REGENERATION AND INNERVATION OF NORMAL AND DYSTROPHIC MUSCLE CULTURED WITH NORMAL AND DYSTROPHIC SPINAL CORD

Teased strips of normal or dystrophic adult mouse muscle were cultured with embryonic normal or 'dystrophic' mouse spinal cord explants, in order to determine whether muscular dystrophy is of primary myopathic or neuropathic origin. When cultured with normal spinal cord, both normal and dystrophic muscle regenerated to form new cross-striated muscle fibres with peripherally located nuclei and showing spontaneous, synchronized contractions. Silver impregnation and acetyl-cholinesterase activity showed that neuromuscular junctions were formed. In marked contrast, both normal and dystrophic muscle cultured with spinal cord from dystrophic animals failed to show such functional regeneration, and neuromuscular junctions could not be identified. Only a few myotubes were formed, and these were rarely cross-striated, had internal nuclei, and only some showed asynchronous fibrillatory contractions. These results strongly suggest that murine muscular dystrophy has a neural pathogenesis. Histochemical reactions were carried out on serial frozen sections of the regenerated muscle fibres, and also of embryonic mouse muscle cultured in cord-myotome explants, to determine whether 'fibre types' were formed. All the fibres gave uniformly strong or medium reactions to phosphorylase, NADH-TR, myofibrillar ATPase, ATPase after acid preincubation, and the PAS reaction for glycogen, except a few fibres in an embryonic muscle which gave weak reactions for all enzymes. Histochemical fibre types were not formed. Two studies of explants of human muscle in association with normal embryonic mouse spinal cord resulted in the establishment of functional neuromuscular contact and the production of well striated myotubes, with peripherally placed nuclei and spontaneous regular contractions.     

Study of neurotrophism in normal/dystrophic parabiotic mice

The trophic influences of nerve and muscle on one another were studied in normal and dystrophic littermates of C57BL/6J dy^2J mice parabiosed at 20 to 23 days after birth. Each parabiont had a soleus muscle cross-reinnervated by a tibial nerve of its partner. Ultrastructural abnormalities of muscle and endplate were quantified and compared 6 to 7 months postoperatively. The dystrophic nerve degenerated despite reinnervation to a normal muscle. The normal muscle did not prevent the dystrophic nerve from degenerating, and the dystrophic nerve induced degenerative changes in the reinnervated normal muscle. Normal nerve did not retard the genetically programmed degeneration of the dystrophic muscle. The dystrophic muscle, however, did not appear to cause normal nerve terminals to degenerate. We conclude that both nerve and muscle cells in dystrophic mice express characteristics of muscular dystrophy. Muscle fibers of a few motor units further suffer from abnormal neurotrophic influence because of the degeneration of the motor neurons. Myotrophic influence on nerve was not observed.     

Muscle regeneration in mdx mouse, and a trial of normal myoblast transfer into regenerating dystrophic muscle

The most ideal therapeutic trial on Duchenne muscular dystrophy (DMD) is a transfer of normal myoblasts into dystrophic muscle which has been attempted on animal models in several institutes. In the process of muscle regeneration, the transferred normal myoblasts are expected to incorporate into the regenerating fibers in host dystrophic mouse. To know the capacity of muscle regeneration in dystrophic muscle, we compared the regenerating process of the normal muscle with that of the dystrophic muscle after myonecrosis induced by 0.25% bupivacaine hydrochloride (BPVC) chronologically. In the present study, C57BL/10ScSn-mdx (mdx) mouse was used as an animal model of DMD and C57BL/10ScSn (B10) mouse as a control. There was no definite difference in the behavior of muscle fiber regeneration between normal and dystrophic muscles. The dystrophic muscle regenerated rapidly at the similar tempo to the normal as to their size and fiber type differentiation. The variation in fiber size diameter of dystrophic muscle, however, was more obvious than that of normal. To promote successful myoblast transfer from B10 mouse into dystrophic mdx mouse at higher ratio, cultured normal myoblasts were transferred into the regenerating dystrophic muscle on the first and the second day after myonecrosis induced by BPVC. Two weeks after the myoblast injection, the muscles were examined with immunohistochemical stain using anti dystrophin antibody. Although dystrophin-positive fibers appeared in dystrophic muscle, the positive fibers were unexpectedly small in number (3.86 +/- 1.50%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycogen cycle enzymes and cyclic nucleotides in avian muscular dystrophy

Fast-frozen pectoralis muscle samples were taken from normal chickens (lines 200 and 412) and chickens having hereditary muscular dystrophy (line 304). The glycogen phosphorylase activity ratio (activity without AMP/activity with AMP) was significantly greater in dystrophic muscles (0.306 +/- 0.046) than it was in normal muscles (0.090 +/- 0.023). Glucagon treatment did not cause any changes in phosphorylase activity ratios. Isoproterenol treatment of both normal and dystrophic muscles raised the phosphorylase activity ratio of normal muscles to 0.446 +/- 0.054, which was not significantly different from that of the dystrophic muscles. The dystrophic muscles had significantly less glycogen than normal muscles (23.3 +/- 2.8 compared with 36.8 +/- 2.8 mumoles glucosyl units/g of muscle). There was no relationship of muscular dystrophy to total phosphorylase activity (measured in the presence of 1 mM AMP) and to glycogen synthase activities measured without and with glucose 6-phosphate. Normal muscles had 28% less cAMP and 49% less cGMP than dystrophic muscles, but these differences were eliminated by treatment of the chickens with glucagon.     

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.     

Tenascin-C expression in dystrophin-related muscular dystrophy

The mdx mouse has a mutated dystrophin gene and is used as a model for the study of Duchenne muscular dystrophy (DMD). We investigated whether regenerating mdx skeletal muscle contains the extracellular matrix protein tenascin-C (TN-C), which is expressed in wound healing and nerve regeneration. Prior to the initiation of muscle degeneration, both normal and mdx mice displayed similar weak staining for TN-C in skeletal muscle, but by 3 weeks of age the mice differed substantially. TN-C was undetectable in normal muscle except at the myotendinous junction, while in dystrophic muscle, TN-C was prominent in degenerating/regenerating areas but absent from undegenerated muscle. With increasing age, TN-C staining declined around stable regenerated mdx myofibers. TN-C was also observed in muscle from dogs with muscular dystrophy and in human boys with DMD. Therefore, in dystrophic muscle, TN-C expression may be stimulated by the degenerative process and remain upregulated unless the tissue undergoes successful regeneration. © 1996 John Wiley & Sons, Inc.     

Lipid changes in Duchenne muscular dystrophy

Thin layer chromatographic analysis of lipid extracts of rectus abdominis and gastrocnemius muscles from controls and patients with severe sex-linked Duchenne muscular dystrophy shows the dystrophic tissue to contain more sphingomyelin, less lecithin plus choline plasmalogen, and more total cholesterol than normal. Comparison of normal, dystrophic, and immature muscle suggests that these observations can be interpreted as showing a similarity between dystrophic and immature muscle and in this respect human Duchenne dystrophy resembles hereditary muscular dystrophy in the mouse. Although sphingomyelin was present in apparently normal amount in muscle biopsies from patients with various other neuromuscular disorders, it was raised in two cases showing evidence of peripheral neuropathy.     

Intracellular elemental content of cardiac and skeletal muscle of normal and dystrophic hamsters

To test the hypothesis that the genetic lesion causing muscular dystrophy might be reflected in an abnormal intracellular elemental content, the elemental content of individual cardiac and skeletal muscle fibers in 50-day-old male control and cardiomyopathic BIO 53.58 hamsters was determined. The technique of electron probe x-ray microanalysis of freeze-dried tissue was employed. No electrolyte content differences were found between control and diseased animals for nuclei, myofibrillar cytoplasm, or mitochondrially-enriched cytoplasm of cardiac myocytes. Sulfur was elevated in dystrophic cardiac myocytes and was the only element significantly different in heart tissue of control and cardiomyopathic animals. Sulfur was also elevated in dystrophic soleus muscle fibers. The pattern of electrolyte content of these cells reflected a mixture of normal cells and damaged cells with altered electrolyte content. In this hamster model, alteration of electrolyte content of myocytes appears to be a result of the disease process and not an inherent characteristic of muscular dystrophy. The elevated sulfur in dystrophic hamster myocytes reflects a biochemical lesion which deserves further study.     

Regeneration of dystrophic muscle following multiple injections of bupivacaine

Regenerated myofibers formed subsequent to orthotopic transplantation of young, dystrophic mouse muscle fail to display the extensive histopathological changes characteristics of murine dystrophy. In order to determine whether this modification of the phenotypic expression of murine dystrophy is unique to the transplantation system or whether it can be found when other extreme trauma induces dystrophic muscle to regenerate, the extensor digitorum longus muscles of 4-6-week-old normal (129 ReJ +/+) and dystrophic (129 ReJ dy/dy) mice were given two series of injections of the myotoxin bupivacaine, spaced 12 hours apart. These injections resulted in necrosis of approximately 90% of the original myofibers. At 100 days after injection, the regenerated normal muscle appeared "healthy," whereas the regenerated dystrophic muscle displayed histopathological changes. It is suggested that the differences in the time course of innervation of the myotubes in the transplantation system as compared with that in the bupivacaine system may be a factor in determining whether regenerated dystrophic myofibers express a dystrophic morphology.     

Altered tissue carnitine levels in animals with hereditary muscular dystrophy

Low levels of muscle carnitine have been found in patients with Duchenne dystrophy, a case possibly of Becker dystrophy, and limb-girdle syndrome as well as in patients with the recently described muscle carnitine deficiency syndrome. Tissues of the mouse, hamster, and chicken were analyzed to determine whether tissue carnitine levels were altered in the animal models of muscular dystrophy. Significantly higher levels of carnitine were found in dystrophic mouse muscle, but carnitine levels in plasma, liver and heart were normal. Histological changes in the skeletal muscle of dystrophic hamsters were relatively mild, and both skeletal muscle and plasma levels were normal. The liver carnitine level was higher than normal levels. The dystrophic hamster also had an inherited cardiomyopathy, and interestingly its heart carnitine level was much lower than normal. The red muscle of the normal chicken contained 5 times the level of carnitine found in white muscle. The dystrophic chicken had higher than normal levels of carnitine in the white muscle, but normal levels in the red muscle. Although all 3 animal models of muscular dystrophy studied have altered levels of carnitine in some tissue, none of the animal models had the same pattern of altered tissue carnitine levels seen in human patients.     

Transplantation of extensor carpi radialis longus muscle in normal and dystrophic chicks

The etiology of avian muscular dystrophy was examined by a cross-transplantation technique. Care was taken for the transplants to regenerate and develop under neural influence, by using the small extensor carpi radialis longus (ECRL) muscle. The ECRL muscles were exchanged between normal and dystrophic chicks 2 to 3 days ex ovo, and the muscle weight, number of muscle fibers, muscle fiber size, and contractile properties of the transplanted muscles were observed 60 to 65 days after operation when the tissue reconstitution was virtually complete. The results obtained for the physiologic, anatomic, and histologic parameters strongly suggested that there exists some failure in the host environment of the dystrophic chicken. The analyses of the histologic parameters suggested that a genetic disorder may also reside in the muscle tissue itself. The myotonic nature of the muscle membrane, however, probably does not contribute significantly to the abnormal behavior of dystrophic chickens. The importance of some neurogenic abnormalities in avian muscular dystrophy is discussed in relation to the results reported by other investigators.     

Is there a maturation defect related to calcium in muscle mitochondria from dystrophic mice and Duchenne and Becker muscular dystrophy patients?

In our study, mitochondria were isolated from skeletal muscle in 2-, 3-, 4-, 6-, 8-, and 12-week-old normal (C57BL6j dy/+), and 4-, 8-, and 12-week-old dystrophic (C57BL6j dy/dy) mice and in normal subjects and patients with Duchenne or Becker muscular dystrophy. A deficit was observed in a calcium-specific mitochondrial protein in the very young control mouse, compared with the adult mouse. In the adult dystrophic mouse this deficit was found in clinically affected hindleg muscles as well as in apparently normal front leg muscles; it was also found in quadriceps muscles from patients with Duchenne and Becker muscular dystrophy. It is not observed in normal adult mice or in normal subjects. The body of our results suggests that in the forms of muscular dystrophy studied there would be a maturation defect in this calcium-binding mitochondrial protein (“calmitine”), a defect which might be generalized in the entire skeletal muscle system and conceivably could be the cause of muscle degeneration in certain myopathies such as Duchenne and Becker muscular dystrophy.     

Slow-type C-protein in dystrophic chicken fast pectoralis muscle

C-protein isoform expression in hereditary dystrophic chicken skeletal muscle was compared with that in normal chicken muscle during postnatal development by immunocytochemical and immunoblot methods. In the pectoralis muscle (PM) of both normal and dystrophic chicken, slow- and fast-type C-proteins were coexpressed in the vast majority of myofibers at neonatal age, but the slow C-protein disappeared, leaving continued expression of only the fast-type C-protein as muscle development progressed up to 2 weeks posthatch. In the dystrophic chicken PM, however, myofibers containing slow-type C-protein reappeared about 1 month posthatch and increased in number with the progression of muscular dystrophy. We conclude that C-protein isoform expression in dystrophic myofibers resembles that in neonatal myofibers and that the expression of slow-type C-protein can be seen as a marker for chicken muscular dystrophy.     

MUSCLE TRANSPLANTATION BETWEEN NORMAL AND DYSTROPHIC MICE. 1. HISTOLOGICAL STUDIES

Minced normal and dystrophic tibialis anterior muscles were transplanted into normal and dystrophic mice in the presence or absence of the peroneal nerve. Transplants were examined at intervals ranging from 0 to over 300 days in normal hosts and 0 to 200 days in dystrophic hosts. In the first 10 days after tranriplantation all muscle minces underwent degeneration followed by proliferation of precursor cells of connective tissue and muscle. By 8 days, myotubes with internal nuclei were abundant. Further differentiation of myotubes into muscle fibres occurred only in normal hosts with an intact peroneal nerve. Transplants in dystrophic mice, and in normal mice in which the peroneal nerves were capped with polydimethyl silicone rubber, underwent progressive degeneration, and by 100 days consisted mainly of connective tissue and fat. In normal hosts innervated dystrophic transplants were histologically similar to auto- and homotransplants of normal muscle. A significant population of regenerated fibres in normal and dystrophic transplants showed internal nuclei, variation in size and splitting, and were smaller in diameter than those from intact normal muscles. Since normal and dystrophic transplants failed to survive in the dystrophic hosts and in the denervated state in normal mice, it is suggested that the failure of dystrophic mice to maintain minced muscle transplants may be due to an inherent inability of dystrophic nerves to establish functional neuromuscular contact with these transplants.     

Active state properties of denervated and immobilized muscle: comparison with dystrophic muscle

Possible roles of neurotrophic mechanisms and muscle activity in the contractile abnormalities of muscular dystrophy were studied by comparing human dystrophic muscle to denervated and immobilized muscle. As evident in denervated muscle from the decreased acceleration of twitch development (decreased active state intensity of shortening), and isoproterenol-induced decrease of twitch with shortened decay of the active state, part of the abnormality in the subcellular calcium transport system in dystrophic muscle seems to be influenced by disordered neural regulation. Other active state abnormalities relating to activation processes and contractile proteins in dystrophic muscle were also demonstrated in both denervated and immobilized muscle, with some being more marked in immobilized muscle. The findings indicate that a neurogenic hypothesis cannot entirely explain the pathogenesis of progressive muscular dystrophy.     

Increased calcium-activated neutral protease activity in muscles of dystrophic hamsters and mice

A Ca²⁺-activated neutral protease activity was examined in muscles of normal and dystrophic hamsters and mice. Light grey and golden brown strains of normal and B10 14.6 strain of dystrophic hamsters were used. Normal and dystrophic mice were of the Bar Harbor 129 ReJ strain. Enzyme activity was measured in the post myofibrillar fraction (homogenate) and in the 75,000 × g pellet (particulate fraction) and supernatant using purified myofibrils.

In normal and dystrophic hamsters or mice, the Ca²⁺-activated neutral protease was most active in the supernatant followed by the homogenate and particulate fractions. As compared to fractions from normal muscle, enzyme activity was significantly elevated in all 3 fractions from dystrophic muscles of hamsters and mice. Both homogenate and supernatant fractions from muscles of normal hamsters had significantly higher enzyme activity than those of normal mice. Enzyme activity was similar in the particulate fraction. Similarly enzyme activity in the 3 fractions from dystrophic hamster and mouse muscles showed no significant difference.

It is suggested that the Ca²⁺-activated neutral protease may be involved in muscle fibre necrosis in muscular dystrophy.     

A new case of Ullrich's disease

A new case of congenital, hypotonic-sclerotic muscular dystrophy is presented. The patient showed congenital hyperlaxity and looseness of distal joints, muscle weakness, and spur-like protrusion of the calcaneus. Afterwards rapid progressive contractures of both knees and hip joints developed. Muscle biopsies revealed unequivocal dystrophic abnormalities and small atrophic fibers with numerous foldings of basal lamina suggestive of a neurogenic lesion. The disease presents clinical variability but the diagnosis is possible when a newborn shows: no dominant family history, slender body, marked distal joint laxity and hyperflexibility, proximal joint contractures and normal or slightly increased serum enzymes.     

Distal muscular dystrophy with autosomal recessive inheritance

Two sisters presented with distal weakness and their muscle biopsy was dystrophic. This distal muscular dystrophy has an autosomal recessive inheritance and its features are somewhat different from the more common autosomal dominant distal muscular dystrophy and include: (a) onset in early adult life: (b) involvement of distal leg muscles and especially peroneal muscles; (c) marked early elevation of serum creatine kinase (CK); (d) brief duration, small amplitude motor units and fibrillation on electromyography; and (e) histologic features of a dystrophic myopathy.     

Blood plasma pyruvate kinase as a marker of muscular dystrophy. Properties in dystrophic chickens and hamsters

Pyruvate kinase activity rises sharply in the blood plasma of the genetically dystrophic chicken, and parallels in its timecourse during the development of the disease the appearance of other known signs. The increase in the dystrophic chicken reaches about 30-fold the normal value; in the genetically dystrophic hamster, a similar rise occurs and reaches 20-fold the normal level. A high correlation exists between the plasma pyruvate kinase and creatine phosphokinase activities in the development of dystrophy. The former appeared in the blood rather faster than the latter, despite the threefold greater molecular size of the former. Chickens heterozygous for muscular dystrophy also had plasma pyruvate kinase elevations, which were much smaller than in the homozygotes, but nevertheless significant: the values were about twofold those of the corresponding normal birds. The isoenzymes of pyruvate kinase were quantitatively analyzed by an isoelectric focusing method: dystrophic chicken muscle contains two isoenzymes, the major one being the M₁ form. It was shown thus that the isoenzymes of normal and of dystrophic chicken muscle were indistinguishable. The pyruvate kinase isoenzyme pattern in the chicken erythrocyte was established, and this, also, was identical in dystrophic and normal animals. The pyruvate kinase accumulating abnormally in the dystrophic blood was not the red cell enzyme but, by the isoelectric focusing evidence, was entirely due to enzyme escaping, unchanged, from the skeletal muscle. All our observations showed plasma pyruvate kinase to be an indicator of muscular dystrophy in these animals, and hence likely to be of value as one of the criteria for assessment of chemotherapeutic effects.