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.     

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.     

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.     

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.     

Conversion of muscle fiber types in regenerating chicken muscles following cross-reinnervation

Summary Slow-tonic anterior latissimus dorsi (ALD) and fast-twitch posterior latissimus dorsi (PLD) muscles of 7 to 10-day-old White Leghorn chickens were (a) crushed and allowed to be reinnervated by their own nerve, or (b) crushed and transplanted to the other side and allowed to be reinnervated by the nerve of the side to which they were transplanted. Following transplantation, changes in the weight of the muscle, fiber-type composition and innervation pattern during regeneration were investigated. Normal growth rate of PLD was about twice that of ALD. Regenerating PLD, however, atrophied rapidly after crushing and denervation whether innervated by its own nerve or the other nerve type, whereas ALD reinnervated by its own nerve showed marked hypertrophy. PLD fibers transformed rapidly to fast-twitch or slow-tonic (ST) fibers when they were reinnervated by PLD or ALD nerve, respectively. When ALD fibers were reinnervated by their own nerve, they differentiated into ST fibers that were surrounded by smaller immature fibers. ALD fibers were, however, resistant to complete control by fast-twitch PLD nerve and contained a large number of slow fibers (ST and ) long after transplantation. Slow fibers in regenerates were initially multiply innervated, but later transformed into fast-twitch fibers that were focally innervated. The mode of differentiation and innervation pattern of different muscle fiber types in regenerating muscles are discussed.Supported by the Muscular Dystrophy Association of America (Dr. T. Kikuchi) and partly by the National Center for Nervous, Mental and Muscular Disorder of Ministry of Health and Welfare, Japan (grant No. 84-04-05)     

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.     

Hypertrophy and hyperplasia of adult chicken anterior latissimus dorsi muscles following stretch with and without denervation

In the anterior latissimus dorsi muscle of the chicken hypertrophy was obtained by stretching, whether the muscle was innervated or not. The teres minor muscle, which was stretched at the same time, also hypertrophied; the posterior latissimus dorsi muscle, consisting of red and white fibers, did not change significantly in weight, but its position in the body is such that it was not stretched adequately.The rate of certain histological changes which accompany hypertrophy keeps pace with the rate of the hypertrophy. These changes include enlargement of nuclei, nuclear proliferation, and invasion with subsequent splitting of the units producing fibers of varying diameters. ATPase differentiation of fibers weakens when hypertrophy exceeds 35–40%, producing muscle fibers exhibiting, in general, a homochromatic, light ATPase response. The amount of hypertrophy and hyperplasia is proportional to the degree of stretch.Excluding an increase caused by fibers splitting, fiber counts of complete cross sections of anterior latissimus dorsi muscles taken at intervals after the onset of stretch showed a steady increase in the total number of fibers. This began promptly under extensive stretch, much later under mild stretch. The presence of new fibers appeared to be a function of both time and the amount of hypertrophy; the degree of hypertrophy had to be at least 70%. The increase in total fibers lagged behind the number of small fibers interpreted as “new.” The fate of many of the new fibers is not known but a sufficient number matured in the 8 wk of the experiment to produce the increase in total number.     

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.     

Myosin expression in hypertrophied fast twitch and slow tonic muscles of normal and dystrophic chickens.

Disruption of the development program of myosin gene expression has been reported in chicken muscular dystrophy. In the present report, the relationship between muscular dystrophy and the ability of muscle to respond to an increased work load with a transition in the myosin phenotype has been investigated. Hypertrophy of slow tonic anterior latissimus dorsi (ALD) and fast twitch patagialis (PAT) muscles was induced by overloading for 35 days and myosin expression was analyzed by electrophoresis and immunocytochemistry. Normal and dystrophic chicken ALD muscles have nearly identical proportions of SM-1 and SM-2 isomyosins and both exhibit an age-related repression of the SM-1 isomyosin which is enhanced and accelerated by overloading. Immunocytochemistry with anti-myosin heavy chain (MHC) antibodies demonstrates the appearance of nascent myofibers in overloaded ALD muscles from both normal and dystrophic chickens. A minor fast twitch fiber population is also identified which doubles in number with overloading in normal ALD muscles. There are only half as many fast twitch fibers in control dystrophic ALD muscles and this number does not increase with overloading. In contrast to ALD muscles, the isomyosin profile of normal and dystrophic PAT muscles is quite different. There is significantly more FM-3 and significantly less FM-1 isomyosin in the dystrophic PAT muscle. However, both normal and dystrophic PAT muscles exhibit an overload-induced accumulation of the FM-3 isomyosin. Immunocytochemistry reveals that, unlike the normal PAT muscle, the dystrophic PAT muscle contains a population of myofibers which express slow MHCs. As in the ALD muscle, overload-induced hypertrophy is associated with a repression of the SM-1 MHC in these fibers. Nascent myofiber formation does not occur in either normal or dystrophic overloaded PAT muscles.     

Restoration of denervated skeletal muscle transplants after reinnervation in rats

The present study describes reinnervation and restoration of rat skeletal muscle denervated for the duration of 3, 6 or 12 months. Denervation of extensor digitorum longus (EDL) muscle was achieved by cutting and ligating the donor rat sciatic nerve in situ. At 3, 6 and 12 months, the denervated EDL muscles were removed and transplanted into an innervated normal leg of another rat. In addition, normal (i.e., no prior denervation) muscles were transplanted as controls for comparison. The muscles were analyzed at 4 and 12 weeks after transplantation. The EDL muscle weight and myofiber size decreased with extended denervation times. After transplantation, the muscles underwent regeneration and reinnervation, and recovered as determined by an increase in muscle mass and myofiber size. The 3-month denervated muscle regenerates recovered completely, and were similar to the non-denervated normal muscle regenerates. Reinnervation, and partial recovery of muscle weight and myofiber size was observed in 6- and 12-month denervated muscle transplants. These results document that while regeneration and reinnervation does occur in denervated muscles after transplantation, the extent of recovery is related to the duration of denervation.     

Studies on the regenerative recovery of long-term denervated muscle in rats

Denervated extensor digitorum longus (EDL) muscles in rats rapidly lose mass and contractile force. After two months of denervation, mass and maximum tetanic force have fallen to 31% and 2% of the values of contralateral control muscles. Our purpose was to determine if grafting a long-term denervated muscle into an innervated site provides an effective means of restoring its structure and function. EDL muscles that had been denervated for periods of 2-12 months were freely grafted into innervated sites of EDL muscles in 4-month inbred host animals. Contralateral normally innervated EDL muscles from the same donors were implanted into the opposite legs of the same hosts. Two months after grafting, the muscles were removed and measurements were made in vitro of isometric contractile properties. The grafts were then prepared for morphological analysts. In all cases, the maximum forces generated by innervated grafts of denervated muscles were greater than those generated by denervated muscles. However, when compared with grafts of control muscles in the contralateral limb, grafts of previously denervated muscles showed a steady decline in structural and functional recovery corresponding to the time of previous denervation. The decline was especially pronounced for muscles denervated between 2 and 7 months prior to grafting. Grafts of 7-month denervated muscles were restored to only 17% of the maximum tetanic force of contralateral control grafts compared with 83% for grafts of 2-month denervated muscles. The longer a muscle had been denervated prior to grafting, the higher proportion of thin atrophic muscle fibers it contained. We conclude that grafting into an innervated site improves the mass and maximum force of a muscle over the denervated state, but the longer the period of prior denervation the poorer the recovery of the grafted muscles.     

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 stretch and denervation on protease activities of normal and dystrophic chicken muscle

Changes of protease activities that follow passive stretch, denervation, and denervation plus stretch were followed in the patagialis muscle of normal and dystrophic chicks between 6 and 7 weeks of age. The baseline activities of cathepsin C, cathepsin D, and leucine aminopeptidase in dystrophic muscle were 2 to 3.5 times higher than in normal muscle. Passive stretch and denervation induced increases in protease activities by 40 to 120% in normal muscle, whereas the same treatments did not significantly affect the activities of the enzymes in dystrophic muscle. We conclude that the level of protease activity in dystrophic chicken muscle at 6 weeks of age had already attained a maximum limit and could not be increased even by denervation. In spite of protease activities, which were not different from control dystrophic muscle, denervated dystrophic muscles lost muscle weight rapidly whether they were stretched or not. They weighed 60% less than the innervated control muscle after 7 days. Inherently high protease activities in dystrophic muscle do not vary at this age regardless of whether or not the muscle is gaining or losing weight.     

Repression of the embryonic myosin heavy chain phenotype in regenerating chicken slow muscle is dependent on innervation.

Ventricular-like and fast myosin heavy chains (VL-MHC and FMHC) are transiently expressed during slow skeletal muscle development. The influence of innervation on repression of these MHC isoforms is investigated over an 84-day time course in: (1) normal anterior latissimus dorsi (N-ALD) muscles, (2) regenerating ALD (R-ALD) muscles, (3) denervated ALD (D-ALD) muscles, and (4) regenerating and denervated ALD (RD-ALD) muscles. Western blotting demonstrates that the VL-MHC is expressed in R-, D-, and RD-ALD muscles, but not in N-ALD muscles. Expression of the VL-MHC is transient in R-ALD muscles. In contrast, VL-MHC expression persists in RD-ALD muscles, and appears with time in D-ALD muscles. FMHC was not detected in N-ALD muscles by Western blotting. Two FMHCs are seen in R-ALD and RD-ALD muscles, and in 13-day embryonic ALD muscles. The slower migrating FMHC (FMHCA) comigrates with developmentally regulated FMHCs in fast pectoralis muscle, while the faster migrating FMHC (FMHCB) comigrates with the faster migrating FMHC in embryonic ALD muscle (13 days in ovo). FMHCB decreases in amount over the time course in R-ALD muscles, while FMHCA persists. In contrast, substantial levels of both FMHCs persist in RD-ALD muscles, and appear with time in D-ALD muscles. The cellular distribution of MHCs is followed by immunocytochemistry. Regenerating cells expressing VL-MHC and FMHC are replaced by a mature population in R-ALD muscles. Some of the mature myofibers in R-ALD muscles express FMHC, but not VL-MHC. In RD-ALD and D-ALD muscles, both regenerating and mature muscle cells are seen which express VL-MHC and FMHC. Our results indicate that innervation is required for the repression of VL-MHC and FMHCB during regeneration of slow muscle.     

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.     

Effect of denervation on overdevelopment of chloroquine-induced autophagic vacuoles in skeletal muscles

We studied how denervation affects the overdevelopment of autophagic vacuoles in muscles of chloroquine-treated rats. The number of autophagic vacuoles increased significantly in the chloroquine-treated soleus muscles after denervation as compared to similarly treated contralateral, innervated muscles. No vacuoles were present in the denervated and innervated muscles of saline-treated rats. After denervation, the autophagic vacuoles in chloroquine-treated muscle contained numerous glycogen particles and various heterogeneous materials. A biochemical study showed no significant difference in the activities of lysosomal proteases and hydrolases in the chloroquine- and salinetreated muscles after denervation, although these activities were markedly increased in comparison to the same activities in the contralateral, innervated muscles. Chloroquine treatment by itself did not, but denervation with or without chloroquine treatment did enhance the biochemical activities of lysosomal enzymes in the animals. We speculate that denervation induces the marked accumulation of autophagic vacuoles in chloroquine-incuced myopathy.     

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.     

Myosin components of the latissimus dorsi and the pectoralis major muscles in the dystrophic chicken

The myosin composition of the anterior latissimus dorsi, the posterior latissimus dorsi, and the pectoralis major muscles was examined in the inbred White Leghorn dystrophic chicken and its isogenic normal line at different ages during development and maturation. Using the biochemical methods of native gel electrophoresis, one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), and peptide mapping, it was found that myosin isozyme changes occurred normally in the anterior latissimus dorsi muscle. However, in the posterior latissimus dorsi muscle, slow myosin components which were not present in the adult normal muscle were present in the adult dystrophic muscle. In addition, the pectoralis major muscle of the dystrophic chicken failed to undergo the neonatal to adult fast myosin isozyme transition. Our data also showed that muscle cell cultures derived from the pectoralis major muscle of dystrophic chickens expressed identical myosin components to cultures derived from normal embryos. However, since these cultures only produced embryonic myosins even after 1 month in culture, it implied that cells in tissue culture were phenotypically normal because present cell culture conditions were insufficient to induce the fetal to adult isozyme changes.     

Glycolipid and glycoprotein sialyltransferase enzyme activity in denervated skeletal muscle

Glycolipid and glycoprotein sialyltransferase enzyme activities were determined in homogenates of control and denervated rat extensor digitorum longus muscles. One week after denervation cytidine monophosphate-N-acetylneuraminic acid (CMP-NANA): lactosyl ceramide and CMP-NANA: fetuin sialyltransferase enzyme activities in denervated muscles were consistently higher in denervated than in control muscles, whether the activities were expressed on a protein, wet weight, or whole muscle basis. These findings support the hypothesis that control of muscle glycoconjugate metabolism, established in the normal relationship between nerve and muscle, is disrupted by denervation.