Treated Mdx Mice Research Articles

VP.86 Effect of a chronic treatment with L-citrulline on funtional, histological and molecular readouts of dystrophic mdx mouse model

The absence of protein dystrophin in Duchenne muscular dystrophy leads to progressive muscle weakness and wasting, paralleled by failing regeneration. Recently, a key role of deregulated nitric oxide production in epigenetic control of regeneration program has been demonstrated. We focused on the potential interest of the amino acid L-citrulline (L-Cit), a precursor of L-arginine, physiologically required for protein turnover and nitric oxide production in muscle and blood vessels. We administered L-citrulline (2 mg/g/die), through diet, alone or in combination with PDN, (1 mg/kg, 5 days/week subcutaneously) to 4-5 week-old mdx mice for 8 weeks. We found that the increment of maximal forelimb strength was improved in all treated groups vs. untreated mdx mice, particularly by L-Cit alone or with PDN. Ex vivo, treated mdx mice displayed a significant increase of specific isometric twitch and tetanic force in diaphragm (DIA), with minor effects in extensor digitorum longus muscle. Additionally, L-Cit, alone or in combination with PDN, reduced fibrosis and inflammation in gastrocnemius and DIA muscles, and decreased plasma levels of lactate dehydrogenase. PCR analysis in muscles is currently ongoing. These data are encouraging regarding the potential benefits of L-Cit supplementation in DMD also in combination with standard therapy (NL-DPP grant).

Serum extracellular vesicles for delivery of CRISPR-CAS9 ribonucleoproteins to modify the dystrophin gene

Extracellular vesicles (EVs) mediate intercellular biomolecule exchanges in the body, making them promising delivery vehicles for therapeutic cargo. Genetic engineering by the CRISPR system is an interesting therapeutic avenue for genetic diseases such as Duchenne muscular dystrophy (DMD). We developed a simple method for loading EVs with CRISPR ribonucleoproteins (RNPs) consisting of SpCas9 proteins and guide RNAs (gRNAs). EVs were first purified from human or mouse serum using ultrafiltration and size-exclusion chromatography. Using protein transfectant to load RNPs into serum EVs, we showed that EVs are good carriers of RNPs invitro and restored the expression of the tdTomato fluorescent protein in muscle fibers of Ai9 mice. EVs carrying RNPs targeting introns 22 and 24 of the DMD gene were also injected into muscles of mdx mice having a non-sense mutation in exon 23. Up to 19% of the cDNA extracted from treated mdx mice had the intended deletion of exons 23 and 24, allowing dystrophin expression in muscle fibers. RNPs alone, without EVs, were inefficient in generating detectable deletions in mouse muscles. This method opens new opportunities for rapid and safe delivery of CRISPR components to treat DMD.

Immunological mechanisms of exosome mediated therapeutic bioactivity in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal myodegenerative disease associated with chronic inflammation. Disruption in the balance between pro‐inflammatory and pro‐regenerative macrophage immunophenotypes contributes to disease progression. Standard‐of‐care is currently limited to anti‐inflammatory steroids, which marginally delay disease progression. We have recently characterized the therapeutic benefits of cardiosphere‐derived cells (CDCs), which are cardiac progenitor/stromal cells with immunomodulatory, pro‐myogenic, and anti‐fibrotic properties. CDCs partially reverse the key abnormalities of heart and skeletal muscle in mdx mice, findings which motivated two clinical trials (HOPE‐Duchenne and HOPE‐2) of CDCs in DMD patients. The bioactivity of CDCs is mediated by the secretion of lipid bilayer nanovesicles called exosomes, which transfer genetic material between cells to modify recipient cell behavior. We previously discovered macrophages are a primary feature in skeletal muscle of CDC‐exosome treated mdx mice, suggesting their presence is not pathological. Here, we sought to elucidate the role macrophages play in the repair of dystrophic skeletal muscle. Circulating monocytes, at least in part, traffic fluorescently‐labeled CDC‐exosomes to damaged muscle after intravenous delivery. Labeled CDC‐exosomes are detected in the interstitium adjacent to non‐muscle cells, macrophages, and surviving muscle fibers. Moreover, CDC‐exosomes confer a unique immunophenotype to mdx macrophages (Mexo). Next generation sequencing and pathway/network analysis indicate Mexo share features of both M1 (pro‐inflammatory) and M2 (pro‐regenerative) macrophages. Indeed, media conditioned by Mexo supports both myoblast proliferation and differentiation in vitro. Antibody chip array indicated Mexo secrete many chemokines and cytokines known to promote inflammatory cell recruitment and modulation. In contrast to M1 and M2 macrophages, Mexo secrete tissue inhibitor of matrix metalloproteinase‐2 (TIMP‐2). Interestingly, TIMP‐2 has been shown to promote myogenic differentiation of C2C12 myoblasts and may function in both ECM remodeling and myogenesis in vivo. Sequencing of small RNAs revealed that several miRNAs with known roles in myogenesis are uniquely enriched in Mexo exosomes. Together, these data suggest CDC‐exosomes are trafficked to areas of muscle damage, in part, by circulating monocytes and modulate the mdx macrophage phenotype to support tissue regeneration. We propose that Mexo macrophages mediate their pleiotropic effects via the secretion of cytokines, chemokines, and possibly exosomes. Modulation of mdx macrophage functions by CDC‐exosomes, to support muscle health, may represent a novel approach to treating DMD.Support or Funding InformationMuscular Dystrophy Association, Coalition Duchenne, NIH R01 to EM

Glycine Enhances Satellite Cell Proliferation, Cell Transplantation, and Oligonucleotide Efficacy in Dystrophic Muscle.

The need to distribute therapy evenly systemically throughout the large muscle volume within the body makes Duchenne muscular dystrophy (DMD) therapy a challenge. Cell and exon-skipping therapies are promising but have limited effects, and thus enhancing their therapeutic potency is of paramount importance to increase the accessibility of these therapies to DMD patients. In this study, we demonstrate that co-administered glycine improves phosphorodiamidate morpholino oligomer (PMO) potency in mdx mice with marked functional improvement and an up to 50-fold increase of dystrophin in abdominal muscles compared to PMO in saline. Glycine boosts satellite cell proliferation and muscle regeneration by increasing activation of mammalian target of rapamycin complex 1 (mTORC1) and replenishing the one-carbon unit pool. The expanded regenerating myofiber population then results in increased PMO uptake. Glycine also augments the transplantation efficiency of exogenous satellite cells and primary myoblasts in mdx mice. Our data provide evidence that glycine enhances satellite cell proliferation, cell transplantation, and oligonucleotide efficacy in mdx mice, and thus it has therapeutic utility for cell therapy and drug delivery in muscle-wasting diseases.

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.

A GDF11/myostatin inhibitor, GDF11 propeptide-Fc, increases skeletal muscle mass and improves muscle strength in dystrophic mdx mice

BackgroundGrowth differentiation factor 11 (GDF11) is a member of the transforming growth factor β superfamily. The GDF11 propeptide, which is derived from the GDF11 precursor protein, blocks the activity of GDF11 and its homolog, myostatin, which are both potent inhibitors of muscle growth. Thus, treatment with GDF11 propeptide may be a potential therapeutic strategy for diseases associated with muscle atrophy like sarcopenia and the muscular dystrophies. Here, we evaluate the impact of GDF11 propeptide-Fc (GDF11PRO-Fc) gene delivery on skeletal muscle in normal and dystrophic adult mice.MethodsA pull-down assay was used to obtain physical confirmation of a protein-protein interaction between GDF11PRO-Fc and GDF11 or myostatin. Next, differentiated C2C12 myotubes were treated with AAV6-GDF11PRO-Fc and challenged with GDF11 or myostatin to determine if GDF11PRO-Fc could block GDF11/myostatin-induced myotube atrophy. Localized expression of GDF11PRO-Fc was evaluated via a unilateral intramuscular injection of AAV9-GDF11PRO-Fc into the hindlimb of C57BL/6J mice. In mdx mice, intravenous injection of AAV9-GDF11PRO-Fc was used to achieve systemic expression. The impact of GDF11PRO-Fc on muscle mass, function, and pathological features were assessed.ResultsGDF11PRO-Fc was observed to bind both GDF11 and myostatin. In C2C12 myotubes, expression of GDF11PRO-Fc was able to mitigate GDF11/myostatin-induced atrophy. Following intramuscular injection in C57BL/6J mice, increased grip strength and localized muscle hypertrophy were observed in the injected hindlimb after 10 weeks. In mdx mice, systemic expression of GDF11PRO-Fc resulted in skeletal muscle hypertrophy without a significant change in cardiac mass after 12 weeks. In addition, grip strength and rotarod latency time were improved. Intramuscular fibrosis was also reduced in treated mdx mice; however, there was no change seen in central nucleation, membrane permeability to serum IgG or serum creatine kinase levels.ConclusionsGDF11PRO-Fc induces skeletal muscle hypertrophy and improvements in muscle strength via inhibition of GDF11/myostatin signaling. However, GDF11PRO-Fc does not significantly improve the dystrophic pathology in mdx mice.

Intermittent PTH treatment improves bone and muscle in glucocorticoid treated Mdx mice: A model of Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy (DMD) is a progressive muscle disorder caused by genetic mutations of the dystrophin encoding gene. In the absence of functional dystrophin, DMD patients suffer from muscle inflammation and wasting, as well as compromised bone health with increased risk of fracture. The use of high dose glucocorticoids (GC) as the standard therapy also contributes to bone fragility. This study examined the effects of intermittent, daily administered parathyroid hormone (iPTH), an approved bone anabolic therapy, on growing bone and dystrophic muscle in the presence and absence of prednisone treatment using the Mdx mouse model of DMD. Five-weeks of prednisone treatment in Mdx mice decreased cortical bone thickness and area (p < 0.001), with a large increase in endocortical osteoclasts that were significantly improved by PTH treatment (p < 0.001). GC-induced decreases in cortical bone toughness and modulus were improved with iPTH therapy (p < 0.05). Mdx mice showed significantly less bone mass in trabecular compartments of lumbar vertebrae and iPTH treatment, with or without glucocorticoids, significantly improved structural and material properties of this bone. Prednisone improved grip strength and endurance of treadmill running, which were maintained and further improved, respectively, in co-treated Mdx mice. Altogether, our study demonstrates that iPTH therapy significantly ameliorated GC-induced bone loss and maintained or further enhanced the positive effects of GCs on dystrophic muscle function. These findings give insight into the potential for use of teriparatide to treat growing bone in children with DMD.

Mineralocorticoid Receptor Antagonists in Muscular Dystrophy Mice During Aging and Exercise.

Mineralocorticoid receptor antagonists added to angiotensin converting enzyme inhibitors have shown preclinical efficacy for both skeletal and cardiac muscle outcomes in young sedentary dystrophin-deficient mdx mice also haploinsufficient for utrophin, a Duchenne muscular dystrophy (DMD) model. The mdx genotypic DMD model has mild pathology, making non-curative therapeutic effects difficult to distinguish at baseline. Since the cardiac benefit of mineralocorticoid receptor antagonists has been translated to DMD patients, it is important to optimize potential advantages for skeletal muscle by further defining efficacy parameters. We aimed to test whether therapeutic effects of mineralocorticoid receptor antagonists added to angiotensin converting enzyme inhibitors are detectable using three different reported methods of exacerbating the mdx phenotype. We tested treatment with lisinopril and the mineralocorticoid receptor antagonist spironolactone in: 10 week-old exercised, 1 year-old sedentary, and 5 month-old isoproterenol treated mdx mice and performed comprehensive functional and histological measurements. None of the protocols to exacerbate mdx phenotypes resulted in dramatically enhanced pathology and no significant benefit was observed with treatment. Since endogenous mineralocorticoid aldosterone production from immune cells in dystrophic muscle may explain antagonist efficacy, it is likely that these drugs work optimally during the narrow window of peak inflammation in mdx mice. Exercised and aged mdx mice do not display prolific damage and inflammation, likely explaining the absence of continued efficacy of these drugs. Since inflammation is more prevalent in DMD patients, the therapeutic window for mineralocorticoid receptor antagonists in patients may be longer.

Macrophages are Required for Recovery from Physiological Muscle Stress in the mdx Mouse Model of Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a lethal genetic disease associated with muscle degeneration and chronic inflammation. Current therapies are limited to corticosteroids which resolve inflammation at the expense of detrimental side effects, and exon skipping which is applicable only to a small subset of patients. Cardiosphere‐derived cells (CDCs) are cardiac progenitor/stromal cells with anti‐inflammatory, anti‐fibrotic, and regenerative properties. Recently, CDCs have been shown to reverse many of the abnormalities of heart and skeletal muscle in mdx mice, motivating the HOPE‐Duchenne clinical trial (Aminzadeh M, Rogers R et al., Stem Cell Reports 2018). Several lines of evidence now support the idea that CDCs exert their bioactivity by secreting nanoparticles called exosomes (CDC‐exos), which mediate cell‐cell transfer of genetic information. A single intravenous dose of CDC‐exos dramatically improves contractile function of the mdx mouse soleus, and restores muscle architecture. Given the anti‐inflammatory effects of CDC‐exos, RNA‐sequencing unexpectedly revealed a striking upregulation of inflammatory pathways in the solei of CDC‐exo treated mdx mice, as confirmed by confocal imaging of tissue macrophages using CD68 immunohistochemistry. Thus, we investigated the role of macrophages in mdx pathology. Clodronate liposomes were used to deplete monocytes and macrophages in vivo, as confirmed in blood by flow cytometry and in tissue by immunohistochemistry. Macrophage‐depleted and control mdx mice were subjected to a single bout of downhill running to induce muscle stress. Maximal exercise capacity was recorded prior to, and every other day for two weeks, following downhill running. Macrophage‐depleted mdx mice failed to recover during the two‐week period, whereas control mdx mice had recovered by the third day. Further, macrophage depletion shifted the force‐frequency relationship to higher frequencies and reduced maximal force developed by the soleus by 24%. Cultured macrophages from mdx bone marrow (BMDMs) express 4‐fold higher levels of the pro‐inflammatory cytokine TNF‐α than those from age‐matched wild‐type mice. In vitro conditioning of mdx BMDMs with CDC‐exos reduced the expression of TNF‐α, suggesting that CDC‐exos may skew mdx macrophage polarization toward a tissue healing phenotype. This interpretation is consistent with the finding that CDC‐exos polarize macrophages to a phenotype with enhanced efferocytosis capacity, minimizing scar formation after acute myocardial infarction. Thus, macrophages are required for recovery from physiological muscle stress in mdx mice, and CDC‐exos decrease pro‐inflammatory gene expression in macrophages. The unexpected requirement for macrophages in mdx skeletal muscle recovery from injury suggests that a macrophage population promotes tissue regeneration. We are currently testing the hypothesis that CDC‐exos may modulate the pro‐inflammatory phenotype of recruited macrophages to stimulate myogenesis and support mdx muscle regeneration.Support or Funding InformationCoalition DuchenneNIH T32 HL116273This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Resveratrol Decreases Oxidative Stress by Restoring Mitophagy and Improves the Pathophysiology of Dystrophin-Deficient mdx Mice.

We previously showed that treatment with resveratrol (3,5,4′-trihydroxy-trans-stilbene), an activator of the NAD+-dependent deacetylase SIRT1 at 4 g/kg food for 32 weeks, significantly decreased the muscular reactive oxygen species (ROS) levels and ameliorated the pathology of mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Here, we treated mdx mice with various doses of resveratrol (0.04, 0.4, and 4 g/kg food) for 56 weeks and examined the effects on serum creatine kinase levels and physical activities. Because resveratrol promotes autophagy, we also investigated whether autophagy including mitochondrial autophagy (mitophagy) is involved in resveratrol's effects. Autophagy/mitophagy-related genes and autophagic flux were downregulated in the muscle of mdx mice, and these phenomena were reversed by resveratrol with significant ROS reduction. Resveratrol at 4 g/kg food reduced the number of immature myofibers containing central nuclei and fine fibers < 400 μm2 and increased that of thicker myofibers in the quadriceps, suggesting that resveratrol decreased myofiber wasting and promoted muscular maturation. Accordingly, resveratrol at 0.4 g/kg food reduced the creatine kinase levels to one-third of those in untreated mdx mice and significantly increased the animals' physical activities. In C2C12 myoblast cells, resveratrol promoted mitophagy and eliminated mitochondria containing high superoxide levels. The clearance of damaged mitochondria and ROS reduction by resveratrol was completely suppressed by an autophagy inhibitor (chloroquine) and by knocking down Atg5 or Pink1, essential genes for autophagy and mitophagy, respectively. Thus, resveratrol is a potential therapeutic agent for DMD, and the clearance of damaged mitochondria probably contributes to its action.

BGP-15 Improves Aspects of the Dystrophic Pathology in mdx and dko Mice with Differing Efficacies in Heart and Skeletal Muscle

Duchenne muscular dystrophy is a severe and progressive striated muscle wasting disorder that leads to premature death from respiratory and/or cardiac failure. We have previously shown that treatment of young dystrophic mdx and dystrophin/utrophin null (dko) mice with BGP-15, a coinducer of heat shock protein 72, ameliorated the dystrophic pathology. We therefore tested the hypothesis that later-stage BGP-15 treatment would similarly benefit older mdx and dko mice when the dystrophic pathology was already well established. Later stage treatment of mdx or dko mice with BGP-15 did not improve maximal force of tibialis anterior (TA) muscles (in situ) or diaphragm muscle strips (invitro). However, collagen deposition (fibrosis) was reduced in TA muscles of BGP-15-treated dko mice but unchanged in TA muscles of treated mdx mice and diaphragm of treated mdx and dko mice. We also examined whether BGP-15 treatment could ameliorate aspects of the cardiac pathology, and in young dko mice it reduced collagen deposition and improved both membrane integrity and systolic function. These results confirm BGP-15's ability to improve aspects of the dystrophic pathology but with differing efficacies in heart and skeletal muscles at different stages of the disease progression. These findings support a role for BGP-15 among a suite of pharmacological therapies for Duchenne muscular dystrophy and related disorders.

The weak combined magnetic field, tuned to ion parametric resonance for Ca2+, stimulates dystrophin synthesis in the skeletal muscles of mdx mice subjected to cell therapy

The possibility of using bone marrow stem cells for treatment of Duchenne muscular dystrophy is intensely studied. Mdx mice are the most widely used laboratory model of Duchenne muscular dystrophy. One approach of cell therapy of muscular dystrophy is substitution of bone marrow in mdx mice after their X-ray irradiation. However, this method does not allow one to increase significantly dystrophin synthesis in muscular fibers of mdx mice. To improve the effect of transplanted cells on muscle regeneration, we additionally treated mdx mice subjected to transplantation of bone marrow cells with a weak combined magnetic field tuned to ion parametric resonance for Ca2+ (Ca2+-CMF). We found that, in irradiated chimeric 3 and 5 Gy mdx mice, additional treatment with Ca2+-CMF for 1 month resulted in significant increases in the portions of dystrophin-positive muscle fibers, by 15.8 and 18.3%, respectively, as compared to the control groups. Furthermore, the share of muscle fibers without centrally located nuclei also increased. We suggest that the magnetic field with these parameters may stimulate functioning of nuclei of donor cells, which were incorporated into muscle fibers.

483. In Vivo DMD Gene Editing in Muscles and Muscle Stem Cells of Dystrophic Mice

Duchenne muscular dystrophy (DMD) is a X-linked genetic disorder that arises from frame-disrupting mutations in the DMD gene, encoding DYSTROPHIN. Lack of DYSTROPHIN expression destabilizes muscle fiber membranes, increases susceptibility to contraction-induced injury and drives muscle degeneration. Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated but still functional protein. In this study, we develop and test a direct gene editing strategy to recover DYSTROPHIN expression in the mdx mouse model of DMD. Coupling clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases delivered via adeno-associated virus (AAV) with paired guide RNAs flanking the mutated Dmd exon 23, we demonstrate precise excision of intervening DNA and restoration of Dystrophin reading frame and protein expression in vivo in both skeletal and cardiac muscles following local or systemic delivery. DYSTROPHIN expression in AAV Dmd-CRISPR treated mdx mice was sufficient to partially recover functional deficiencies of dystrophic muscle. Finally, we demonstrate in vivo targeting of the mdx mutation in endogenous muscle stem cells, suggesting that AAV-CRISPR may provide a means to support ongoing repair of dystrophic fibers with corrected muscle precursors. This study provides proof-of-concept evidence supporting the feasibility and efficacy of in vivo genome editing to correct frame-disrupting mutations in DMD.View Large Image | Download PowerPoint Slide

In vivo treatment with the NF-κB inhibitor ursodeoxycholic acid (UDCA) improves tension development in the isolated mdx costal diaphragm.

Previous experiments have indicated that in vivo administration of ursodeoxycholic acid (UDCA) inhibits nuclear NF-κB activation and has beneficial effects on the structure and function of dystrophic (mdx) muscle. We examined the effect of UDCA on tension development in dystrophic muscle. Isometric tension development was examined in costal diaphragms that were freshly isolated from vehicle and UDCA treated mdx mice. Percent recovery scores were obtained by directly comparing these measurements to those obtained from age-matched nondystrophic mice. Vehicle treated mdx mice exhibited significantly reduced optimal muscle lengths (lo ) and specific twitch and tetanic tensions compared with age-matched nondystrophic mice. UDCA treated preparations exhibited significantly improved tension development with a 33% recovery score. Because UDCA is used in treating certain clinical disorders, these results provide a rationale for human clinical trials using this and related drugs for treatment of Duchenne and related muscular dystrophies.

G.P.90: Effects of S48168/Arm210, a new rycal® compound, on pathology related signs of exercised dystrophic mdx mouse

The progressive myofiber degeneration in Duchenne muscular dystrophy (DMD) is caused by the complex cascade of events triggered by the absence of dystrophin. Studies in the mdx mouse model of DMD have shown that pathology-related post-translational modifications of the Ryanodine receptor subtype 1 (RyR1) result in the dissociation of the stabilizing subunit calstabin1, leading to leaky channels and altered Ca2+homeostasis, a key event in DMD (Bellinger, Nat Med 2009). S48168, also known as ARM210, is a small molecule stabilizer of the calstabin-RyR channel complex that prevents Ca 2+ leak without altering RyR1 post-translational modifications. We assessed the effects of 4weeks oral administration of S48168 (10 and 50mg/kg/day) on treadmill-exercised mdx mice, starting at 4–5weeks of age. A functional improvement was observed in vivo in S48168 -treated mdx mice, with a 50% recovery score of maximal forelimb force at 50mg/kg. Both doses prevented the decline in running performance, observed in vehicle-treated mdx mice, as assessed in an exhaustion test. Ex vivo assessment showed a dose-dependent improvement of diaphragm specific force, with a significant 30% increase of maximal force (100–140Hz) at 50mg/kg, while minor, if any, effects were observed in hind-limb EDL muscle. However, and consistently with the S48168 mechanism of action, the mechanical threshold EDL myofibers, an index of calcium handling during contraction, was significantly shifted toward the wild type (wt) values at the highest dose. In parallel, the resting cytosolic calcium level in FDB myofibers was significantly reduced by 25% in the S48168-treated mice. S48168 did not lower CK or LDH plasma levels. However a reduction of damaged area was detected in diaphragm and in gastrocnemius muscle of 50mg/kg treated mdx mice. Dose-proportional increases in plasma and muscle drug content were also observed, without any signs of toxicity. These results support S48168 as a potential therapy for DMD.

G.P.111: Development of an ultrasensitive ELISA method for the determination of phosphorodiamidate morpholino oligonucleotide (PMO) levels in biological samples

Antisense oligonucleotide (AON) induced exon skipping is one of the most promising strategies for treating Duchene muscular dystrophy (DMD) and other rare genetic diseases, with the first generation of AONs having been applied in phase 3 clinical trials. Several different chemistries of AONs are available with 2-O-methyl phosphorothioate (2′OMe) and phosphorodiamidate morpholino oligonucleotide (PMO) being the most advanced AONs in development. In addition to determining the efficacy of AON induced dystrophin expression, detecting the levels of AON in blood and tissue is essential for determining the pharmacokinetic/pharmacodynamic (PK/PD) relationship. In comparison with other AON detection methods such as high-performance liquid chromatography or liquid chromatography–mass spectrometry, an ELISA based approach has demonstrated far greater sensitivity with detection levels in the picomolar range. As a result the ELISA is the method of choice for 2′OMe detection in pre-clinical and clinical development. However, to the best of our knowledge, no such assay has been developed for PMO and therefore a novel PMO ELISA assay is currently being developed in our lab with encouraging results. The hybridisation-based assay is currently able to detect PMO levels in buffer with a linear range of detection between 30 and 1000 pM (R2 = 0.99). More importantly the sensitivity is unaffected by various biological samples such as mouse serum, plasma and tissue lysate. The present level of sensitivity should be sufficient for both pre-clinical and clinical analysis. In addition, the assay can be easily adapted for detecting peptide-conjugated PMOs (i.e. PPMOs) which are the next generation of PMO based AONs in pre-clinical development. Here we present the results of studies from in PMO and PPMO treated mdx mice.

Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice

BackgroundPresently, there is no effective treatment for the lethal muscle wasting disease Duchenne muscular dystrophy (DMD). Here we show that increased sphingosine-1-phoshate (S1P) through direct injection or via the administration of the small molecule 2-acetyl-4(5)-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, has beneficial effects in acutely injured dystrophic muscles of mdx mice.MethodsWe treated mdx mice with and without acute injury and characterized the histopathological and functional effects of increasing S1P levels. We also tested exogenous and direct administration of S1P on mdx muscles to examine the molecular pathways under which S1P promotes regeneration in dystrophic muscles.ResultsShort-term treatment with THI significantly increased muscle fiber size and extensor digitorum longus (EDL) muscle specific force in acutely injured mdx limb muscles. In addition, the accumulation of fibrosis and fat deposition, hallmarks of DMD pathology and impaired muscle regeneration, were lower in the injured muscles of THI-treated mdx mice. Furthermore, increased muscle force was observed in uninjured EDL muscles with a longer-term treatment of THI. Such regenerative effects were linked to the response of myogenic cells, since intramuscular injection of S1P increased the number of Myf5nlacz/+ positive myogenic cells and newly regenerated myofibers in injured mdx muscles. Intramuscular injection of biotinylated-S1P localized to muscle fibers, including newly regenerated fibers, which also stained positive for S1P receptor 1 (S1PR1). Importantly, plasma membrane and perinuclear localization of phosphorylated S1PR1 was observed in regenerating muscle fibers of mdx muscles. Intramuscular increases of S1P levels, S1PR1 and phosphorylated ribosomal protein S6 (P-rpS6), and elevated EDL muscle specific force, suggest S1P promoted the upregulation of anabolic pathways that mediate skeletal muscle mass and function.ConclusionsThese data show that S1P is beneficial for muscle regeneration and functional gain in dystrophic mice, and that THI, or other pharmacological agents that raise S1P levels systemically, may be developed into an effective treatment for improving muscle function and reducing the pathology of DMD.

Reducing CTGF/CCN2 slows down mdx muscle dystrophy and improves cell therapy

In Duchenne muscular dystrophy (DMD) and the mdx mouse model, the absence of the cytoskeletal protein dystrophin causes defective anchoring of myofibres to the basal lamina. The resultant myofibre degeneration and necrosis lead to a progressive loss of muscle mass, increased fibrosis and ultimately fatal weakness. Connective tissue growth factor (CTGF/CCN-2) is critically involved in several chronic fibro-degenerative diseases. In DMD, the role of CTGF might extend well beyond replacement fibrosis secondary to loss of muscle fibres, since its overexpression in skeletal muscle could by itself induce a dystrophic phenotype. Using two independent approaches, we here show that mdx mice with reduced CTGF availability do indeed have less severe muscular dystrophy. Mdx mice with hemizygous CTGF deletion (mdx-Ctgf+/-), and mdx mice treated with a neutralizing anti-CTGF monoclonal antibody (FG-3019), performed better in an exercise endurance test, had better muscle strength in isolated muscles and reduced skeletal muscle impairment, apoptotic damage and fibrosis. Transforming growth factor type-β (TGF-β), pERK1/2 and p38 signalling remained unaffected during CTGF suppression. Moreover, both mdx-Ctgf+/- and FG-3019 treated mdx mice had improved grafting upon intramuscular injection of dystrophin-positive satellite cells. These findings reveal the potential of targeting CTGF to reduce disease progression and to improve cell therapy in DMD.

Ibuprofen plus isosorbide dinitrate treatment in the mdx mice ameliorates dystrophic heart structure

Background: Co-administration of ibuprofen (IBU) and isosorbide dinitrate (ISDN) provides synergistic beneficial effects on dystrophic skeletal muscle. Whether this treatment has also cardioprotective effects in this disease was still unknown. Aims: To evaluate the effects of co-administration of IBU and ISDN (a) on left ventricular (LV) structure and function, and (b) on cardiac inflammatory response and fibrosis in mdx mice. Methods: Three groups of mice were studied: mdx mice treated with IBU (50mgkg−1)+ISDN (30mgkg−1) administered daily in the diet, mdx mice that received standard diet without drugs and wild type aged-matched mice. Animals were analysed after 10–11 months of treatment. Structural and functional parameters were evaluated by echocardiography while histological analyses were performed to evaluate inflammatory response, collagen deposition, cardiomyocyte number and area. Results: Treatment for 10–11 months with IBU+ISDN preserved LV wall thickness and LV mass. Drug treatment also preserved the total number of cardiomyocytes in the LV and attenuated the increase in cardiomyocyte size, when compared to untreated mdx mice. Moreover, a trend towards a decreased number of inflammatory cells, a reduced LV myocardial interstitial fibrosis and an enhanced global LV function response to stress was observed in treated mdx mice. Conclusions: Treatment for 10–11 months with IBU+ISDN is effective in preventing the alterations in LV morphology of mdx mice while not reaching statistical significance on LV function and cardiac inflammation.

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.