Plasma phosphoglycerate mutase as a marker of muscular dystrophy

An elevation of phosphoglycerate mutase (PMG) has been detected in the blood plasma of the genetically dystrophic chicken and in Duchenne muscular dystrophy (DMD) patients. In the dystrophic chicken, plasma PGM in the pectoral muscle was simultaneously depressed to less than one-half that of the normal chicken. In a group of 9 DMD patients, plasma PGM activity was found to be significantly raised above the normal range. A survey of a small group of plasma specimens from human fetuses at risk for muscular dystrophy also suggested that PGM merits investigation as a potential adjunct to other diagnostic indices.     

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.     

Serum CK,calcium, magnesium,and oxidative phosphorylation in mdx mouse muscular dystrophy

Serum creatine kinase (CK) activity, calcium (Ca) and magnesium (Mg) contents of skeletal muscle and isolated mitochondria, as well as oxidative phosphorylation of X-linked muscular dystrophic (mdx) mice were compared with normal control animals at ages 5, 10, and 23 weeks. Serum CK is elevated in mdx mice at all ages, with highest activities at 5 weeks. The Ca content of dystrophic skeletal muscle is increased at all ages, whereas no clearly abnormal trend in muscle Mg levels was observed. Noncollagen protein (NCP), which was used as a reference base, is significantly diminished in muscle from 10- and 23-week-old mdx animals. Isolated mitochondria from mdx mice have elevated calcium content and decreased respiratory control ratios with NAD-linked substrates pyruvate/malate. The findings are distinct from those in dystrophic mice, strain 129/ReJ, but similar to observations in dystrophic hamsters and Duchenne muscular dystrophy and reflect the occurrence of overt muscle cell necrosis.     

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.     

Cyclic neucleotides in progressive muscular dystrophy.

The authors radioimmunoassayed cyclic nucleotide concentrations in plasma and biopsied muscles of muscular dystrophy and muscles of chicken embryo. c-AMP concentrations in plasma were significantly lowered in Duchenne-type muscular dystrophy and this lowered degree was correlated with the stage of progression. Plasma c-GMP levels were also depressed in Duchenne-type dystrophy. In biopsied muscles, c-AMP concentrations per milligram of non-collagen protein were within normal limits. Therefore, the decrease of plasma c-AMP concentrations might be an expression of total metabolic changes rather than a pathologic process of the muscle itself. As for the dystrophic chicken embryo, both c-AMP and GMP concentrations were decreasing in the pectoral muscles in parallel with the advancement of hatching stages.     

Plasma acetylcholinesterase in Duchenne muscular dystrophy

Muscle acetylcholinesterase (AChE) in unregulated in animal and human muscular dystrophies and its activity is elevated in plasma of dystrophic chickens, probably due to a leakage from affected muscles. It is possible to measure AChE activity in human plasma in spite of high butyrylcholinesterase activity if acetyl-beta-methylcholine is used as the substrate and butyrylcholinesterase is inhibited by iso-OMPA. It has been found that, unlike in chickens, the plasma AChE activity in human newborns is not higher than that in adults. The AChE activity in plasma of children afflicted by Duchenne muscular dystrophy does not differ from that found in plasma of normal boys of the same age. In this respect Duchenne muscular dystrophy differs from chicken muscular dystrophy as well as from a neurogenic muscle disease (amyotrophic lateral sclerosis) in man.     

Baclofen,procainamide, verapamil,and prenylamine in hereditary muscular dystrophy of the chicken

Two “myotonia antagonists” (baclofen and procainamide) and two “calcium antagonists” (verapamil and prenylamine) were evaluated for their effects on hereditary muscular dystrophy of the chicken. Righting ability of dystrophic chicks was improved only by baclofen and procainamide. Plasma creatine kinase activity of dystrophic chicks was reduced 45% after chronic administration of baclofen, but was still nearly nine-fold greater than activity in plasma of normal chicks. Baclofen and verapamil both reduced acetylcholinesterase activity in pectoralis major muscles of dystrophic chicks by about 40%, but these values were still significantly greater than those measured in muscles of normal chicks. The data provide further support for the concept that impaired righting ability of young dystrophic chickens is associated with the presence of myotonia in affected muscles, but do not rule out the possibility that certain of the biochemical features of the avian dystrophy may involve calcium.     

A freeze-fracture analysis of intramembrane particle densities on dystrophic hamster heart sarcolemma

The intramembrane particle (IMP) profile of control and dystrophic (Bio 14.6) hamster cardiac muscle plasma membrane was assessed in freeze-fracture replicas to determine whether this animal model of muscular dystrophy exhibits the same membrane characteristics found in skeletal muscle from other more thoroughly studied dystrophic animals, and to test the hypothesis that the plasma membrane of the cardiac muscle cell is the site of a defect associated with the disease. Samples of cardiac muscle tissue from hamsters ranging in age from 1 to 13 months were freeze-fractured. Intramembrane particle numbers were determined for all tissue samples by counting randomly selected areas of P- and E-face surfaces. Up to the age of 1 month, the particle density was the same in both strains of hamster, after which time, the population of IMPs was about 30% lower in dystrophic than in normal heart sarcolemma. This 30% difference in particle frequency in dystrophic hamster heart membrane is consistent with values published for cell membrane from other muscular dystrophies and supports the theory that there is a defect in the plasma membrane of dystrophic cells. In addition, this study has shown for the first time that a presumed membrane defect related to muscular dystrophy (reduced number of IMPs) may be present throughout the life of the animal (1-13 months), and expressed in every cell sampled.     

Distribution of acetylcholinesterase activity in normal,dystrophic, and denervated muscles of the chicken

One distinctive property of denervated and genetically dystrophic muscles of chickens is high acetylcholinesterase activity found in the fibers. The distribution of AChE activity in single fibers from these muscles was studied by using fresh frozen serial sections, a specific histochemical stain, and photodensitometry. The results confirm the findings of a previous report (22) that high AChE activity occurs only around the neuromuscular junction region in fibers from biceps muscles of 6-week-old dystrophic chickens. In contrast, both normal and dystrophic biceps muscle of 6-week old chickens that had been denervated for 5 days exhibited high AChE activity throughout the length of the fibers. These results suggest that changes in AChE activity due to denervation are superimposed on the activity already present in dystrophy. The data support the idea that inherited muscular dystrophy in the chicken involves a specific defect in the regulation of AChE and perhaps other molecules around the neuromuscular junction.     

Delayed degeneration of dystrophic and normal muscle cell cultures treated with pepstatin, leupeptin, and antipain

The effect of the proteinase inhibitors, pepstatin, leupeptin, and antipain, on dystrophic and normal embryonic chicken muscle cells growing in tissue culture was determined. The three inhibitors are effective against lysosomal cathepsins as well as other proteinases. The inhibitors appeared to delay atrophy and degeneration of dystrophic muscle fibers markedly; the effect on the normal muscle fibers was less striking. Catheptic activity and acidic autolysis are known to increase in the dystrophic chicken. These results support the suggestion that lysosomal proteases are involved, by an unknown mechanism, in the degradative process in dystrophic tissue. Delay in the process of degradation of muscle tissue suggests that these low molecular weight, nontoxic inhibitors offer some prospects as therapeutic agents for treatment of muscular dystrophy and other degenerative muscle diseases.     

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.     

An examination of some factors influencing creatine kinase in the blood of patients with muscular dystrophy

The natural variability of plasma creatine kinase activity has been examined in patients suffering from muscular dystrophy and in normal subjects. The coefficient of variation of the plasma creatine kinase activities was found to be large (approximately 35%) in both patients with Duchenne muscular dystrophy and normal control subjects. A comparison of the plasma activities of creatine kinase with other muscle-derived enzymes suggests that the cause of this variability is changes in the release of enzymes from muscle. Data obtained concerning the effect of physical activity on plasma creatine kinase activity are contradictory, but several young patients with Duchenne muscular dystrophy and a very high creatine kinase activity (greater than 5000 IU/liter) showed a decreased activity following admission to hospital. An estimate of the rate of efflux of certain kinase from muscle has been made, indicating that young ambulant patients with Duchenne muscular dystrophy have a grossly elevated muscle creatine kinase efflux (495.0 +/- 61.3 IU/kg muscle/hr) compared to control subjects (1.4 +/- 0.5 IU/kg muscle/hr).     

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.     

Evidence against a lymphocyte-mediated autoimmune etiology for murine muscular dystrophy

The hypothesis that murine muscular dystrophy (MMD) is a lymphocyte-mediated autoimmune disease was tested by orthotopically transplanting normal and dystrophic muscle into normal or dystrophic hosts immunosuppressed with antilymphocyte serum (ALS). Normal serum (NS)-treated animals served as controls. Allograft conditions revealed that both normal and dystrophic muscle were antigenic and were rejected by NS-treated hosts, with the myofiber component of the muscle implant being rejected before the fibroblast portion. New muscle regenerated in a host receiving ALS therapy and was retained by the host until 30 days after the withdrawal of the ALS therapy. Normal muscle isografted into dystrophic hosts regenerated irrespective of whether the host was treated with ALS or NS. In the reciprocal experiment, dystrophic muscle regenerated in normal hosts, but the myofibers were gradually eliminated and replaced by connective tissue, thus behaving as they would have in the donor animal. These observations are incompatible with a lymphocyte-mediated autoimmune etiology for MMD. Furthermore, they raise some question about the claims of muscular dystrophy being attributable to a neural or vascular lesion, and imply that the lesion may be intrinsic to the muscle.     

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.     

Appearance of acetylcholinesterase and creatine kinase in plasma of normal chickens after denervation.

Evidence that acetylcholinesterase (AChE) activity is released from normal chick embryonic muscle fibers and from muscles of chickens with inherited muscular dystrophy suggested that denervated chick muscles, which have AChE properties similar to dystrophic muscles, would also release AChE. Bilateral denervation of the breast and wing muscles of normal chickens was followed by the appearance of AChE activity, distinguished from plasma cholinesterase by differential substrate hydrolysis, inhibitor sensitivity, and electrophoretic migration. Plasma creatine kinase (CK) activity was also elevated after denervation.     

A preparation for studying dystrophic avian muscle and neuromuscular junctions

The extensor of the second digit of the chicken wing (EDII) is a small, fast-twitch muscle susceptible to chicken muscular dystrophy and well-suited for correlated studies of the morphology and physiology of identified nerve and muscle fibers. In cross section, the dystrophic EDII show morphological abnormalities common to other well-studied dystrophic chicken muscles. However, in contrast to other dystrophic chicken muscles, living endplates can be viewed in the EDII, facilitating electrophysiological studies. A survey of electrophysiological properties of the EDII muscle in birds 5–16 weeks ex ovo revealed that compared to normal preparations, the dystrophic preparations had: (1) lower resting potentials, (2) lower miniature endplate potential frequency, (3) abortive nerve-evoked action potentials recorded extrajunctionally, and (4) multiple muscle fiber action potentials to a single depolarizing current pulse.     

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.     

Righting ability and skeletal muscle properties of phenytoin-treated dystrophic chickens

Chickens with an inherited muscular dystrophy (line 413) and genetically related normal controls (line 412) were treated with phenytoin (diphenylhydantoin, DPH) on Days 1 through 40 ex ovo. DPH (20 mg/kg, intraperitoneally, b.i.d.) and a combination of DPH and intermittent “exercise” (twice-daily testing of righting ability) beneficially affected muscle function, morphology, and biochemistry. DPH dramatically improved the righting ability of dystrophic chicks, and the exhaustion scores of many treated birds were still as high as those of normal chicks after 30 days of treatment. Muscles from chicks killed after 27 to 40 days of treatment had decreased fiber diameters, increased lactic dehydrogenase (LDH) activity, and reduced acetylcholinesterase and butyrylcholinesterase activities. The effects of DPH were more pronounced in the posterior latissimus dorsi than in the pectoral muscle and were potentiated by exercise. Exercise alone produced a transient increase in righting ability and increased LDH activity of dystrophic muscles. The abnormal, rounded appearance of dystrophic muscle fibers was not altered by treatments. Research reported elsewhere showed that DPH partially corrected abnormal electromyographic activity of dystrophic chicken muscles. The data presented here show that other symptoms of avian dystrophy are also alleviated by DPH and suggest that abnormal membrane activity plays an important role in this myopathy.     

High molecular weight kininogen, the extracellular inhibitor of thiol proteases, is deficient in hamsters with muscular dystrophy

High molecular weight kininogen has been shown to be the principal plasma inhibitor of cellular thiol proteases including cathepsins B, H and L and calpains 1 and 2. Since these same enzymes have been reported to be elevated in animals with muscular dystrophy, we studied plasmas from hamsters with muscular dystrophy and compared these to normal hamster plasma. The ability of plasma to inhibit purified platelet calpain was assayed and found to be 62% of normal. Since low molecular weight kininogen can also inhibit calpain, the coagulant activity of kininogen, an activity unique for high molecular weight kininogen, was determined in dystrophic hamster plasma and found to be 69% of normal in close agreement with the calpain inhibitory activity. The contribution of the other plasma calpain inhibitor alpha 2-macroglobulin appeared small since inactivation with methylamine did not alter the ability to inhibit calpain in either normal or dystrophic plasma. We conclude that there is a selective deficiency of plasma high molecular weight kininogen in dystrophic hamsters, an abnormality which could play a role in the pathogenesis of this disorder.