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.     

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.     

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.     

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.     

Further studies of mitochondrial and lipid storage myopathies

Further observations on a family with facioscapulohumeral (FSH) muscular dystrophy due to mitochondrial myopathy, and on a case with lipid storage myopathy are reported. One member of the family with FSH muscular dystrophy died due to a viral pneumonia, during which she developed gross hyperlacticacidaemia and acidosis. Autopsy examination showed that the mitochondrial morphological abnormality was restricted to the skeletal muscle. Two other members of the family, who also had mitochondrial myopathy, have developed a cerebellar syndrome. The skeletal muscle carnitine level in the propositus of this family was normal. A woman with lipid storage myopathy has been shown to have skeletal muscle carnitine deficiency, the plasma carnitine level being only slightly lower than normal.     

3H]nitrendipine receptors as markers of a class of putative voltage-sensitive Ca2+ channels in normal human skeletal muscle and in muscle from Duchenne muscular dystrophy patients

Properties of nitrendipine receptors have been analyzed in skeletal muscle from normal young boys and boys with Duchenne muscular dystrophy (DMD). The dissociation constant (Kd) of the complex formed by nitrendipine with its specific receptors was 0.5 +/- 0.1 nM in dystrophic muscle and 0.4 +/- 0.1 nM in normal muscle. Maximum binding capacities Bmax were 403 +/- 80 and 460 +/- 60 fmol/mg protein in DMD and normal muscle, respectively. These results suggest that nitrendipine binding sites on nitrendipine-sensitive Ca2+ channel binding sites are not altered in Duchenne muscular dystrophy.     

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.     

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.     

Some factors affecting spontaneous transmitter release in dystrophic mice

Neuromuscular junctions of slow- and fast-twitch skeletal muscles from dystrophic (dy2J/dy2J) and control mice of the C57BL/6J strain were used to investigate the effect of muscular dystrophy on nerve-terminal regulation of their intracellular concentration of free calcium ions. The frequency of spontaneous miniature endplate potentials (MEPPs) was taken as an indicator of the intraterminal free calcium ion concentration. Dicoumarol, 2,4-dinitrophenol, ruthenium red, and the calcium ionophore A-23187 all potentiated the MEPP frequency in dystrophic muscles at concentrations which had negligible effects on normal muscles. Dystrophic muscle preparations were also more sensitive to an increased extracellular calcium concentration. Usually, these manipulations had more effect on the nerve terminals of dystrophic slow muscle than on those of dystrophic fast muscle. We conclude that muscular dystrophy alters the nerve terminal's ability to regulate the concentration of intracellular free calcium ions.     

Expression profiling in stably regenerating skeletal muscle of dystrophin-deficient mdx mice

The mdx mouse is comparable to Duchenne muscular dystrophy in having an absence of dystrophin. While dystrophic human skeletal muscle undergoes progressive degeneration, in the mdx mouse regeneration and tissue remodeling substantially compensate for the lack of dystrophin. To better understand the molecular events leading to active muscle regeneration in mdx muscles, we have determined the gene expression profiles of wild-type and mdx hind limb muscles using oligonucleotide arrays. Compared to wild-type, 58 genes were found to be differentially expressed in mdx. The molecular signature of actively regenerating skeletal muscle in young adult mdx mice showed upregulation of muscle development genes and genes involved in immune response, proteolysis and extracellular matrix remodeling. Moreover, energy metabolism and mitochondrial function were not compromised. Insights into the processes activated in the mdx muscle to compensate for chronic degeneration may have important implications for therapy in patients with muscular dystrophy.     

Myotonic ADR-MDX mutant mice show less severe muscular dystrophy than MDX mice

In Duchenne muscular dystrophy (DMD) and its murine model, the dystrophic mouse (MDX), the skeletal musculature lacks dystrophin. The presumed function of this cytoskeletal protein is to protect the sarcolemma against mechanical stress during muscle activity. To test this hypothesis in vivo, we bred a double mutant mouse that combines two genetic defects: the dystrophin-deficiency of the MDX mouse and the Cl- channel myotonia of the arrested development of righting response (ADR) mouse. We hypothesized that high mechanical muscle activity would aggravate muscular dystrophy in double mutant ADR-MDX mice. On the contrary, ADR-MDX mice showed fewer signs of muscle fiber necrosis and fibrosis than MDX mice at all ages. Plasma creatine kinase levels were slightly increased in ADR-MDX, but significantly lower when compared to MDX mice. Sections of ADR-MDX muscle showed a uniform pattern of oxidative muscle fibers. Similar findings have been obtained in dystrophin-positive ADR mice, they result from a complete fiber-type IIB to IIA transformation in myotonic muscle. Our results suggest that small, oxidative fibers of myotonic mice are less sensitive to dystrophin deficiency. Therefore, ADR-MDX mice develop less severe muscular dystrophy than MDX mice do, although their muscles are continually stressed. The new ADR-MDX double mutant mouse is the first animal model combining both a dystrophinopathy and a channelopathy. The results presented here give new insights into the pathomechanism of muscular dystrophy and may be helpful for the therapeutic management of DMD.     

Myotonic ADR-MDX mutant mice show less severe muscular dystrophy than MDX mice

In Duchenne muscular dystrophy (DMD) and its murine model, the dystrophic mouse (MDX), the skeletal musculature lacks dystrophin. The presumed function of this cytoskeletal protein is to protect the sarcolemma against mechanical stress during muscle activity. To test this hypothesis in vivo, we bred a double mutant mouse that combines two genetic defects: the dystrophin-deficiency of the MDX mouse and the Cl⁻ channel myotonia of the arrested development of righting response (ADR) mouse. We hypothesized that high mechanical muscle activity would aggravate muscular dystrophy in double mutant ADR-MDX mice. On the contrary, ADR-MDX mice showed fewer signs of muscle fiber necrosis and fibrosis than MDX mice at all ages. Plasma creatine kinase levels were slightly increased in ADR-MDX, but significantly lower when compared to MDX mice. Sections of ADR-MDX muscle showed a uniform pattern of oxidative muscle fibers. Similar findings have been obtained in dystrophin-positive ADR mice, they result from a complete fiber-type IIB to IIA transformation in myotonic muscle. Our results suggest that small, oxidative fibers of myotonic mice are less sensitive to dystrophin deficiency. Therefore, ADR-MDX mice develop less severe muscular dystrophy than MDX mice do, although their muscles are continually stressed. The new ADR-MDX double mutant mouse is the first animal model combining both a dystrophinopathy and a channelopathy. The results presented here give new insights into the pathomechanism of muscular dystrophy and may be helpful for the therapeutic management of DMD.     

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.     

Fukutin gene mutations cause dilated cardiomyopathy with minimal muscle weakness

OBJECTIVE: The fukutin gene (FKTN) is the causative gene for Fukuyama-type congenital muscular dystrophy, characterized by rather homogeneous clinical features of severe muscle wasting and hypotonia from early infancy with mental retardation. In contrast with the severe dystrophic involvement of skeletal muscle, cardiac insufficiency is quite rare. Fukuyama-type congenital muscular dystrophy is one of the disorders associated with glycosylation defects of alpha-dystroglycan, an indispensable molecule for intra-extra cell membrane linkage. METHODS: Protein and functional analyses of alpha-dystroglycan and mutation screening of FKTN and other associated genes were performed. RESULTS: Surprisingly, we identified six patients in four families showing dilated cardiomyopathy with no or minimal limb girdle muscle involvement and normal intelligence, associated with a compound heterozygous FKTN mutation. One patient died by rapid progressive dilated cardiomyopathy at 12 years old, and the other patient received cardiac implantation at 18 years old. Skeletal muscles from the patients showed minimal dystrophic features but have altered glycosylation of alpha-dystroglycan and reduced laminin binding ability. One cardiac muscle that underwent biopsy showed altered glycosylation of alpha-dystroglycan similar to that observed in a Fukuyama-type congenital muscular dystrophy patient. INTERPRETATION: FKTN mutations could cause much wider spectrum of clinical features than previously perceived, including familial dilated cardiomyopathy and mildest limb girdle 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.     

Carnitine levels in normal children and adults and in patients with diseased muscle

An assay for evaluating carnitine levels in normal children and adults is described. After a 12-hour period of fasting, individual variations in 24-hour urinary excretion of carnitine were observed in adults. In children, there was a significant decrease in excretion from the 10th month to the third year, and then an increase until the 10th year. There was no significant difference between children and adults in the serum and skeletal muscle levels. Muscle carnitine levels were also studied in 12 cases of lipid-storage myopathy and in cases of other muscle diseases, including Duchenne muscular dystrophy in children.     

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.     

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.     

3H leucine incorporation into myofibrils of normal and dystrophic mouse skeletal muscle.

The study of 3H leucine incorporation into skeletal muscle of mouse muscular dystrophy (129 ReJ/dy Bar Harbour strain) shows the uptake of isotope into myofibrils. The techniques employed were light and EM autoradiography before and after glycerination (Szent-Gyorgyi 1947). The results indicate a marked drop in uptake of the 3H-Leucine into myofibrils in the dystrophic animals, supporting the contention of Nihei et al (1971) that reduced myosin synthesis occurs in mouse muscular dystrophy.