Sensorineural hearing loss in the mdx mouse: a model of Duchenne muscular dystrophy

Sensorineural hearing loss has been identified in several types of muscular dystrophy, but few studies have investigated any relationship between Duchenne muscular dystrophy and hearing. An animal model of Duchenne muscular dystrophy, the mdx mouse, exhibits the same genetic defect as humans. We performed brainstem auditory evoked responses on mdx and control mice in order to assess sensorineural hearing loss. The amplitude and latency of wave I for each animal were measured at increasing sound pressure levels. A significant increase in threshold and a decrease in wave I amplitude were found in the mdx mice. These results indicate that significant sensorineural hearing loss is associated with muscular dystrophy in the mdx mouse. Possible cellular mechanisms contributing to the hearing deficit are presented.     

Skeletal muscle fiber degeneration in mdx mice induced by electrical stimulation

We present an in vitro model in which mouse skeletal muscle fibers undergo degeneration by increasing the current strength of tetanic stimulation. To understand the mechanisms of muscle fiber necrosis in Duchenne muscular dystrophy patients, the process of fiber degeneration was compared between mdx and control mice. The process consisted of four steps, beginning with muscle fiber contraction and extending to onset of myofibril disruption. The four processes were not observed in fibers in Krebs-HEPES (-Ca2+) buffer, nor in the presence of L-type Ca2+ channel blockers. These results suggest that this degenerative phenomenon is regulated by intracellular Ca2+, which moved into fibers mainly through voltage-dependent L-type Ca2+ channels. With the exception of myofibril disruption, mdx mice also exhibited the three other steps, but at a significantly lower current strength than in the fibers in the control mice. We postulate that excess Ca2+ flux occurs in fibers, mainly through abnormal L-type Ca2+ channels, and that the excessively accumulated calcium results in premature degeneration of the fibers by tetanic contraction. This study would provide a clue to investigate and prevent the degeneration processes in Duchenne muscular dystrophy.     

Dystrophin acts as a transplantation rejection antigen in dystrophin-deficient mice: implication for gene therapy

Duchenne muscular dystrophy is a lethal and common X-linked recessive disease caused by a defect in dystrophin. Normal myoblast transplantation and dystrophin gene transfer have been expected to correct the deficiency in the muscles, but their clinical application has been hampered by the limited preservation of dystrophin-positive myofibers. In this study we investigated the mechanism for immunologic rejection of normal C57BL/10 (B10) myoblasts transplanted into dystrophin-deficient mdx mice, an animal model of Duchenne muscular dystrophy. We found that mdx mice develop CTL specific for dystrophin itself, which were CD8 dominant and restricted by H-2Kb. We identified several antigenic peptides derived from dystrophin that bind to H-2Kb and are recognized by the mdx anti-B10 CTL. Immunologic tolerance against dystrophin was successfully induced by i.v. injection of these peptides before B10 myoblast transplantation, which resulted in sustained preservation of dystrophin-expressing myofibers in mdx mice. These results demonstrate that dystrophin is antigenic in dystrophin-deficient mice and that immunologic regimen would be necessary to achieve the persistent expression of introduced dystrophin in the muscles of dystrophin-deficient individuals.     

Apoptotic myonuclei in human Duchenne muscular dystrophy

The current view that apoptosis precedes necrosis in death of dystrophin-deficient muscle fibers of the mdx mouse has been well substantiated. Moreover, apoptotic myonuclei have been reported to increase in dystrophin-deficient mice 2 days after spontaneous exercise. To investigate the role of apoptosis in human muscular dystrophy, muscles from 11 patients of different ages with Duchenne muscular dystrophy were analyzed for apoptosis. The amount of apoptosis was assessed by terminal deoxynucleotidyl transferase assay, and the expression of bcl-2 and bax was examined by immunohistochemistry. Although very rare in normal muscles (less than 0.1%), apoptotic nuclei were detected in dystrophic muscles, particularly at the interstitial level. Nevertheless, few dystrophin-deficient myofibers with centrally located nuclei showed a positive reaction for DNA fragmentation. A mosaic pattern of bcl-2/bax-positive myofibers characterized dystrophic muscles, thus the relative proportion of pro- and antiapoptotic proteins differs among muscle fibers in correlation with the presence of apoptotic myonuclei. In the interstitium, apoptotic cells were identified as macrophages and activated satellite cells. This is the first study to show an apoptotic process in adult muscle fibers of patients with Duchenne muscular dystrophy, thereby shedding new light on muscle damage and its progression in dystrophinopathies.     

Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy

The absence of dystrophin at the muscle membrane leads to Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease that is inevitably fatal in early adulthood. In contrast, dystrophin-deficient mdx mice appear physically normal despite their underlying muscle pathology. We describe mice deficient for both dystrophin and the dystrophin-related protein utrophin. These mice show many signs typical of DMD in humans: they show severe progressive muscular dystrophy that results in premature death, they have ultrastructural neuromuscular and myotendinous junction abnormalities, and they aberrantly coexpress myosin heavy chain isoforms within a fiber. The data suggest that utrophin and dystrophin have complementing roles in normal functional or developmental pathways in muscle. Detailed study of these mice should provide novel insights into the pathogenesis of DMD and provide an improved model for rapid evaluation of gene therapy strategies.     

Molecular forms of acetylcholinesterase in dystrophic (mdx) mouse tissues

We analyzed the activity of acetylcholinesterase (AChE) and its molecular forms in the tissues of normal and dystrophic (mdx) mice, at different developmental stages. We studied the brain, the heart and the serum, in addition to four predominantly fast-twitch muscles (tibialis, plantaris, gastrocnemius and extensor digitorum longus (EDL)) and the slow-twitch, soleus muscle. We found no difference between mdx and control mice in the AChE activity of the brain and the heart. The skeletal muscles affected by the disease undergo active degeneration counterbalanced by regeneration between 3 and 14 weeks after birth. The distribution of AChE patches associated with neuromuscular junctions was abnormally scattered in mdx muscles, and in some cases (tibialis and soleus), the number of endplates was more than twice that of normal muscles. There were only minor differences in the concentration and pattern of AChE molecular forms during the acute phase of muscle degeneration and regeneration. After this period, however, we observed a marked deficit in the membrane-bound G4 molecular form of AChE in adult mdx tibialis, gastrocnemius and EDL but not in the plantaris or in the soleus, as compared with their normal counterparts. Whereas the amount of AChE markedly decreased in the serum of normal mice during the first weeks of life, it remained essentially unchanged in the serum of mdx mice. It is likely that this excess of AChE activity in serum originates from the muscles. A deficit in muscle G4 was also reported in other forms of muscular dystrophy in the mouse and chicken. Since it is not correlated to the acute phase of the disease in mdx and also occurs in genetically different dystrophies, it probably represents a secondary effect of the dystrophy.     

Neurotransmitter transporters as molecular targets for addictive drugs

The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca2+ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.     

Cyclophosphamide immunosuppression does not permit successful myoblast allotransplantation in mouse

Cyclophosphamide immunosuppression does not permit successful myoblast allotransplantation in mouse. Myoblast transplantation is a potential treatment for Duchenne muscular dystrophy. In one clinical trial, Duchenne patients were immunosuppressed with cyclophosphamide. We report here that myoblasts from transgenic mice expressing the beta-galactosidase reporter gene transplanted in mdx mice failed to form new muscle fibres when cyclophosphamide (2 or 10 mg kg-1 per day) was used for immunosuppression. At the lowest dose of cyclophosphamide (2 mg kg-1 per day), some mdx recipient mice formed antibodies against donor myoblasts; however, no humoral immune reaction was observed at the highest dose (10 mg kg-1 per day). The failure of transplantation under cyclophosphamide treatment was attributed to the low immunosuppressive activity at a low dose and to the toxic action of a high dose of this drug. These results could explain the lack of success of myoblast transplantation in a previous clinical trial.     

Magnetic resonance imaging of regenerating and dystrophic mouse muscle

Magnetic resonance imaging allows serial visualization of living muscle. Clinically magnetic resonance imaging would be the first step in selecting a region of interest for assessment of muscle disease state and treatment effects by magnetic resonance spectroscopy. In this study, magnetic resonance imaging was used to follow dystrophy and regeneration in the mdx mouse, a genetic homologue to human Duchenne muscular dystrophy. It was hypothesized that images would distinguish normal control from mdx muscle and that regenerating areas (spontaneous and after an imposed injury) would be evident and evolve over time. T2-weighted images of hind-limb muscles were obtained on anaesthetized mice in a horizontal bore 7.1-T experimental magnet. Magnetic resonance images of mdx muscle appeared heterogeneous in comparison to homogeneous images of control muscle. Foci of high intensity in mdx images corresponded to dystrophic lesions observed in the histologic sections of the same muscles. In addition, it was possible to follow chronologically the extent of injury and repair after an imposed crush injury to mdx muscle. These results should make it possible to obtain meaningful magnetic resonance spectra from particular regions of interest in muscle as viewed in magnetic resonance images (i.e., regenerating, degenerating, normal muscle) acquired during neuromuscular diseases and treatment regimens.     

A continuous and pulsatile flow circulation system for evaluation of cardiovascular devices

In a previous report we suggested that muscle fibers in distal myopathy with rimmed vacuoles (DMRV) were degraded by both lysosomal proteolysis (cathepsins) and Ca2+-dependent, nonlysosomal proteolysis (calpain). Given recent evidence of abnormal ubiquitin accumulation in rimmed vacuoles, we examined the role of the ATP-ubiquitin-dependent proteolytic pathway (proteasomes) in myofiber degradation in this myopathy. Immunohistochemically, proteasomes (26S) were located in the cytoplasm in normal human muscle, but the staining intensity was weak. Quantitative analysis showed more reactivity for proteasomes in DMRV muscles and, to a lesser extent, in muscles from muscular dystrophy, polymyositis, and amyotrophic lateral sclerosis patients. In DMRV, proteasomes often were located within or on the rim of rimmed vacuoles, and in the cytoplasm of atrophic fibers. Ubiquitin accumulation was marked within rimmed vacuoles and was seen less extensively in the cytoplasm of atrophic fibers. The latter proteins colocalized well. In other diseased muscles, proteasomes and ubiquitin showed a positive reaction in the atrophic or necrotic fibers. The results indicate increased proteasome and ubiquitin in these muscle fibers as well as in other diseased muscle fibers. We suggest that the ATP-ubiquitin-proteasome proteolytic pathway as well as the nonlysosomal calpain and the lysosomal proteolytic pathway may participate in the muscle fiber degradation in DMRV.     

In situ analysis of a variant surface glycoprotein expression-site promoter region in Trypanosoma brucei

Duchenne muscular dystrophy is frequently associated with a non-progressive cognitive deficit attributed to the absence of 427,000 mol. wt brain dystrophin, or to altered expression of other C-terminal products of this protein, Dp71 and/or Dp140. To further explore the role of these membrane cytoskeleton-associated proteins in brain function, we studied spatial learning and ex vivo synaptic plasticity in the mdx mouse, which lacks 427,000 mol. wt dystrophin, and in the mdx3cv mutant, which shows a dramatically reduced expression of all the dystrophin gene products known so far. We show that reference and working memories are largely unimpaired in the two mutant mice performing a spatial discrimination task in a radial maze. However, mdx3cv mice showed enhanced emotional reactivity and developed different strategies in learning the task, as compared to control mice. We also showed that both mutants display apparently normal levels of long-term potentiation and paired-pulse facilitation in the CA1 field of the hippocampus. On the other hand, an increased post-tetanic potentiation was shown by mdx, but not mdx3cv mice, which might be linked to calcium-regulatory defects. Otherwise, immunoblot analyses suggested an increased expression of a 400,000 mol. wt protein in brain extracts from both mdx and mdx3cv mice, but not in those from control mice. This protein might correspond to the dystrophin-homologue utrophin. The present results suggest that altered expression of dystrophin or C-terminal dystrophin proteins in brain did not markedly affect hippocampus-dependent spatial learning and CA1 hippocampal long-term potentiation in mdx and mdx3cv mice. The role of these membrane cytoskeleton-associated proteins in normal brain function and pathology remains to be elucidated. Furthermore, the possibility that redundant mechanisms could partially compensate for dystrophins' deficiency in the mdx and mdx3cv models should be further considered.     

Dihydropyridine receptor gene expression in skeletal muscle from mdx and control mice

The expression of isoform-specific dihydropyrine receptor-calcium channel (DHPR) alpha 1-subunit genes was investigated in mdx and control mouse diaphragm (DIA) and tibialis anterior (TA). RNase protection assays were carried out with a rat DHPR cDNA probe specific for skeletal muscle and a mouse DHPR cDNA probe specific for cardiac muscle. The level of expression of the gene encoding the cardiac DHPR was very weak in TA muscle from both control and mdx mice. Compared to TA, DIA expressed mRNA for the cardiac isoform at significantly higher levels, but mdx and control mouse DIA levels were similar to one another. In contrast, mRNA expression levels for the DHPR skeletal muscle isoform were lower in control DIA than TA. However, there was a dramatic increase in the expression for the DHPR skeletal muscle isoform in mdx DIA compared with control DIA, reaching the TA expression level, whereas dystrophy did not affect TA expression. [3H]-PN200-110 binding was used to further assess DIA DHPR expression at the protein level. The density of binding sites for the probe was not significantly affected in DIA muscles of mdx vs. control mice, but it was reduced in older mdx and control mice. The increase in DHPR mRNA levels without a consequent increase in DHPR protein expression could be secondary to possible enhanced protein degradation which occurs in mdx DIA. The altered DHPR expression levels found here do not appear to be responsible for the severe deficits in contractile function of the mdx DIA.     

Mechanism of increasing dystrophin-positive myofibers by myoblast transplantation: study using mdx/beta-galactosidase transgenic mice

Female mdx/mdx mice were crossed with non-dystrophic transgenic males expressing the beta-galactosidase (beta-gal) gene under a muscle-specific promoter (TnILacZ1/29). All male offspring were mdx mice and about 50% of them also expressed the beta-gal gene. The beta-gal/mdx mice were selected as recipients for the transplantation of myoblasts from non-transgenic normal BALB/c mice. When host muscles were not irradiated before myoblast transplantation, 4.6% of the muscle fibers in host muscles were dystrophin positive 1 month after transplantation. Most of these dystrophin-positive muscle fibers were also beta-gal positive. About one quarter of these fibers are the result of reverse mutations; most of them have, however, been produced by fusion of donor myoblasts with host muscle fibers or with host myoblasts. The virtual absence of beta-gal-negative fibers indicates that there were no exclusively donor-donor fusions. When host muscles were irradiated before myoblast transplantation, roughly the same percentage (5.5%) of dystrophin-positive fibers were formed in the injected muscle, but 42% of them were beta-gal negative. These beta-gal-negative dystrophin-positive muscle fibers were formed by the exclusive fusion of donor myoblasts with one another rather than with host cells. This clearly indicates that myoblast transplantation can form completely new muscle fibers or muscle fiber segments when host satellite cell proliferation is reduced by irradiation. These newly formed muscle fibers had, however, a small diameter and additional myoblast transplantation may be required to increase their size. This situation has some similarities with findings in Duchenne muscular dystrophy patients of more than 6 years of age, who also have a limited proliferation capacity of their satellite cells.     

Absence of a calmitine-specific protease inhibitor in skeletal muscle mitochondria of patients with Duchenne's muscular dystrophy

We studied the effect of mitochondrial extracts from skeletal muscle of patients with Duchenne's muscular dystrophy (DMD) on calmitine from the skeletal muscle of normal mice and control subjects. Our results clearly show the existence of an abnormal proteolytic activity of mitochondria from patients with DMD on calmitine from the normal mouse. This proteolytic activity was not found on calmitine from the control subject. Overall, our observations suggest that calmitine concentration in the muscle of the control subject remains elevated because of the presence of a calmitine-specific protease and an inhibitor of this protease which regulates and/or suppresses the activity of the enzyme according to the requirements of the muscle cell. Conversely, the calmitine deficiency observed in the muscle of patients with DMD would be due to the absence of this inhibitor. This would account for the continual activity of the enzyme in degrading calmitine as soon as it is synthesized. The identification of this inhibitor is currently being investigated in our laboratory.     

Back pain following epidural anaesthesia in labour

For palliative therapy of Duchenne muscular dystrophy (DMD), corticosteroids have been tried since 1970. According to recent reports, corticosteroids maintained muscular strength to some extent and prolonged period of ambulation. However, their mechanism of action is mostly unclear. In the present study, mdx mice were injected with 0.6 mg prednisolone 3 times a week for 30 weeks. Serum creatine kinase (CK) values remained 23% of controls. In muscle pathology of the quadriceps muscle, fibers with peripheral nuclei were increased, suggesting reduction of muscle necrosis. In pathological examination of liver, pyknotic cytoplasmic masses and formation of vacuoles were observed. Present study showed that prednisolone might attenuate muscle fiber necrosis at least for 30 weeks. Prednisolone may reduce secondary tissue reactions, which develop more serious muscle damage.     

Progressive muscular dystrophy in alpha-sarcoglycan-deficient mice

Limb-girdle muscular dystrophy type 2D (LGMD 2D) is an autosomal recessive disorder caused by mutations in the alpha-sarcoglycan gene. To determine how alpha-sarcoglycan deficiency leads to muscle fiber degeneration, we generated and analyzed alpha-sarcoglycan- deficient mice. Sgca-null mice developed progressive muscular dystrophy and, in contrast to other animal models for muscular dystrophy, showed ongoing muscle necrosis with age, a hallmark of the human disease. Sgca-null mice also revealed loss of sarcolemmal integrity, elevated serum levels of muscle enzymes, increased muscle masses, and changes in the generation of absolute force. Molecular analysis of Sgca-null mice demonstrated that the absence of alpha-sarcoglycan resulted in the complete loss of the sarcoglycan complex, sarcospan, and a disruption of alpha-dystroglycan association with membranes. In contrast, no change in the expression of epsilon-sarcoglycan (alpha-sarcoglycan homologue) was observed. Recombinant alpha-sarcoglycan adenovirus injection into Sgca-deficient muscles restored the sarcoglycan complex and sarcospan to the membrane. We propose that the sarcoglycan-sarcospan complex is requisite for stable association of alpha-dystroglycan with the sarcolemma. The Sgca-deficient mice will be a valuable model for elucidating the pathogenesis of sarcoglycan deficient limb-girdle muscular dystrophies and for the development of therapeutic strategies for this disease.     

The membrane-cytoskeleton interface: the role of dystrophin and utrophin

Recent studies with transgenic animals have considerably advanced our knowledge of the roles of dystrophin and utrophin in both muscle and non-muscle tissues. Rigorous analyses of the roles of the various mdx mutations in mice, as well as the use of artificial transgenes in an mdx background, are beginning to define the functional importance of various regions of the dystrophin protein in normal muscle. Furthermore, recent biochemical analyses have revealed new insights into the role and organization of dystrophin at the membrane-cytoskeleton interface. Transgenic approaches have also revealed surprising and encouraging results with respect to utrophin. Against expectations, the long-awaited utrophin knockout mice have a remarkably mild phenotype with only subtle changes in neuromuscular junction architecture. On the other hand, mdx mice transgenic for a mini-utrophin construct showed rescue of the muscular dystrophy phenotype, clearly an encouraging finding with obvious therapeutic possibilities. These and other recent findings are discussed in the context of the structure and function of dystrophin and utrophin at the membrane-cytoskeleton interface.     

Exercise metabolism in Duchenne muscular dystrophy: a biochemical and [31P]-nuclear magnetic resonance study of mdx mice

Intracellular pH, ratios of phosphocreatine (PCr) to ATP and PCr to inorganic phosphate (Pi) as well as isometric tension were measured during 1 Hz sciatic nerve stimulation and during recovery in the calf muscles of mdx (a model of Duchenne muscular dystrophy) and control mice. Tension did not decline significantly in either strain. The ratio of PCr/(PCr + Pi) was significantly reduced in mdx as against control muscle during exercise and recovery, but the ratio of PCr/ATP and the half-time for PCr recovery were similar in both strains. A reduction in the maximal activities of succinate dehydrogenase and succinate-cytochrome c reductase suggests that mitochondrial metabolism may be impaired. The similarity in PCr recovery times suggests that the muscle has adapted, making any impairment of oxidative metabolism negligible in the intact system. The rate of pH recovery is prolonged in mdx muscle and provides strong evidence for a decline in the capacity of dystrophic muscle to extrude proton equivalents. These data are compared with a previous study which used 10 Hz stimulation and also observed a slow pH recovery. The slow pH recovery could be explained by an elevation in intracellular sodium.     

Does utrophin expression in muscles of mdx mice during postnatal development functionally compensate for dystrophin deficiency?

We correlated utrophin expression with the physiopathological course in mdx mice. Evolution of the pathology was assessed by monitoring expression of developmental MHC in mdx mice versus control. Utrophin expression is detected by dystrophin/utrophin cross-reacting antibodies and can only be evaluated in mdx mouse muscles (in absence of dystrophin). This protein was expressed at the periphery of all myotubes and myofibers during the first postnatal week. It began declining in fast muscles before the third week and disappeared from the soleus between the 3rd and the 4th week. The decrease was concomitant with a sudden degenerative/regenerative process affecting slow muscle earlier and more massively than fast muscles. The pathological process became stable in all muscle types (except the diaphragm), with greater utrophin expression in the soleus. These results in mdx mice along with observed utrophin expression in severely affected DMD patients suggest that overexpression of utrophin is not enough to explain the stability of regenerated fibers in mdx mice.