Twenty-one days of low-intensity eccentric training improve morphological characteristics and function of soleus muscles of mdx mice.

Duchene muscular dystrophy (DMD) is caused by the absence of the protein dystrophin, which leads to muscle weakness, progressive degeneration, and eventually death due to respiratory failure. Low-intensity eccentric training (LIET) has been used as a rehabilitation method in skeletal muscles after disuse. Recently, LIET has also been used for rehabilitating dystrophic muscles, but its effects are still unclear. The purpose of this study was to investigate the effects of 21 days of LIET in dystrophic soleus muscle. Thirty-six male mdx mice were randomized into six groups (n = 6/each): mdx sedentary group; mdx training group-3 days; mdx training group-21 days; wild-type sedentary group; wild-type training group-3 days and wild-type training group-21 days. After the training sessions, animals were euthanized, and fragments of soleus muscles were removed for immunofluorescence and histological analyses, and measurements of active force and Ca2+ sensitivity of the contractile apparatus. Muscles of the mdx training group-21 days showed an improvement in morphological characteristics and an increase of active force when compared to the sedentary mdx group. The results show that LIET can improve the functionality of dystrophic soleus muscle in mice.

Degenerative and regenerative features of myofibers differ among skeletal muscles in a murine model of muscular dystrophy.

Skeletal muscle myofibers constantly undergo degeneration and regeneration. Histopathological features of 6 skeletal muscles (cranial tibial [CT], gastrocnemius, quadriceps femoris, triceps brachii [TB], lumbar longissimus muscles, and costal part of the diaphragm [CPD]) were compared using C57BL/10ScSn-Dmd mdx (mdx) mice, a model for muscular dystrophy versus control, C57BL/10 mice. Body weight and skeletal muscle mass were lower in mdx mice than the control at 4 weeks of age; these results were similar at 6-30 weeks. Additionally, muscular lesions were observed in all examined skeletal muscles in mdx mice after 4 weeks, but none were noted in the controls. Immunohistochemical staining revealed numerous paired box 7-positive satellite cells surrounding the embryonic myosin heavy chain-positive regenerating myofibers, while the number of the former and staining intensity of the latter decreased as myofiber regeneration progressed. Persistent muscular lesions were observed in skeletal muscles of mdx mice between 4 and 14 weeks of age, and normal myofibers decreased with age. Number of muscular lesions was lowest in CPD at all ages examined, while the ratio of normal myofibers was lowest in TB at 6 weeks. In CT, TB, and CPD, Iba1-positive macrophages, the main inflammatory cells in skeletal muscle lesions, showed a significant positive correlation with the appearance of regenerating myofibers. Additionally, B220-positive B-cells showed positive and negative correlation with regenerating and regenerated myofibers, respectively. Our data suggest that degenerative and regenerative features of myofibers differ among skeletal muscles and that inflammatory cells are strongly associated with regenerative features of myofibers in mdx mice.

Phenotypic differences between mdx black mice and mdx albino mice. Comparison of cytokine levels in the blood.

In mdx mice, mutation in the muscle protein dystrophin gene results in the development of chronic degeneration of the muscle tissue. We performed a comparative analysis of blood cytokine levels in mdx mice, classical black mice and mice with additional genetic defect responsible for the manifestations of oculocutaneous albinism. In mdx albino mice, the total pool of cytokines (IL-10, IL-6, IL-5, IL-2, IL-1α, IL-4, IL-17, granulocyte-macrophage growth factor, TNF-α, and IFN-γ) was increased. This increase was not associated with selective release of one of the above cytokines into the blood. The fraction of pro-inflammatory cytokines (IL-6, IL-1α, TNF-α) was increased in the total pool and the percentage of antiinflammatory cytokines (IL-4) was reduced. Changes in cytokine pool probably reflect the differences in the severity of the pathological process in the muscle tissue of both genetic variations of mdx mice.

Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma.

Duchenne muscular dystrophy (DMD) is the most common, lethal genetic disorder of children. A number of animal models of muscular dystrophy exist, but the most effective model for characterizing the structural and functional properties of dystrophin and therapeutic interventions has been the mdx mouse. Despite the approximately 20 years of investigations of the mdx mouse, the impact of the disease on the life span of mdx mice and the cause of death remain unresolved. Consequently, a life span study of the mdx mouse was designed that included cohorts of male and female mdx and wild-type C57BL/10 mice housed under specific pathogen-free conditions with deaths restricted to natural causes and with examination of the carcasses for pathology. Compared with wild-type mice, both mdx male and female mice had reduced life spans and displayed a progressively dystrophic muscle histopathology. Surprisingly, old mdx mice were prone to develop muscle tumors that resembled the human form of alveolar rhabdomyosarcoma, a cancer associated with poor prognosis. Rhabdomyosarcomas have not been observed previously in nontransgenic mice. The results substantiate the mdx mouse as an important model system for studies of the pathogenesis of and potential remedies for DMD.

Electromyographic studies in mdx and wild-type C57 mice.

The electromyographic (EMG) characteristics of human Duchenne muscular dystrophy (DMD) have been well-described. However, to our knowledge, no prior needle electromyographic (EMG) studies of motor unit morphology have been undertaken in muscles from the mdx mouse, an animal that is genetically homologous to DMD. There are significant phenotypic differences between the human and murine dystrophic conditions, bringing into question whether the mdx mouse is an appropriate animal model for DMD. This study was done in order to characterize the EMG findings in mdx mice, compared to normal wild-type mice, and to assess for similarities to DMD. The tibialis anterior and gastrocnemius/soleus muscles from 34 mice (16 C57 wild-type and 18 mdx), divided into four age groups (3, 12, 18, and 24 months), were examined. Wild-type muscles showed normal insertional activity and no abnormal activity at rest. Motor unit action potential (MUAP) parameters were characterized. In contrast to wild-type muscles, mdx muscles showed increased insertional activity, abnormal spontaneous potentials, and the presence of complex repetitive discharges (CRDs). MUAPs showed increased numbers of phases (4.0 +/- 0.6, P < 0.001) and duration (7.1 +/- 1.2 ms, P < 0.02), as well as late components (15%). These EMG data indicate that mdx muscles display EMG characteristics similar to those found in muscles from boys with DMD, lending credence to the mdx mouse as an animal model for this disease. The data obtained in this study indicate a potential role for EMG as an in vivo, objective measurement tool that could be used longitudinally to monitor the effects of therapeutic interventions in mdx mice. This is important as there are few objective measures of muscle function in murine models that do not require killing the animal.

The ontogeny of soleus muscles in mdx and wild type mice.

The satellite cell, the organotypic muscle stem cell, is the key element in ontogenetic and load induced muscle fibre growth and repair. It is therefore possible that the satellite pool becomes exhausted with age, especially in mdx mice where dystrophin deficiency results in skeletal muscle degeneration. We compared structural criteria and satellite cell frequencies in soleus muscles of 26 mdx and 23 wild type mice aged between 26 and 720 days. The total number of muscle fibres was similar in both groups and remained stable throughout life, except for an early increase in wild type mice. However, in mdx muscles there was always a proportion of small-diameter fibres which resulted in a reduction in the effective myogenic area on cross-section, whereas total cross-sectional area and muscle weights were increased relative to controls throughout life. In adult animals, the frequency and numbers of satellite cells remained stable with age and were similar in both animal groups. Satellite cell numbers showed some considerable variation between individual animals, although with a markedly smaller variability between results of the same animal, pointing to the satellite cell pool being an individual variant.

Distal mdx muscle groups exhibiting up-regulation of utrophin and rescue of dystrophin-associated glycoproteins exemplify a protected phenotype in muscular dystrophy.

Unique unaffected skeletal muscle fibres. unlike necrotic torso and limb muscles, may pave the way for a more detailed understanding of the molecular pathogenesis of inherited neuromuscular disorders and help to develop new treatment strategies for muscular dystrophies. The sparing of extraocular muscle in Duchenne muscular dystrophy is mostly attributed to the special protective properties of extremely fast-twitching small-diameter fibres, but here we show that distal muscles also represent a particular phenotype that is more resistant to necrosis. Immunoblot analysis of membranes isolated from the well established dystrophic animal model mdx shows that, in contrast to dystrophic limb muscles, the toe musculature exhibits an up-regulation of the autosomal dystrophin homologue utrophin and a concomitant rescue of dystrophin-associated glycoproteins. Thus distal mdx muscle groups provide a cellular system that naturally avoids myofibre degeneration which might be useful in the search for naturally occurring compensatory mechanisms in inherited skeletal muscle diseases.

Effect of zinc-carnosine chelate compound on muscle function in mdx mouse.

The effect of antioxidant Z-103, catena-(S)-[mu-[N(alpha)-(3-aminopropinyl)histidinnato-(2-)N(1),N(2),O:N(tau)]-zinc], on muscle function in the muscular dystrophy (mdx) mouse was examined by repetitive intraperitoneal administration in subjects aged 4 to 12 weeks. Z-103 administration at a dose of 150 mg/kg increased the load resistant time (LRT), during which the animal with a load holds itself upright on a wire net. The Z-103 administration reduced hypertrophy, the ratio of centronucleated myofibers, and the rate of decay for magnitude of twitch force elicited by 0.5 Hz of electricity to the extensor digitorum longus (EDL) muscle of 12-week-old mdx mice, with little effect on the magnitude of twitch force. The administration of Z-103 (100 mg/kg) had a lesser effect on LRT and the other characteristics examined for EDL muscles. The constituent of Z-103, Zn(2+) applied in the form of ZnSO(4) (5 mg/kg), carnosine (100 mg/kg), and the combination of the two had no beneficial effect on mdx mice. Z-103 (150 mg/kg) administered to normal mice increased LRT with little effect on the contractile properties of EDL muscles. These results suggest that the administration of Z-103 ameliorates muscle function in the mdx mouse.

Running endurance abnormality in mdx mice.

The mdx mouse lacks dystrophin and has histological features of Duchenne muscular dystrophy but little weakness in the first year of life. We report here an early deficit in voluntary wheel running, as assayed with a computerized wheel. All mdx mice showed an intermittent running pattern, in contrast to the continuous running seen in controls. The average continuous running time differed significantly between mdx and control mice at all ages tested (5-21 weeks). This assay is noninvasive, has the advantage of unbiased automatic data collection, and should be useful for quantifying the mdx deficit in therapeutic studies.

Effect of zinc-carnosine complex on muscular function in frail distrophin-deficient (mdx) mice.

The effect of zinc-carnosine complex (Z-103) on muscle function in dystrophin-deficient (mdx) mice was examined using several different courses of repetitive administration. Z-103 at a dose of 100 mg/kg increased the load resistant time (LRT), during which an animal bearing a load holds himself upright on a wire net, in mdx mice when administered at an age of less than about 4 months. The effect of Z-103 on LRT was independent of sex when given by intraperitoneal (I.P.) administration between 4 and 8 weeks of age. Administration of Z-103 from the age of 4 to 9 weeks had no significant effect on wet weight, magnitude or rate of rise of twitch force, or rate of decay of twitch force over time with twitch elicited by 0.5 Hz of electricity in the extensor digitorum longus muscle or calcium content in the gastrocnemius muscle, while it increased the magnitude of twitch force in the soleus muscle. These results suggest that Z-103 reduces fatigability of the whole body in mdx mice, possibly by increasing the contractility of slow fibers.

Expression of truncated utrophin improves pH recovery in exercising muscles of dystrophic mdx mice: a 31P NMR study.

31P NMR spectroscopy was used to study the energy metabolism of dystrophin-deficient skeletal muscle of mdx mice, an animal model of Duchenne muscular dystrophy, in which expression of a truncated form of utrophin has been obtained through transgenesis technology. Measurements of ATP, phosphocreatine (PCr), inorganic phosphates (Pi) and intracellular pH (pHi) were made at rest, during a fatigue protocol and during the subsequent recovery. Mechanical fatigue of transgenic muscles was similar to normal muscle, while mdx muscle showed larger force loss. At rest, muscles of all groups had similar values for [ATP], [PCr], [Pi] and pHi. During fatigue, [PCr] decreases mirrored [Pi] increases and were similar in all groups. The major difference between mdx muscles and the group of normal and trc-utrophin muscles concerned the values and evolution of pHi. The mdx muscles showed a more severe intracellular acidosis during exercise and a slower and incomplete post-exercise recovery of normal pHi. In contrast, in trc-utrophin muscles, the kinetics and amplitude of pHi changes were remarkably close to normal behaviour. We conclude that the impaired proton washout which is present in mdx muscles, is corrected to a great extent by the expression of trc-utrophin.

Muscular dystrophy in mdx mice despite lack of neuronal nitric oxide synthase.

Neuronal nitric oxide synthase (nNOS) is a component of the dystrophin complex in skeletal muscle. The absence of dystrophin protein in Duchenne muscular dystrophy and in mdx mouse causes a redistribution of nNOS from the plasma membrane to the cytosol in muscle cells. Aberrant nNOS activity in the cytosol can induce free radical oxidation, which is toxic to myofibers. To test the hypothesis that derangements in nNOS disposition mediate muscle damage in Duchenne dystrophy, we bred dystrophin-deficient mdx male mice and female mdx heterozygote mice that lack nNOS. We found that genetic deletion of nNOS does not itself cause detectable pathology and that removal of nNOS does not influence the extent of increased sarcolemmal permeability in dystrophin-deficient mice. Thus, histological analyses of nNOS-dystrophin double mutants show pathological changes similar to the dystrophin mutation alone. Taken together, nNOS defects alone do not produce muscular dystrophy in the mdx model.

The effect of age and temperature on mdx muscle fatigue.

We have studied the in vitro contractile and fatigue characteristics of extensor digitorum longus (EDL) muscles from 8- and 62-week-old dystrophin-deficient (mdx) and control mice at 20 degrees C and 35 degrees C. There were no differences in fatigability at 20 degrees C, but at 35 degrees C the dystrophin-deficient muscles demonstrated increased fatigability compared to controls, with the older mice exhibiting the greatest fatigue. These results suggest a temperature-related mechanism of myofibrillar fatigue in dystrophin-deficient EDL muscles.

Evidence of mdx mouse skeletal muscle fragility in vivo by eccentric running exercise.

Duchenne muscular dystrophy is an X-linked devastating disease due to the lack of expression of a functional dystrophin. Unfortunately, the dystrophin-deficient mdx mouse model does not present clinical signs of dystrophy before the age of 18 months, and the role of dystrophin in fiber integrity is not fully understood. The fragility of the skeletal muscle fibers was investigated in transgenic mice expressing beta-galactosidase under the control of a muscle specific promoter. Adult mdx/beta-galactosidase (dystrophin-negative) and normal/beta-galactosidase (dystrophin-positive) mice were submitted to one short session of eccentric, downhill running exercise. The leakage of muscle enzymes creatine kinase and beta-galactosidase was investigated before, 1 h after, and 3 days after the running session. A significant and transient rise in the level of these enzymes was noted in the serum of mdx mice following the exercise session. Thus, the lack of dystrophin in the mdx model led to local microdamages to the exercised muscle allowing leakage of proteins from the fibers. The peak leakage was transient, suggesting that muscle fiber lesions were rapidly repaired following this short, noninvasive eccentric running session.

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.

Distribution of intramembranous particle size in the muscle plasma membrane of the mdx mouse.

We investigated whether the reduced intramembranous particles (IMP) in the muscle plasma membrane in mdx mice reflects a preferential depletion of a particular size of the IMP. The experiments were performed using the freeze-fracture method to analyze the frequency distribution of the size of IMP, the density of orthogonal array and caveolae in the extensor digitorum longus (EDL) and soleus (SOL) muscles obtained from mdx and control mice. We detected a reduced density of IMP and orthogonal array, and the increased density of caveolae in EDL muscle but not in SOL in the mdx mouse compared with those of the same muscles in control animals. The reduction of IMP was, however, not limited to any specific size of IMP. Our results suggest that the dystrophin associated glycoprotein present in the membrane does not reflect a specific size of IMP. Therefore, our findings indicate that the mechanism of reduced IMP in dystrophinopathy may be different from that of diminished dystrophin binding glycoprotein associated with dystrophin deficiency in Duchenne muscular dystrophy.

Contractile properties of myocardium are altered in dystrophin-deficient mdx mice.

The objective of this study was to determine whether cardiac contractile force is altered in the dystrophin-deficient mdx mouse model of muscular dystrophy. Left atria from 12-14-week-old control and mdx mice were paced at 1 Hz in 1.25 mM external Ca2+ buffer. Twitch properties and effects of interposing intervals of 0.3 to 600 s on the force of subsequent beats (force-interval curves) were examined. Peak force and time-to-peak force were similar in both groups, but half-relaxation time was significantly prolonged in mdx heart. In control hearts, force-interval curves increased to an inflection point at about 1 s, then rose to a second peak near 60 s. In mdx heart, curves reached the early inflection more quickly, the second peak was diminished in magnitude and force was greatly depressed at long intervals. Curves were fitted to a four-parameter equation to quantify differences in shape. The parameter a, which reflects rate of rise to the first inflection, was significantly increased in mdx atria, while the parameter B, which reflects amplitude of the late peak, was significantly reduced. These differences in force production were more marked when external Ca2+ was raised to 2.5 mM. Results show contractile properties are markedly altered in atria from dystrophin-deficient mdx mice. These findings are consistent with the hypothesis that dystrophin deficiency affects cardiac contractile function, possibly through effects on SR function.

Spatial learning and hippocampal long-term potentiation are not impaired in mdx mice.

Moderate non-progressive cognitive impairment is a consistent feature of Duchenne muscular dystrophy (DMD), although few central nervous system abnormalities have yet been identified. A model for DMD is provided by the mdx mouse which fails to produce full length dystrophin in muscle and brain. In this study we have compared performances in a hippocampal-dependent spatial learning task, the Morris water maze, in mdx mice and in age-matched normal (C57BL/10) mice. There was no difference in acquisition rates or in retention between the two groups. We also found no difference in the magnitude of long-term potentiation (LTP) between the two groups, either in the dentate gyrus or in area CA. These experiments demonstrate that neither spatial learning nor hippocampal synaptic plasticity are significantly affected by the lack of full-length dystrophin.