Dystrophic Mdx Mice Research Articles

Genetic disruption of the inflammasome adaptor ASC has minimal impact on the pathogenesis of Duchenne muscular dystrophy in mdx mice

AimUp-regulation of inflammasome proteins was reported in dystrophin-deficient muscles. However, it remains to be determined whether inflammasome activation plays a role in the pathogenesis of Duchenne muscular dystrophy. This study was therefore set out to investigate whether genetic disruption of the inflammasome pathway impacts the disease progression in mdx mice. Main methodsMice deficient in both dystrophin and ASC (encoded by Pycard [PYD And CARD Domain Containing]) were generated. The impact of ASC deficiency on muscular dystrophy of mdx mice were assessed by measurements of serum cytokines, Western blot, real-time PCR and histopathological staining. Key findingsThe pro-inflammatory cytokines such as TNF-α, IL-6, KC/GRO and IL-10 were markedly increased in the sera of 8-week-old mdx mice compared to WT. Western blotting showed that P2X7, caspase-1, ASC and IL-18 were upregulated. Disruption of ASC and dystrophin expression in the mdx/ASC−/− mice was verified by Western blot analysis. Histopathological analysis did not find significant alterations in the muscular dystrophy phenotype in mdx/ASC−/− mice as compared to mdx mice. SignificanceTaken together, our results show that disruption of the central adaptor ASC of the inflammasome is insufficient to alleviate muscular dystrophy phenotype in mdx mice.

Desmin prevents muscle wasting, exaggerated weakness and fragility, and fatigue in dystrophic mdx mouse.

Desmin, similar to dystrophin, is associated with costameric structures bridging sarcomeres to the extracellular matrix. Deletion of the desmin gene in mdx mice [double knockout (DKO) mice] induces marked muscle weakness and fatigue resistance compared to mdx mice. Muscle fragility (higher susceptibility to contraction-induced injury) was also aggravated in DKO mice compared to mdx mice. By contrast to mdx mice, the DKO mice did not undergo muscle hypertrophy. Desmin cDNA transfer with adeno-associated virus in newborn mdx mice reduced muscle weakness. Overall, desmin plays important and beneficial roles in muscle wasting, performance and fragility in dystrophic muscle. Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease caused by dystrophin deficiency. Desmin, similar to dystrophin, is associated with costameric structures bridging sarcomeres to the extracellular matrix that contributes to muscle function. In the present study, we attempted to provide further insight into the roles of desmin, for which the expression is increased in the muscle from the mouse mdx DMD model. We show that a deletion of the desmin gene (Des) in mdx mice [double knockout (DKO) mice, mdx:desmin-/-] induces a marked muscle weakness; namely, a reduced absolute maximal force production and increased fatigue compared to that in mdx mice. Fragility (i.e. higher susceptibility to contraction-induced injury) was also aggravated in DKO mice compared to mdx mice, despite the promotion of supposedly less fragile muscle fibres in DKO mice, and this worsening of fragility was related to a decreased muscle excitability. Moreover, in contrast to mdx mice, the DKO mice did not undergo muscle hypertrophy, as indicated by smaller and fewer fibres, with a reduced percentage of centronucleated fibres, potentially explaining the severe muscle weakness. Notably, Desmin cDNA transfer with adeno-associated virus in newborn mdx mice improved specific maximal force normalized to muscle weight. Overall, desmin plays important and beneficial roles in muscle wasting, performance and fragility in dystrophic mdx mice, which differ, at least in part, from those observed in healthy muscle.

Nox4 – RyR1 – Nox2: Regulators of micro-domain signaling in skeletal muscle

The ability for skeletal muscle to perform optimally can be affected by the regulation of Ca2+ within the triadic junctional space at rest. Reactive oxygen species impact muscle performance due to changes in oxidative stress, damage and redox regulation of signaling cascades. The interplay between ROS and Ca2+ signaling at the triad of skeletal muscle is therefore important to understand as it can impact the performance of healthy and diseased muscle. Here, we aimed to examine how changes in Ca2+ and redox signaling within the junctional space micro-domain of the mouse skeletal muscle fibre alters the homeostasis of these complexes. The dystrophic mdx mouse model displays increased RyR1 Ca2+ leak and increased NAD(P)H Oxidase 2 ROS. These alterations make the mdx mouse an ideal model for understanding how ROS and Ca2+ handling impact each other. We hypothesised that elevated t-tubular Nox2 ROS increases RyR1 Ca2+ leak contributing to an increase in cytoplasmic Ca2+, which could then initiate protein degradation and impaired cellular functions such as autophagy and ER stress. We found that inhibiting Nox2 ROS did not decrease RyR1 Ca2+ leak observed in dystrophin-deficient skeletal muscle. Intriguingly, another NAD(P)H isoform, Nox4, is upregulated in mice unable to produce Nox2 ROS and when inhibited reduced RyR1 Ca2+ leak. Our findings support a model in which Nox4 ROS induces RyR1 Ca2+ leak and the increased junctional space [Ca2+] exacerbates Nox2 ROS; with the cumulative effect of disruption of downstream cellular processes that would ultimately contribute to reduced muscle or cellular performance.

Matrix Metalloproteinase 13 from Satellite Cells is Required for Efficient Muscle Growth and Regeneration.

Cell migration and extracellular matrix remodeling underlie normal mammalian development and growth as well as pathologic tumor invasion. Skeletal muscle is no exception, where satellite cell migration replenishes nuclear content in damaged tissue and extracellular matrix reforms during regeneration. A key set of enzymes that regulate these processes are matrix metalloproteinases (MMP)s. The collagenase MMP-13 is transiently upregulated during muscle regeneration, but its contribution to damage resolution is unknown. The purpose of this work was to examine the importance of MMP-13 in muscle regeneration and growth in vivo and to delineate a satellite cell specific role for this collagenase. Mice with total and satellite cell specific Mmp13 deletion were utilized to determine the importance of MMP-13 for postnatal growth, regeneration after acute injury, and in chronic injury from a genetic cross with dystrophic (mdx) mice. We also evaluated insulin-like growth factor 1 (IGF-1) mediated hypertrophy in the presence and absence of MMP-13. We employed live-cell imaging and 3D migration measurements on primary myoblasts obtained from these animals. Outcome measures included muscle morphology and function. Under basal conditions, Mmp13-/- mice did not exhibit histological or functional deficits in muscle. However, following acute injury, regeneration was impaired at 11 and 14 days post injury. Muscle hypertrophy caused by increased IGF-1 was blunted with minimal satellite cell incorporation in the absence of MMP-13. Mmp13-/- primary myoblasts displayed reduced migratory capacity in 2D and 3D, while maintaining normal proliferation and differentiation. Satellite cell specific deletion of MMP-13 recapitulated the effects of global MMP-13 ablation on muscle regeneration, growth and myoblast movement. These results show that satellite cells provide an essential autocrine source of MMP-13, which not only regulates their migration, but also supports postnatal growth and resolution of acute damage.

Nonmyeloablative bone marrow cells transplantation restores dystrophin synthesis in the muscles of MDX mice

Duchenne muscular dystrophy is an X-linked recessive muscular dystrophy associated with a mutations in the dystrophin protein gene. The most common laboratory model of Duchenne muscular dystrophy is mdx mice. The striated muscle fibers of mdx mice are characterized by the absence of dystrophin, the presence of centrally located nuclei, and the high level of renewal of the striated muscle fibers. In addition, mdx mice show a morphological aberrations at neuromuscular junctions, expressed in the breakdown of large clusters of acetylcholine receptors in the form of branches into small clusters in the form of islets. One approach to treating muscular dystrophy in mdx mice may be the nonmyeloablative transplantation of wild-type bone marrow cells after X-ray irradiation of mdx mice at a dose of 3 Gy. The aim of this work is to evaluate the effect of nonmyeloabla-tive transplantation of wild-type bone marrow cells on dystrophin synthesis and the structure of neuromuscular junctions of mdx mice. Mdx mice were irradiated with X-rays at a dose of 3 Gy, after 24 hours was performed intravenous transplantation of bone marrow cells of C57BL/6 mice. The m. quariceps femoris and diaphragm were examined 2, 4, 6, 9, 12 months after transplantation. Muscle studies were performed using immunohisto-chemical methods of study (immunohistochemical staining with antibodies to dystrophin). The neuromuscular junctions were stained with tetramethylrodamine-a-bungarotoxin. After intravenous bone marrow cells transplantation, the part of dystrophin-positive muscle fibers in the muscle quadriceps femoris was shown to increase to a 27,6±6,7% 6 months after transplantation. After 12 months, the part of dystrophin-positive muscle fibers decreased to 5,1±1,1%. There was also an increase in the proportion of striated muscle fibers without centrally located nuclei and a decrease in the part of dead striated muscle fibers. Similar changes were found in the striated muscle fibers of the diaphragm of mdx mice. In addition, transplantation of bone marrow cells after irradiation at a dose of 3 Gy increases the part of neuromuscular junctions with normal structure. Thus, nonmy-eloablative transplantation of wild-type bone marrow cells can be considered as one way to treat monogenic disease of striated muscle fibers muscular dystrophy of mdx mice.

Treatment of Dystrophic mdx Mice with an ADAMTS-5 Specific Monoclonal Antibody Increases the Ex Vivo Strength of Isolated Fast Twitch Hindlimb Muscles

Aberrant extracellular matrix synthesis and remodeling contributes to muscle degeneration and weakness in Duchenne muscular dystrophy (DMD). ADAMTS-5, a secreted metalloproteinase with catalytic activity against versican, is implicated in myogenesis and inflammation. Here, using the mdx mouse model of DMD, we report increased ADAMTS-5 expression in dystrophic hindlimb muscles, localized to regions of regeneration and inflammation. To investigate the pathophysiological significance of this, 4-week-old mdx mice were treated with an ADAMTS-5 monoclonal antibody (mAb) or IgG2c (IgG) isotype control for 3 weeks. ADAMTS-5 mAb treatment did not reduce versican processing, as protein levels of the cleaved versikine fragment did not differ between hindlimb muscles from ADAMTS-5 mAb or IgG treated mdx mice. Nonetheless, ADAMTS-5 blockade improved ex vivo strength of isolated fast extensor digitorum longus, but not slow soleus, muscles. The underpinning mechanism may include modulation of regenerative myogenesis, as ADAMTS-5 blockade reduced the number of recently repaired desmin positive myofibers without affecting the number of desmin positive muscle progenitor cells. Treatment with the ADAMTS-5 mAb did not significantly affect markers of muscle damage, inflammation, nor fiber size. Altogether, the positive effects of ADAMTS-5 blockade in dystrophic muscles are fiber-type-specific and independent of versican processing.

Contribution of TRPC Channels to Intracellular Ca2 + Dyshomeostasis in Smooth Muscle From mdx Mice.

Duchenne muscular dystrophy (DMD) is an irreversible muscle disease characterized by a progressive loss of muscle function, decreased ambulation, and ultimately death as a result of cardiac or respiratory failure. DMD is caused by the lack of dystrophin, a protein that is important for membrane stability and signaling in excitable cells. Although vascular smooth muscle cells (VSMCs) dysfunction occurs in many pathological conditions, little is known about vascular smooth muscle function in DMD. We have previously shown that striated muscle cells, as well as neurons isolated from dystrophic (mdx) mice have higher intracellular Ca2+ ([Ca2+]i) and Na+ ([Na+]i) concentrations and decreased cell viability in comparison with wild type (Wt). Experiments were carried out in isolated VSMCs from mdx (a murine model of DMD) and congenic C57BL/10SnJ Wt mice. We found elevated [Ca2+]i and [Na+]i in VSMCs from mdx mice compared to Wt. Exposure to 1-oleoyl-2-acetyl-sn-glycerol (OAG), a TRPC3 and TRPC6 channel activator, induced a greater elevation of [Ca2+]i and [Na+]i in mdx than Wt VSMCs. The OAG induced increases in [Ca2+]i could be abolished by either removal of extracellular Ca2+ or by SAR7334, a blocker of TRPC3 and TRPC 6 channels in both genotypes. Mdx and Wt VSMCs were susceptible to muscle cell stretch-induced elevations of [Ca2+]i and [Na+]i which was completely inhibited by GsMTx-4, a mechanosensitive ion channel inhibitor. Western blots showed a significant upregulation of TRPC1 -3, -6 proteins in mdx VSMCs compare to age-matched Wt. The lack of dystrophin in mdx VSMCs produced a profound alteration of [Ca2+]i and [Na+]i homeostasis that appears to be mediated by TRPC channels. Moreover, we have been able to demonstrate pharmacologically that the enhanced stretch-induced elevation of intracellular [Ca2+] and concomitant cell damage in mdx VSMCs also appears to be mediated through TRPC1, -3 and -6 channel activation.

Triazine-cored polymeric vectors for antisense oligonucleotide delivery in vitro and in vivo

BackgroundThe polymer-based drug/gene delivery is promising for the treatment of inherent or acquire disease, because of the polymer’s structural flexibility, larger capacity for therapeutic agent, low host immunogenicity and less cost. Antisense therapy is an approach to fighting genetic disorders or infections using antisense oligonucleotides (AOs). Unfortunately, the naked AOs showed low therapeutic efficacy in vivo and in clinical trial due to their poor cellular uptake and fast clearance in bloodstream. In this study, a series of triazine-cored amphiphilic polymers (TAPs) were investigated for their potential to enhance delivery of AOs, 2′-O-methyl phosphorothioate RNA (2′-OMePS) and phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo.ResultsTAPs significantly enhanced AO-induced exon-skipping in a GFP reporter-based myoblast and myotube culture system, and observed cytotoxicity of the TAPs were lower than Endoporter, Lipofectamine-2000 or PEI 25K. Application of optimized formulations of TAPs with AO targeted to dystrophin exon 23 demonstrated a significant increase in exon-skipping efficiency in dystrophic mdx mice. The best ones for PMO and 2′-OMePS delivery have reached to 11-, 15-fold compared with the AO only in mdx mice, respectively.ConclusionThe study of triazine-cored amphiphilic polymers for AO delivery in vitro and in mdx mice indicated that the carrier’s performances are related to the molecular size, compositions and hydrophilic-lipophilic balance (HLB) of the polymers, as well as the AO’s structure. Improved exon-skipping efficiency of AOs observed in vitro and in mdx mice accompanied with low cytotoxicity demonstrated TAP polymers are potentials as safe and effective delivery carrier for gene/drug delivery.

Isolated Extensor Digitorum Longus Muscles from Old mdx Dystrophic Mice Show Little Force Recovery 120 Minutes after Eccentric Damage

Duchenne muscular dystrophy is characterized by progressive wasting and cycles of regeneration in skeletal muscle. Our laboratory work suggests, branched fibres, could be responsible for the terminal phase of muscle damage in old (58-112 weeks) dystrophic mice. A recent study in 12 week old dystrophic mice reported that the majority of force loss produced by a series of eccentric contractions (EC) in extensor digitorum longus (EDL) muscles recovers (65%) within 120 minutes, concluding this is incompatible with the assumption that EC force loss is due to mechanical damage (Olthoff et al. 2018). The aim of this project was to assess the recovery post EC damage in adult (∼16 weeks) and old (∼88 weeks) dystrophic muscles. Male mdx mice and littermate controls were euthanized, EDL muscles dissected from the hind limbs and maintained in Krebs solution at room temperature. The muscles were maximally stimulated, and a series of 6x EC performed to assess force loss. Muscles were left to rest for up to 120 minutes whilst measuring recovery. Single muscle fibres were isolated enzymatically to assess the degree of fibre branching. Our findings replicated force recovery in young mdx mice with simple fibre branches. However, minimal recovery (∼23%) in muscles of 88 week old mdx mice at 120 minutes post EC was observed. This data supports our “tipping point” hypothesis and shows a distinct pathophysiology in aged mdx mice with EC force loss due to acute fibre rupture at branch nodes that occurs in “old” dystrophic EDL muscles with >70% complex branched fibres. These findings have important implications for pre-clinical drug studies that use protection from EC damage in young mdx mice as a marker for drug efficacy.

Spatiotemporal Mapping Reveals Regional Gastrointestinal Dysfunction in mdx Dystrophic Mice Ameliorated by Oral L-arginine Supplementation.

Background/AimsPatients with Duchenne muscular dystrophy exhibit significant, ongoing impairments in gastrointestinal (GI) function likely resulting from dysregulated nitric oxide production. Compounds increasing neuronal nitric oxide synthase expression and/or activity could improve GI dysfunction and enhance quality of life for dystrophic patients. We used video imaging and spatiotemporal mapping to identify GI dysfunction in mdx dystrophic mice and determine whether dietary intervention to enhance nitric oxide could alleviate aberrant colonic activity in muscular dystrophy.MethodsFour-week-old male C57BL/10 and mdx mice received a specialized diet either with no supplementation (control) or supplemented (1 g/kg/day) with L-alanine, L-arginine, or L-citrulline for 8 weeks. At the conclusion of treatment, mice were sacrificed by cervical dislocation and colon motility examined by spatiotemporal (ST) mapping ex vivo.ResultsST mapping identified increased contraction number in the mid and distal colon of mdx mice on control and L-alanine supplemented diets relative to C57BL/10 mice (P < 0.05). Administration of either L-arginine or L-citrulline attenuated contraction number in distal colons of mdx mice relative to C57BL/10 mice.ConclusionsGI dysfunction in Duchenne muscular dystrophy has been sadly neglected as an issue affecting quality of life. ST mapping identified regional GI dysfunction in the mdx dystrophic mouse. Dietary interventions to increase nitric oxide signaling in the GI tract reduced the number of colonic contractions and alleviated colonic constriction at rest. These findings in mdx mice reveal that L-arginine can improve colonic motility and has potential therapeutic relevance for alleviating GI discomfort, improving clinical care, and enhancing quality of life in Duchenne muscular dystrophy.

Histological features of masticatory muscles after botulinum toxin A injection into the right masseter muscle of dystrophin deficient (mdx-) mice

Objective/backgroundThe most frequently used animal model for human DMD (Duchenne muscular dystrophy) research is the mdx mouse. In both species, characteristic histological changes like inflammation, muscle fiber degeneration and fibrosis are the same, but in contrast to humans, in mdx mice, phases of muscle fiber degeneration are compensated by regeneration processes. AimTherefore, the interest of this study was to evaluate histological features in masticatory muscles after BTX-A injection into the right masseter muscle of wild type and dystrophic (mdx) mice, illustrating de- and regeneration processes induced by this substance. Material and methodsThe right masseter muscle of 100 days old healthy and mdx mice were selectively paralyzed by a single intramuscular BTX-A injection. Masseter as well as temporal muscle of injection and non-injection side were carefully dissected 21 days and 42 days after injection, respectively, and fiber diameter, cell nuclei position, necrosis and collagen content were analyzed histomorphologically in order to evaluate de- and regeneration processes in these muscles. Statistical analysis was performed using SigmaStat Software and Mann Whitney U-test (significance level: p < 0.05). ResultsAt both investigation periods and in both mouse strains fiber diameter was significantly reduced and collagen content was significantly increased in the right injected masseter muscle whereas fiber diameters in mdx mice were much smaller, and these differences were even more apparent at the second investigation period. Necrosis and central located nuclei could generally be found in all mdx mice muscles investigated with an amount of centronucleation exceeding 60%, and a significant increase of necrosis six weeks after injection. In wild type mice central located nuclei could primarily be found in the treated masseter muscle with a portion of 2.7%, and this portion decreased after six weeks, whereas in mdx mice a decrease could also be seen in the non-injected muscles. In contrast, in wild type mice necrosis was not apparent at any time and in all muscles investigated. ConclusionFrom our results it can be concluded that in mdx mice masticatory muscles de- and regeneration processes were extended, triggered by a selective BTX-A injection, or mdx mice at this age, independently of BTX-A treatment, went through another cycle of de- and regeneration as a characteristic of this disease.

Blockade of IGF2R improves muscle regeneration and ameliorates Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a debilitating fatal X‐linked muscle disorder. Recent findings indicate that IGFs play a central role in skeletal muscle regeneration and development. Among IGFs, insulinlike growth factor 2 (IGF2) is a key regulator of cell growth, survival, migration and differentiation. The type 2 IGF receptor (IGF2R) modulates circulating and tissue levels of IGF2 by targeting it to lysosomes for degradation. We found that IGF2R and the store‐operated Ca2+ channel CD20 share a common hydrophobic binding motif that stabilizes their association. Silencing CD20 decreased myoblast differentiation, whereas blockade of IGF2R increased proliferation and differentiation in myoblasts via the calmodulin/calcineurin/NFAT pathway. Remarkably, anti‐IGF2R induced CD20 phosphorylation, leading to the activation of sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA) and removal of intracellular Ca2+. Interestingly, we found that IGF2R expression was increased in dystrophic skeletal muscle of human DMD patients and mdx mice. Blockade of IGF2R by neutralizing antibodies stimulated muscle regeneration, induced force recovery and normalized capillary architecture in dystrophic mdx mice representing an encouraging starting point for the development of new biological therapies for DMD.

Cultured hippocampal neurons of dystrophic mdx mice respond differently from those of wild type mice to an acute treatment with corticosterone

Duchenne muscular dystrophy is a lethal genetic disease characterised by progressive degeneration of skeletal muscles induced by deficiency of dystrophin, a cytoskeletal protein expressed in myocytes and in certain neuron populations. The severity of the neurological disorder varies in humans and animal models owing to dysfunction in numerous brain areas, including the hippocampus. Cyclic treatments with high-dose glucocorticoids remain a major pharmacological approach for treating the disease; however, elevated systemic levels of either stress-induced or exogenously administered anti-inflammatory molecules dramatically affect hippocampal activity. In this study, we analysed and compared the response of hippocampal neurons isolated from wild-type and dystrophic mdx mice to acute administration of corticosterone in vitro, without the influence of other glucocorticoid-regulated processes. Our results showed that in neurons of mdx mice, both the genomic and intracellular signalling-mediated responses to corticosterone were affected compared to those in wild-type animals, evoking the characteristic response to detrimental chronic glucocorticoid exposure. Responsiveness to glucocorticoids is, therefore, another function of hippocampal neurons possibly affected by deficiency of Dp427 since embryonic development. Knowing the pivotal role of hippocampus in stress hormone signalling, attention should be paid to the effects that prolonged glucocorticoid treatments may have on this and other brain areas of DMD patients.

The panniculus carnosus muscle: A novel model of striated muscle regeneration that exhibits sex differences in the mdx mouse

The dermal striated muscle panniculus carnosus (PC), prevalent in lower mammals with remnants in humans, is highly regenerative, and whose function is purported to be linked to defence and shivering thermogenesis. Given the heterogeneity of responses of different muscles to disease, we set out to characterize the PC in wild-type and muscular dystrophic mdx mice. The mouse PC contained mainly fast-twitch type IIB myofibers showing body wide distribution. The PC exemplified heterogeneity in myofiber sizes and a prevalence of central nucleated fibres (CNFs), hallmarks of regeneration, in wild-type and mdx muscles, which increased with age. PC myofibers were hypertrophic in mdx compared to wild-type mice. Sexual dimorphism was apparent with a two-fold increase in CNFs in PC from male versus female mdx mice. To evaluate myogenic potential, PC muscle progenitors were isolated from 8-week old wild-type and mdx mice, grown and differentiated for 7-days. Myogenic profiling of PC-derived myocytes suggested that male mdx satellite cells (SCs) were more myogenic than female counterparts, independent of SC density in PC muscles. Muscle regenerative differences in the PC were associated with alterations in expression of calcium handling regulatory proteins. These studies highlight unique aspects of the PC muscle and its potential as a model to study mechanisms of striated muscle regeneration in health and disease.

Coenzyme Q10 supplementation acts as antioxidant on dystrophic muscle cells.

Increased oxidative stress is a frequent feature in Duchenne muscular dystrophy (DMD). High reactive oxygen species (ROS) levels, associated with altered enzyme antioxidant activity, have been reported in dystrophic patients and mdx mice, an experimental model of DMD. In this study, we investigated the effects of coenzyme Q10 (CoQ10) on oxidative stress marker levels and calcium concentration in primary cultures of dystrophic muscle cells from mdx mice. Primary cultures of skeletal muscle cells from C57BL/10 and mdx mice were treated with coenzyme Q10 (5 μM) for 24 h. The untreated mdx and C57BL/10 muscle cells were used as controls. The MTT and live/dead cell assays showed that CoQ10 presented no cytotoxic effect on normal and dystrophic muscle cells. Intracellular calcium concentration, H2O2 production, 4-HNE, and SOD-2 levels were higher in mdx muscle cells. No significant difference in the catalase, GPx, and Gr levels was found between experimental groups. This study demonstrated that CoQ10 treatment was able to reduce levels of oxidative stress markers, such as H2O2, acting as an antioxidant, as well as decreasing abnormal intracellular calcium influx in dystrophic muscles cells. This study demonstrated that CoQ10 treatment was able to reduce levels of oxidative stress markers, such as H2O2, acting as an antioxidant, as well as decreasing abnormal intracellular calcium influx in dystrophic muscles cells. Our findings also suggest that the decrease of oxidative stress reduces the need for upregulation of antioxidant pathways, such as SOD and GSH.

Glycine administration attenuates progression of dystrophic pathology in prednisolone-treated dystrophin/utrophin null mice

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by progressive muscle wasting and weakness and premature death. Glucocorticoids (e.g. prednisolone) remain the only drugs with a favorable impact on DMD patients, but not without side effects. We have demonstrated that glycine preserves muscle in various wasting models. Since glycine effectively suppresses the activity of pro-inflammatory macrophages, we investigated the potential of glycine treatment to ameliorate the dystrophic pathology. Dystrophic mdx and dystrophin-utrophin null (dko) mice were treated with glycine or L-alanine (amino acid control) for up to 15 weeks and voluntary running distance (a quality of life marker and strong correlate of lifespan in dko mice) and muscle morphology were assessed. Glycine increased voluntary running distance in mdx mice by 90% (P < 0.05) after 2 weeks and by 60% (P < 0.01) in dko mice co-treated with prednisolone over an 8 week treatment period. Glycine treatment attenuated fibrotic deposition in the diaphragm by 28% (P < 0.05) after 10 weeks in mdx mice and by 22% (P < 0.02) after 14 weeks in dko mice. Glycine treatment augmented the prednisolone-induced reduction in fibrosis in diaphragm muscles of dko mice (23%, P < 0.05) after 8 weeks. Our findings provide strong evidence that glycine supplementation may be a safe, simple and effective adjuvant for improving the efficacy of prednisolone treatment and improving the quality of life for DMD patients.

Muscle specific kinase protects dystrophic mdx mouse muscles from eccentric contraction-induced loss of force-producing capacity.

Adeno-associated viral vector was used to elevate the expression of muscle specific kinase (MuSK) and rapsyn (a cytoplasmic MuSK effector protein) in the tibialis anterior muscle of wild-type and dystrophic (mdx) mice. In mdx mice, enhanced expression of either MuSK or rapsyn ameliorated the acute loss of muscle force associated with strain injury. Increases in sarcolemmal immunolabelling for utrophin and β-dystroglycan suggest a mechanism for the protective effect of MuSK in mdx muscles. MuSK also caused subtle changes to the structure and function of the neuromuscular junction, suggesting novel roles for MuSK in muscle physiology and pathophysiology. Muscle specific kinase (MuSK) has a well-defined role in stabilizing the developing mammalian neuromuscular junction, but MuSK might also be protective in some neuromuscular diseases. In the dystrophin-deficient mdx mouse model of Duchenne muscular dystrophy, limb muscles are especially fragile. We injected the tibialis anterior muscle of 8-week-old mdx and wild-type (C57BL10) mice with adeno-associated viral vectors encoding either MuSK or rapsyn (a cytoplasmic MuSK effector protein) fused to green fluorescent protein (MuSK-GFP and rapsyn-GFP, respectively). Contralateral muscles injected with empty vector served as controls. One month later mice were anaesthetized with isoflurane and isometric force-producing capacity was recorded from the distal tendon. MuSK-GFP caused an unexpected decay in nerve-evoked tetanic force, both in wild-type and mdx muscles, without affecting contraction elicited by direct electrical stimulation of the muscle. Muscle fragility was probed by challenging muscles with a strain injury protocol consisting of a series of four strain-producing eccentric contractions in vivo. When applied to muscles of mdx mice, eccentric contraction produced an acute 27% reduction in directly evoked muscle force output, affirming the susceptibility of mdx muscles to strain injury. mdx muscles overexpressing MuSK-GFP or rapsyn-GFP exhibited significantly milder force deficits after the eccentric contraction challenge (15% and 14%, respectively). The protective effect of MuSK-GFP in muscles of mdx mice was associated with increased immunolabelling for utrophin and β-dystroglycan in the sarcolemma. Elevating the expression of MuSK or rapsyn revealed several distinct synaptic and extrasynaptic effects, suggesting novel roles for MuSK signalling in muscle physiology and pathophysiology.

MiR-146a deficiency does not aggravate muscular dystrophy in mdx mice

Duchenne muscular dystrophy (DMD) is a genetic disease evoked by a mutation in the dystrophin gene. It is associated with progressive muscle degeneration and increased inflammation. Up to this date, mainly anti-inflammatory treatment is available for patients suffering from DMD. miR-146a is known to diminish inflammation and fibrosis in different tissues by downregulating the expression of proinflammatory cytokines. However, its role in DMD has not been studied so far.In our work, we have generated mice globally lacking both dystrophin and miR-146a (miR-146a−/−mdx) and examined them together with wild-type, single miR-146a knockout and dystrophic (mdx—lacking dystrophin) mice in a variety of aspects associated with DMD pathophysiology (muscle degeneration, inflammatory reaction, muscle satellite cells, muscle regeneration, and fibrosis).We have shown that miR-146a level is increased in dystrophic muscles in comparison to wild-type mice. Its deficiency augments the expression of proinflammatory cytokines (IL-1β, CCL2, TNFα). However, muscle degeneration was not significantly worsened in mdx mice lacking miR-146a up to 24 weeks of age, although some aggravation of muscle damage and inflammation was evident in 12-week-old animals, though no effect of miR-146a deficiency was visible on quantity, proliferation, and in vitro differentiation of muscle satellite cells isolated from miR-146a−/−mdx mice vs. mdx. Similarly, muscle regeneration and collagen deposition were not changed by miR-146a deficiency. Nevertheless, the lack of miR-146a is associated with decreased Vegfa and increased Tgfb1.Overall, the lack of miR-146a did not aggravate significantly the dystrophic conditions in mdx mice, but its effect on DMD in more severe conditions warrants further investigation.

AAV9 Edits Muscle Stem Cells in Normal and Dystrophic Adult Mice.

CRISPR editing of muscle stem cells (MuSCs) with adeno-associated virus serotype-9 (AAV9) holds promise for sustained gene repair therapy for muscular dystrophies. However, conflicting evidence exists on whether AAV9 transduces MuSCs. To rigorously address this question, we used a muscle graft model. The grafted muscle underwent complete necrosis before regenerating from its MuSCs. We injected AAV9.Cre into Ai14 mice. These mice express tdTomato upon Cre-mediated removal of a floxed stop codon. About 28%-47% and 24%-89% of Pax7+ MuSCs expressed tdTomato in pre-grafts and regenerated grafts (p > 0.05), respectively, suggesting AAV9 efficiently transduced MuSCs, and AAV9-edited MuSCs renewed successfully. Robust MuSC transduction was further confirmed by delivering AAV9.Cre to Pax7-ZsGreen-Ai14 mice in which Pax7+ MuSCs are genetically labeled by ZsGreen. Next, we co-injected AAV9.Cas9 and AAV9.gRNA to dystrophic mdx mice to repair the mutated dystrophin gene. CRISPR-treated and untreated muscles were grafted to immune-deficient, dystrophin-null NSG.mdx4cv mice. Grafts regenerated from CRISPR-treated muscle contained the edited genome and yielded 2.7-fold more dystrophin+ cells (p= 0.015). Importantly, increased dystrophin expression was not due to enhanced formation of revertant fibers or de novo transduction by residual CRISPR vectors in the graft. We conclude that AAV9 effectively transduces MuSCs. AAV9 CRISPR editing of MuSCs may provide enduring therapy.

Proof-of-concept validation of the mechanism of action of Src tyrosine kinase inhibitors in dystrophic mdx mouse muscle: in vivo and in vitro studies

Src tyrosine kinase (TK), a redox-sensitive protein overexpressed in dystrophin-deficient muscles, can contribute to damaging signaling by phosphorylation and degradation of β-dystroglycan (β-DG). We performed a proof-of-concept preclinical study to validate this hypothesis and the benefit-safety ratio of a pharmacological inhibition of Src-TK in Duchenne muscular dystrophy (DMD).Src-TK inhibitors PP2 and dasatinib were administered for 5 weeks to treadmill-exercised mdx mice. The outcome was evaluated in vivo and ex vivo on functional, histological and biochemical disease-related parameters. Considering the importance to maintain a proper myogenic program, the potential cytotoxic effects of both compounds, as well as their cytoprotection against oxidative stress-induced damage, was also assessed in C2C12 cells.In line with the hypothesis, both compounds restored the level of β-DG and reduced its phosphorylated form without changing basal expression of genes of interest, corroborating a mechanism at post-translational level. The histological profile of gastrocnemius muscle was slightly improved as well as the level of plasma biomarkers. However, amelioration of in vivo and ex vivo functional parameters was modest, with PP2 being more effective than dasatinib. Both compounds reached appreciable levels in skeletal muscle and liver, supporting proper animal exposure. Dasatinib exerted a greater concentration-dependent cytotoxic effect on C2C12 cells than the more selective PP2, while being less protective against H2O2 cytotoxicity, even though at concentrations higher than those experienced during in vivo treatments.Our results support the interest of Src-TK as drug target in dystrophinopathies, although further studies are necessary to assess the therapeutic potential of inhibitors in DMD.