Myosin isozymes in developing chicken muscles.
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Serial frozen sections of longissimus dorsi muscles from seven pigs at different live weights (13 to 127 kg) were reacted for ATPase by the calcium method at an alkaline pH and for NADH oxidative activity. One hundred muscle fibres from each animal were identified individually in serial sections and their staining intensity was measured with a microscope photometer at 600 nm. For each section, staining intensity of fibres (% tranmission) was measured and converted to the nearest one-tenth unit of the range from the darkest to the lightest staining fibres. Frequency of occurrence of fibre types was plotted on a 10 X 10 grid using the range co-ordinates for NADH oxidative activity (vertical) and ATPase activity (horizontal). The commonly recognized histochemical fibre types in this muscle appeared as crowded areas in the grid but, in many cases, these areas were part of a continuous 'L' shaped range, a continuous but skewed distribution with regard to NADH oxidative activity was detected. In fibres with NADH oxidative activity of 0.6 to 1.0 units of the range, a continuous but irregular distribution with regard to ATPase activity was detected. Within this range, there was some evidence of a growth-related shift towards weaker ATPase activity.
Specific developmental changes in smooth muscle were studied in gizzards obtained from 6-, 8-, 10-, 12-, 14-, 16-, 18-, and 20-day chick embryos and from 1- and 7-day posthatch chicks. Myoblasts were actively replicating in tissue from 6-day embryos. Cytoplasmic dense bodies (CDBs) first appeared at Embryonic Day 8 (E8) and were recognized as patches of increased electron density that consisted of actin filaments (AFs), intermediate filaments (IFs), and cross-connecting filaments (CCFs). Although the assembly of CDBs was not synchronized within a cell, the number, size, and electron density of CDBs increased as age increased. Membrane-associated dense bodies (MADBs) also could be recognized at E8. The number and size of MADBs increased as age increased, especially after E16. Filaments with the diameter of thick filaments first appeared at E12. Smooth muscle cells were able to divide as late as E20. The axial intermediate filament bundle (IFB) could first be identified in 1-day posthatch cells and became larger and more prominent in 7-day posthatch cells. Immunogold labeling of 1- and 7-day posthatch cells with anti-desmin showed that the IFB contained desmin IFs. The developmental events during this 23-day period were classified into seven stages, based primarily on the appearance and the growth of contractile and cytoskeletal elements. These stages are myoblast proliferation, dense body appearance, thick filament appearance, dense body growth, muscle cell replication, IFB appearance, and appearance of adult type cells. Smooth muscle cells in each stage express similar developmental characteristics. The mechanism of assembly of myofilaments and cytoskeletal elements in smooth muscle in vivo indicates that myofilaments (AFs and thick filaments) and filament attachment sites (CDBs and MADBs) are assembled before the axial IFB, a major cytoskeletal element.
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Calpains are Ca2+ -dependent thiol proteases which have been identified in various tissues of eucaryotes, but their physiological function in the cell is uncertain. In the muscle fiber, two types of calpains are present which differ by their calcium sensitivity: calpain 1 and calpain 2, which require for their activity micro and millimolar concentrations of calcium respectively. These calpains are associated with protein kinase C activities in the differentiated fiber. The multinucleate myotube is formed by fusion of mononucleated precursor cells, myoblasts. Calpains have been reported to appear in myoblasts at around the time of fusion. Moreover, an apparent synthesis of 1,2 diacylglycerol, an activator of protein kinase C, was observed during fusion of myoblasts. However, more information is required to incriminate totally protein kinase C and calpains in the mechanism of myoblast fusion.
Two separate genes encode fast-twitch and slow-twitch/cardiac muscle forms of the Ca2+ ATPase of sarcoplasmic reticulum. Full length Ca2+ ATPase clones have been isolated from adult rabbit fast-twitch, slow-twitch, and cardiac muscles. Segments of these clones containing unique sequences have been used as probes to study developmental changes in Ca2+ ATPase transcripts. The fast-twitch Ca2+ ATPase transcript undergoes developmentally regulated alternative splicing in which a penultimate 42-base pair exon is retained in the adult transcript but is excised in the neonatal transcript. This additional exon shifts the exon encoding the neonatal carboxyl-terminal sequence, -Asp-Pro-Glu-Asp-Glu-Arg-Arg-Lys (Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H. (1986) Cell 44, 597-607) into a nontranslated region and results in the expression of an adult isoform with a carboxyl-terminal -Gly. The neonatal form of the fast-twitch Ca2+ ATPase represents 72% of the fast-twitch Ca2+ ATPase transcripts just prior to birth but only 17% by 14 days of age and 4% in adult fast-twitch muscle. Adult slow-twitch, adult cardiac, and neonatal skeletal muscles express an identical Ca2+ATPase mRNA transcript which is distinct from either of the fast-twitch forms. The slow-twitch/cardiac Ca2+ ATPase is the predominant form expressed in late fetal and early neonatal rabbit skeletal muscle, but this form is lost as the skeletal muscle differentiates into a fast-twitch state. Three or more alternative polyadenylation signals exist for this mRNA in all tissues with the most 3' signal predominating.
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The development of muscle spindles, with respect to the expression of myosin heavy chain isoforms was studied in rat hind limbs from 17 days of gestation up to seven days after birth. Serial cross-sections were labelled with antibodies against slow tonic, slow twitch and neonatal isomyosins, myomesin, laminin and neurofilament protein. At 17-18 days of gestation, a small population of primary myotubes expressing slow tonic myosin were identified as the earliest spindle primordia. These myotubes also expressed slow twitch and, to a lesser extent, neonatal myosin. At 19-20 days of gestation a second myotube became apparent; this staining strongly with anti-neonatal myosin. A day later this secondary myotube acquired reactivity to anti-slow tonic and anti-slow twitch myosins. By birth, a third myotube was present; this staining strongly with anti-neonatal myosin but otherwise unreactive with the other antibodies against myosin heavy chains. Three days after birth a fourth myotube, with identical reactivity to the third one, became apparent. Regional variation in the expression of isomyosins, which was present since birth in the two nuclear bag fibers was further enhanced: the nuclear bag staining strongly with anti-slow tonic and antineonatal in the equatorial region and with decreasing intensity towards the poles, whilst with anti-slow twitch the stainability was low in the equatorial and high in the polar region. The nuclear bag fiber showed a homogeneous staining: high with anti-slow tonic, moderate with anti-neonatal, and displayed stainability to anti-slow twitch myosin in the polar regions only. No regional variation was found along the chain fiber/myotube.(ABSTRACT TRUNCATED AT 250 WORDS)
The influence of muscle (vastus lateralis) length on the muscle fibre conduction velocity (MFCV) and on muscle fatigue was studied in 8 healthy volunteers. In experiment 1, the electromyographic (EMG) responses were evoked by electrical stimulation of the motor point and recorded by a surface electrode array aligned along the muscle fibre direction. The MFCV (determined by cross-correlation) was measured at knee flexions of 5 degrees (full extension), 45 degrees, 90 degrees and 120 degrees with 3 different extension torques. The MFCV declined with increasing muscle length and increased with increasing background torque at knee flexions from 5 degrees to 90 degrees. From 90 degrees to 120 degrees knee flexion of MFCV tended to increase. In experiment 2, the EMG activity at a static fatiguing contraction (80% MVC) was measured at 45 degrees and 90 degrees knee flexion. The EMG was measured until the subject gave up contracting the muscle (endurance). The largest increase in the RMS amplitude and the fastest decreases in the mean power frequency (MPF) and MFCV were found at 90 degrees flexion. The MVC at 45 degrees knee flexion was 35% lower than at 90 degrees and the time until endurance was approximately twice as long for the 45 degrees contraction. The results indicate that muscle length is an important parameter for the propagation velocity of action potentials and for the development of static muscle fatigue.
The soleus muscles of fetal rats were examined by electron microscopy to determine whether the early differentiation of muscle spindles is dependent upon sensory innervation, motor innervation, or both. Simple unencapsulated afferent-muscle contacts were observed on the primary myotubes at 17 and 18 days of gestation. Spindles, encapsulations of muscle fibers innervated by afferents, could be recognized early on day 18 of gestation. The full complement of spindles in the soleus muscle was present at day 19, in the region of the neuromuscular hilum. More afferents innervated spindles at days 18 and 19 of gestation than at subsequent developmental stages, or in adult rats; hence, competition for available myotubes may exist among afferents early in development. Some of the myotubes that gave rise to the first intrafusal (bag2) fiber had been innervated by skeletomotor (alpha) axons prior to their incorporation into spindles. However, encapsulated intrafusal fibers received no motor innervation until fusimotor (gamma) axons innervated spindles 3 days after the arrival of afferents and formation of spindles, at day 20. The second (bag1) intrafusal fiber was already formed when gamma axons arrived. Thus, the assembly of bag1 and bag2 intrafusal fibers occurs in the presence of sensory but not gamma motor innervation. However, transient innervation of future bag2 fibers by alpha axons suggests that both sensory and alpha motor neurons may influence the initial stages of bag2 fiber assembly. The confinement of nascent spindles to a localized region of the developing muscle and the limited number of spindles in developing muscles in spite of an abundance of afferents raise the possibility that afferents interact with a special population of undifferentiated myotubes to form intrafusal fibers.
An immunocytochemical study was done on the skeletal muscles of human fetuses (19-36 weeks gestation), infants and adults using a new monoclonal antibody (McAb) ALD-47. The antibody was generated against slow myosin of chicken and is specific for myosin heavy chain (MHC). In human infants and adults the type I muscle fibres are strongly reactive with this McAb and the type II fibres uniformly non-reactive. In the fetuses from 19-20 weeks gestation (in whom the fibre types are not distinguishable by the histochemical myosin ATPase test) a proportion of muscle fibres react specifically with ALD-47. Other muscle fibres at this stage react positively with a fast specific MHC McAb HM-1.2 or are negative to both ALD-47 and HM-1.2 antibodies. These McAbs, thus, identify three distinct fibre populations in the early fetal muscle which by histochemical staining appears homogeneous. The percentage of ALD-47 positive fibres increases in fetuses at later gestational periods; at all stages these fibres lack reactivity with the HM-1.2 antibody. Because of its selective fibre type reactivity in differentiating muscles, the McAb ALD-47 in conjunction with HM-1.2 should be useful in immunoaffinity fractionation and biochemical studies of myosin isoforms in developing human muscles.
The entire process of normal development of a muscle nerve to a muscle (middle interradial muscle) in the tail region of the medaka (Oryzias latipes) is briefly reported. The nerve was stained immunohistochemically by using anti-neurofilament protein antibodies or stained by HRP and DiI labeling methods. The muscle was stained immunohistochemically by using anti-troponin T and anti-desmin antibodies. The smallness and transparency of the medaka embryos provide us with an opportunity to examine nerve-muscle development in whole-mount specimens. Our observations suggest that prior to the appearance of the middle interradial muscle a neural pathway has established, extending from the starting point to the 'door step' of the muscle.
During early stages of postnatal development skeletal muscle fibres of mammals are contacted by several axons. The transition from poly- to mononeuronal innervation has been extensively studied on the rat soleus. The role of activity in this process has been acknowledged but the mechanisms leading to synapse remodelling are not understood. The participation of the muscle has to be taken into account; if muscles are paralysed by alpha-bungarotoxin, the elimination of terminals is arrested. Changes in Ca2+ also influence the rate of removal of terminals. Calcium seems to act through a calcium-activated neutral protease (CANP) present in nerve endings. If CANP is inhibited, elimination fails to take place. Thus Ca2+ enters the terminal and activates the CANP. Release of K+ ions from active muscle could link muscle activity and synapse elimination. Excess K+ was found to reduce nerve-muscle contacts, by depolarizing terminals and allowing Ca2+ entry. A greater increase of Ca2+ concentration in smaller terminals would be expected, because of their surface-to-volume ratio, and they are preferentially eliminated. Thus elimination depends on the unequal size of terminals at the endplate. Therefore the 'survivability' of individual nerve endings may already be determined at the time of synapse elimination.
Muscle fibers specialized for fast or slow contraction are arrayed in characteristic patterns within developing limbs. Clones of myoblasts analyzed in vitro express fast and slow myosin isoforms typical of the muscle from which they derive. As a result, it has been suggested that distinct myoblast lineages generate and maintain muscle fiber pattern. We tested this hypothesis in vivo by using a retrovirus to label myoblasts genetically so that the fate of individual clones could be monitored. Both myoblast clones labeled in muscle in situ and clones labeled in tissue culture and then injected into various muscles contribute progeny to all fiber types encountered. Thus, extrinsic signals override the intrinsic commitment of myoblast nuclei to particular programs of gene expression. We conclude that in postnatal development, pattern is not dictated by myoblast lineage.
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The morphogenesis and innervation of the ventral abdominal musculature in Xenopus embryos was examined using microscopic techniques. Muscle development begins at Nieuwkoop and Faber Stage 31, when aggregates of undifferentiated cells form on the ventrolateral margins of rostral trunk myotomes. During subsequent stages, aggregates form and detach from progressively more caudal myotomes to form a series of seven discrete cell clusters (anlagen). The anlagen migrate ventrally in a cell-free space between the epidermis and a subepidermal layer of pigment cells. Extracellular aggregates of 30-nm granules are evident transiently between the migrating anlagen and the epidermis. During stages 39 and 40, each anlage transforms into a sheet of myotubes which attaches rostrally and caudally to adjacent sheets to form a seven-segmented muscle. The series of broad segments, approximately one fiber thick, extends from the pericardium to the level of the proctodeum. The embryonic muscle is innervated by the ventral rami of spinal nerves 2 to 9. The major nerve trunks to the muscle develop between stages 35/36 and 40. Axons initially grow ventrally along the paths taken by the muscle anlagen. When the anlagen become muscle segments, the nerves are deep to the narrow boundaries between the segments. Spinal nerve 2 ramifies in the first muscle segment and sends fibers rostrally to the geniohyoid muscle. The findings represent the first description of the development of this muscle in Xenopus and the first account of the development of the abdominal motor nerves in an amphibian embryo.