Dystrophin-deficient Mdx Mice Research Articles

Dystrophin-deficient dogs with reduced myostatin have unequal muscle growth and greater joint contractures.

BackgroundMyostatin (Mstn) is a negative regulator of muscle growth whose inhibition promotes muscle growth and regeneration. Dystrophin-deficient mdx mice in which myostatin is knocked out or inhibited postnatally have a less severe phenotype with greater total mass and strength and less fibrosis and fatty replacement of muscles than mdx mice with wild-type myostatin expression. Dogs with golden retriever muscular dystrophy (GRMD) have previously been noted to have increased muscle mass and reduced fibrosis after systemic postnatal myostatin inhibition. Based partly on these results, myostatin inhibitors are in development for use in human muscular dystrophies. However, persisting concerns regarding the effects of long-term and profound myostatin inhibition will not be easily or imminently answered in clinical trials.MethodsTo address these concerns, we developed a canine (GRippet) model by crossbreeding dystrophin-deficient GRMD dogs with Mstn-heterozygous (Mstn+/−) whippets. A total of four GRippets (dystrophic and Mstn+/−), three GRMD (dystrophic and Mstn wild-type) dogs, and three non-dystrophic controls from two litters were evaluated.ResultsMyostatin messenger ribonucleic acid (mRNA) and protein levels were downregulated in both GRMD and GRippet dogs. GRippets had more severe postural changes and larger (more restricted) maximal joint flexion angles, apparently due to further exaggeration of disproportionate effects on muscle size. Flexors such as the cranial sartorius were more hypertrophied on magnetic resonance imaging (MRI) in the GRippets, while extensors, including the quadriceps femoris, underwent greater atrophy. Myostatin protein levels negatively correlated with relative cranial sartorius muscle cross-sectional area on MRI, supporting a role in disproportionate muscle size. Activin receptor type IIB (ActRIIB) expression was higher in dystrophic versus control dogs, consistent with physiologic feedback between myostatin and ActRIIB. However, there was no differential expression between GRMD and GRippet dogs. Satellite cell exhaustion was not observed in GRippets up to 3 years of age.ConclusionsPartial myostatin loss may exaggerate selective muscle hypertrophy or atrophy/hypoplasia in GRMD dogs and worsen contractures. While muscle imbalance is not a feature of myostatin inhibition in mdx mice, findings in a larger animal model could translate to human experience with myostatin inhibitors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0085-7) contains supplementary material, which is available to authorized users.

Electrical Impedance Myography to Detect the Effects of Electrical Muscle Stimulation in Wild Type and Mdx Mice.

ObjectiveTools to better evaluate the impact of therapy on nerve and muscle disease are needed. Electrical impedance myography (EIM) is sensitive to neuromuscular disease progression as well as to therapeutic interventions including myostatin inhibition and antisense oligonucleotide-based treatments. Whether the technique identifies the impact of electrical muscle stimulation (EMS) is unknown.MethodsTen wild-type (wt) C57B6 mice and 10 dystrophin-deficient (mdx) mice underwent 2 weeks of 20 min/day EMS on left gastrocnemius and sham stimulation on the right gastrocnemius. Multifrequency EIM data and limb girth were obtained before and at the conclusion of the protocol. Muscle weight, in situ force measurements, and muscle fiber histology were also assessed at the conclusion of the study.ResultsAt the time of sacrifice, muscle weight was greater on the EMS-treated side than on the sham-stimulated side (p = 0.018 for wt and p = 0.007 for mdx). Similarly, in wt animals, EIM parameters changed significantly compared to baseline (resistance (p = 0.009), reactance (p = 0.0003) and phase (p = 0.002); these changes were due in part to reductions in the EIM values on the EMS-treated side and elevations on the sham-simulated side. Mdx animals showed analogous but non-significant changes (p = 0.083, p = 0.064, and p = 0.57 for resistance, reactance and phase, respectively). Maximal isometric force trended higher on the stimulated side in wt animals only (p = 0.06). Myofiber sizes in wt animals were also larger on the stimulated side than on the sham-stimulated side (p = 0.034); no significant difference was found in the mdx mice (p = 0.79).ConclusionEIM is sensitive to stimulation-induced muscle alterations in wt animals; similar trends are also present in mdx mice. The mechanisms by which these EIM changes develop, however, remains uncertain. Possible explanations include longer-term trophic effects and shorter-term osmotic effects.

In vivo neutralization of IL-6 receptors ameliorates gastrointestinal dysfunction in dystrophin-deficient mdx mice.

Duchenne muscular dystrophy (DMD) is a fatal disease characterized by progressive deterioration and degeneration of striated muscle. A mutation resulting in the loss of dystrophin, a structural protein which protects cells from contraction-induced damage, underlies DMD pathophysiology. Damage to muscle fibers results in chronic inflammation and elevated levels of proinflammatory cytokines such as interleukin-6 (IL-6). However, loss of cellular dystrophin also affects neurons and smooth muscle in the gastrointestinal (GI) tract with complaints such as hypomotility, pseudo-obstruction, and constipation reported in DMD patients. Using dystrophin-deficient mdx mice, studies were carried out to examine colonic morphology and function compared with wild-type mice. Treatment with neutralizing IL-6 receptor antibodies (xIL-6R) and/or the corticotropin-releasing factor (CRF) 2 receptor agonist, urocortin 2 (uro2) was tested to determine if they ameliorated GI dysfunction in mdx mice. Mdx mice exhibited thickening of colonic smooth muscle layers and delayed stress-induced defecation. In organ bath studies, neurally mediated IL-6-evoked contractions were larger in mdx colons. In vivo treatment of mdx mice with xIL-6R normalized defecation rates and colon lengths. Uro2 treatment did not affect motility or morphology. The potentiated colonic contractile response to IL-6 was attenuated by treatment with xIL-6R. These findings confirm the importance of dystrophin in normal GI function and implicate IL-6 as an important regulator of GI motility in the mdx mouse. Inhibition of IL-6 signaling may offer a potential new therapeutic strategy for treating DMD-associated GI symptoms.

Diaphragm degeneration and cardiac structure in mdx mouse: potential clinical implications for Duchenne muscular dystrophy.

We examined the effects of exercise on diaphragm degeneration and cardiomyopathy in dystrophin-deficient mdx mice. Mdx mice (11months of age) were exercised (swimming) for 2months to worsen diaphragm degeneration. Control mdx mice were kept sedentary. Morphological evaluation demonstrated increased fibrosis in the diaphragm of exercised mdx mice (33.3±6.0% area of fibrosis) compared with control mdx mice (20.9±1.7% area of fibrosis). Increased (26%) activity of MMP-2, a marker of fibrosis, was detected in the diaphragms from exercised mdx mice. Morphological evaluation of the heart demonstrated a 45% increase in fibrosis in the right ventricle (8.3±0.6% in sedentary vs. 12.0±0.6% of fibrosis in exercised) and in the left ventricle (35% increase) in the exercised mdx mice. The density of inflammatory cells-degenerating cardiomyocytes increased 95% in the right ventricle (2.3±0.6 in sedentary vs. 4.5±0.8 in exercised) and 71% in the left ventricle (1.4±0.6 sedentary vs. 2.4±0.5 exercised). The levels of both active MMP-2 and the pro-fibrotic factor transforming growth factor beta were elevated in the hearts of exercised compared with sedentary mdx mice. The wall thicknesstolumen diameter ratio of the pulmonary trunk was significantly increased in the exercised mdx mice (0.11±0.04 in sedentary vs. 0.28±0.12 in exercised), as was the thickness of the right ventricle wall, which suggests the occurrence of pulmonary hypertension in those animals. It is suggested that diaphragm degeneration is a main contributor to right ventricle dystrophic pathology. These findings may be relevant for future interventional studies for Duchenne muscular dystrophy-associated cardiomyopathy.

The Role of Sulforaphane on Duchenne Muscular Dystrophy by Activation of Nrf2

Sulforaphane (SFN) possesses powerful chemo-preventive effects and plays a crucial role on oxidative stress and inflammatory. In our recent study, SFN treatment could relieve muscular dystrophy in mdx mice by activating Nrf2 (NF-E2 related factor 2). Moreover, our findings indicated that SFN-activated Nrf2 alleviated muscle inflammation in dystrophin-deficient mdx mice through suppressing NF-κB signaling pathway. Collectively, SFNinduced Nrf2 molecular pathway might be a promising approach for treatment of the patients with Duchenne muscular dystrophy.

PC-FACS

PC-FACS

Wnt signaling pathway improves central inhibitory synaptic transmission in a mouse model of Duchenne muscular dystrophy

The dystrophin-associated glycoprotein complex (DGC) that connects the cytoskeleton, plasma membrane and the extracellular matrix has been related to the maintenance and stabilization of channels and synaptic receptors, which are both essential for synaptogenesis and synaptic transmission. The dystrophin-deficient (mdx) mouse model of Duchenne muscular dystrophy (DMD) exhibits a significant reduction in hippocampal GABA efficacy, which may underlie the altered synaptic function and abnormal hippocampal long-term plasticity exhibited by mdx mice. Emerging studies have implicated Wnt signaling in the modulation of synaptic efficacy, neuronal plasticity and cognitive function. We report here that the activation of the non-canonical Wnt-5a pathway and Andrographolide, improves hippocampal mdx GABAergic efficacy by increasing the number of inhibitory synapses and GABAA receptors or GABA release. These results indicate that Wnt signaling modulates GABA synaptic efficacy and could be a promising novel target for DMD cognitive therapy.

Comparison of Experimental Protocols of Physical Exercise for mdx Mice and Duchenne Muscular Dystrophy Patients

Duchenne Muscular Dystrophy (DMD) is caused by mutations in the gene coding for dystrophin and leads to muscle degeneration, wheelchair dependence and death by cardiac or respiratory failure. Physical exercise has been proposed as a palliative therapy for DMD to maintain muscle strength and prevent contractures for as long as possible. However, its practice remains controversial because the benefits of training may be counteracted by muscle overuse and damage.The effects of physical exercise have been investigated in muscles of dystrophin-deficient mdx mice and in patients with DMD. However, a lack of uniformity among protocols limits comparability between studies and translatability of results from animals to humans. In the present review, we summarize and discuss published protocols used to investigate the effects of physical exercise on mdx mice and DMD patients, with the objectives of improving comparability between studies and identifying future research directions.

Altered social behavior and ultrasonic communication in the dystrophin-deficient mdx mouse model of Duchenne muscular dystrophy.

BackgroundThe Duchenne and Becker muscular dystrophies (DMD, BMD) show significant comorbid diagnosis for autism, and the genomic sequences encoding the proteins responsible for these diseases, the dystrophin and associated proteins, have been proposed as new candidate risk loci for autism. Dystrophin is expressed not only in muscles but also in central inhibitory synapses in the cerebellum, hippocampus, amygdala, and cerebral cortex, where it contributes to the organization of autism-associated trans-synaptic neurexin-neuroligin complexes and to the clustering of synaptic gamma-aminobutyric acid (GABA)A receptors. While brain defects due to dystrophin loss are associated with deficits in cognitive and executive functions, communication skills and social behavior, only a subpopulation of DMD patients meet the criteria for autism, suggesting that mutations in the dystrophin gene may confer a vulnerability to autism. The loss of dystrophin in the mdx mouse model of DMD has been associated with cognitive and emotional alterations, but social behavior and communication abilities have never been studied in this model.MethodsHere, we carried out the first in-depth analysis of social behavior and ultrasonic communication in dystrophin-deficient mdx mice, using a range of socially relevant paradigms involving various degrees of executive and cognitive demands, from simple presentation of sexual olfactory stimuli to social choice situations and direct encounters with female and male mice of various genotypes.ResultsWe identified context-specific alterations in social behavior and ultrasonic vocal communication in mdx mice during direct encounters in novel environments. Social behavior disturbances depended on intruders’ genotype and behavior, suggesting alterations in executive functions and adaptive behaviors, and were associated with selective alterations of the development, rate, acoustic properties, and use of the ultrasonic vocal repertoire.ConclusionsThis first evidence that a mutation impeding expression of brain dystrophin affects social behavior and communication sheds new light on critical cognitive, emotional, and conative factors contributing to the development of autistic-like traits in this disease model.Electronic supplementary materialThe online version of this article (doi:10.1186/s13229-015-0053-9) contains supplementary material, which is available to authorized users.

G.P.363 - Glucose uptake and mitochondrial function following 8 weeks of dietary nitrate supplementation in the dystrophin-deficient mdx mouse

G.P.363 - Glucose uptake and mitochondrial function following 8 weeks of dietary nitrate supplementation in the dystrophin-deficient mdx mouse

Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy.

During embryonic development, skeletal muscles arise from somites, which derive from the presomitic mesoderm (PSM). Using PSM development as a guide, we establish conditions for the differentiation of monolayer cultures of mouse embryonic stem (ES) cells into PSM-like cells without the introduction of transgenes or cell sorting. We show that primary and secondary skeletal myogenesis can be recapitulated in vitro from the PSM-like cells, providing an efficient, serum-free protocol for the generation of striated, contractile fibers from mouse and human pluripotent cells. The mouse ES cells also differentiate into Pax7(+) cells with satellite cell characteristics, including the ability to form dystrophin(+) fibers when grafted into muscles of dystrophin-deficient mdx mice, a model of Duchenne muscular dystrophy (DMD). Fibers derived from ES cells of mdx mice exhibit an abnormal branched phenotype resembling that described in vivo, thus providing an attractive model to study the origin of the pathological defects associated with DMD.

Muscle hypertrophy induced by myostatin inhibition accelerates degeneration in dysferlinopathy.

Myostatin is a secreted signaling molecule that normally acts to limit muscle growth. As a result, there is extensive effort directed at developing drugs capable of targeting myostatin to treat patients with muscle loss. One potential concern with this therapeutic approach in patients with muscle degenerative diseases like muscular dystrophy is that inducing hypertrophy may increase stress on dystrophic fibers, thereby accelerating disease progression. To investigate this possibility, we examined the effect of blocking the myostatin pathway in dysferlin-deficient (Dysf−/−) mice, in which membrane repair is compromised, either by transgenic expression of follistatin in skeletal muscle or by systemic administration of the soluble form of the activin type IIB receptor (ACVR2B/Fc). Here, we show that myostatin inhibition by follistatin transgene expression in Dysf−/− mice results in early improvement in histopathology but ultimately exacerbates muscle degeneration; this effect was not observed in dystrophin-deficient (mdx) mice, suggesting that accelerated degeneration induced by follistatin transgene expression is specific to mice lacking dysferlin. Dysf−/− mice injected with ACVR2B/Fc showed significant increases in muscle mass and amelioration of fibrotic changes normally seen in 8-month-old Dysf−/− mice. Despite these potentially beneficial effects, ACVR2B/Fc treatment caused increases in serum CK levels in some Dysf−/− mice, indicating possible muscle damage induced by hypertrophy. These findings suggest that depending on the disease context, inducing muscle hypertrophy by myostatin blockade may have detrimental effects, which need to be weighed against the potential gains in muscle growth and decreased fibrosis.

Lmo7 is dispensable for skeletal muscle and cardiac function.

Emery-Dreifuss muscular dystrophy (EDMD) is a degenerative disease primarily affecting skeletal muscles in early childhood as well as cardiac muscle at later stages. EDMD is caused by a number of mutations in genes encoding proteins associated with the nuclear envelope (e.g., Emerin, Lamin A/C, and Nesprin). Recently, a novel protein, Lim-domain only 7 (lmo7) has been reported to play a role in the molecular pathogenesis of EDMD. Prior in vitro and in vivo studies suggested the intriguing possibility that Lmo7 plays a role in skeletal or cardiac muscle pathophysiology. To further understand the in vivo role of Lmo7 in striated muscles, we generated a novel Lmo7-null (lmo7(-/-)) mouse line. Using this mouse line, we examined skeletal and cardiac muscle physiology, as well as the role of Lmo7 in a model of muscular dystrophy and regeneration using the dystrophin-deficient mdx mouse model. Our results demonstrated that lmo7(-/-) mice had no abnormalities in skeletal muscle morphology, physiological function, or regeneration. Cardiac function was also unaffected. Moreover, we found that ablation of lmo7 in mdx mice had no effect on the observed myopathy and muscular regeneration exhibited by mdx mice. Molecular analyses also showed no changes in dystrophin complex factors, MAPK pathway components, and Emerin levels in lmo7 knockout mice. Taken together, we conclude that Lmo7 is dispensable for skeletal muscle and cardiac physiology and pathophysiology.

The effects of resveratrol and SIRT1 activation on dystrophic cardiomyopathy.

The muscular dystrophies, which cause progressive weakening of the skeletal muscles, are frequently associated with cardiomyopathy. In fact, the leading cause of mortality in patients with Duchenne muscular dystrophy, the most common and most severe type of muscular dystrophy, is heart failure due to cardiomyopathy. Therefore, more effective methods for treating cardiomyopathy are expected to improve long-term outcomes for patients with Duchenne muscular dystrophy. Our recent preclinical data show that treatment with the SIRT1 activator resveratrol is beneficial for dystrophic cardiomyopathy. We examined the effects of resveratrol treatment in two different models of muscular dystrophy: dystrophin-deficient mdx mice and δ-sarcoglycan-deficient TO-2 hamsters. In both models, resveratrol suppressed cardiac hypertrophy, preserved cardiac function, and reduced tissue fibrosis in the diseased heart. Importantly, resveratrol significantly improved survival in TO-2 hamsters. Resveratrol also attenuated skeletal muscle pathology in mdx mice. These promising results indicate resveratrol's potential for clinical translation to treat cardiomyopathy in patients with muscular dystrophies.

559. Spell Checking Nature: A Therapeutic Use of the CRISPR/Cas9 System in Duchenne Muscular Dystrophy

Recently the high precision genome-modifying technology, originated from a prokaryotic defense mechanism called the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system, has been harnessed as a tool for programmable, high precision gene modification in eukaryotes. Thus far it has been shown to be highly effective in inactivating genes, however little is known about the gene editing and regulation capabilities of this system. As a proof of concept we utilized CRISPR/Cas9 system to study three different therapeutic approaches for Duchenne Muscular Dystrophy (DMD), a devastating X-linked neuromuscular disorder.The first approach is aimed at approximately 26% of patients that have point mutations in DMD gene. Here we used gene editing to correct DMD E2035X, a pathogenic mutation in DMD patient myoblasts by designing short RNA sequences complementary to the locus called guides, paired with the Streptococcus pyogenes Cas9. We were able to generate targeted double stranded DNA breaks that were corrected through homology directed repair using single stranded oligos carrying the corrected DNA sequence. Other pathogenic DMD mutations are duplications, which are found in approximately 13% of DMD patients. Utilizing a modified CRISPR strategy that involves a double nuclease approach we deleted a 145 kb (Exons 18-30) duplication and restored dystrophin expression in patient cells. Furthermore, as an universal, mutation-independent treatment approach, we have also utilized a third modified CRISPR system to upregulate utrophin expression to compensate for the loss of dystrophin. In DMD patient myoblasts, we utilized S.pyogenes Cas9, fused to transcriptional activator VP160 to target utrophin promoter. We demonstrated 2.5-5 fold utrophin upregulation resulting in increased expression of β-dystroglycan, providing evidence for functional significance of this strategy.Lastly, in order to determine if this tool is of potential clinical significance, we have began to interrogate CRISPR/Cas9 system in dystrophin-deficient mdx mouse. We have developed strategies for the delivery of CRISPR machinery via rAAV with the goal to achieve dystrophin editing and utrophin upregulation simultaneously in vivo. Taken together, our proof of concept studies provide insights into the potentially far-reaching impact of the therapeutic benefits of the CRISPR system that can be adapted to target a variety of mutations in DMD.

Targeting Mechanically‐Activated NOS Signaling in Dystrophic Cardiomyopathy

Patients deficient in dystrophin, a protein that links the cytoskeleton to the extracellular matrix via the dystrophin‐glycoprotein complex (DGC), exhibit muscular dystrophy, cardiomyopathy, and impaired muscle nitric oxide (NO) production. Our objective was to identify the mechanisms of how the DGC regulates cardiac muscle nitric oxide synthase (NOS) activity. Isolated adult wild‐type (WT) or dystrophin‐deficient mdx mouse cardiomyocytes were plated on deformable membranes, and live‐cell DAF‐FM dye fluorescence and biochemical approaches were used to investigate how the DGC regulates mechanically‐activated NO signaling. 1Hz cyclic stretch of WT cardiomyocytes significantly increased NO production and neuronal NOS (nNOS) S1412 phosphorylation. In contrast, stretch‐induced NO production and nNOS phosphorylation were impaired in mdx cardiomyocytes, suggesting that dystrophin regulates the mechanical activation of NOS. We found that inhibition of AMP‐activated protein kinase (AMPK) inhibited stretch‐induced NO production in WT cardiomyocytes, indicating that AMPK plays a critical role in mechanoactivation of NOS. Importantly, direct pharmacological activation of AMPK increased nNOS S1412 phosphorylation and was sufficient to enhance NO production in mdx cardiomyocytes. We conclude that the DGC regulates the mechanical activation of cardiac NOS, and that pharmacological AMPK activation may be a therapeutic strategy to restore NOS activity in dystrophin‐deficient cardiomyocytes. Supported by the Muscular Dystrophy Association, the University of Michigan CVRE Training Program, and NIH T32 GM‐08322.

What has the mdx mouse model of Duchenne muscular dystrophy contributed to our understanding of this disease?

Duchenne muscular dystrophy (DMD) is a fatal X-chromosome linked recessive disorder caused by the truncation or deletion of the dystrophin gene. The most widely used animal model of this disease is the dystrophin-deficient mdx mouse which was first discovered 30 years ago. Despite its extensive use in DMD research, no effective treatment has yet been developed for this devastating disease. This review explores what we have learned from this mouse model regarding the pathophysiology of DMD and asks if it has a future in providing a better more thorough understanding of this disease or if it will bring us any closer to improving the outlook for DMD patients.

Low intensity training of mdx mice reduces carbonylation and increases expression levels of proteins involved in energy metabolism and muscle contraction

High intensity training induces muscle damage in dystrophin-deficient mdx mice, an animal model for Duchenne muscular dystrophy. However, low intensity training (LIT) rescues the mdx phenotype and even reduces the level of protein carbonylation, a marker of oxidative damage. Until now, beneficial effects of LIT were mainly assessed at the physiological level. We investigated the effects of LIT at the molecular level on 8-week-old wild-type and mdx muscle using 2D Western blot and protein–protein interaction analysis. We found that the fast isoforms of troponin T and myosin binding protein C as well as glycogen phosphorylase were overcarbonylated and downregulated in mdx muscle. Some of the mitochondrial enzymes of the citric acid cycle were overcarbonylated, whereas some proteins of the respiratory chain were downregulated. Of functional importance, ATP synthase was only partially assembled, as revealed by Blue Native PAGE analysis. LIT decreased the carbonylation level and increased the expression of fast isoforms of troponin T and of myosin binding protein C, and glycogen phosphorylase. In addition, it increased the expression of aconitate hydratase and NADH dehydrogenase, and fully restored the ATP synthase complex. Our study demonstrates that the benefits of LIT are associated with lowered oxidative damage as revealed by carbonylation and higher expression of proteins involved in energy metabolism and muscle contraction. Potentially, these results will help to design therapies for DMD based on exercise mimicking drugs.

Peptide Nucleic Acid Promotes Systemic Dystrophin Expression and Functional Rescue in Dystrophin-deficient mdx Mice.

Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise for Duchenne muscular dystrophy (DMD) patients. However, recent failure with drisapersen, an AO candidate drug in phase 3 trial, highlights the importance of exploring other effective AO chemistries for DMD. Previously, we demonstrated the appreciable biological activity of peptide nucleic acid (PNA) AOs in restoring dystrophin expression in dystrophin-deficient mdx mice intramuscularly. Here, we further explore the systemic potential and feasibility of PNA AOs in mediating exon skipping in mdx mice as a comprehensive systemic evaluation remains lacking. Systemic delivery of PNA AOs resulted in therapeutic level of dystrophin expression in body-wide peripheral muscles and improved dystrophic pathology in mdx mice without any detectable toxicity. Up to 40% of dystrophin restoration was achieved in gastrocnemius, to a less extent with other skeletal muscles, with no dystrophin in heart. Notably, comparable systemic activity was obtained between PNA AOs and phosphorodiamidate morpholino oligomer, a DMD AO chemistry in phase 3 clinical trial, under an identical dosing regimen. Overall, our data demonstrate that PNA is viable for DMD exon-skipping therapeutics with 20 mer showing the best combination of activity, solubility, and safety and further modifications to increase PNA aqueous solubility can enable longer, more effective therapeutics without the associated toxicity.

Defects in mitochondrial ATP synthesis in dystrophin-deficient mdx skeletal muscles may be caused by complex I insufficiency.

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