Young Mdx Mice Research Articles

Notch signaling deficiency underlies age-dependent depletion of satellite cells in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating disease characterized by muscle wasting, loss of mobility and death in early adulthood. Satellite cells are muscle-resident stem cells responsible for the repair and regeneration of damaged muscles. One pathological feature of DMD is the progressive depletion of satellite cells, leading to the failure of muscle repair. Here, we attempted to explore the molecular mechanisms underlying satellite cell ablation in the dystrophin mutant mdx mouse, a well-established model for DMD. Initial muscle degeneration activates satellite cells, resulting in increased satellite cell number in young mdx mice. This is followed by rapid loss of satellite cells with age due to the reduced self-renewal ability of mdx satellite cells. In addition, satellite cell composition is altered even in young mdx mice, with significant reductions in the abundance of non-committed (Pax7+ and Myf5−) satellite cells. Using a Notch-reporter mouse, we found that the mdx satellite cells have reduced activation of Notch signaling, which has been shown to be necessary to maintain satellite cell quiescence and self-renewal. Concomitantly, the expression of Notch1, Notch3, Jag1, Hey1 and HeyL are reduced in the mdx primary myoblast. Finally, we established a mouse model to constitutively activate Notch signaling in satellite cells, and show that Notch activation is sufficient to rescue the self-renewal deficiencies of mdx satellite cells. These results demonstrate that Notch signaling is essential for maintaining the satellite cell pool and that its deficiency leads to depletion of satellite cells in DMD.

Role of toll-like receptors in the pathogenesis of dystrophin-deficient skeletal and heart muscle

Although the cause of Duchenne muscular dystrophy (DMD) is known, the specific factors that initiate and perpetuate disease progression are not well understood. We hypothesized that leaky dystrophin-deficient skeletal muscle releases endogenous danger signals (TLR ligands), which bind to Toll-like receptors (TLRs) on muscle and immune cells and activate downstream processes that facilitate degeneration and regeneration in dystrophic skeletal muscle. Here, we demonstrate that dystrophin-deficient mouse muscle cells show increased expression of several cell-surface and endosomal TLRs. In vitro screening identified ssRNA as a relevant endogenous TLR7 ligand. TLR7 activation led to myd88-dependent production of pro-inflammatory cytokines in dystrophin-deficient muscle cells, and cause significant degeneration/regeneration in vivo in mdx mouse muscle. Also, knockout of the central TLR adaptor protein, myd88 in mdx mice significantly improved skeletal and cardiac muscle function. Likewise, proof-of-concept experiments showed that treating young mdx mice with a TLR7/9 antagonist significantly reduced skeletal muscle inflammation and increased muscle force, suggesting that blocking this pathway may have therapeutic potential for DMD.

Distinct roles of TRAF6 at early and late stages of muscle pathology in the mdx model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal genetic disorder caused by loss of functional dystrophin protein. Accumulating evidence suggests that the deficiency of dystrophin leads to aberrant activation of many signaling pathways which contribute to disease progression. However, the proximal signaling events leading to the activation of various pathological cascades in dystrophic muscle remain less clear. TNF receptor-associated factor 6 (TRAF6) is an adaptor protein which acts as a signaling intermediate for several receptor-mediated signaling events leading to the context-dependent activation of a number of signaling pathways. TRAF6 is also an E3 ubiquitin ligase and an important regulator of autophagy. However, the role of TRAF6 in pathogenesis of DMD remains unknown. Here, we demonstrate that the levels and activity of TRAF6 are increased in skeletal muscle of mdx (a mouse model of DMD) mice. Targeted deletion of TRAF6 improves muscle strength and reduces fiber necrosis, infiltration of macrophages and the activation of proinflammatory transcription factor nuclear factor-kappa B (NF-κB) in 7-week-old mdx mice. Ablation of TRAF6 also increases satellite cells proliferation and myofiber regeneration in young mdx mice. Intriguingly, ablation of TRAF6 exacerbates muscle injury and increases fibrosis in 9-month-old mdx mice. TRAF6 inhibition reduces the markers of autophagy and Akt signaling in dystrophic muscle of mdx mice. Collectively, our study suggests that while the inhibition of TRAF6 improves muscle structure and function in young mdx mice, its continued inhibition causes more severe myopathy at later stages of disease progression potentially through repressing autophagy.

P.13.11 Proteomic analysis of cardiomyopathic tissue from the aged mdx model of Duchenne muscular dystrophy

Senescent heart tissue from the mdx mouse model of Duchenne muscular dystrophy exhibits distinct pathological aspects of X-linked cardiomyopathy. In order to establish proteome-wide alterations in dystrophin-deficient hearts during aging, cardiomyopathic tissue from young versus aged mdx mice was examined by label-free LC–MS/MS analysis. Significant age-dependent alterations were established for 67 proteins, of which 28 proteins were found to be decreased and 39 proteins were shown to be increased in their expression levels. Drastic changes were demonstrated for proteins involved in ion transportation, the immune response, the cellular stress response, muscle contraction, metabolic regulation and the extracellular matrix. An immunoblotting survey of young and old wild type versus mdx hearts confirmed these proteomic findings and illustrated the effects of natural aging versus dystrophin deficiency. These proteome-wide alterations suggest a disintegration of the basal lamina structure and cytoskeletal network in dystrophin-deficient cardiac fibres, increased levels of antibodies in a potential autoimmune reaction of the degenerating heart, compensatory binding of excess iron and a general perturbation of metabolic pathways in dystrophy-associated cardiomyopathy. The proteomic technologies and analytical tools used in this study to identify significant proteins of interest have the potential to be brought forward and provide new therapeutic and diagnostic biomarkers for dystrophinopathy-related cardiomyopathy.

Age-dependent muscle dystrophy related changes of craniofacial morphology in mdx mice

ObjectiveMasticatory muscle weakness in patients with Duchenne muscular dystrophy may influence cranial bone development and cause changes in cranio- and dentofacial morphology. To examine whether masticatory muscle weakness and thus disturbed muscle function is accompanied by changes of geometry of the craniofacial complex, we compared craniofacial morphology of dystrophic mdx mice with those of controls during growth and adulthood. DesignFor that 30, 100 and 365 days old control and mdx mice were investigated. Standardized radiographs and cone-beam computed tomographies of each mouse head were taken; cephalometric landmarks identified and processed by the computer programs FR-WIN and Osirix. ResultsIn 30 days old mdx mice, which display muscle weakness, all measured dimensions were smaller compared to controls with exception of incisor length. Only four of eight analysed dimensions were found to be smaller through all age groups investigated when compared to controls. ConclusionThe reduction of measurements in young mdx mice suggests that muscle function may influence the geometry of the craniofacial complex during growth. Muscle regeneration and functional recovery that occur in older mice may compensate for bone growth deficiencies during earlier stages of development.

Proteomic profiling of cardiomyopathic tissue from the aged mdx model of Duchenne muscular dystrophy reveals a drastic decrease in laminin, nidogen and annexin

The majority of patients afflicted with Duchenne muscular dystrophy develop cardiomyopathic complications, warranting large-scale proteomic studies of global cardiac changes for the identification of new protein markers of dystrophinopathy. The aged heart from the X-linked dystrophic mdx mouse has been shown to exhibit distinct pathological aspects of cardiomyopathy. In order to establish age-related alterations in the proteome of dystrophin-deficient hearts, cardiomyopathic tissue from young versus aged mdx mice was examined by label-free LC-MS/MS. Significant age-dependent alterations were established for 67 proteins, of which 28 proteins were shown to exhibit a lower abundance and 39 proteins were found to be increased in their expression levels. Drastic changes were demonstrated for 17 proteins, including increases in Ig chains and transferrin, and drastic decreases in laminin, nidogen and annexin. An immunblotting survey of young and old wild-type versus mdx hearts confirmed these proteomic findings and illustrated the effects of natural aging versus dystrophin deficiency. These proteome-wide alterations suggest a disintegration of the basal lamina structure and cytoskeletal network in dystrophin-deficient cardiac fibres, increased levels of antibodies in a potential autoimmune reaction of the degenerating heart, compensatory binding of excess iron and a general perturbation of metabolic pathways in dystrophy-associated cardiomyopathy.

Protective effect of female gender–related factors on muscle force‐generating capacity and fragility in the dystrophic mdx mouse

The dystrophic features in hindlimb skeletal muscles of female mdx mice are unclear. We analyzed force-generating capacity and force decline after lengthening contraction-induced damage (fragility). Young (6-month-old) female mdx mice displayed reduced force-generating capacity (-18%) and higher fragility (23% force decline) compared with female age-matched wild-type mice. These 2 dystrophic features were less accentuated in young female than in young male mdx mice (-32% and 42% force drop). With advancing age, force-generating capacity decreased and fragility increased in old (20 month) female mdx mice (-21% and 57% force decline), but they were unchanged in old male mdx mice. Moreover, estradiol treatment had no effect in old female mdx mice. Female gender-related factors mitigate dystrophic features in young but not old mdx mice. Further studies are warranted to identify the beneficial gender-related factor in dystrophic muscle.

Fibroadipogenic progenitors mediate the ability of HDAC inhibitors to promote regeneration in dystrophic muscles of young, but not old Mdx mice

HDAC inhibitors (HDACi) exert beneficial effects in mdx mice, by promoting endogenous regeneration; however, the cellular determinants of HDACi activity on dystrophic muscles have not been determined. We show that fibroadipogenic progenitors (FAP) influence the regeneration potential of satellite cells during disease progression in mdx mice and mediate HDACi ability to selectively promote regeneration at early stages of disease. FAPs from young mdx mice promote, while FAPs from old mdx mice repress, satellite cell-mediated formation of myotubes. In young mdx mice HDACi inhibited FAP adipogenic potential, while enhancing their ability to promote differentiation of adjacent satellite cells, through upregulation of the soluble factor follistatin. By contrast, FAPs from old mdx mice were resistant to HDACi-mediated inhibition of adipogenesis and constitutively repressed satellite cell-mediated formation of myotubes. We show that transplantation of FAPs from regenerating young muscles restored HDACi ability to increase myofibre size in old mdx mice. These results reveal that FAPs are key cellular determinants of disease progression in mdx mice and mediate a previously unappreciated stage-specific beneficial effect of HDACi in dystrophic muscles.

Exclusive skeletal muscle correction does not modulate dystrophic heart disease in the aged mdx model of Duchenne cardiomyopathy

Duchenne muscular dystrophy (DMD) is characterized by severe degeneration and necrosis of both skeletal and cardiac muscle. While many experimental therapies have shown great promise in treating skeletal muscle disease, an effective therapy for Duchenne cardiomyopathy remains a challenge in large animal models and human patients. The current views on cardiac consequences of skeletal muscle-centered therapy are controversial. Studies performed in young adult mdx mice (a mild DMD mouse model) have yielded opposing results. Since mdx mice do not develop dystrophic cardiomyopathy until ≥21 months of age, we reasoned that old mdx mice may represent a better model to assess the impact of skeletal muscle rescue on dystrophic heart disease. Here, we aged skeletal muscle-specific micro-dystrophin transgenic mdx mice to 23 months and examined the cardiac phenotype. As expected, transgenic mdx mice had minimal skeletal muscle disease and they also outperformed original mdx mice on treadmill running. On cardiac examination, the dystrophin-null heart of transgenic mdx mice displayed severe cardiomyopathy matching that of non-transgenic mdx mice. Specifically, both the strains showed similar heart fibrosis and cardiac function deterioration in systole and diastole. Cardiac output and ejection fraction were also equally compromised. Our results suggest that skeletal muscle rescue neither aggravates nor alleviates cardiomyopathy in aged mdx mice. These findings underscore the importance of treating both skeletal and cardiac muscles in DMD therapy.

Effects of Dantrolene Therapy on Disease Phenotype in Dystrophin Deficient mdx Mice

Dystrophin deficiency causes contraction-induced injury and damage to the muscle fiber, resulting in sustained increase in intracellular calcium levels, activation of calcium-dependent proteases and cell death. It is known that the Ryanodine receptor (RyR1) on the sarcoplasmic reticular (SR) membrane controls calcium release. Dantrolene, an FDA approved skeletal muscle relaxant, inhibits the release of calcium from the SR during excitation-contraction and suppresses uncontrolled calcium release by directly acting on the RyR complex to limit its activation. This study examines whether Dantrolene can reduce the disease phenotype in the mdx mouse model of muscular dystrophy. We treated mdx mice (4 weeks old) with daily intraperitoneal injections of 40mg/kg of Dantrolene for 6 weeks and measured functional (grip strength, in vitro force contractions), behavioral (open field digiscan), imagining (optical imaging for inflammation), histological (H&E), and molecular (protein and RNA) endpoints in a blinded fashion. We found that treatment with Dantrolene resulted in decreased grip strength and open field behavioral activity in mdx mice. There was no significant difference in inflammation either by optical imaging analysis of cathepsin activity or histological (H&E) analysis. In vitro force contraction measures showed no changes in EDL muscle-specific force, lengthening-contraction force deficit, or fatigue resistance. We found Dantrolene treatment significantly reduces serum CK levels. Further, Dantrolene-treated mice showed decreased SERCA1 but not RyR1 expression in skeletal muscle. These results suggest that Dantrolene treatment alone has no significant beneficial effects at the tested doses in young mdx mice.

Injection of Vessel-Derived Stem Cells Prevents Dilated Cardiomyopathy and Promotes Angiogenesis and Endogenous Cardiac Stem Cell Proliferation in mdx/utrn−/− but Not Aged mdx Mouse Models for Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy. DMD patients lack dystrophin protein and develop skeletal muscle pathology and dilated cardiomyopathy (DCM). Approximately 20% succumb to cardiac involvement. We hypothesized that mesoangioblast stem cells (aorta-derived mesoangioblasts [ADMs]) would restore dystrophin and alleviate or prevent DCM in animal models of DMD. ADMs can be induced to express cardiac markers, including Nkx2.5, cardiac tropomyosin, cardiac troponin I, and α-actinin, and adopt cardiomyocyte morphology. Transplantation of ADMs into the heart of mdx/utrn(-/-) mice prior to development of DCM prevented onset of cardiomyopathy, as measured by echocardiography, and resulted in significantly higher CD31 expression, consistent with new vessel formation. Dystrophin-positive cardiomyocytes and increased proliferation of endogenous Nestin(+) cardiac stem cells were detected in ADM-injected heart. Nestin(+) striated cells were also detected in four of five mdx/utrn(-/-) hearts injected with ADMs. In contrast, when ADMs were injected into the heart of aged mdx mice with advanced fibrosis, no functional improvement was detected by echocardiography. Instead, ADMs exacerbated some features of DCM. No dystrophin protein, increase in CD31 expression, or increase in Nestin(+) cell proliferation was detected following ADM injection in aged mdx heart. Dystrophin was observed following transplantation of ADMs into the hearts of young mdx mice, however, suggesting that pathology in aged mdx heart may alter the fate of donor stem cells. In summary, ADMs delay or prevent development of DCM in dystrophin-deficient heart, but timing of stem cell transplantation may be critical for achieving benefit with cell therapy in DMD cardiac muscle.

Hierarchical accumulation of RyR post-translational modifications drives disease progression in dystrophic cardiomyopathy

Duchenne muscular dystrophy (DMD) is a muscle disease with serious cardiac complications. Changes in Ca(2+) homeostasis and oxidative stress were recently associated with cardiac deterioration, but the cellular pathophysiological mechanisms remain elusive. We investigated whether the activity of ryanodine receptor (RyR) Ca(2+) release channels is affected, whether changes in function are cause or consequence and which post-translational modifications drive disease progression. Electrophysiological, imaging, and biochemical techniques were used to study RyRs in cardiomyocytes from mdx mice, an animal model of DMD. Young mdx mice show no changes in cardiac performance, but do so after ∼8 months. Nevertheless, myocytes from mdx pups exhibited exaggerated Ca(2+) responses to mechanical stress and 'hypersensitive' excitation-contraction coupling, hallmarks of increased RyR Ca(2+) sensitivity. Both were normalized by antioxidants, inhibitors of NAD(P)H oxidase and CaMKII, but not by NO synthases and PKA antagonists. Sarcoplasmic reticulum Ca(2+) load and leak were unchanged in young mdx mice. However, by the age of 4-5 months and in senescence, leak was increased and load was reduced, indicating disease progression. By this age, all pharmacological interventions listed above normalized Ca(2+) signals and corrected changes in ECC, Ca(2+) load, and leak. Our findings suggest that increased RyR Ca(2+) sensitivity precedes and presumably drives the progression of dystrophic cardiomyopathy, with oxidative stress initiating its development. RyR oxidation followed by phosphorylation, first by CaMKII and later by PKA, synergistically contributes to cardiac deterioration.

D.P.16 Metabolic and hemodynamic alterations in the mdx skeletal muscle revisited using multi-parametric functional NMR

The mdx mouse remains a popular model of the Duchenne muscular dystrophy. While criticized for the mild impairment of muscle function and structure, the mdx remains highly relevant in terms of energy and microvascular control disturbances associated to muscle dystrophy. In this study, a non-invasive multi-parametric functional NMR approach (mpf) was used to revisit these anomalies. This NMR approach interleaved arterial spin labeled imaging and 31P spectroscopy to measure perfusion, capillary oxygenation and muscle energetics of the gastrocnemius of C57Bl/10wt and mdx mice (between 10 and 65weeks of age). Hind-limb ischemia (30min) or repeated bouts of exercises (2min) and recovery were investigated. After ischemia, phosphocreatine (PCr) depleted more in 28-week-old mdx vs wt mice (ΔPCr: 82±6% vs 62±10%). Resynthesis of PCr at reactive hyperemia appeared prolonged (TCr: 88.7 ±21.5s vs 53±23.3s). In parallel, vasodilatation lasted less at reactive hyperemia in mdx mice compared to wt mice. Similarly, a shorter reperfusion profile was visible in 65-week old mdx mice following exercise, confirming a default in maintaining vasodilatation in old mdx vs old wt mice but also compared to young mdx mice. Moreover capillary oxygenation at reactive hyperemia showed reduced O2 extraction in mdx mice. In this study, the mpf-NMR protocol proved again its capacity to characterize in vivo functional anomalies, this time in dystrophic muscle. The underlying mechanisms require further investigation, with neuronal NO synthase (nNOS) deficiency most likely playing a direct role in the perfusion profile alterations. The defective nNOS might also contribute indirectly, through PFK inhibition, to the faster PCr depletion during ischemia, which could be indicative of impaired glycolysis. On the other hand, the slower PCr resynthesis and lower oxygen extraction help to appreciate and quantify the real impact in vivo of mitochondrial dysfunction in mdx.

Resveratrol decreases inflammation and oxidative stress in the mdx mouse model of duchenne muscular dystrophy

Duchenne Muscular Dystrophy (DMD) is a genetic disease characterized by muscle damage, oxidative stress, chronic inflammation, and fibrosis. Resveratrol (RES) is an antioxidant and anti‐inflammatory. We have shown that RES improves muscle function in the mdx mouse model of DMD, and others have shown resveratrol decreases fibrosis and oxidative stress in older mdx mice. However, its effect on pathology in young mdx mice is unknown. The purpose of this study was to investigate the effect of resveratrol on muscle pathology in young mdx mice. RES (100 mg/kg) or vehicle was administered to 4–5 week old mdx mice everyday for 10 days or every other day for 8 weeks. Muscle fiber integrity, inflammation, and oxidative stress were assessed by H&E staining and 4‐HNE content. Total inflammation was reduced 21 ± 6% (p < 0.05) after 10 days of treatment with no change in oxidative stress. After 8 weeks of RES treatment, centrally located nuclei were reduced 12 ± 4% (p < 0.05), oxidative stress measured through 4‐HNE content decreased 2 ± 0.13 fold (p < 0.05), and total inflammation and fibrosis did not change. We conclude that RES enhances muscle membrane integrity by reducing inflammation during the peak pathological period and long term oxidative stress. Therefore, resveratrol could be a treatment for boys with DMD. This project was funded by The Center for Alternative Medicine at The University of South Carolina School of Medicine.Grant Funding Source : Center of Alternative Medicine University of South Carolina School of Medicine

Therapeutic potential of PEGylated insulin-like growth factor I for skeletal muscle disease evaluated in two murine models of muscular dystrophy

ObjectiveDuchenne muscular dystrophy (DMD) is a fatal monogenetic disease with affected males displaying severe and progressive muscle wasting and weakness eventually leading to premature death. Possible therapeutic benefits of insulin-like growth factor I (IGF-I) have been studied extensively in various models of muscle disease and DMD with IGF-I as a mediator of improved skeletal muscle regeneration by enhancing myoblast proliferation and differentiation. DesignWe tested the efficacy of a novel IGF-I analogue, a polyethylene glycol modified IGF-I (PEG-IGF-I), to ameliorate the pathophysiology of muscular dystrophy in two mouse models of DMD. We used mdx mice which lack dystrophin (as in DMD) but exhibit only a relatively mild phenotype, and the dko mouse which is a transgenic model lacking utrophin in addition to dystrophin, and which exhibits a more severe, lethal phenotype like that in DMD. ResultsIn young mdx mice, twice-weekly PEG-IGF-I s.c. injections for 6weeks protected the diaphragm muscle against fatigue and the tibialis anterior (TA) muscle against contraction-induced injury. However, this beneficial effect of PEG-IGF-I was less pronounced in mdx mice when treatment was initiated later in adulthood. In severely affected dko mice PEG-IGF-I treatment did not affect pathophysiological parameters including animal survival. ConclusionsThese data suggest a therapeutic benefit with PEG-IGF-I treatment only in mild muscle pathologies, since its potential to ameliorate the pathophysiology in models of severe muscular dystrophies was limited. Treatment should be initiated only for mild muscle pathologies if functional benefits are to be realised and therefore may be relevant as a short-term therapy to hasten the functional repair of otherwise healthy muscles after injury.

Effect of uphill running on myocardium T2 in mdx mice

Background Cardiac dysfunction is a major cause of death in Duchenne muscular dystrophy. In mdx mice, the lack of functional dystrophin localized to the cell membrane leads to increased susceptibility to muscle damage and enhanced muscle degeneration. In this study we examined the effect of an uphill running protocol (Michele et al. Circ Res. 105(10):984-93, 2009) on myocardium transverse relaxation time (T2) in young adult mdx mice (16 weeks).

AAV-based shRNA silencing of NF-κB ameliorates muscle pathologies in mdx mice

Chronic inflammation, promoted by an upregulated NF-kappa B (NF-κB) pathway, has a key role in Duchenne muscular dystrophy (DMD) patients' pathogenesis. Blocking the NF-κB pathway has been shown to be a viable approach to diminish chronic inflammation and necrosis in the dystrophin-defective mdx mouse, a murine DMD model. In this study, we used the recombinant adeno-associated virus serotype 9 (AAV9) carrying an short hairpin RNA (shRNA) specifically targeting the messenger RNA of NF-κB/p65 (p65-shRNA), the major subunit of NF-κB associated with chronic inflammation in mdx mice. We examined whether i.m. AAV9-mediated delivery of p65-shRNA could decrease NF-κB activation, allowing for amelioration of muscle pathologies in 1- and 4-month-old mdx mice. At 1 month after treatment, NF-κB/p65 levels were significantly decreased by AAV gene transfer of p65-shRNA in the two ages of treatment groups, with necrosis significantly decreased compared with controls. Quantitative analysis revealed that central nucleation (CN) of the myofibers of p65-shRNA-treated 1-month-old mdx muscles was reduced from 67 to 34%, but the level of CN was not significantly decreased in treated 4-month-old mdx mice. Moreover, delivery of the p65-shRNA enhanced the capacity of myofiber regeneration in old mdx mice treated at 4 months of age when the dystrophic myofibers were most exhausted; however, such p65 silencing diminished the myofiber regeneration in young mdx mice treated at 1 month of age. Taken together, these findings demonstrate that the AAV-mediated delivery of p65-shRNA has the capacity to ameliorate muscle pathologies in mdx mice by selectively reducing NF-κB/p65 activity.

Motor performance of young dystrophic mdx mice treated with long-circulating prednisolone liposomes

For Duchenne muscular dystrophy (DMD), a common myopathy that leads to severe disability, no causal therapy is available. Glucocorticosteroids improve patients' muscle strength, but their long-term use is limited by negative side effects. Thus, pharmacological modifications of glucocorticosteroids are required to increase the efficacy by drug targeting. Liposomal encapsulation augments systemic half-life and local tissue concentrations of glucocorticosteroids and, at the same time, reduces systemic side effects. In this study, the efficacy of novel, long-circulating, polyethylene-glycol-coated liposomes encapsulating prednisolone was compared with free prednisolone in the treatment of mdx mice, a well-established animal model for DMD. Using an objective and sensitive computerized 24-hr detection system of voluntary wheel-running in single cages, we demonstrate a significant impairment of the running performance in mdx compared with black/10 control mice aged 3-6 weeks. Treatment with liposomal or free prednisolone did not improve running performance compared with saline control or empty liposomes. Histopathological parameters, including the rate of internalized nuclei and fiber size variation, and mRNA and protein expression levels of transforming growth factor (TGF)-β and monocytes chemotactic protein (MCP)-1 also remained unchanged. Bioactivity in skeletal muscle of liposomal and free prednisolone was demonstrated by elevated mRNA expression of muscle ring finger protein 1 (MuRF1), a mediator of muscle atrophy, and its forkhead box transcription factors (Foxo1/3). Our data support the assessment of voluntary running to be a robust and reproducible outcome measure of skeletal muscle performance during the early disease course of mdx mice and suggest that liposomal encapsulation is not superior in treatment efficacy compared with conventional prednisolone. Our study helps to improve the future design of experimental treatment in animal models of neuromuscular diseases.

Insights into RyRs Dysfunctions via Studies of Intracellular Calcium Signals

Duchenne muscular dystrophy (DMD) is a striated muscle disease with severe cardiac manifestations. The mdx mouse, an animal model of DMD, develops dilated cardiomyopathy. Several studies associated changes in Ca2+ homeostasis and oxidative stress with DMD. In particular, posttranslational modifications of Ca2+ release channels (RyRs) increase their sensitivity, leading to augmented Ca2+ responses during mechanical challenges, and to cellular and mitochondrial Na+ overload, dysfunction and cell death. We examined whether changes in RyR function were causal for or a consequence of cardiac failure and which posttranslational modifications of RyRs drive the development of the pathology. Fluorescent indicators and imaging techniques make it possible to study the function of RyR channels in the natural cellular environment on a near-molecular level. Young mdx mice show no changes in cardiac performance, but do so after ∼8 months. However, even myocytes from 1 month old mdx mice produced exaggerated Ca2+ sparks and waves after osmotic shock, and exhibited “hypersensitive” excitation-contraction coupling. Both were nearly abolished by antioxidants and NOX inhibitors and reduced by CaMKII but not by NOS- and PKA-inhibitors. SR Ca2+ load, leak or resting [Ca2+]i were unchanged in young mdx cells. However, by the age of 4-5 months and in senescence, load was reduced, leak and resting [Ca2+]i increased, indicating disease progression. By this age, all agents listed above reduced intracellular Ca2+ responses and prevented changes in ECC, Ca2+ load and leak. Thus 1) increased Ca2+ sensitivity of RyRs precedes and presumably contributes to the development of dystrophic cardiomyopathy and 2) oxidative stress drives its development. RyR oxidation, nitrosylation and phosphorylation, first by CaMKII followed by PKA, lead to even further sensitization. This synergistic sensitization of RyRs by several pathways results in cardiac muscle deterioration and heart failure.

Age may Influence Dystrophin Expression for Bone Marrow Cells Transplantation in mdx Mice

Objective: To investigate if age influence dystrophin expression for bone marrow cells (BMCs) transplantation in mdx mice. Methods: BMCs of C57BL/6 mice(new born) ,1.2 × 107 cells ,were injected intravenously into mdx mice of 6 weeks and 12 weeks of age, the mice were preconditioned with 8 Gy γ ray . Result: 12 weeks after the transplantation, 16% skeletal muscles fibers of 6 weeks and 7% those of 12 weeks age mdx mice have dystrophin protein expression at the sarcolemma of muscle fibers. RT-PCR and Western blot proved the results. Conclusion: Young mdx mice transplantated with BMCs show better effect than elder one.