Subcutaneous injection,from birth,of epigallocatechin-3-gallate,a component of green tea,limits the onset of muscular dystrophy in <Emphasis Type="Italic">mdx</Emphasis> mice: a quantitative histological,immunohistochemical and electrophysiological study

Dystrophic muscles suffer from enhanced oxidative stress. We have investigated whether administration of an antioxidant, epigallocatechin-3-gallate (EGCG), a component of green tea, reduces their oxidative stress and pathophysiology in mdx mice, a mild phenotype model of human Duchenne-type muscular dystrophy. EGCG (5 mg/kg body weight in saline) was injected subcutaneously 4× a week into the backs of C57 normal and dystrophin-deficient mdx mice for 8 weeks after birth. Saline was injected into normal and mdx controls. EGCG had almost no observable effects on normal mice or on the body weights of mdx mice. In contrast, it produced the following improvements in the blood chemistry, muscle histology, and electrophysiology of the treated mdx mice. First, the activities of serum creatine kinase were reduced to normal levels. Second, the numbers of fluorescent lipofuscin granules per unit volume of soleus and diaphragm muscles were significantly decreased by about 50% compared to the numbers in the corresponding saline-treated controls. Third, in sections of diaphragm and soleus muscles, the relative area occupied by histologically normal muscle fibres increased significantly 1.5- to 2-fold whereas the relative areas of connective tissue and necrotic muscle fibres were substantially reduced. Fourth, the times for the maximum tetanic force of soleus muscles to fall by a half increased to almost normal values. Fifth, the amount of utrophin in diaphragm muscles increased significantly by 17%, partially compensating for the lack of dystrophin expression.     

Muscle Structure Influences Utrophin Expression in mdx Mice

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the dystrophin gene. To examine the influence of muscle structure on the pathogenesis of DMD we generated mdx^4cv:desmin double knockout (dko) mice. The dko male mice died of apparent cardiorespiratory failure at a median age of 76 days compared to 609 days for the desmin^−/− mice. An ∼2.5 fold increase in utrophin expression in the dko skeletal muscles prevented necrosis in ∼91% of 1a, 2a and 2d/x fiber-types. In contrast, utrophin expression was reduced in the extrasynaptic sarcolemma of the dko fast 2b fibers leading to increased membrane fragility and dystrophic pathology. Despite lacking extrasynaptic utrophin, the dko fast 2b fibers were less dystrophic than the mdx^4cv fast 2b fibers suggesting utrophin-independent mechanisms were also contributing to the reduced dystrophic pathology. We found no overt change in the regenerative capacity of muscle stem cells when comparing the wild-type, desmin^−/−, mdx^4cv and dko gastrocnemius muscles injured with notexin. Utrophin could form costameric striations with α-sarcomeric actin in the dko to maintain the integrity of the membrane, but the lack of restoration of the NODS (nNOS, α-dystrobrevin 1 and 2, α1-syntrophin) complex and desmin coincided with profound changes to the sarcomere alignment in the diaphragm, deposition of collagen between the myofibers, and impaired diaphragm function. We conclude that the dko mice may provide new insights into the structural mechanisms that influence endogenous utrophin expression that are pertinent for developing a therapy for DMD.     

Difference in the expression pattern of dystrophin on the surface membrane between the skeletal and cardiac muscles of mdx carrier mice

Summary We examined the expression of dystrophin by immunohistochemical and immunoblot analyses in the skeletal and cardiac muscles of X^mdx/X⁺ heterozygous mice, which were obtained by mating male mdx mice (X^mdx/Y) with female wild type mice (X⁺/X⁺). Dystrophin was expressed on the surface membrane in both muscles, but the mode of expression was different between the two muscles. In cardiac muscle, dystrophin positive and negative cells were present in roughly equal numbers intermingled in a mosaic pattern; this was considered to reflect the random inactivation of X-chromosomes in early development. In skeletal muscle, most of the surface membrane was dystrophin positive. There were little signs of fiber necrosis or regeneration, and serum creatine kinase levels were normal. We are at present of opinion that the predominance of dystrophin-positive area in skeletal muscle is due to intracellular diffusion of dystrophin. On leave from The Department of Pediatrics, Tokyo Women's Medical College     

Label‐free mass spectrometric analysis of the mdx‐4cv diaphragm identifies the matricellular protein periostin as a potential factor involved in dystrophinopathy‐related fibrosis

Proteomic profiling plays a decisive role in the identification of novel biomarkers of muscular dystrophy and the elucidation of new pathobiochemical mechanisms that underlie progressive muscle wasting. Building on the findings of recent comparative analyses of tissue samples and body fluids from dystrophic animals and patients afflicted with Duchenne muscular dystrophy, we have used here label‐free MS to study the severely dystrophic diaphragm from the not extensively characterized mdx‐4cv mouse. This animal model of progressive muscle wasting exhibits less dystrophin‐positive revertant fibers than the conventional mdx mouse, making it ideal for the future monitoring of experimental therapies. The pathoproteomic signature of the mdx‐4cv diaphragm included a significant increase in the fibrosis marker collagen and related extracellular matrix proteins (asporin, decorin, dermatopontin, prolargin) and cytoskeletal proteins (desmin, filamin, obscurin, plectin, spectrin, tubulin, vimentin, vinculin), as well as decreases in proteins of ion homeostasis (parvalbumin) and the contractile apparatus (myosin‐binding protein). Importantly, one of the most substantially increased proteins was identified as periostin, a matricellular component and apparent marker of fibrosis and tissue damage. Immunoblotting confirmed a considerable increase of periostin in the dystrophin‐deficient diaphragm from both mdx and mdx‐4cv mice, suggesting an involvement of this matricellular protein in dystrophinopathy‐related fibrosis.     

Satellite cells and utrophin are not directly correlated with the degree of skeletal muscle damage in mdx mice

To determine whether muscle satellite cells and utrophin are correlated with the degree of damage in mdx skeletal muscles, we measured the area of the degenerative region as an indicator of myofiber degeneration in the masseter, gastrocnemius, soleus, and diaphragm muscles of mdx mice. Furthermore, we analyzed the expression levels of the paired box homeotic gene 7 (pax7), m-cadherin (the makers of muscle satellite cells), and utrophin mRNA. We also investigated the immunolocalization of m-cadherin and utrophin proteins in the muscles of normal C57BL/10J (B10) and mdx mice. The expression level of pax7 mRNA and the percentage of m-cadherin-positive cells among the total number of cell nuclei in the muscle tissues in all four muscles studied were greater in the mdx mice than in the B10 mice. However, there was no significant correlation between muscle damage and expression level for pax7 mRNA (R = –0.140), nor was there a correlation between muscle damage and the percentage of satellite cells among the total number of cell nuclei (R = –0.411) in the mdx mice. The expression level of utrophin mRNA and the intensity of immunostaining for utrophin in all four muscles studied were greater in the mdx mice than in the B10 mice. However, there also was not a significant correlation between muscle damage and expression level of utrophin mRNA (R = 0.231) in the mdx mice, although upregulated utrophin was incorporated into the sarcolemma. These results suggest that satellite cells and utrophin are not directly correlated with the degree of skeletal muscle damage in mdx mice. dystrophy; pax7; m-cadherin; dystrophin-related proteins     

Increased neointimal thickening in dystrophin-deficient mdx mice

Background

The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), encodes a large cytoskeletal protein present in muscle fibers. While dystrophin in skeletal muscle has been extensively studied, the function of dystrophin in vascular smooth muscle is less clear. Here, we have analyzed the role of dystrophin in injury-induced arterial neointima formation.

Methodology/Principal Findings

We detected a down-regulation of dystrophin, dystroglycan and β-sarcoglycan mRNA expression when vascular smooth muscle cells de-differentiate in vitro. To further mimic development of intimal lesions, we performed a collar-induced injury of the carotid artery in the mdx mouse, a model for DMD. As compared with control mice, mdx mice develop larger lesions with increased numbers of proliferating cells. In vitro experiments demonstrate increased migration of vascular smooth muscle cells from mdx mice whereas the rate of proliferation was similar in cells isolated from wild-type and mdx mice.

Conclusions/Significance

These results show that dystrophin deficiency stimulates neointima formation and suggest that expression of dystrophin in vascular smooth muscle cells may protect the artery wall against injury-induced intimal thickening.     

Characterization of Dystrophin Deficient Rats: A New Model for Duchenne Muscular Dystrophy

A few animal models of Duchenne muscular dystrophy (DMD) are available, large ones such as pigs or dogs being expensive and difficult to handle. Mdx (X-linked muscular dystrophy) mice only partially mimic the human disease, with limited chronic muscular lesions and muscle weakness. Their small size also imposes limitations on analyses. A rat model could represent a useful alternative since rats are small animals but 10 times bigger than mice and could better reflect the lesions and functional abnormalities observed in DMD patients. Two lines of Dmd mutated-rats (Dmd^mdx) were generated using TALENs targeting exon 23. Muscles of animals of both lines showed undetectable levels of dystrophin by western blot and less than 5% of dystrophin positive fibers by immunohistochemistry. At 3 months, limb and diaphragm muscles from Dmd^mdx rats displayed severe necrosis and regeneration. At 7 months, these muscles also showed severe fibrosis and some adipose tissue infiltration. Dmd^mdx rats showed significant reduction in muscle strength and a decrease in spontaneous motor activity. Furthermore, heart morphology was indicative of dilated cardiomyopathy associated histologically with necrotic and fibrotic changes. Echocardiography showed significant concentric remodeling and alteration of diastolic function. In conclusion, Dmd^mdx rats represent a new faithful small animal model of DMD.     

Localisation and quantification of dehydrogenase activities in single muscle fibres of mdx gastrocnemius

The kinetics of succinate (SDH) and lactate (LDH) dehydrogenases were determined in single muscle fibres in unfixed sections of the gastrocnemius of dystrophic mdx mice (with an X-linked genetic disorder lacking a cytoskeletal protein, dystrophin) and age-matched C57BL/10 control mice. Quantitative gel substrate-film techniques and a real-time image analysis system were used. Three main fibre types were observed in regenerated mdx gastrocnemius and in corresponding controls: small fibres (S) with high SDH and LDH initial reaction velocities and activities, large fibres (L) with low activities of these dehydrogenases and intermediate-sized fibres (I) with intermediate enzyme activities. The small and intermediate fibres in both mdx and control muscles exhibited respectively high and moderate subsarcolemmal SDH and LDH activities attributable to accumulated mitochondria. The ratios of the initial velocities of the intrinsic enzyme reactions in the sarcoplasm, excluding the subsarcolemmal regions, of mdx muscle fibres compared to those in control fibres were 0.958 (S), 1.09 (I) and 0.959 (L) for SDH, and 1.03 (S), 1.06 (I) and 1.07 (L) for LDH. A parameter a, a measure of the diffusion of LDH out of muscle sections during incubation on gel substrate films, was found to be 0.981 and 1.00 in mdx and control muscles, respectively. Thus there are no significant differences in the activities and microenvironments of the enzymes between regenerated mdx muscle fibres and normal control muscle fibres. These data suggest that dystrophin deficiency in mdx muscles has no effects on the interactions of LDH with cytoskeletal proteins or on SDH activities in mitochondria whose number and morphology differ in mdx muscle fibres compared to those in normal controls. SDH and LDH activities were also found in the mitochondria clustered on two longitudinally directed poles of each central nucleus in regenerated mdx muscle fibres. They were proportional to the activities in the sarcoplasm excluding the subsarcolemmal regions. Accepted: 12 October 1999     

Genetic correction of splice site mutation in purified and enriched myoblasts isolated from mdx5cv mice

Background  

Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disorder that results in the production of a dysfunctional form of the protein, dystrophin. The mdx^5cv mouse is a model of DMD in which a point mutation in exon 10 of the dystrophin gene creates an artificial splice site. As a result, a 53 base pair deletion of exon 10 occurs with a coincident creation of a frameshift and a premature stop codon. Using primary myoblasts from mdx^5cv mice, single-stranded DNA oligonucleotides were designed to correct this DNA mutation.     

Several dystrophin-glycoprotein complex members are present in crude surface membranes but they are sodium dodecyl sulphate invisible in KCl-washed microsomes from <Emphasis Type="Italic">mdx</Emphasis> mouse muscle

The dystrophin-glycoprotein complex (DGC) is a large trans-sarcolemmal complex that provides a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. In skeletal muscle, it consists of the dystroglycan, sarcoglycan and cytoplasmic complexes, with dystrophin forming the core protein. The DGC has been described as being absent or greatly reduced in dystrophin-deficient muscles, and this lack is considered to be involved in the dystrophic phenotype. Such a decrease in the DGC content was observed in dystrophin-deficient muscle from humans with muscular dystrophy and in mice with X-linked muscular dystrophy (mdx mice). These deficits were observed in total muscle homogenates and in partially membrane-purified muscle fractions, the so-called KCl-washed microsomes. Here, we report that most of the proteins of the DGC are actually present at normal levels in the mdx mouse muscle plasma membrane. The proteins are detected in dystrophic animal muscles when the immunoblot assay is performed with crude surface membrane fractions instead of the usually employed KCl-washed microsomes. We propose that these proteins form SDS-insoluble membrane complexes when dystrophin is absent.     

Non-Invasive MRI and Spectroscopy of mdx Mice Reveal Temporal Changes in Dystrophic Muscle Imaging and in Energy Deficits

In Duchenne muscular dystrophy (DMD), a genetic disruption of dystrophin protein expression results in repeated muscle injury and chronic inflammation. Magnetic resonance imaging shows promise as a surrogate outcome measure in both DMD and rehabilitation medicine that is capable of predicting clinical benefit years in advance of functional outcome measures. The mdx mouse reproduces the dystrophin deficiency that causes DMD and is routinely used for preclinical drug testing. There is a need to develop sensitive, non-invasive outcome measures in the mdx model that can be readily translatable to human clinical trials. Here we report the use of magnetic resonance imaging and spectroscopy techniques for the non-invasive monitoring of muscle damage in mdx mice. Using these techniques, we studied dystrophic mdx muscle in mice from 6 to 12 weeks of age, examining both the peak disease phase and natural recovery phase of the mdx disease course. T2 and fat-suppressed imaging revealed significant levels of tissue with elevated signal intensity in mdx hindlimb muscles at all ages; spectroscopy revealed a significant deficiency of energy metabolites in 6-week-old mdx mice. As the mdx mice progressed from the peak disease stage to the recovery stage of disease, each of these phenotypes was either eliminated or reduced, and the cross-sectional area of the mdx muscle was significantly increased when compared to that of wild-type mice. Histology indicates that hyper-intense MRI foci correspond to areas of dystrophic lesions containing inflammation as well as regenerating, degenerating and hypertrophied myofibers. Statistical sample size calculations provide several robust measures with the ability to detect intervention effects using small numbers of animals. These data establish a framework for further imaging or preclinical studies, and they support the development of MRI as a sensitive, non-invasive outcome measure for muscular dystrophy.     

A Highlights from MBoC Selection: Calpain-mediated proteolysis of tropomodulin isoforms leads to thin filament elongation in dystrophic skeletal muscle

Duchenne muscular dystrophy (DMD) induces sarcolemmal mechanical instability and rupture, hyperactivity of intracellular calpains, and proteolytic breakdown of muscle structural proteins. Here we identify the two sarcomeric tropomodulin (Tmod) isoforms, Tmod1 and Tmod4, as novel proteolytic targets of m-calpain, with Tmod1 exhibiting ∼10-fold greater sensitivity to calpain-mediated cleavage than Tmod4 in situ. In mdx mice, increased m-calpain levels in dystrophic soleus muscle are associated with loss of Tmod1 from the thin filament pointed ends, resulting in ∼11% increase in thin filament lengths. In mdx/mTR mice, a more severe model of DMD, Tmod1 disappears from the thin filament pointed ends in both tibialis anterior (TA) and soleus muscles, whereas Tmod4 additionally disappears from soleus muscle, resulting in thin filament length increases of ∼10 and ∼12% in TA and soleus muscles, respectively. In both mdx and mdx/mTR mice, both TA and soleus muscles exhibit normal localization of α-actinin, the nebulin M1M2M3 domain, Tmod3, and cytoplasmic γ-actin, indicating that m-calpain does not cause wholesale proteolysis of other sarcomeric and actin cytoskeletal proteins in dystrophic skeletal muscle. These results implicate Tmod proteolysis and resultant thin filament length misspecification as novel mechanisms that may contribute to DMD pathology, affecting muscles in a use- and disease severity–dependent manner.     

Defects in Mitochondrial ATP Synthesis in Dystrophin-Deficient Mdx Skeletal Muscles May Be Caused by Complex I Insufficiency

Duchenne Muscular Dystrophy is a chronic, progressive and ultimately fatal skeletal muscle wasting disease characterised by sarcolemmal fragility and intracellular Ca²⁺ dysregulation secondary to the absence of dystrophin. Mounting literature also suggests that the dysfunction of key energy systems within the muscle may contribute to pathological muscle wasting by reducing ATP availability to Ca²⁺ regulation and fibre regeneration. No study to date has biochemically quantified and contrasted mitochondrial ATP production capacity by dystrophic mitochondria isolated from their pathophysiological environment such to determine whether mitochondria are indeed capable of meeting this heightened cellular ATP demand, or examined the effects of an increasing extramitochondrial Ca²⁺ environment. Using isolated mitochondria from the diaphragm and tibialis anterior of 12 week-old dystrophin-deficient mdx and healthy control mice (C57BL10/ScSn) we have demonstrated severely depressed Complex I-mediated mitochondrial ATP production rate in mdx mitochondria that occurs irrespective of the macronutrient-derivative substrate combination fed into the Kreb’s cycle, and, which is partially, but significantly, ameliorated by inhibition of Complex I with rotenone and stimulation of Complex II-mediated ATP-production with succinate. There was no difference in the MAPR response of mdx mitochondria to increasing extramitochondrial Ca²⁺ load in comparison to controls, and 400 nM extramitochondrial Ca²⁺ was generally shown to be inhibitory to MAPR in both groups. Our data suggests that DMD pathology is exacerbated by a Complex I deficiency, which may contribute in part to the severe reductions in ATP production previously observed in dystrophic skeletal muscle.     

Glucocorticoid-treated mice are an inappropriate positive control for long-term preclinical studies in the mdx mouse

Background

Dmd^mdx (mdx) mice are used as a genetic and biochemical model of dystrophin deficiency. The long-term consequences of glucocorticoid (GC) treatment on dystrophin-deficient skeletal and heart muscle are not yet known. Here we used systematic phenotyping to assess the long-term consequences of GC treatment in mdx mice. Our investigation addressed not only the effects of GC on the disease phenotype but also the question of whether GCs can be used as a positive control for preclinical drug evaluations.

Methods and Findings

We performed nine pre-clinical efficacy trials (treated N = 129, untreated N = 106) of different durations in 9-to-50-week-old dystrophic mdx mice over a 3-year time period using standardized methods. In all these trials, we used either 1 mg/kg body weight of prednisone or 5 mg/kg body weight of prednisolone as positive controls to compare the efficacy of various test drugs. Data from untreated controls and GC-treated mice in the various trials have been pooled and analyzed to assess the effects of GCs on dystrophin-deficient skeletal and cardiac muscles of mdx mice. Our results indicate that continuous GC treatment results in early (e.g., at 50 days) improvements in normalized parameters such as grip strength, motor coordination and maximal in vitro force contractions on isolated EDL muscle, but these initial benefits are followed by a progressive loss of muscle strength after 100 days. We also found a significant increase in heart fibrosis that is reflected in a significant deterioration in cardiac systolic function after 100 days of treatment.

Conclusion

Continuous administration of prednisone to mdx mice initially improves skeletal muscle strength, but further therapy result in deterioration of muscle strength and cardiac function associated with enhanced cardiac fibrosis. These results suggest that GCs may not serve as an appropriate positive control for long-term mdx mouse preclinical trials.     

ACE2 Is Augmented in Dystrophic Skeletal Muscle and Plays a Role in Decreasing Associated Fibrosis

Duchenne muscular dystrophy (DMD) is the most common inherited neuromuscular disease and is characterized by absence of the cytoskeletal protein dystrophin, muscle wasting, and fibrosis. We previously demonstrated that systemic infusion or oral administration of angiotensin-(1-7) (Ang-(1-7)), a peptide with opposing effects to angiotensin II, normalized skeletal muscle architecture, decreased local fibrosis, and improved muscle function in mdx mice, a dystrophic model for DMD. In this study, we investigated the presence, activity, and localization of ACE2, the enzyme responsible for Ang-(1-7) production, in wild type (wt) and mdx skeletal muscle and in a model of induced chronic damage in wt mice. All dystrophic muscles studied showed higher ACE2 activity than wt muscle. Immunolocalization studies indicated that ACE2 was localized mainly at the sarcolemma and, to a lesser extent, associated with interstitial cells. Similar results were observed in the model of chronic damage in the tibialis anterior (TA) muscle. Furthermore, we evaluated the effect of ACE2 overexpression in mdx TA muscle using an adenovirus containing human ACE2 sequence and showed that expression of ACE2 reduced the fibrosis associated with TA dystrophic muscles. Moreover, we observed fewer inflammatory cells infiltrating the mdx muscle. Finally, mdx gastrocnemius muscles from mice infused with Ang-(1-7), which decreases fibrosis, contain less ACE2 associated with the muscle. This is the first evidence supporting ACE2 as an important therapeutic target to improve the dystrophic skeletal muscle phenotype.     

Green tea extract and its major polyphenol (-)-epigallocatechin gallate improve muscle function in a mouse model for Duchenne muscular dystrophy

Duchenne muscular dystrophy is a frequent muscular disorder caused by mutations in the gene encoding dystrophin, a cytoskeletal protein that contributes to the stabilization of muscle fiber membrane during muscle activity. Affected individuals show progressive muscle wasting that generally causes death by age 30. In this study, the dystrophic mdx^5Cv mouse model was used to investigate the effects of green tea extract, its major component (–)-epigallocatechin gallate, and pentoxifylline on dystrophic muscle quality and function. Three-week-old mdx^5Cv mice were fed for either 1 or 5 wk a control chow or a chow containing the test substances. Histological examination showed a delay in necrosis of the extensor digitorum longus muscle in treated mice. Mechanical properties of triceps suræ muscles were recorded while the mice were under deep anesthesia. Phasic and tetanic tensions of treated mice were increased, reaching values close to those of normal mice. The phasic-to-tetanic tension ratio was corrected. Finally, muscles from treated mice exhibited 30–50% more residual force in a fatigue assay. These results demonstrate that diet supplementation of dystrophic mdx^5Cv mice with green tea extract or (–)-epigallocatechin gallate protected muscle against the first massive wave of necrosis and stimulated muscle adaptation toward a stronger and more resistant phenotype. pharmacotherapy; muscular disorders; dystrophic mdx^5cv mouse; muscle mechanical properties; muscle histology     

Nifedipine Treatment Reduces Resting Calcium Concentration,Oxidative and Apoptotic Gene Expression,and Improves Muscle Function in Dystrophic mdx Mice

Duchenne Muscular Dystrophy (DMD) is a recessive X-linked genetic disease, caused by mutations in the gene encoding dystrophin. DMD is characterized in humans and in mdx mice by a severe and progressive destruction of muscle fibers, inflammation, oxidative/nitrosative stress, and cell death. In mdx muscle fibers, we have shown that basal ATP release is increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, depolarization-induced ATP release is blocked by nifedipine, leading us to study the potential therapeutic effect of nifedipine in mdx muscles and its relation with extracellular ATP signaling. Acute exposure to nifedipine (10 µM) decreased [Ca²⁺]_r, NF-κB activity and iNOS expression in mdx myotubes. In addition, 6-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1 mg/Kg for 1 week. This treatment lowered the [Ca²⁺]_r measured in vivo in the mdx vastus lateralis. We demonstrated that extracellular ATP levels were higher in adult mdx flexor digitorum brevis (FDB) fibers and can be significantly reduced after 1 week of treatment with nifedipine. Interestingly, acute treatment of mdx FDB fibers with apyrase, an enzyme that completely degrades extracellular ATP to AMP, reduced [Ca²⁺]_r to a similar extent as was seen in FDB fibers after 1-week of nifedipine treatment. Moreover, we demonstrated that nifedipine treatment reduced mRNA levels of pro-oxidative/nitrosative (iNOS and gp91^phox/p47^phox NOX2 subunits) and pro-apoptotic (Bax) genes in mdx diaphragm muscles and lowered serum creatine kinase (CK) levels. In addition, nifedipine treatment increased muscle strength assessed by the inverted grip-hanging test and exercise tolerance measured with forced swimming test in mdx mice. We hypothesize that nifedipine reduces basal ATP release, thereby decreasing purinergic receptor activation, which in turn reduces [Ca²⁺]_r in mdx skeletal muscle cells. The results in this work open new perspectives towards possible targets for pharmacological approaches to treat DMD.     

Evaluation of Skeletal and Cardiac Muscle Function after Chronic Administration of Thymosin β-4 in the Dystrophin Deficient Mouse

Thymosin beta-4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. We studied the effects of chronic administration of Tβ4 on the skeletal and cardiac muscle of dystrophin deficient mdx mice, the mouse model of Duchenne muscular dystrophy. Female wild type (C57BL10/ScSnJ) and mdx mice, 8–10 weeks old, were treated with 150 µg of Tβ4 twice a week for 6 months. To promote muscle pathology, mice were exercised for 30 minutes twice a week. Skeletal and cardiac muscle function were assessed via grip strength and high frequency echocardiography. Localization of Tβ4 and amount of fibrosis were quantified using immunohistochemistry and Gomori''s tri-chrome staining, respectively. Mdx mice treated with Tβ4 showed a significant increase in skeletal muscle regenerating fibers compared to untreated mdx mice. Tβ4 stained exclusively in the regenerating fibers of mdx mice. Although untreated mdx mice had significantly decreased skeletal muscle strength compared to untreated wild type, there were no significant improvements in mdx mice after treatment. Systolic cardiac function, measured as percent shortening fraction, was decreased in untreated mdx mice compared to untreated wild type and there was no significant difference after treatment in mdx mice. Skeletal and cardiac muscle fibrosis were also significantly increased in untreated mdx mice compared to wild type, but there was no significant improvement in treated mdx mice. In exercised dystrophin deficient mice, chronic administration of Tβ4 increased the number of regenerating fibers in skeletal muscle and could have a potential role in treatment of skeletal muscle disease in Duchenne muscular dystrophy.     

Testing of SHIRPA, a mouse phenotypic assessment protocol, on Dmd mdx and Dmd mdx3cv dystrophin-deficient mice

The SHIRPA protocol was proposed as a rapid, comprehensive screening method for qualitatively abnormal phenotypes in the mouse (Rogers et al., Mamm Genome 8, 711, 1997). This screening technique is currently being used to identify mutants induced by N-ethylnitrosourea (ENU) mutagenesis (Brown and Nolan, Hum Mol Genet 7, 1627, 1998). SHIRPA can be used to identify mutants with neuromuscular abnormalities, but the sensitivity of the protocol is unknown. We tested two dystrophin-deficient mutants Dmd ^mdx and Dmd ^mdx3cv , both of which are indistinguishable from wild-type by a simple visual assessment, at different ages, using the primary screen of the SHIRPA protocol. The most dramatic observation was that both Dmd ^mdx and Dmd ^mdx3cv mice showed extreme fatigue after testing, while mice from the same C57BL strains appeared unaffected. Each strain of dystrophin-deficient mice showed a different profile in locomotor activity and deficiencies in the wire maneuver, righting reflex, and negative geotaxis tests. Furthermore, the wire maneuver test indicated an earlier onset of muscular impairment in Dmd ^mdx than Dmd ^mdx3cv mice. These data suggest that the SHIRPA primary screen is effective not only in identifying subtle neuromuscular mutants, but also in distinguishing qualitative differences between mutants with neuromuscular abnormalities. Received: 5 August 1999 / Accepted: 14 April 2000