Target For Duchenne Muscular Dystrophy Research Articles

Diapocynin treatment induces functional and structural improvements in an advanced disease state in the mdx5Cv mice

Duchenne muscular dystrophy (DMD) is the most common muscular disorder affecting children. It affects nearly 1 male birth over 5000. Oxidative stress is a pervasive feature in the pathogenesis of DMD. Recent work shows that the main generators of ROS are NADPH oxidases (NOX), suggesting that they are an early and promising target in DMD. In addition, skeletal muscles of mdx mice, a murine model of DMD, overexpress NOXes. We investigated the impact of diapocynin, a dimer of the NOX inhibitor apocynin, on the chronic disease phase of mdx5Cv mice. Treatment of these mice with diapocynin from 7 to 10 months of age resulted in decreased hypertrophy of several muscles, prevented force loss induced by tetanic and eccentric contractions, improved muscle and respiratory functions, decreased fibrosis of the diaphragm and positively regulated the expression of disease modifiers. These encouraging results ensure the potential role of diapocynin in future treatment strategies.

CB2 Receptor as Emerging Anti-Inflammatory Target in Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy (DMD) is a very severe X-linked dystrophinopathy. It is due to a mutation in the DMD gene and causes muscular degeneration in conjunction with several secondary co-morbidities, such cardiomyopathy and respiratory failure. DMD is characterized by a chronic inflammatory state, and corticosteroids represent the main therapy for these patients. To contradict drug-related side effects, there is need for novel and more safe therapeutic strategies. Macrophages are immune cells stringently involved in both physiological and pathological inflammatory processes. They express the CB2 receptor, one of the main elements of the endocannabinoid system, and have been proposed as an anti-inflammatory target in several inflammatory and immune diseases. We observed a lower expression of the CB2 receptor in DMD-associated macrophages, hypothesizing its involvement in the pathogenesis of this pathology. Therefore, we analyzed the effect of JWH-133, a CB2 receptor selective agonist, on DMD-associated primary macrophages. Our study describes the beneficial effect of JWH-133 in counteracting inflammation by inhibiting pro-inflammatory cytokines release and by directing macrophages' phenotype toward the M2 anti-inflammatory one.

Pharmacological inhibition of HDAC6 improves muscle phenotypes in dystrophin-deficient mice by downregulating TGF-β via Smad3 acetylation

The absence of dystrophin in Duchenne muscular dystrophy disrupts the dystrophin-associated glycoprotein complex resulting in skeletal muscle fiber fragility and atrophy, associated with fibrosis as well as microtubule and neuromuscular junction disorganization. The specific, non-conventional cytoplasmic histone deacetylase 6 (HDAC6) was recently shown to regulate acetylcholine receptor distribution and muscle atrophy. Here, we report that administration of the HDAC6 selective inhibitor tubastatin A to the Duchenne muscular dystrophy, mdx mouse model increases muscle strength, improves microtubule, neuromuscular junction, and dystrophin-associated glycoprotein complex organization, and reduces muscle atrophy and fibrosis. Interestingly, we found that the beneficial effects of HDAC6 inhibition involve the downregulation of transforming growth factor beta signaling. By increasing Smad3 acetylation in the cytoplasm, HDAC6 inhibition reduces Smad2/3 phosphorylation, nuclear translocation, and transcriptional activity. These findings provide in vivo evidence that Smad3 is a new target of HDAC6 and implicate HDAC6 as a potential therapeutic target in Duchenne muscular dystrophy.

Cholesterol metabolism is a potential therapeutic target in Duchenne muscular dystrophy.

BackgroundDuchenne muscular dystrophy (DMD) is a lethal muscle disease detected in approximately 1:5000 male births. DMD is caused by mutations in the DMD gene, encoding a critical protein that links the cytoskeleton and the extracellular matrix in skeletal and cardiac muscles. The primary consequence of the disrupted link between the extracellular matrix and the myofibre actin cytoskeleton is thought to involve sarcolemma destabilization, perturbation of Ca2+ homeostasis, activation of proteases, mitochondrial damage, and tissue degeneration. A recently emphasized secondary aspect of the dystrophic process is a progressive metabolic change of the dystrophic tissue; however, the mechanism and nature of the metabolic dysregulation are yet poorly understood. In this study, we characterized a molecular mechanism of metabolic perturbation in DMD.MethodsWe sequenced plasma miRNA in a DMD cohort, comprising 54 DMD patients treated or not by glucocorticoid, compared with 27 healthy controls, in three groups of the ages of 4–8, 8–12, and 12–20 years. We developed an original approach for the biological interpretation of miRNA dysregulation and produced a novel hypothesis concerning metabolic perturbation in DMD. We used the mdx mouse model for DMD for the investigation of this hypothesis.ResultsWe identified 96 dysregulated miRNAs (adjusted P‐value <0.1), of which 74 were up‐regulated and 22 were down‐regulated in DMD. We confirmed the dysregulation in DMD of Dystro‐miRs, Cardio‐miRs, and a large number of the DLK1‐DIO3 miRNAs. We also identified numerous dysregulated miRNAs yet unreported in DMD. Bioinformatics analysis of both target and host genes for dysregulated miRNAs predicted that lipid metabolism might be a critical metabolic perturbation in DMD. Investigation of skeletal muscles of the mdx mouse uncovered dysregulation of transcription factors of cholesterol and fatty acid metabolism (SREBP‐1 and SREBP‐2), perturbation of the mevalonate pathway, and the accumulation of cholesterol in the dystrophic muscles. Elevated cholesterol level was also found in muscle biopsies of DMD patients. Treatment of mdx mice with Simvastatin, a cholesterol‐reducing agent, normalized these perturbations and partially restored the dystrophic parameters.ConclusionsThis investigation supports that cholesterol metabolism and the mevalonate pathway are potential therapeutic targets in DMD.

DMD – ANIMAL MODELS & PRECLINICAL TREATMENT: P.211 Src tyrosine kinase as potential target in Duchenne muscular dystrophy: assessment of a novel dasatinib formulation in the mdx mouse model

The overexpression and the increased activity of redox-sensitive protein Src-Tyrosine Kinase (TK) in dystrophin-deficient muscles can contribute to β-dystroglycan (β-DG) degradation and in reinforcing proinflammatory damaging signalling. According to the hypothesis that Src-TK is a feasible drug target for ameliorating Duchenne Muscular Dystrophy (DMD), we have recently focused on preclinical studies for repurposing dasatinib, a Src-TK's competitive inhibitor. We have shown that a 4-week subcutaneous treatment with dasatinib (5mg/kg, 3 times/week) induced an increment in β-DG at muscle level, although a parallel limited efficacy on pathology-related functional, structural and biochemical indices was observed. Considering the occurrence of possible pharmacokinetic or time related issues, we presently tested in mdx mouse a new oral formulation (dasatinib-hydroxypropyl-β-cyclodextrin inclusion complex), administered at 10 or 20mg/kg for 12 weeks, in drinking water. The new formulation allowed the drug to reach plasma, liver and skeletal muscle level in a dose-related fashion. Our results showed that, in vivo, dasatinib contrasted the impaired exercise performance during an exhaustion treadmill test, with a recovery score (R.S.) toward WT value of 28% (10mg/kg) and 17% (20mg/kg), but did not exert any effect on mdx mice weakness, assessed by forelimb grip test. Ex vivo, dasatinib ameliorated tetanic isometric force of fast-twitch extensor digitorum longus muscle, with a R.S. of 31% for both doses, while at 10mg/kg it increased tetanic isometric force of diaphragm muscle (R.S. 45%), and partially recovered the elastic properties of this latter during eccentric contraction. Moreover, dasatinib, either 10 or 20mg/kg, notably decreased plasma levels of pathology-related biomarkers creatine kinase (R.S. 28% and 47%) and lactate dehydrogenase (R.S. 36% and 46%). These preliminary data support the interest of this new oral dasatinib formulation for DMD treatment. However, further experiments are needed in order to better establish the therapeutic value of dasatinib in the light of a future repurposing as potential treatment for DMD patients.

Reduction of circulating sphingosine-1-phosphate worsens mdx soleus muscle dystrophic phenotype.

What is the central question of the study? What are the consequences of reducing circulating sphingosine-1-phosphate (S1P) for muscle physiology in the murine model of Duchenne muscular dystrophy (DMD)? What is the main result and its importance? Reduction of the circulating S1P level in mdx mice aggravates the dystrophic phenotype, as seen by an increase in fibre atrophy, fibrosis and loss of specific force, suggesting that S1P signalling is a potential therapeutic target in DMD. Although further studies are needed, plasma S1P levels have the intriguing possibility of being used as a biomarker for disease severity, an important issue in DMD. Sphingosine-1-phosphate (S1P) is an important regulator of skeletal muscle properties. The dystrophin-deficient mdx mouse possesses low levels of S1P (∼50%) compared with wild type. Increased S1P availability was demonstrated to ameliorate the dystrophic phenotype in Drosophila and in mdx mice. Here, we analysed the effects produced by further reduction of S1P availability on the mass, force and regenerative capacity of dystrophic mdx soleus. Circulating S1P was neutralized by a specific anti-S1P antibody (S1P-Ab) known to lower the extracellular concentration of this signalling lipid. The S1P-Ab was administered intraperitoneally in adult mdx mice every 2days for the duration of experiments. Soleus muscle properties were analysed 7 or 14days after the first injection. The decreased availability of circulating S1P after the 14day treatment reduced mdx soleus fibre cross-sectional area (-16%, P<0.05), an effect that was associated with an increase in markers of proteolytic (MuRF1 and atrogin-1) and autophagic (p62 and LC3-II/LC3-I ratio) pathways. Moreover, an increase of fibrosis was also observed (+26%, P<0.05). Notably, the treatment also caused a reduction of specific tetanic tension (-29%, P<0.05). The mdx soleus regenerative capacity was only slightly influenced by reduced S1P. In conclusion, neutralization of circulating S1P reduces the mass and specific force and increases fibrosis of mdx soleus muscle, thus worsening the dystrophic phenotype. The results confirm that active, functional S1P signalling might counteract the progression of soleus mdx pathology and validate the pathway as a potential therapeutic target for muscular dystrophies.

Leveraging Quantitative Systems Pharmacology Approach into Development of Human Recombinant Follistatin Fusion Protein for Duchenne Muscular Dystrophy.

Quantitative understanding about the dynamics of drug–target interactions in biological systems is essential, especially in rare disease programs with small patient populations. Follistatin, by antagonism of myostatin and activin, which are negative regulators of skeletal muscle and inflammatory response, is a promising therapeutic target for Duchenne Muscular Dystrophy. In this study, we constructed a quantitative systems pharmacology model for FS‐EEE‐Fc, a follistatin recombinant protein to investigate its efficacy from dual target binding, and, subsequently, to project its human efficacious dose. Based on model simulations, with an assumed efficacy threshold of 7–10% muscle volume increase, 3–5 mg/kg weekly dosing of FS‐EEE‐Fc is predicted to achieve meaningful clinical outcome. In conclusion, the study demonstrated an application of mechanism driven approach at early stage of a rare disease drug development to support lead compound optimization, enable human dose, pharmacokinetics, and efficacy predictions.

P.315Src tyrosine kinase as a drug target in Duchenne muscular dystrophy: a proof-of-concept study in the exercised mdx mouse

P.315Src tyrosine kinase as a drug target in Duchenne muscular dystrophy: a proof-of-concept study in the exercised mdx mouse

NADPH oxidase mediates microtubule alterations and diaphragm dysfunction in dystrophic mice.

Skeletal muscle from mdx mice is characterized by increased Nox2 ROS, altered microtubule network, increased muscle stiffness, and decreased muscle/respiratory function. While microtubule de-tyrosination has been suggested to increase stiffness and Nox2 ROS production in isolated single myofibers, its role in altering tissue stiffness and muscle function has not been established. Because Nox2 ROS production is upregulated prior to microtubule network alterations and ROS affect microtubule formation, we investigated the role of Nox2 ROS in diaphragm tissue microtubule organization, stiffness and muscle/respiratory function. Eliminating Nox2 ROS prevents microtubule disorganization and reduces fibrosis and muscle stiffness in mdx diaphragm. Fibrosis accounts for the majority of variance in diaphragm stiffness and decreased function, implicating altered extracellular matrix and not microtubule de-tyrosination as a modulator of diaphragm tissue function. Ultimately, inhibiting Nox2 ROS production increased force and respiratory function in dystrophic diaphragm, establishing Nox2 as a potential therapeutic target in Duchenne muscular dystrophy.

Levels of S100B protein drive the reparative process in acute muscle injury and muscular dystrophy

Regeneration of injured skeletal muscles relies on a tightly controlled chain of cellular and molecular events. We show that appropriate levels of S100B protein are required for timely muscle regeneration after acute injury. S100B released from damaged myofibers and infiltrating macrophages expands the myoblast population, attracts macrophages and promotes their polarization into M2 (pro-regenerative) phenotype, and modulates collagen deposition, by interacting with RAGE (receptor for advanced glycation end-products) or FGFR1 (fibroblast growth factor receptor 1) depending on the muscle repair phase and local conditions. However, persistence of high S100B levels compromises the regeneration process prolonging myoblast proliferation and macrophage infiltration, delaying M1/M2 macrophage transition, and promoting deposition of fibrotic tissue via RAGE engagement. Interestingly, S100B is released in high abundance from degenerating muscles of mdx mice, an animal model of Duchenne muscular dystrophy (DMD), and blocking S100B ameliorates histopathology. Thus, levels of S100B differentially affect skeletal muscle repair upon acute injury and in the context of muscular dystrophy, and S100B might be regarded as a potential molecular target in DMD.

P.317 - Src tyrosine kinase as potential drug target in Duchenne muscular dystrophy: in vivo and in vitro preclinical studies

Src Tyrosine Kinase (TK), a redox-sensitive protein, is overexpressed in dystrophin-deficient muscles and can be overactive due to excessive production of reactive oxygen species, contributing to β-dystroglycan degradation and reinforcing damaging signaling. Thus, the pharmacological inhibition of Src TK seems a feasible strategy to ameliorate Duchenne muscular dystrophy (DMD). We focused on two Src TK inhibitors, PP2 and dasatinib. We performed a proof-of-concept preclinical study in treadmill exercised mdx mice by in vivo administration of PP2 and dasatinib at the same doses of 5 mg/kg, three times a week s.c.. The treatments, although well tolerated, had no significant efficacy on pathology-related in vivo and ex vivo parameters, even though a trend of amelioration for some functional and morphological parameters of PP2-treated mice was observed. However, an increased expression of β-dystroglycan was found by western blot for both treatments. The lack of efficacy may be related to PK issues (under evaluation) or to muscle specific drug toxicity affecting the myogenic program during regeneration. Thus we conducted in vitro studies on C2C12 murine muscle satellite cell line to evaluate the effects of the drugs on cell viability and their potential protection against oxidative stress-induced cytotoxicity. We tested increasing concentrations of PP2 (0.1–300 µM) and dasatinib (0.1–150 µM). PP2 from 3 µM, exerted a significant cytotoxic effect which was not concentration-dependent. By contrast, dasatinib showed a marked concentration-dependent decrease of cell viability. The cytotoxic effect of 300 µM H2O2 was significantly counteracted by 0.1 µM dasatinib and by 0.3–100 µM PP2. Higher concentrations of dasatinib (0.5 µM and 1 µM) were necessary to exert cytoprotection against 1 mM H2O2, while no protection was found with PP2. Our results support the interest of further studies of PP2 and dasatinib as potential treatments for DMD (Supported by Duchenne Parent Project NL_DPP).

Repression of phosphatidylinositol transfer protein α ameliorates the pathology of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by X-linked inherited mutations in the DYSTROPHIN (DMD) gene. Absence of dystrophin protein from the sarcolemma causes severe muscle degeneration, fibrosis, and inflammation, ultimately leading to cardiorespiratory failure and premature death. Although there are several promising strategies under investigation to restore dystrophin protein expression, there is currently no cure for DMD, and identification of genetic modifiers as potential targets represents an alternative therapeutic strategy. In a Brazilian golden retriever muscular dystrophy (GRMD) dog colony, two related dogs demonstrated strikingly mild dystrophic phenotypes compared with those typically observed in severely affected GRMD dogs despite lacking dystrophin. Microarray analysis of these "escaper" dogs revealed reduced expression of phosphatidylinositol transfer protein-α (PITPNA) in escaper versus severely affected GRMD dogs. Based on these findings, we decided to pursue investigation of modulation of PITPNA expression on dystrophic pathology in GRMD dogs, dystrophin-deficient sapje zebrafish, and human DMD myogenic cells. In GRMD dogs, decreased expression of Pitpna was associated with increased phosphorylated Akt (pAkt) expression and decreased PTEN levels. PITPNA knockdown by injection of morpholino oligonucleotides in sapje zebrafish also increased pAkt, rescued the abnormal muscle phenotype, and improved long-term sapje mutant survival. In DMD myotubes, PITPNA knockdown by lentiviral shRNA increased pAkt and increased myoblast fusion index. Overall, our findings suggest PIPTNA as a disease modifier that accords benefits to the abnormal signaling, morphology, and function of dystrophic skeletal muscle, and may be a target for DMD and related neuromuscular diseases.

CDK3 is a major target of miR-150 in cell proliferation and anti-cancer effect

MiR-150, a member of small non-coding RNAs, has been proven to dysregulate in different types of tumor and bear on carcinogenesis and cancer prognosis by regulating the expression of a series of gene including utrophin. Given that utrophin can compensate for dystrophin's absence and be regarded as a promising therapeutic target for Duchenne Muscular Dystrophy (DMD), we further detected the deep role of miR-150 in dystrophic muscle. Using a range of bioinformatic, molecular and cell biology techniques, we declared that miR-150 directly targets cyclin-dependent kinase 3 (CDK3) and leads to the regulation of CDK3 gene expression in both muscle-derived and non-muscle cells. The results indicated the expression of miR-150 was upregulated in mdx muscle and closely related to the lower level of CDK3. Transient transfection of miR-150 into cultured C2C12 cells led to significant decrease in cell proliferation, which is partly mediated via the 3′-UTRs of CDK3 mRNA. Targeting of CDK3 could also play a role, at least in part, in the anti-cancer activity suggested for miR-150 in previous studies. Consistently, the analysis of tumor and matched normal lung tissues indicates that miR-150 downregulation in lung tumors correlates with higher CDK3 levels. In addition, miR-150 transfection experiments with cancer-derived cell lines reveal that miR-150-mediated CDK3 suppression directly induces to growth inhibition. Collectively, our results highlight a novel activity for CDK3 in myoblast cell proliferation and confirm CDK3 as a key target that further enhances the tumor suppressor function proposed for miR-150.

Association Study of Exon Variants in the NF-κB and TGFβ Pathways Identifies CD40 as a Modifier of Duchenne Muscular Dystrophy

The expressivity of Mendelian diseases can be influenced by factors independent from the pathogenic mutation: in Duchenne muscular dystrophy (DMD), for instance, age at loss of ambulation (LoA) varies between individuals whose DMD mutations all abolish dystrophin expression. This suggests the existence of trans-acting variants in modifier genes. Common single nucleotide polymorphisms (SNPs) in candidate genes (SPP1, encoding osteopontin, and LTBP4, encoding latent transforming growth factor β [TGFβ]-binding protein 4) have been established as DMD modifiers. We performed a genome-wide association study of age at LoA in a sub-cohort of European or European American ancestry (n = 109) from the Cooperative International Research Group Duchenne Natural History Study (CINRG-DNHS). We focused on protein-altering variants (Exome Chip) and included glucocorticoid treatment as a covariate. As expected, due to the small population size, no SNPs displayed an exome-wide significant p value (< 1.8× 10-6). Subsequently, we prioritized 438 SNPs in the vicinities of 384 genes implicated in DMD-related pathways, i.e., the nuclear-factor-κB and TGFβ pathways. The minor allele at rs1883832, in the 5'-untranslated region of CD40, was associated with earlier LoA (p = 3.5× 10-5). This allele diminishes the expression of CD40, a co-stimulatory molecule for Tcell polarization. We validated this association in multiple independent DMD cohorts (United Dystrophinopathy Project, Bio-NMD, and Padova, total n = 660), establishing this locus as a DMD modifier. This finding points to cell-mediated immunity as a relevant pathogenetic mechanism and potential therapeutic target in DMD.

Elucidating the Role of Mineralocorticoid Receptors in Skeletal Muscle as a Potential Therapeutic Target for Duchenne Muscular Dystrophy

Our lab identified a potential new treatment for Duchenne Muscular Dystrophy using the mineralocorticoid receptor (MR) antagonist spironolactone and ACE inhibitor lisinopril. Drug studies using dystrophic Het (utrn+/-;mdx) mice showed a dramatic improvement in both respiratory and limb muscle force and a reduction of ongoing muscle damage, in addition to preventing cardiomyopathy. We show MR is present in both skeletal muscle tissue and myogenic cultures, supporting a direct affect by MR antagonists on skeletal muscle. Global analysis of gene expression between treated and untreated dystrophic mice identified potential molecular targets to unravel the drugs' mode of action. Preliminary microarray data comparing quadriceps muscle from lisinopril and spironolactone treated het mice to untreated controls revealed changes in the expression of several gene targets with known roles in striated muscle, Gene Name Gene Symbol Fold-change (Treated/Control) Role in Striated Muscle ankyrin repeat domain 1 Ankrd1 -2.15634 Marker of skeletal muscle pathological remodeling tight junction protein 2 Tjp2 -2.02372 Important molecular target in vascular remodeling myosin VA Myo5a -2.12497 Involved in maturation of NMJs in regenerating muscle mid1 interacting protein 1 Mid1ip1 -2.48061 Potential biomarker of skeletal myopathy myosin binding protein H Mybph 2.059236 Muscle remodeling in response to injury thyroid hormone responsive SPOT14 homolog Thrsp 2.138901 Modulator of fatty acid synthesis which have been confirmed using immunofluorescence and western blot analysis. Myogenic cultures treated with MR agonists and antagonists are being used to test whether these potential downstream targets are affected in a cell autologous manner in skeletal muscle and represent bona fide MR gene targets. NIH 1 R01 NS082868; T32 NS077984

Toll-like receptor 4 ablation in mdx mice reveals innate immunity as a therapeutic target in Duchenne muscular dystrophy.

Toll-like receptor 4 (TLR4) recognizes specific structural motifs associated with microbial pathogens and also responds to certain endogenous host molecules associated with tissue damage. In Duchenne muscular dystrophy (DMD), inflammation plays an important role in determining the ultimate fate of dystrophic muscle fibers. In this study, we used TLR4-deficient dystrophic mdx mice to assess the role of TLR4 in the pathogenesis of DMD. TLR4 expression was increased and showed enhanced activation following agonist stimulation in mdx diaphragm muscle. Genetic ablation of TLR4 led to significantly increased muscle force generation in dystrophic diaphragm muscle, which was associated with improved histopathology including decreased fibrosis, as well as reduced pro-inflammatory gene expression and macrophage infiltration. TLR4 ablation in mdx mice also altered the phenotype of muscle macrophages by inducing a shift toward a more anti-inflammatory (iNOS(neg) CD206(pos)) profile. In vitro experiments confirmed that lack of TLR4 is sufficient to influence macrophage activation status in response to classical polarizing stimuli such as IFN-gamma and IL-4. Finally, treatment of dystrophic mice with glycyrrhizin, an inhibitor of the endogenous TLR4 ligand, high mobility group box (HMGB1), also pointed to involvement of the HMGB1-TLR4 axis in promoting dystrophic diaphragm pathology. Taken together, our findings reveal TLR4 and the innate immune system as important players in the pathophysiology of DMD. Accordingly, targeting either TLR4 or its endogenous ligands may provide a new therapeutic strategy to slow disease progression.

Oral 2022-1 - Defective skeletal muscle nNOS-cGMP signaling as a therapeutic target in Duchenne muscular dystrophy

Oral 2022-1 - Defective skeletal muscle nNOS-cGMP signaling as a therapeutic target in Duchenne muscular dystrophy

G.P.120: The Sodium/Hydrogen Exchanger (NHE-1): A promising novel target for DMD

Abnormal intracellular calcium load is one of the most consistent findings in Duchenne muscular dystrophy (DMD) but previous attempts to improve the dystrophic pathology with calcium antagonists have been unsuccessful. However, there is an intriguing possibility of restoring calcium homeostasis indirectly by exploiting the relationship between the Sodium/Hydrogen Exchanger (NHE-1) and the Sodium/Calcium Exchanger (NCX). NHE-1 has been reported to be abnormally overactive in DMD models, leading to an increased influx of sodium which in turn switches the NCX into reserve mode leading to calcium influx. Using selective NHE-1 inhibitors the influx of sodium can be reduced thereby allowing the NCX to revert back to normal mode, where calcium is extruded, thus reducing the cellular calcium load. In collaboration with our industrial partner Peacock Pharma, we have demonstrated the potential of NHE-1 inhibition in the mdx mouse model for DMD by chronically treating animals with a novel NHE-1 inhibitor. The pilot study showed significantly reduced calcium uptake in treated mice (<i>n</i>=6) using manganese enhanced MRI (MEMRI). Manganese acts as a contrast agent in T1 weighted MRI and is thought to be taken up by cardiac and skeletal muscle by the same receptors as calcium. There was also an increase in functional grip strength, although not statistically significant the results are nevertheless encouraging and indicate the potential of this novel approach in DMD. This is currently being investigated more thoroughly using a greater number of animals (<i>n</i>=12) treated chronically via drug in chow over 6-months and assessed using MEMRI and grip test. In addition, in vitro calcium-flux assays (e.g. Fluo-4) are used to determine the potency and investigate the mechanism of action of this drug. The objective of our study is to provide the requisite pre-clinical data that would enable these drugs to progress towards clinical trials.

Angiogenic Impairment of the Vascular Endothelium

Objective— Dystrophin, the missing or defective protein in Duchenne muscular dystrophy, is expressed not only in muscle cells but also in vascular endothelial cells (ECs). In this study, we assessed the effects of dystrophin deficiency on the angiogenic capacities of ECs. Approach and Results— We isolated vascular ECs from mdx mice, the murine equivalent of Duchenne muscular dystrophy in humans, and wild-type controls, and we found that mdx -derived ECs have impaired angiogenic properties, in terms of migration, proliferation, and tube formation. They also undergo increased apoptosis in vitro compared with wild-type cells and have increased senescence-associated β-galactosidase activity. Mdx -derived ECs also display reduced ability to support myoblast proliferation when cocultured with satellite cell–derived primary myoblasts. These endothelial defects are mirrored by systemic impairment of angiogenesis in vivo, both on induction of ischemia, stimulation with growth factors in the corneal model and matrigel plug assays, and tumor growth. We also found that dystrophin forms a complex with endothelial NO synthase and caveolin-1 in ECs, and that NO production and cGMP formation are compromised in ECs isolated from mdx mice. Interestingly, treatment with aspirin enhances production of both cGMP and NO in dystrophic ECs, whereas low-dose aspirin improves the dystrophic phenotype of mdx mice in vivo, in terms of resistance to physical exercise, muscle fiber permeability, and capillary density. Conclusions— These findings demonstrate that impaired angiogenesis is a novel player and potential therapeutic target in Duchenne muscular dystrophy.

Identification of FHL1 as a therapeutic target for Duchenne muscular dystrophy

Utrophin is a potential therapeutic target for the fatal muscle disease, Duchenne muscular dystrophy (DMD). In adult skeletal muscle, utrophin is restricted to the neuromuscular and myotendinous junctions and can compensate for dystrophin loss in mdx mice, a mouse model of DMD, but requires sarcolemmal localization. NFATc1-mediated transcription regulates utrophin expression and the LIM protein, FHL1 which promotes muscle hypertrophy, is a transcriptional activator of NFATc1. By generating mdx/FHL1-transgenic mice, we demonstrate that FHL1 potentiates NFATc1 activation of utrophin to ameliorate the dystrophic pathology. Transgenic FHL1 expression increased sarcolemmal membrane stability, reduced muscle degeneration, decreased inflammation and conferred protection from contraction-induced injury in mdx mice. Significantly, FHL1 expression also reduced progressive muscle degeneration and fibrosis in the diaphragm of aged mdx mice. FHL1 enhanced NFATc1 activation of the utrophin promoter and increased sarcolemmal expression of utrophin in muscles of mdx mice, directing the assembly of a substitute utrophin-glycoprotein complex, and revealing a novel FHL1-NFATc1-utrophin signaling axis that can functionally compensate for dystrophin.