Quantitative freeze-fracture studies of membrane changes in chicken muscular dystrophy

Muscular dystrophy induces extensive changes in the patterning of sarcolemmal caveolae of fast-twitch fibers from the chicken posterior latissimus dorsi (PLD) muscle, which in healthy fibers are arranged in striking bands over the myofibrillar I-bands. In dystrophic fibers the caveolae lack this patterned arrangement, and instead are dispersed over the entire sarcolemma, are irregular in shape, and are more numerous in older birds. Quantitative analysis of these differences provides three independent numerical indices of the dystrophic state and suggests that constraints responsible for normal patterning are lost in diseased fibers. These observations support theories that defects of the muscle plasma membrane are important for dystrophic pathogenesis. In contrast, the sarcolemma of slow tonic fibers from anterior latissimus dorsi (ALD) and metapatagialis latissimus dorsi (MLD) muscles have randomly dispersed caveolae whose appearance and distribution are unaffected by the disease.     

Effects of denervation on spectrin concentration in avian skeletal muscle

The effect of denervation on avian muscle alpha-spectrin was examined in fast and slow muscles. Using immunofluorescence, the surgically denervated fast-twitch posterior latissimus dorsi (PLD) exhibited a significant increase in spectrin antigen associated with the sarcolemma and within the sarcoplasm compared with the contralateral innervated control muscle. Using gel electrophoresis followed by immunoblotting, we found a two- to three-fold increase in the levels of spectrin in the denervated PLD over that found in the innervated PLD. These levels were comparable to those found previously in slow and dystrophic muscle. The intrafiber distribution of spectrin is similar between the denervated PLD and the slow-tonic anterior latissimus dorsi (ALD). When spectrin was examined in dystrophic PLD muscle, denervation was found to have no effect. These results support our hypothesis that the concentration of spectrin within muscle fibers reflects the physiological state of those fibers. Changes in spectrin concentration may be a useful probe to study the various alterations in physical parameters found among fast, slow, dystrophic, and denervated fibers.     

Innervation of avian latissimus dorsi muscles and axonal outgrowth pattern in the posterior latissimus dorsi motor nerve during embryonic development

The distribution of the innervation to the anterior latissimus dorsi (ALD) and posterior latissimus dorsi (PLD) muscles of the chicken are described on the day of hatching and 6 weeks later using electron microscopy. In the ALD muscle, there are 5,000 muscle fibres and 374,400 endplates supplied by about 169 skeletomotor axons; in the PLD muscle, there are 12,000 focally innervated muscle fibers supplied by about 20 skeletomotor axons. On the cell surface of the muscle fibers the mean total subsynaptic area contacted by each motor axon is comparable in the ALD and PLD muscles. The growth pattern of the axons in the PLD motor nerve was described from the ninth day in ovo up to 6 weeks after hatching. The axons arrive in the PLD muscle in two successive waves: first, the large somatic axons which are already present before the ninth day in ovo and second, the small autonomic axons which continue to accumulate until hatching. The total number of somatic axons decreases from the ninth day until the hatching day when it reaches its definitive value. This decrease takes place during a period when the numbers of myofibers and of endplates dramatically increase, and it coincides with the axonal segregation by the Schwann cells. The myelination of the axons starts on the 15th day in ovo and is essentially complete upon hatching. Despite the decreasing number of somatic axons in the PLD nerve, the decrease in number of nerve endings per PLD endplate and the increasing number of PLD endplates per PLD muscle, it was found that between the 16th day in ovo and 6 weeks after hatching the mean number of axonal branches per PLD motor axon does not decrease.     

Noncoordinate expression of M band proteins in slow and fast embryonic chick muscles

The expression of the myosin-associated M band proteins myomesin and M protein in differentiating muscle fibers in the anterior latissimus dorsi (ALD) and posterior latissimus dorsi (PLD) muscles during embryonic chicken development was examined by immunocytochemistry using monoclonal antibodies. Early in the embryonic development of both muscles, both myomesin and M protein are expressed in primary and secondary myotubes. However, beginning at 10 days in ovo, M protein is gradually suppressed first in primary, then in secondary, presumptive slow-tonic type 3 fibers. M protein is transiently suppressed in presumptive fast-twitch type 2 fibers derived from primary myotubes but continuously expressed in those derived from secondary myotubes. Thus, initially all myotubes have a common intrinsic M band composition with respect to myomesin and M protein, whereas at later stages the expression of M protein is fiber-type specific. Intrafusal spindle fibers, which are segregated from extrafusal fibers around 14 days in ovo, have a heterogeneous M band composition atypical of extrafusal fibers.     

Energy reserves and chemical changes in denervated anterior and posterior latissimus dorsi muscles of the chicken

Postdenervation changes in most muscles, whether experimentally produced in animals or resulting from disease or injury in man, are characterized by a largely irreversible atrophy. One notable exception is the prolonged hypertrophy exhibited by the denervated anterior latissimus dorsi (ALD) muscle of the chicken. We have determined the concentrations of several chemical compounds and nitrogen fractions in the innervated and denervated latissimus dorsi muscles of the young chicken at various intervals after denervation. The posterior latissimus dorsi (PLD), a fast muscle, has significantly decreased ATP, phosphocreatine (PC), and total creatine (TC) concentrations 1 week after denervation. It also has less noncollagen nitrogen (NCN), as well as sarcoplasmic and fibrillar nitrogen; however, collagen nitrogen is increased and the muscle weighs 15% less than its contralateral control. The slow ALD, on the other hand, has not yet decreased its level of energy stores (ATP or PC) or TC at 3 weeks after denervation; nor are there any changes in NCN or any individual nitrogen fraction at 1 week. The hypertrophy of the denervated ALD reaches its maximum by the end of the first week, when it is 43% heavier than the control muscle. The weight gain in both innervated and denervated ALD are similar after that with the denervated muscle still 41% heavier at 3 weeks.     

Transplantation of the anterior latissimus dorsi muscle in normal and dystrophic chickens.

The anterior latissimus dorsi, a slow-tonic muscle, and the posterior latissimus dorsi, a fast-twitch muscle, were auto-transplanted to the site of the biceps brachii, a fast-twitch muscle. The regenerate muscle contained only α (fast-twitch) fibers when the posterior latissimus dorsi was the donor muscle. The regenerate contained both α and β (slow-twitch) fibers when the anterior latissimus dorsi was the donor muscle. No differences were observed between regenerates in normal and dystrophic chicks.     

Influence of spinal cord stimulation on the innervation pattern of muscle fibers in vivo.

In chick embryo, chronic stimulation of the brachial spinal cord at a fast rhythm from days 7 to 18 of development induced an increase in AChE activity sites and ACh receptor (AChR) clusters in slow anterior latissimus dorsi (ALD) muscle. Most AChR clusters and AChE spots were contacted by nerve endings. A previous study showed that such spinal cord stimulation causes changes in ALD muscle properties, especially the appearance of a high proportion of fast type II fibers (Fournier Le Ray et al., 1989). Analysis of the synaptic pattern in different fiber types of experimental ALD muscle indicated a decrease in the distance between successive AChE spots in slow type III fibers compared to controls, whereas the intersynaptic distance in fast type II fibers was very similar to that in the rare fast fibers developing in control ALD. Fast fibers of experimental muscles exhibited less AChR than did slow fibers. The increased number of neuromuscular junctions in ALD muscle after spinal cord stimulation appeared to be preferentially located in slow fibers. Electron microscopy showed no change in the number of axons in ALD nerve after spinal cord stimulation. The activity imposed on brachial motoneurons apparently caused terminal sprouting of ALD nerve in target muscle, thus accounting for the increase in neuromuscular contacts in ALD muscle fibers. Differences in the distribution of nerve contacts indicate that the type of muscle fiber innervated may play a critical role in the synaptic pattern during chick embryogenesis.     

Biochemistry of adenosine triphosphatase activity of avian fast and slow muscle

The posterior latissimus dorsi (PLD), a pure twitch muscle, and the anterior latissimus dorsi (ALD), a pure tonus muscle, of the chicken were examined for a comparison of their actomyosin adenosine triphosphatase (AM-ATPase) activity. Other investigators have determined that the PLD contracts six to eight times faster than the ALD. The AM-ATPase activity of the PLD is only 1.4 times that of the ALD, which does not account for the difference in contraction times. Rather, the morphological differences between the two muscles are proposed as the basis for the difference in speeds of contraction. The well-developed SR and T system in the PLD provide a more rapid and efficient excitation-contraction coupling than in the ALD. The effects of cyclic adenosine 3′,5′-monophosphate (cAMP) on these muscles were studied, and found to inhibit the AM-ATPase and lower the optimal ATP concentration. Rabbit skeletal muscle was studied for comparison, and cAMP was again found to inhibit the AM-ATPase. However, there was a differential inhibition of the fast and slow twitch muscles; the fast twitch muscles were inhibited to a greater degree than the slow twitch.     

Role of nerve and tension in maturation of posthatching slow-tonic muscle in chicken

The role of motor innervation and muscle tension in the posthatching maturation of the slow-tonic anterior latissimus dorsi (ALD) muscle of the chicken has been investigated. Modification of the muscle tension was obtained either by maintaining ALD in a shortened state or by stretching, after or without denervation. In denervated as well as in innervated ALD, shortening resulted in atrophy and inhibition of developmental change in muscle fiber population. In contrast, stretch causes hypertrophy, transformation of all 3B fibers, increase in SM2 isomyosin expression, and decrease in Ca2+-activated myosin ATPase in innervated or denervated ALD. On the other hand oxidative activity in ALD fibers was strikingly reduced after denervation even in presence of stretch-induced hypertrophy. This study suggests that a passive stretch can be involved in some, but not all, changes in ALD characteristics occurring after denervation and may be also involved in normal posthatching development of the slow-tonic muscle. Possible clinical implications of these results in relation to treatments for preventing muscle atrophy resulting from immobilization or disuse are suggested.     

Ultrastructure of chicken slow muscle after nerve cross union

The slow anterior latissimus dorsi (ALD) of the chick was cross innervated by a predominantly fast superior brachialis nerve which had been implanted into the muscle immediately after hatching. Six to eight months after the operation, the structure of cross-innervated muscles (ALD-X) was investigated and compared with that of the control slow (ALD-C) and fast (PLD-C) muscles. Sixty to eighty percent of muscle fibers of the ALD-X were found to be innervated focally by end-plates of the fast type, the remaining fibers being supplied by multiple end-plates of the slow type. The ultrastructure of most ALD-X muscle fibers appeared to be changed toward the fast type. A sample of maximally altered ALD-X fibers was selected for quantitative evaluation. The number of triads was significantly increased in the ALD-X fibers becoming similar to the number in the control PLD-C fibers. This change is probably related to the significant shortening of the contraction time of the ALD-X, found in analogous experiments earlier. The Z lines became significantly narrower in width, but still remained considerably wider than in the PLD-C muscles. The mean content of mitochondria was reduced by 60% in ALD-X fibers in comparison to ALD-C fibers and was comparable to the mean mitochondrial content of the PLD-C; this indicates a change in the oxidative metabolism of the cross-innervated fibers. Thus, in contrast to adult birds, the ultrastructure of slow multiply innervated muscle fibers becomes altered toward the fast type if nerve cross union is performed at an early stage of development.     

Heterogeneity of spectrin distribution among avian muscle fiber types

Muscle spectrin has been examined in avian fast, slow, and mixed muscles using the techniques of immunofluorescence microscopy and immunoautoradiography. By immunofluorescence, fibers of the fast-twitch pectoralis major (PM) are seen to contain alpha-spectrin antigen primarily at the sarcolemma, while in the slow-tonic anterior latissimus dorsi (ALD), alpha-spectrin antigen is found in high concentrations throughout the sarcoplasm as well as being present in association with the sarcolemma. In mixed (fast- and slow-twitch) muscles of the leg, two populations of fibers can be distinguished: those which resemble the fibers of the PM and another group which displays interior staining similar to the fibers of the ALD. Histochemical staining for actomyosin ATPase reveals that the fibers of mixed muscles which contain the most spectrin antigen correspond to the slow-twitch fibers. Supportive data were obtained using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoautoradiography. In slow muscle, an approximate threefold increase in alpha-spectrin concentration relative to other proteins is evident. These results suggest that the distribution of alpha-spectrin may be modified by the physiological state of the myofiber.     

Heterogeneity of the chicken ALD muscle: Evidence for a minor, twitch fiber type

Examination of the anterior latissimus dorsi muscle of the chicken using histochemical, immunofluorescent, and ultrastructural techniques revealed the presence of a minor muscle fiber type which was similar but not identical to the fast-twitch fibers of the posterior latissimus dorsi muscle. Heterogeneity of muscle fiber type appeared within the 1st week after hatching.     

Effect of activity on the selective stabilization of the motor innervation of fast muscle posterior latissimus dorsi from chick embryo

The role of neuromuscular activity in the maturation of the motor innervation was investigated in the fast focally innervated posterior latissimus dorsi (PLD) muscle of the chick embryo. The axonal supply in the PLD motor nerve, and the focal multiple innervation of the endplates were described on days 15 and 16 of embryonic life in normal and experimental embryos. In the first series of experiments, chick embryos were paralyzed by repeated injections between days 4 and 10 in ovo of the curare-like agent, flaxedil. Twice more axons in the PLD motor nerve and about twice more nerve terminal profiles at the endplates in the PLD muscles were found in paralyzed than in control embryos. In a second series of experiments, electrodes were implanted around the spinal cord of 7-day-old embryos and electric pulses delivered at 0.5 Hz frequency from day 10 to days 15-16 of incubation. At day 15.5, no change was observed in the axonal supply in the PLD motor nerve of stimulated embryos, while a two-fold decrease was observed in the number of motor nerve terminal profiles per endplate in the corresponding PLD muscle. The statistical distribution of the number of motor nerve terminal profiles per endplate was described from complete semi-serial sections in the PLD muscle from normal, paralyzed and stimulated chick embryos. In these three cases, the distribution of supernumerary nerve terminal profiles followed a Poisson law after one nerve ending had been subtracted from the number of nerve endings counted per endplate.     

Membrane cable properties of normal and dystrophic chicken muscle fibers

Values of fiber radius obtained by square pulse analysis and histological measurements indicated that the innervated dystrophic fibers which had been examined with microelectrodes were hypertrophic. The specific membrane resistance of dystrophic fibers was also greater than normal. In addition, experimentally induced compensatory hypertrophy of innervated nondystrophic (normal) fibers of the posterior latissimus dorsi led to alterations in several membrane characteristics which resulted in values resembling those of innervated dystrophic fibers. Twenty-one days after denervation, the values for the cable properties of normal and dystrophic fibers were increased, yet similar values were attained for the space constant, specific membrane resistance, and membrane capacitance. In both normal and dystrophic muscles which were denervated for 21 days the fiber radius decreased 40%. To study the mechanism underlying the increase of the specific membrane resistance after denervation, the resting membrane conductance was selectively altered. In solutions of low pH (5.0) where chloride conductance was presumably reduced, the space constant, time constant and specific membrane resistance of innervated normal and dystrophic fibers were increased and approached values obtained from 21-day denervated muscles. In contrast, solutions of low pH had no marked effects on 21-day denervated normal and dystrophic muscles. It is suggested that the increased values for these cable properties from denervated normal and dystrophic posterior latissimus dorsi muscles may be partially due to reduced potassium and chloride conductances. Furthermore, the presence of hypertorphic fibers may be a significant morphological adaptation in dystrophic muscles.     

Intracytoplasmic vacuoles in αW fibers of dystrophic chicken muscle —Probable early pathologic event initiates massive fiber necrosis

Summary An electron-microscopic study on dystrophic chicken white muscle, posterior latissimus dorsi (PLD), was performed with histochemical identification of three fiber types of R (red), R and W (white) fibers to evaluate the pathophysiology in fiber necrosis. As seen in histochemically stained sections, vacuolar formation in the cytoplasm, an outstanding pathologic feature in chicken dystrophy, was recognized in the W fibers by electron microscopy. The vacuole was membrane-bound and thought to originate from coalescence or dilatation of extensively proliferated sarcotubular system. There was evidence of a delay in fiber type transformation from R to W in dystrophic white muscle, while the initial pathologic event of sarcotubular system proliferation might be expressed only after muscle fibers had attained histochemical characteristics of W fibers. Localized myofibrillar degeneration was encountered in the vicinity of the vacuole with focal membrane defect. An influx of extracellular fluid through the vacuolated sarcotubular system into the sarcoplasm may activate certain proteases, such as calcium-dependent protease because the extracellular fluid contains high concentration of calcium ion. The activated protease then degrades structural protein, especially Z-line protein, followed by fiber necrosis with phagocytosis.Supported in part by grant no. 4, 1980, from the National Center for Nervous, Mental, and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare, Japan     

Heterogeneity of type 1 skeletal muscle fibers revealed by monoclonal antibody to slow myosin

Monoclonal antibodies (McAbs) were generated against slow myosin from the chicken anterior latissimus dorsi (ALD) muscle and their reactivity was checked against fast (pectoralis), slow (ALD), cardiac (ventricular), and smooth (gizzard) myosins by radioimmunoassay (RIA), immunoautoradiography (immunoblots), and indirect immunofluorescence techniques. In RIAs, the McAb (ALD-58) described in this article reacted specifically with slow myosin, with only a weak cross-reactivity to cardiac myosin. In immunoblots against whole muscle homogenates and purified myosins, it bound selectively to the 200 Kd myosin heavy chain band. The ALD-58 antibody stained the fibers of the ALD muscle uniformly but gave three grades of reactions (strong, weak, and negative), with histochemically identified type 1 fibers of sartorius and gastrocnemius muscles demonstrating the immunological heterogeneity of myosins in type 1 skeletal muscle fibers.     

Electrical, mechanical, and physical properties of denervated latissimus dorsi muscles of the chicken

The anterior (ALD) and posterior (PLD) latissimus dorsi muscles of Cornish-cross chickens were examined after 1 wk of denervation to determine changes which might occur in their electrical and mechanical properties. Both muscles exhibited increased percentage of water and intracellular sodium concentrations, and decreased intracellular potassium concentrations. Although the denervated PLD had a relative increase in the volume of its extracellular space, the ALD experienced a relative decrease in extracellular space. The normal resting membrane potential dropped from ?70 mv (ALD) and ?74 mv (PLD) to ?57 mv after denervation. The mechanical threshold of the ALD, determined by potassium contracture studies, occurred at a lower [K]₀ in the denervated than in the control muscle; nevertheless, the resting potentials at threshold were the same. Similarly, the denervated ALD approached its maximum mechanical response at a lower [K]₀ than the control muscle, but at identical membrane potentials. This implies that the role of the membrane in excitation-contraction coupling was not eliminated by denervation. However, the denervated muscle developed only one-half the tension of the control muscle which suggests a deficiency in the excitation-contraction coupling process within the muscle fiber. The relationship between the resting potential and [K]₀ for the control ALD followed the Nernst equation, whereas potentials obtained from the denervated muscles were lower than predicted. This may be attributed to the effects of other ions on the resting potential, changes in the membrane permeability characteristics, or possible intracellular potassium binding.     

Some observations on the innervation patterns of different fiber types of chick muscle

Muscle fibers from the tonic anterior latissimus dorsi and the phasic sartorius and complexus muscles of chicks were examined for their terminal innervation patterns. Advantage was taken of a recently developed cytochemical technique which allows simultaneous visualization of muscle fiber types and their end plates. This technique increases the sensitivity of the acetycholinesterase assay. It was found that both fiber types in the anterior latissimus dorsi as well as the β fiber in the phasic muscles have similar patterns of innervation. All are multiply innervated with major end plates spaced at regular intervals. In addition, many minor end plates are randomly scattered along these fiber types. Despite similar innervation patterns, all three types of fibers exhibit different myofibrillar adenosine triphosphatase characteristics.     

Influence of chronic spinal cord stimulation upon differentiation of beta muscle fibers in a fast muscle (posterior latissimus dorsi) of the chick embryo

The aim of this work was to study the influence upon differentiation of muscle fiber types of the multiple innervation induced in a fast muscle by chronic spinal cord stimulation. In previous work, we showed that low-frequency stimulation applied to the spinal cord of the chick embryo caused a distributed innervation of muscle fibers in the posterior latissimus dorsi (PLD). In normal development, some beta fibers differentiate within this muscle, the maximal number being attained by 14 to 15 days of embryonic development. Later, the numbers of beta fibers decreased with age. In spinal cord-stimulated embryos the beta muscle fibers within the PLD were stabilized and did not disappear. After the cessation of spinal cord stimulation, the number of beta fibers within the PLD muscle did not decrease. There are possible explanations of the influence of chronic spinal cord stimulation and muscular activity upon formation and persistence of beta fibers within a fast muscle.     

Inhibition of lysosomal function in red and white skeletal muscles by chloroquine

In chickens treated 7 days with chloroquine, morphological observations and chemical analyses were in agreement with our hypothesis that lysosomes degrade some fraction of skeletal muscle mitochondria, plasma membrane, and glycogen but apparently not normal myofibrillar proteins. After chloroquine inhibition of lysosomal digestion, autophagy was apparent in anterior (ALD) and posterior (PLD) latissimus dorsi muscles. Membrane-limited vacuoles (autophagic vacuoles) contained mitochondria, membranes, and glycogen. To help identify the vacuolar constituents, a variety of marker enzyme activities and chemical analyses were monitored for the ALD and PLD muscles. Parallel increases in 5′-nucleotidase and cytochrome oxidase specific activities correlated with the increase in observable inclusions. Autolytic rates were increased in acidic (pH 4.0) but not in alkaline (pH 8.5) or neutral (pH 7.0) conditions when homogenates were prepared from muscles of chloroquine-treated chickens. The removal of chloroquine inhibition in vitro by the preparation of tissue homogenates resulted in increased proteolysis by lysosomal enzymes presumably due to an increase in available substrates derived from mitochondria and plasma membranes.