of two muscle proteins, myosin heavy chain (MyHC) and parvalbumin (PV), that affect contractile properties. We hypothesized that the relative contribution of slow and fast MyHC isoforms will modulate V(max) in these fishes, while the presence of PV in muscle will enhance rates of muscle relaxation. Myotomal muscle displays an increase in sMyHC expression with growth, in agreement with its physiological properties. Feeding muscles such as epaxial and sternohyoideus show no change or a decrease in sMyHC expression with growth, again as predicted from contractile properties. PV expression in myotomal muscle decreases with growth in both species, as has been seen in other fishes. The feeding muscles again show no change or an increase in PV expression with growth, contributing to faster contractile properties in these fishes. Both MyHC and PV appear to play important roles in modulating muscle contractile properties of swimming and feeding muscles in centrarchid fishes.     

Histochemical characterization of myotomal muscle in the roach, Rutilus rutilus (L.)

A histochemical study of the myotomal muscles in the roach revealed three main muscle regions: red, intermediate and white. These were distinguished on the basis of glycogen content, succinate dehydrogenase (SDH), and myofibrillar ATPase (mATPase) activity. Except for the red fibre region, none of these described regions is homogeneous. The principal new findings are the toniclike fibre, the presence of a transitional zone with two fibre types, and the mosaic organization of the white fibre region. The significance of this type of myotome architecture in relation to the locomotion of the species is discussed.     

Fine structural study of the abdominal muscle receptor organs of the crayfish (Procambarus clarkii). Fast and slow receptor muscles

Receptor muscles of the abdominal muscle receptor organs of the crayfish, Procambarus clarkii, were examined by electron microscopy. Both the fast and the slow receptor strand comprises a single muscle fibre which is divided by invagination of the cell membrane into numerous cytoplasmic processes in its intermediate region (the so-called intercalated tendon). Most of these myofibrillar processes insert in this region, but some of them pass through the intermediate region without interruption and join the other portion of the fibre. Thus the receptor muscles, whilst maintaining cytoplasmic continuity throughout their whole length, are modified in their intermediate regions, becoming fasciculated and providing spaces which are occupied by the connective tissue and the dendrites of the sensory neurone. Clear-cut differences in fine structure are shown between the muscle of the two types of receptor unit. The fast receptor muscle shows the typical features of arthropod fast muscles, including short sarcomere length (on average 3.3 μm), cylindrical myofibrils, well-developed sarcoplasmic reticulum, and regular hexagonal array of the myofilaments. By contrast, the slow receptor muscle fibre is characterized by long sarcomeres (average 6.5 μm) and unique organization of the myofilaments, with very thick ‘thick’ filaments having diameters in the range of 25–36 nm surrounded by about 12 thin filaments.     

Regional variations in fibre growth dynamics of myotomal and caudal fin muscles in relation to body size of a freshwater teleost, Barbus sarana (Cuv. & Val.)

The growth of red fibres in anterior and middle myotomal regions of B. sarana was mainly by hyperplasia in smaller size classes. In higher size classes, growth by hyperplasia was greater in posterior myotomal region compared to the other two myotomal regions. The growth of pink fibres in anterior myotomal regions was mainly by hypertrophy. The middle and posterior myotomal regions showed fibre growth by hyperplasia. The growth dynamics of white fibres revealed more or less similar pattern in all three myotomal regions against the somatic development. White fibres grew by hyperplasia up to 8 cm F.L. size classes and thereafter by hypertrophy. However, in > 12 cm F.L. size classes, the mean diameter of white fibres did not increase significantly. Similar pattern of growth was found in the white fibres of caudal fin muscle. It is interesting to note that the hyperplasia was mostly completed in the white fibres of the smallest fish studies, whereas, it continued to quite larger fish size in red and pink fibres. Thus, hyperplasia and hypertrophy may be responsible for growth in all fibre types in all myotomal regions in relation to somatic development in this small and medium growing species.     

Differential expression of two MyoD genes in fast and slow muscles of gilthead seabream ( Sparus aurata)

Members of the myogenic regulatory gene family, including MyoD, Myf5, Myogenin and MRF4, are specifically expressed in myoblast and skeletal muscle cells and play important roles in regulating skeletal muscle development and growth. They are capable of converting a variety of non-muscle cells into myoblasts and myotubes. To better understand their roles in the development of fish muscles, we have isolated the MyoD genomic genes from gilthead seabream (Sparus aurata), analyzed the genomic structures, patterns of expression and the regulation of muscle-specific expression. We have demonstrated that seabream contain two distinct non-allelic MyoDgenes, MyoD1 and MyoD2. Sequence analysis revealed that these two MyoD genes shared a similar gene structure. Expression studies demonstrated that they exhibited overlapping but distinct patterns of expression in seabream embryos and adult slow and fast muscles. MyoD1 was expressed in adaxial cells that give rise to slow muscles, and lateral somitic cells that give rise to fast muscles. Similarly, MyoD2 was initially expressed in both slow and fast muscle precursors. However, MyoD2 expression gradually disappeared in the adaxial cells of 10- to 15-somite-stage embryos, whereas its expression in fast muscle precursor cells was maintained. In adult skeletal muscles, MyoD1 was expressed in both slow and fast muscles, whereas MyoD2 was specifically expressed in fast muscles. Treating seabream embryos with forskolin, a protein kinase A activator, inhibited MyoD1 expression in adaxial cells, while expression in fast muscle precursors was not affected. Promoter analysis demonstrated that both MyoD1 and MyoD2 promoters could drive green fluorescence protein expression in muscle cells of zebrafish embryos. Together, these data suggest that the two non-allelic MyoD genes are functional in seabream and their expression is regulated differently in fast and slow muscles. Hedgehog signaling is required for induction of MyoDexpression in adaxial cells.     

Neuromuscular relationships in the abdomen of the Californian shore crab Pachygrapsus crassipes

There are two pairs of muscles in each abdominal segment of the crab; one pair of flexors and one pair of extensors. In the early larval stages the muscles have short sarcomeres--a property of fast fibers--and high thin to thick filament ratios--a property of slow fibers. In the adult the abdominal muscles are intermediate and slow, since they have fibers with intermediate and long sarcomeres, high thin to thick filament ratios, low myofibrillar ATPase activity, and high NADH diaphorase activity. The different fiber types are regionally distributed within the flexor muscle. Microelectrode recordings from single flexor muscle fibers in the adult showed that most fibers are supplied by three excitatory motor axons, although some are supplied by as many as five efferents. One axon supplies all of the flexor muscle fibers in its own hemisegment, and the evoked junctional potentials exhibit depression. This feature together with the innervation patterns of the fibers are similar to those reported for the deep flexor muscles of crayfish and lobsters. Therefore, in the adult crab, the abdominal flexor muscles have some features in common with the slow superficial flexors of crayfish and other features in common with the fast deep flexor muscles.     

Growth of inhibitory innervation in a lobster muscle

The fine structure of inhibitory innervation to a limb muscle was examined in larval, juvenile, and adult lobsters. The innervation is essentially similar in qualitative features among these different stages, although there are some marked quantitative changes associated with growth. From being localized to discrete regions in the larval muscle, the inhibitory innervation spreads to groups of muscle fibers in the early juvenile muscle and to single fibers in the late juvenile and adult muscles. Concurrently, its neuromuscular synapses enlarge in area, become perforated, and acquire more active sites of transmitter release. Inhibitory nerve terminals occur in close proximity to their excitatory counterparts in the muscles of larval and early juvenile stages, although in later stages this juxtaposition occurs preferentially in some muscle fibers but not others. The inhibitory innervation is, nevertheless, much more restricted in occurrence than is the excitatory innervation.     

The fate of specific motoneurons and sensory neurons of the pregenital abdominal segments inTenebrio molitor (Insecta : Coleoptera) during metamorphosis

The anatomy and innervation of the lateral external muscle and sensory cells located in the ventral region of pregenital abdominal segments were examined at the larval and adult stages ofTenebrio molitor (Coleoptera). All seven muscles located in this region degenerate during the pupal stage, whilst only the lateral external median (lem) appears in the adult. Backfillings of the motor nerve innervating this muscle reveal that, at both larval and adult stages, it is innervated by ten neurons. Intracellular records from the muscle fibres show that two neurons are inhibitory, and at least five are excitatory. There are also two unpaired neurons. A variety of sensory organs are located in the ventral region of the larvae, whilst only campaniform sensilla are found in the adult. At both stages, the innervation pattern of the sensory nerve branches is very similar. Also, the central projections of the sensory cells occupy similar neuropilar areas. Finally, prolonged intracellular records from the lem muscle revealed that, at the larval stage, it participates only in segmental or intersegmental reflexes, whilst in the adult it has a primary expiratory role in ventilation. The results show that extensive changes occur in the number of muscles located in the ventral region of the pregenital abdominal segments, as well as in the arrangement and number of sensory neurons, in the structure of the exoskeleton, and even in the central nervous system. In contrast, only minor changes are observed in the sensory and motor nerve branches, in the sensory projections, and in the number and the location of the motoneurons innervating the lateral external median muscle. Correspondence to: G. Theophilidis     

Histochemical characterization of myotomal muscle in the grass pickerel, Esox americanus vermiculatus (LeSeuer), and the muskellunge, E. masquinongy (Mitchell)

A histochemical study of the myotomal muscles in the grass pickerel, Esox americanus vermiculatus , and the muskellunge, E. masquinongy , was performed using actomyosin ATPase and NADH diaphorase activities. Three fibre types, i.e., red, white and pink were distinguished on the basis of their enzyme activities. White muscle fibres comprised the bulk of the myotomal musculature. The relative proportion of red muscle fibres was greater in the caudal region than in more anterior regions of the body. Pink fibres formed only a few layers between red and white. These findings are discussed in relation to the possible functional significance of the muscle fibre types in swimming and feeding behaviour in these species.     

Myofibrillar gene expression in differentiating lobster claw muscles

Lobster claw muscles undergo a process of fiber switching during development, where isomorphic muscles containing a mixture of both fast and slow fibers, become specialized into predominantly fast, or exclusively slow, muscles. Although this process has been described using histochemical methods, we lack an understanding of the shifts in gene expression that take place. In this study, we used several complementary techniques to follow changes in the expression of a number of myofibrillar genes in differentiating juvenile lobster claw muscles. RNA probes complementary to fast and slow myosin heavy chain (MHC) mRNA were used to label sections of 7th stage (approximately 3 months old) juvenile claw muscles from different stages of the molt cycle. Recently molted animals (1-5 days postmolt) had muscles with distinct regions of fast and slow gene expression, whereas muscles from later in the molt cycle (7-37 days postmolt) had regions of fast and slow MHC expression that were co-mingled and indistinct. Real-time PCR was used to quantify several myofibrillar genes in 9th and 10th stages (approximately 6 months old) juvenile claws and showed that these genes were expressed at significantly higher levels in the postmolt claws, as compared with the intermolt and premolt claws. Finally, Western blot analyses of muscle fibers from juvenile lobsters approximately 3 to 30 months in age showed a shift in troponin-I (TnI) isoform expression as the fibers differentiated into the adult phenotypes, with expression of the adult fast fiber TnI pattern lagging behind the adult slow fiber TnI pattern. Collectively, these data show that juvenile and adult fibers differ both qualitatively and quantitative in the expression of myofibrillar proteins and it may take as much as 2 years for juvenile fibers to achieve the adult phenotype.     

Junctional acetylcholine receptor channel open time is not presynaptically regulated in developing muscle

The role of motor innervation in controlling the development of acetylcholine receptor (AChR) channel open time was tested by examining synaptic current durations in transplanted muscles of Xenopus tadpoles. The presumptive lower jaw region, which gives rise to the interhyoideus muscle, was transplanted to the tail, overlying the myotomal muscle cells. The transplanted muscles became innervated, presumably by spinal nerves which normally innervate myotomal muscle. Despite development in the presence of foreign innervation, synaptic currents in the transplanted interhyoideus were predominantly long in duration and resembled those in the normally innervated interhyoideus. They did not resemble those in the myotomal muscle, where synaptic currents are brief. The apparent lack of neural influence on development of AChR function in muscle contrasts with the evidence for presynaptic control of AChR open time in frog sympathetic ganglia. This may reflect a fundamental difference between nerve and muscle in the regulation of postsynaptic function.     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. IX. Genes for muscle structural proteins

Ascidians are simple chordates that are related to, and may resemble, vertebrate ancestors. Comparison of ascidian and vertebrate genomes is expected to provide insight into the molecular genetic basis of chordate/vertebrate evolution. We annotated muscle structural (contractile protein) genes in the completely determined genome sequence of the ascidian Ciona intestinalis, and examined gene expression patterns through extensive EST analysis. Ascidian muscle protein isoform families are generally of similar, or lesser, complexity in comparison with the corresponding vertebrate isoform families, and are based on gene duplication histories and alternative splicing mechanisms that are largely or entirely distinct from those responsible for generating the vertebrate isoforms. Although each of the three ascidian muscle types - larval tail muscle, adult body-wall muscle and heart - expresses a distinct profile of contractile protein isoforms, none of these isoforms are strictly orthologous to the smooth-muscle-specific, fast or slow skeletal muscle-specific, or heart-specific isoforms of vertebrates. Many isoform families showed larval-versus-adult differential expression and in several cases numerous very similar genes were expressed specifically in larval muscle. This may reflect different functional requirements of the locomotor larval muscle as opposed to the non-locomotor muscles of the sessile adult, and/or the biosynthetic demands of extremely rapid larval development.     

The myotomal basement membrane

The importance of laminin-containing basement membranes (BM) for adult muscle function is well established, in particular due to the severe phenotype of congenital muscular dystrophies in patients with mutations disrupting the BM-muscle cell interaction. Developing muscles in the embryo are also dependent on an intact BM. However, the processes controlled by BM-muscle cell interactions in the embryo are only beginning to be elucidated. In this review, we focus on the myotomal BM to illustrate the critical role of laminin-111 in BM assembly and function at the surface of embryonic muscle cells. The myotomal BM provides also an interesting paradigm to study the complex interplay between laminins-containing BM and growth factor-mediated signaling and activity.     

The developmental program of fast myosin heavy chain expression in avian skeletal muscles

We have examined the types of fast myosin heavy chains (MHCs) expressed in a number of different developing chicken skeletal muscles by combining peptide mapping and immunoblotting to identify fast MHC-specific peptides among the total mixture of MHC digestion products. Using this technique, we have identified three different fast MHC patterns among the different fast and mixed (i.e., fast and slow) fiber type muscles of the adult. While the different muscles all underwent sequential changes in fast MHC isoform expression during their development, the exact sequence of these changes and the isoform patterns expressed varied from muscle to muscle. During late embryonic or fetal development, all muscles expressed a similar fast MHC pattern (designated here as the fetal pattern) which was replaced shortly after hatching with a different fast MHC pattern (the neonatal pattern). During the transition from the neonatal to the adult state that occurred sometime in the first year after hatching, many of the muscles underwent additional changes in fast MHC isoform expression. In muscles such as the pectoralis major and pectoralis minor, a new fast MHC isoform pattern was seen in the adult so that the developmental program of isoform switching in these muscles involved the sequential appearance of distinct fetal, neonatal, and adult fast MHCs. Other muscles, such as the sartorius and posterior latissimus dorsi, underwent a qualitatively different program of isoform switching and expressed as an adult a fast MHC pattern that was indistinguishable from that expressed during fetal development. Finally, in some muscles, such as the superficial biceps, no change in isoform pattern was detected during the neonatal to adult transition--in these muscles, expression of the neonatal MHC isoform pattern apparently persisted into the adult state. These data indicate that no single scheme or program of fast MHC isoform switching can describe all the developmental changes that occur in fast MHC isoform expression in the chicken and that at least three different programs of isoform switching and expression can be identified.     

EAST and Chromator control the destruction and remodeling of muscles during Drosophila metamorphosis

Metamorphosis involves the destruction of larval, the formation of adult and the transformation of larval into adult tissues. In this study, we demonstrate the role of the Drosophila nuclear proteins EAST and Chromator in tissue destruction and remodeling. To better understand the function of east, we performed a yeast two-hybrid screen and identified the euchromatin associated protein Chromator as a candidate interactor. To analyze the functional significance of our two-hybrid data, we generated a set of novel pupal lethal Chro alleles by P-element excision. The pupal lethal Chro mutants resemble lethal east alleles as homozygous mutants develop into pharates with normal looking body parts, but fail to eclose. The eclosion defect of the Chro alleles is rescued in an east heterozygous background, indicating antagonistic genetic interactions between the two genes. Live cell imaging was applied to study muscle development during metamorphosis. Consistent with the eclosion defects, mutant pharates of both genes show loss and abnormal differentiation of adult eclosion muscles. The two genes have opposite effects on the destruction of larval muscles in metamorphosis. While Chro mutants show incomplete histolysis, muscles degenerate prematurely in east mutants. Moreover east mutants affect the remodeling of abdominal larval muscles into adult eclosion muscles. During this process, loss of east interferes with the spatial coordination of thinning of the larval muscles. Overexpression of EAST-GFP can prevent the disintegration of polytene chromosomes during programmed cell death. We propose that Chro activates and east inhibits processes and genes involved in tissue destruction and remodeling.     

Immunochemical analysis of troponin T isoforms in adult, embryonic, regenerating, and denervated chicken fast skeletal muscles

Using monoclonal antibodies (McAbs) which can distinguish between breast- and leg-type troponin T (TnT), we studied the spatial distribution of TnT isoforms in adult chicken fast skeletal muscles. The breast (pectoralis major) and leg (iliotibialis posterior) muscles were composed predominantly of homogeneous fibers containing breast- and leg-type TnT, respectively. The posterior latissimus dorsi muscle was composed of heterogeneous fibers of at least two types, namely breast and leg types. In developing and regenerating fast muscles, only leg-type TnT was expressed at early stages, and later breast-type TnT appeared either transiently or permanently. This led ultimately to several distinct adult fast muscle breast/leg TnT isoform profiles. Since both types of TnT were synthesized in embryonic and regenerating muscles with nerves intact as well as in regenerating muscles with nerves resected, the switching on of their expression during fast muscle development appears to be independent of nerves. However, its full development ("fine tuning" of the protein isoform distribution within the fast fiber types) and the maintenance of the adult state are presumed to be dependent on the nerves, since, although regenerating fibers in denervated muscles could exhibit the early and then the later embryonic stainabilities, they again returned to the early embryonic state; further, the denervation of adult muscles caused the replacement of TnT isoform from the adult to the early embryonic state.