Mitochondrial alterations and oxidative stress in an acute transient mouse model of muscle degeneration: implications for muscular dystrophy and related muscle pathologies.

Muscular dystrophies (MDs) and inflammatory myopathies (IMs) are debilitating skeletal muscle disorders characterized by common pathological events including myodegeneration and inflammation. However, an experimental model representing both muscle pathologies and displaying most of the distinctive markers has not been characterized. We investigated the cardiotoxin (CTX)-mediated transient acute mouse model of muscle degeneration and compared the cardinal features with human MDs and IMs. The CTX model displayed degeneration, apoptosis, inflammation, loss of sarcolemmal complexes, sarcolemmal disruption, and ultrastructural changes characteristic of human MDs and IMs. Cell death caused by CTX involved calcium influx and mitochondrial damage both in murine C2C12 muscle cells and in mice. Mitochondrial proteomic analysis at the initial phase of degeneration in the model detected lowered expression of 80 mitochondrial proteins including subunits of respiratory complexes, ATP machinery, fatty acid metabolism, and Krebs cycle, which further decreased in expression during the peak degenerative phase. The mass spectrometry (MS) data were supported by enzyme assays, Western blot, and histochemistry. The CTX model also displayed markers of oxidative stress and a lowered glutathione reduced/oxidized ratio (GSH/GSSG) similar to MDs, human myopathies, and neurogenic atrophies. MS analysis identified 6 unique oxidized proteins from Duchenne muscular dystrophy samples (n = 6) (versus controls; n = 6), including two mitochondrial proteins. Interestingly, these mitochondrial proteins were down-regulated in the CTX model thereby linking oxidative stress and mitochondrial dysfunction. We conclude that mitochondrial alterations and oxidative damage significantly contribute to CTX-mediated muscle pathology with implications for human muscle diseases.

Grafting of a single donor myofibre promotes hypertrophy in dystrophic mouse muscle.

Skeletal muscle has a remarkable capability of regeneration following injury. Satellite cells, the principal muscle stem cells, are responsible for this process. However, this regenerative capacity is reduced in muscular dystrophies or in old age: in both these situations, there is a net loss of muscle fibres. Promoting skeletal muscle muscle hypertrophy could therefore have potential applications for treating muscular dystrophies or sarcopenia. Here, we observed that muscles of dystrophic mdx nude host mice that had been acutely injured by myotoxin and grafted with a single myofibre derived from a normal donor mouse exhibited increased muscle area. Transplantation experiments revealed that the hypertrophic effect is mediated by the grafted fibre and does not require either an imposed injury to the host muscle, or the contribution of donor cells to the host muscle. These results suggest the presence of a crucial cross-talk between the donor fibre and the host muscle environment.

Inhibition of prostaglandin D synthase suppresses muscular necrosis.

Duchenne muscular dystrophy is a fatal muscle wasting disease that is characterized by a deficiency in the protein dystrophin. Previously, we reported that the expression of hematopoietic prostaglandin D synthase (HPGDS) appeared in necrotic muscle fibers from patients with either Duchenne muscular dystrophy or polymyositis. HPGDS is responsible for the production of the inflammatory mediator, prostaglandin D(2). In this paper, we validated the hypothesis that HPGDS has a role in the etiology of muscular necrosis. We investigated the expression of HPGDS/ prostaglandin D(2) signaling using two different mouse models of muscle necrosis, that is, bupivacaine-induced muscle necrosis and the mdx mouse, which has a genetic muscular dystrophy. We treated each mouse model with the HPGDS-specific inhibitor, HQL-79, and measured both necrotic muscle volume and selected cytokine mRNA levels. We confirmed that HPGDS expression was induced in necrotic muscle fibers in both bupivacaine-injected muscle and mdx mice. After administration of HQL-79, necrotic muscle volume was significantly decreased in both mouse models. Additionally, mRNA levels of both CD11b and transforming growth factor beta1 were significantly lower in HQL-79-treated mdx mice than in vehicle-treated animals. We also demonstrated that HQL-79 suppressed prostaglandin D(2) production and improved muscle strength in the mdx mouse. Our results show that HPGDS augments inflammation, which is followed by muscle injury. Furthermore, the inhibition of HPGDS ameliorates muscle necrosis even in cases of genetic muscular dystrophy.

Degeneration of dystrophic or injured skeletal muscles induces high expression of Galectin-1.

Muscle degenerative diseases such as Duchenne Muscular Dystrophy are incurable and treatment options are still restrained. Understanding the mechanisms and factors responsible for muscle degeneration and regeneration will facilitate the development of novel therapeutics. Several recent studies have demonstrated that Galectin-1 (Gal-1), a carbohydrate-binding protein, induces myoblast differentiation and fusion in vitro, suggesting a potential role for this mammalian lectin in muscle regenerative processes in vivo. However, the expression and localization of Gal-1 in vivo during muscle injury and repair are unclear. We report the expression and localization of Gal-1 during degenerative-regenerative processes in vivo using two models of muscular dystrophy and muscle injury. Gal-1 expression increased significantly during muscle degeneration in the murine mdx and in the canine Golden Retriever Muscular Dystrophy animal models. Compulsory exercise of mdx mouse, which intensifies degeneration, also resulted in sustained Gal-1 levels. Furthermore, muscle injury of wild-type C57BL/6 mice, induced by BaCl(2) treatment, also resulted in a marked increase in Gal-1 levels. Increased Gal-1 levels appeared to localize both inside and outside the muscle fibers with significant extracellular Gal-1 colocalized with infiltrating CD45(+) leukocytes. By contrast, regenerating muscle tissue showed a marked decrease in Gal-1 to baseline levels. These results demonstrate significant regulation of Gal-1 expression in vivo and suggest a potential role for Gal-1 in muscle homeostasis and repair.

Flk-1+ adipose-derived mesenchymal stem cells differentiate into skeletal muscle satellite cells and ameliorate muscular dystrophy in mdx mice.

Duchenne muscular dystrophy (DMD) is a severe hereditary disease characterized by the absence of dystrophin on the sarcolemma of muscle fiber. This absence results in widespread muscle damage and satellite cell activation. After depletion of the satellite cell pool, skeletal muscle is then invariably replaced by connective tissue, leading to progressive muscle weakness. Herein, we isolated Flk-1(+) mesenchymal stem cells (MSCs) from adult adipose tissue and induced them to differentiate into skeletal muscle cells in culture. Within mdx mice, an animal model of DMD, adipose tissue-derived Flk-1(+) MSCs (AD-MSCs) homed to and differentiated into cells that repaired injured muscle tissue. This repair correlated with reconstitution of dystrophin expression on the damaged fibers. Flk-1(+) AD-MSCs also differentiated into muscle satellite cells. This differentiation may have accounted for long-term reconstitution. These cells also differentiated into endothelial cells, thereby possibly improving fiber regeneration as a result of the induced angiogenesis. Therefore, Flk-1(+) AD-MSC transplants may repair muscular dystrophy.

Ca2+-independent phospholipase A2 enhances store-operated Ca2+ entry in dystrophic skeletal muscle fibers.

Duchenne muscular dystrophy is caused by deficiency of dystrophin and leads to progressive weakness. It has been proposed that the muscle degeneration occurring in this disease is caused by increased Ca2+ influx due to enhanced activity of cationic channels that are activated either by stretch of the plasma membrane (stretch-activated channels) or by Ca2+-store depletion (store-operated channels). Using both cytosolic Ca2+ measurements with Fura-2 and the manganese quench method, we show here that store-operated Ca2+ entry is greatly enhanced in dystrophic skeletal flexor digitorum brevis fibers isolated from mdx(5cv) mice, a mouse model of Duchenne muscular dystrophy. Moreover, we show for the first time that store-operated Ca2+ entry in these fibers is under the control of the Ca2+-independent phospholipase A2 and that the exaggerated Ca2+ influx can be completely attenuated by inhibitors of this enzyme. Enhanced store-operated Ca2+ entry in dystrophic fibers is likely to be due to a near twofold overexpression of Ca2+-independent phospholipase A2. The Ca2+-independent phospholipase A2 pathway therefore appears as an attractive target to reduce excessive Ca2+ influx and subsequent degeneration occurring in dystrophic fibers.

Identification of the RAG-1 as a suitable mouse model for mitochondrial DNA disease.

Previous studies have shown that transfer of human myoblasts carrying a mitochondrial DNA mutation into muscles of the severe combined immunodeficient mouse may provide an important animal model for mitochondrial myopathy. However, a major drawback of this mouse is its extreme sensitivity to ionising radiation, a pre-treatment which enhances the efficiency of myoblast transfer success. We implanted human myoblasts into the tibialis anterior muscles of another immunodeficient mouse, mutated in the recombinase activating gene-1 (RAG-1), to determine if this mouse could be an alternative to the severe combined immunodeficient for our mitochondrial myoblast transfer model. We also examined several different methods of muscle degeneration prior to myoblast transfer to determine which method resulted in the greatest amount of human tissue in implanted muscles. Our results show that the RAG-1 mouse displayed no sensitivity to the irradiation process compared to the high sensitivity in the severe combined immunodeficient mouse which resulted in early termination of the study. We also show that degeneration of host muscles by the myotoxin barium chloride (BaCl(2)) resulted in the greatest amount of regenerating human muscle fibres in both the severe combined immunodeficient and RAG-1 mice. In addition, the maximum amount of human fibres observed in transplanted muscles was similar in each mouse strain. The average number of fibres throughout muscles was significantly greater in severe combined immunodeficient mice injured by BaCl(2), but was similar between all other muscle groups. This study suggests that the RAG-1 mouse is a suitable host for the mitochondrial myoblast transfer model and may also prove valuable for other myoblast transfer models such as muscular dystrophy.

[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)

Polyamine biosynthetic decarboxylases in muscles of rats with different experimental myopathies.

The activities of the two polyamine biosynthetic decarboxylases (PBD), L-ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (SAMD), have been measured in quadriceps femoris of rats killed at different times after the induction of calciphylaxis- or serotonin(5-HT)-induced myopathy. Decreases in both PBD levels were observed at early times after both myotoxic treatments. Subsequent progressive increases in both enzyme levels were observed to nearly control values by 4 days after 5-HT administration. In the 5-HT-treated rats, the effects on the myocardial PBD activities were different from those in skeletal muscle, with no effect on ODC but much on SAMD, when rats were killed shortly after 5-HT injection. These results demonstrate that the time-course of the changes in PBD activities in quadriceps femoris mirrors quite well the successive occurrence of degenerative and regenerative processes during the calciphylaxis-induced myopathy and the 5-HT-induced myopathy; it is 5-HT that is mainly responsible for the decreases in PBD levels observed in both experimental myopathies, since dihydrotachysterol alone was without any effect on PBD activity levels and 5-HT alone was effective; myocardial ODC reacts more slowly to 5-HT than quadriceps femoris ODC.

Characterization of a myopathy caused by prostaglandin dysfunction.

Administration of inhibitors of prostaglandin synthetase to chicken embryos produced myopathies in their skeletal muscles which were characterized by ringbinden, loss of Z-discs, M-bands, and thick and thin filaments and decreased myoblast proliferation and type 2 myotube formation. The effect of administration of prostaglandins on myoblast proliferation was also examined and PGE was found to suppress proliferation. There was also a tendency for PGF2 alpha to suppress and PGI2 to stimulate proliferation, although neither of these effects were statistically significant. PGA, PGB and PGD did not affect myoblast proliferation.

The effect of the myotoxic agent iodoacetate on dystrophic mice 129/Re.

Three groups of dystrophic and non-dystrophic mice 129/Re were used for studying the effect of the myotoxic agent iodoacetate on dystrophic muscle. The mice of the first group were given intramuscular injections of iodoacetate. The mice of the second group were injected with normal saline and the third group was maintained as untreated controls. The most severe histopathological changes were found in the dystrophic mice treated with iodoacetate. The non-dystrophic mice of the same group showed a significant increase in the number of internal nuclei. Moderate changes were observed in saline-treated dystrophic controls. There was no significant decrease in the life expectancy in any of the groups. The body weight of dystrophic mice was reduced throughout the experiment. On the contrary the non-dystrophic group showed an increased in weight, regardless of the treatment. The aggravation of the histopathological changes of dystrophic mice by iodoacetate would probably give support to the cyclical necrosis/abnormal regeneration theory of pathogenesis of muscular dystrophy.

Lymphocyte abnormality in human myotonic dystrophy and experimental drug-induced myotonia.

In the cytoplasm of peripheral blood lymphocytes in 3 of 13 patients with myotonic dystrophy, myelin-like structures were observed electronmicroscopically. Some were connected to the cytoplasmic membranes, and some were surrounded by a limiting membrane possessing acid phosphatase activity. These findings suggest an aberration of cytoplasmic membranes in lymphocytes. Similar structures had appeared in lymphocytes of normal rats that became myotonic with 20, 25-diazacholesterol. These findings suggest that a primary genetic defect in human myotonic dystrophy, which participates in the formation of myelin-like structures in lymphocytes, might be also responsible for the occurrence of a myotonic phenomenon.

Morphometric comparison of Duchenne's muscular dystrophy and experimental bupivacaine myopathy.

The findings of this study confirm the role of the hyaline fibre in D.M.D. and support the concept of continuing necrosis with progressive failure of regeneration as the major factor in the aetiology of the pathological changes. The persistence of internal nuclei , "split" fibres and progressive fibrosis are secondary phenomenon. Comparison of the changes in D.M.D. with those seen in experimental Bupivacaine myopathy shows that the changes in the experimental myopathy are comparable to those seen in D.M.D. between the ages of 4 and 6 years. It is suggested that Bupivacaine myopathy is an acceptable animal model for D.M.D.

Sarcolemmal membrane changes related to enzyme release in the imipramine/serotonin experimental animal model.

We report specific findings in the imipramine/serotonin animal model that are consistent with sarcolemmal membrane alterations. Among these findings are cytoplasmic enzyme release, diminished uptake of alpha-aminoisobutyrate (an amino acid analog), decreased oxygen consumption in isolated rat diaphragm, and ribosuria. Furthermore, we describe for the first time the release of the MB isoenzyme of creatine kinase from a source other than cardiac tissue; that is, isolated diaphragms from imipramine/serotonin-treated animals release increased amounts of MB isoenzyme as compared to diaphragms from control animals. We believe the similarities between this animal model and the human disease (Duchenne muscular dystrophy) support a genetically determined generalized membrane abnormality in the pathogenesis of this form of muscular dystrophy.

Imipramine-serotonin induced myopathy.

Imipramine and serotonin (5-HT) were used to produce a myopathy in rats. Imipramine was used to stimulate a defect in transport of 5-HT observed in the platelets of Duchenne's dystrophy patients. The most effective dosage schedule was imipramine, 10 mg per kilogram, for 7 days followed by 5-HT, 100 mg per kilogram, 6 hours after the final imipramine dose. A single series of injections produced focal groups of necrotic and regenerating muscle fibers. In some rats, multiple series of injections resulted in a chronic myopathy with a predilection for proximal muscles, particularly quadriceps. In addition to skeletal muscle lesions, focal areas of myocardial damage were seen. The affected rats had a marked elevation of plasma creatine phosphokinase (including MB isoenzyme), serum glutamic oxaloacetic transaminase, and lactic dehydrogenase. Femoral nerve section did not affect the development of muscle lesions.

Inhibition of actomyosin ATPase by high concentrations of 5-hydroxytryptamine. Possible basis of lesion in 5HT-induced experimental myopathy.

The effect of 5-hydroxytryptamine (5HT) on the ATPase activity and sulphydryl group reactivity of mammalian skeletal muscle actomyosin has been studied. 5HT inhibited the Mg2+-activated but not the Ca2+-activated ATPase activity of actomyosin. It slightly activated myosin ATPase. The sulphydryl groups of actomyosin reacting with 5,5'-dithiobis-(2-nitrobenzoic acid) were blocked by concentrations of 5HT which inhibited the Mg2+-activated ATPase. The significance of the results are discussed in relation to the muscle lesions in the experimental myopathy induced by 5HT and imipramine.

Precipitation of a selenium deficiency by high dietary levels of copper and zinc.

High mortality and a high incidence of exudative diathesis and muscular dystrophy were observed in chicks fed a diet supplemented with either 800 or 1600 ppm copper. Adding 0.5 ppm selenium to a basal diet containing 0.2 ppm prevented mortality and selenium deficiency signs. Dietary zinc levels of 2100 to 4100 ppm also resulted in high mortality, exudative diathesis, and muscular dystrophy. A selenium supplement of 0.5 ppm completely prevented the deficiency signs and markedly reduced mortality. The results demonstrate that both copper and zinc can induce a selenium deficiency in chicks when a diet relatively low in this element is fed.