Adult Mdx Mice Research Articles

Aspects of Respiratory Control in the Dystrophin‐deficient mdx Mouse

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease characterised by skeletal muscle weakness which extends to the respiratory musculature. Although deficits in the diaphragm muscle of dystrophin‐deficient mdx mice are well described, there is a paucity of information pertaining to central control of breathing in dystrophinopathies. We have previously reported hypoventilation in mdx mice with reserved capacity to increase ventilation in response to chemoactivation (Burns et al., 2017). Systemic and muscle inflammation are reported in mdx mice and human DMD. We sought to examine aspects of respiratory system control in the mdx mouse model of DMD including electrical activity of the diaphragm muscle and markers of inflammation in putative phrenic motor nuclei.Young adult (8 week old) wild‐type (WT) and mdx mice were studied. Inspiratory (Ti) and expiratory (Te) durations were assessed in conscious WT and mdx mice using whole‐body plethysmograpghy. Diaphragm EMG activity was assessed in WT and mdx mice using fine concentric needle electrodes in anaesthetised spontaneously breathing mice. Basal EMG and responsiveness to chemoactivation were determined. Diaphragm muscle function was examined ex vivo. Cervical spinal cord (C3–C5 region) segments of WT and mdx mice were examined for immunohistochemical and gene expression analyses.Poincaré analysis of Ti and Te revealed no evidence of perturbed respiratory rhythm in young mdx mice based on short‐ and long‐term variability of breathing. Diaphragm muscle force was severely depressed in mdx diaphragm within the frequency range of both ventilatory and airway protective behaviours. Mdx diaphragm had potentiated EMG activity during chemoactivation compared to WT mice. Dystrophin mRNA is expressed in the C3–C5 region of WT spinal cords and expression is significantly reduced in mdx mice, with no indication of altered inflammatory gene expression. Putative phrenic motornuclei in mdx mice had unaltered expression of microglia and astrocytes compared to WT.Our study revealed a potentiated neural drive to breathe in mdx mice during chemoactivation suggesting compensatory neuroplasticity facilitating respiratory motor output. Although systemic and muscle inflammation are present in mdx mice, there is no evidence of neuroinflammation in the cervical spinal cord (C3–C5) of young mdx mice. If neuroplasticity is a feature of DMD, it may be possible to boost motor facilitation of breathing through safe interventions and thus preserve or limit deficiencies in respiratory capacity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Calcium current properties in dystrophin-deficient ventricular cardiomyocytes from aged mdx mice.

Duchenne muscular dystrophy (DMD), caused by mutations in the gene encoding for the cytoskeletal protein dystrophin, is linked with severe cardiac complications including cardiomyopathy development and cardiac arrhythmias. We and others recently reported that currents through L‐type calcium (Ca) channels were significantly increased, and channel inactivation was reduced in dystrophin‐deficient ventricular cardiomyocytes derived from the mdx mouse, the most commonly used animal model for human DMD. These gain‐of‐function Ca channel abnormalities may enhance the risk of Ca‐dependent arrhythmias and cellular Ca overload in the dystrophic heart. All studies, which have so far investigated L‐type Ca channel properties in dystrophic cardiomyocytes, have used hearts from either neonatal or young adult mdx mice as cell source. In consequence, the dimension of the Ca channel abnormalities present in the severely‐diseased aged dystrophic heart has remained unknown. Here, we have studied potential abnormalities in Ca currents and intracellular Ca transients in ventricular cardiomyocytes derived from aged dystrophic mdx mice. We found that both the L‐type and T‐type Ca current properties of mdx cardiomyocytes were similar to those of myocytes derived from aged wild‐type mice. Accordingly, Ca release from the sarcoplasmic reticulum was normal in cardiomyocytes from aged mdx mice. This suggests that, irrespective of the presence of a pronounced cardiomyopathy in aged mdx mice, Ca currents and Ca release in dystrophic cardiomyocytes are normal. Finally, our data imply that dystrophin‐ regulation of L‐type Ca channel function in the heart is lost during aging.

Treatment of Adult mdx Mice with Phosphorodiamidate Morpholino Oligomer Restores Cardiac Mitochondrial Energetics and Prevents the Dystrophic Cardiomyopathy

Duchenne muscular dystrophy (DMD) occurs due to the absence of dystrophin, and is associated with a fatal dilated cardiomyopathy. DMD-associated cardiomyopathy is underpinned by disrupted cytoskeletal architecture and poor energy metabolism. The L-type Ca2+ channel (LTCC) plays a critical role facilitating Ca2+ influx required for contraction, but also regulates mitochondrial function. This crosstalk is mediated via the cytoskeleton, and is disrupted in a mouse model of DMD (mdx). We previously demonstrated that treatment of pre-cardiomyopathic mdx mice with 10mg/kg/wk phosphorodiamidate morpholino oligomer (PMO) to induce skipping of exon 23 (M23D) for 24wks partially restored increases in mitochondrial membrane potential (Ψm) and metabolic activity in response to activation of LTCC in myocytes isolated from pre-cardiomyopathic mice. Here we investigated the effect of a short-term, but high PMO treatment dose, namely 120mg/kg/wk for 3wks, on alterations in Ψm and metabolic activity. Treatment resulted in partial restoration of Ψm and metabolic activity. Based on these findings we investigated whether a long-term and high PMO treatment dose would more effectively restore Ψm and metabolic activity to wt levels. 24wk old pre-cardiomyopathic mdx mice were treated with 120mg/kg/wk for 19wks. Positive exon skipping was verified via RT-PCR, and immunohistochemistry confirmed the presence of dystrophin positive fibers within mdx heart tissue. PMO treatment completely restored Ψm to wt levels (wt untreated: 30.9±4.2%, n=11; mdx untreated: 1.6±0.8%, n=8; mdx treated: 31.6±2.6%, n=7). PMO treatment also completely restored metabolic activity (assessed as formation of formazan from tetrazolium salt) (wt untreated: 48.9±6.4%, n=15; mdx untreated: 3.0±1.3%, n=11; mdx treated: 64.9±14.0%, n=26). Furthermore, PMO treatment prevented development of the mdx cardiomyopathy assessed using serial echocardiography. These data indicate that treatment with M23D restores cardiac mitochondrial energetics in adult mdx mice. We propose that restoring mitochondrial function may play a vital role in mediating PMO-induced improvements in contractility.

Systemic Antisense Therapeutics for Dystrophin and Myostatin Exon Splice Modulation Improve Muscle Pathology of Adult mdx Mice.

Antisense-mediated exon skipping is a promising approach for the treatment of Duchenne muscular dystrophy (DMD), a rare life-threatening genetic disease due to dystrophin deficiency. Such an approach can restore the disrupted reading frame of dystrophin pre-mRNA, generating a truncated form of the protein. Alternatively, antisense therapy can be used to induce destructive exon skipping of myostatin pre-mRNA, knocking down myostatin expression to enhance muscle strength and reduce fibrosis. We have reported previously that intramuscular or intraperitoneal antisense administration inducing dual exon skipping of dystrophin and myostatin pre-mRNAs was beneficial in mdx mice, a mouse model of DMD, although therapeutic effects were muscle type restricted, possibly due to the delivery routes used. Here, following systemic intravascular antisense treatment, muscle strength and body activity of treated adult mdx mice increased to the levels of healthy controls. Importantly, hallmarks of muscular dystrophy were greatly improved in mice receiving the combined exon-skipping therapy, as compared to those receiving dystrophin antisense therapy alone. Our results support the translation of antisense therapy for dystrophin restoration and myostatin inhibition into the clinical setting for DMD.

Eliminating Nox2 reactive oxygen species production protects dystrophic skeletal muscle from pathological calcium influx assessed in vivo by manganese-enhanced magnetic resonance imaging.

Inhibiting Nox2 reactive oxygen species (ROS) production reduced in vivo calcium influx in dystrophic muscle. The lack of Nox2 ROS production protected against decreased in vivo muscle function in dystrophic mice. Manganese-enhanced magnetic resonance imaging (MEMRI) was able to detect alterations in basal calcium levels in skeletal muscle and differentiate disease status. Administration of Mn2+ did not affect muscle function or the health of the animal, and Mn2+ was cleared from skeletal muscle rapidly. We conclude that MEMRI may be a viable, non-invasive technique to monitor molecular alterations in disease progression and evaluate the effectiveness of potential therapies for Duchenne muscular dystrophy. Duchenne muscular dystrophy (DMD) is an X-linked progressive degenerative disease resulting from a mutation in the gene that encodes dystrophin, leading to decreased muscle mechanical stability and force production. Increased Nox2 reactive oxygen species (ROS) production and sarcolemmal Ca2+ influx are early indicators of disease pathology, and eliminating Nox2 ROS production reduces aberrant Ca2+ influx in young mdx mice, a model of DMD. Various imaging modalities have been used to study dystrophic muscle in vivo; however, they are based upon alterations in muscle morphology or inflammation. Manganese has been used for indirect monitoring of calcium influx across the sarcolemma and may allow detection of molecular alterations in disease progression in vivo using manganese-enhanced magnetic resonance imaging (MEMRI). Therefore, we hypothesized that eliminating Nox2 ROS production would decrease calcium influx in adult mdx mice and that MEMRI would be able to monitor and differentiate disease status in dystrophic muscle. Both in vitro and in vivo data demonstrate that eliminating Nox2 ROS protected against aberrant Ca2+ influx and improved muscle function in dystrophic muscle. MEMRI was able to differentiate between different pathological states in vivo, with no long-term effects on animal health or muscle function. We conclude that MEMRI is a viable, non-invasive technique to differentiate disease status and might provide a means to monitor and evaluate the effectiveness of potential therapies in dystrophic muscle.

Validation of ultrasonography for non-invasive assessment of diaphragm function in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, degenerative muscle disease that is commonly studied using the mdx mouse. The mdx diaphragm muscle closely mimics the pathophysiological changes in DMD muscles. mdx diaphragm force is commonly assessed ex vivo, precluding time course studies. Here we used ultrasonography to evaluate time-dependent changes in diaphragm function in vivo, by measuring diaphragm movement amplitude. In mdx mice, diaphragm amplitude decreased with age and values were much lower than for wild-type mice. Importantly, diaphragm amplitude strongly correlated with ex vivo specific force values. Micro-dystrophin administration increased mdx diaphragm amplitude by 26% after 4weeks. Diaphragm amplitude correlated positively with ex vivo force values and negatively with diaphragm fibrosis, a major cause of DMD muscle weakness. These studies validate diaphragm ultrasonography as a reliable technique for assessing time-dependent changes in mdx diaphragm function in vivo. This technique will be valuable for testing potential therapies for DMD. Duchenne muscular dystrophy (DMD) is a severe, degenerative muscle disease caused by dystrophin mutations. The mdx mouse is a widely used animal model of DMD. The mdx diaphragm muscle most closely recapitulates key features of DMD muscles, including progressive fibrosis and considerable force loss. Diaphragm function in mdx mice is commonly evaluated by specific force measurements ex vivo. While useful, this method only measures force from a small muscle sample at one time point. Therefore, accurate assessment of diaphragm function in vivo would provide an important advance to study the time course of functional decline and treatment benefits. Here, we evaluated an ultrasonography technique for measuring time-dependent changes of diaphragm function in mdx mice. Diaphragm movement amplitude values for mdx mice were considerably lower than those for wild-type, decreased from 8 to 18months of age, and correlated strongly with ex vivo specific force. We then investigated the time course of diaphragm amplitude changes following administration of an adeno-associated viral vector expressing Flag-micro-dystrophin (AAV-μDys) to young adult mdx mice. Diaphragm amplitude peaked 4weeks after AAV-μDys administration, and was 26% greater than control mdx mice at this time. This value decreased slightly to 21% above mdx controls after 12weeks of treatment. Importantly, diaphragm amplitude again correlated strongly with ex vivo specific force. Also, diaphragm amplitude and specific force negatively correlated with fibrosis levels in the muscle. Together, our results validate diaphragm ultrasonography as a reliable technique for assessing time-dependent changes in dystrophic diaphragm function in vivo, and for evaluating potential therapies for DMD.

Optimization of Internally Deleted Dystrophin Constructs.

Duchenne muscular dystrophy (DMD) is a severe, genetic muscle disease caused by the absence of the sarcolemmal protein dystrophin. Gene replacement therapy is considered a potential strategy for the treatment of DMD, aiming to restore the missing protein. Although the elements of the dystrophin molecule have been identified and studies in transgenic mdx mice have explored the importance of a number of these structural domains, the resulting modified dystrophin protein products that have been developed so far are only partially characterized in relation to their structure and function in vivo. To optimize a dystrophin cDNA construct for therapeutic application we designed and produced four human minidystrophins within the packaging capacity of lentiviral vectors. Two novel minidystrophins retained the centrally located neuronal nitric oxide synthase (nNOS)-anchoring domain in order to achieve sarcolemmal nNOS restoration, which is lost in most internally deleted dystrophin constructs. Functionality of the resulting truncated dystrophin proteins was investigated in muscle of adult dystrophin-deficient mdx mice followed by a battery of detailed immunohistochemical and morphometric tests. This initial assessment aimed to determine the overall suitability of various constructs for cloning into lentiviral vectors for ex vivo gene delivery to stem cells for future preclinical studies.

Increased levels of interleukin-6 exacerbate the dystrophic phenotype in mdx mice.

Duchenne muscular dystrophy (DMD) is characterized by progressive lethal muscle degeneration and chronic inflammatory response. The mdx mouse strain has served as the animal model for human DMD. However, while DMD patients undergo extensive necrosis, the affected muscles of adult mdx mice rapidly regenerates and regains structural and functional integrity. The basis for the mild effects observed in mice compared with the lethal consequences in humans remains unknown. In this study, we provide evidence that interleukin-6 (IL-6) is causally linked to the pathogenesis of muscular dystrophy. We report that forced expression of IL-6, in the adult mdx mice, recapitulates the severe phenotypic characteristics of DMD in humans. Increased levels of IL-6 exacerbate the dystrophic muscle phenotype, sustaining inflammatory response and repeated cycles of muscle degeneration and regeneration, leading to exhaustion of satellite cells. The mdx/IL6 mouse closely approximates the human disease and more faithfully recapitulates the disease progression in humans. This study promises to significantly advance our understanding of the pathogenic mechanisms that lead to DMD.

Respiratory Function in the Mdx Mouse Model of Duchenne Muscular Dystrophy: Role of Hypoxia, Stress and Immune Factors

Duchenne muscular dystrophy (DMD) is a genetic disease caused by defects in the dystrophin gene. Patients have severe progressive respiratory muscle dysfunction due to progressive weakness and fibrosis culminating in hypoxaemic periods due to hypoventilation. We examined respiratory function in young adult male Mdx (dystrophin deficient) mice (C57BL/10ScSnMdx/J, n=11) and wild type (WT) controls (C57BL/10ScSn, n=10). During normoxic breathing, Mdx mice had significantly reduced breathing frequency, tidal volume and minute ventilation compared to WT. Mdx mice had a significantly increased ventilatory response to acute hypoxic challenge. We also determined that young adult Mdx mice have reduced diaphragm functional capacity compared to WT, with significant decreases in tetanic force, work and power. Interestingly, Mdx diaphragm displayed tolerance to an acute (6hr) in vivo hypoxic stress, which was not observed in WT. In separate studies, co‐treatment with a monoclonal IL‐6 receptor antibody (MR1‐61; 0.3mg/kg) and the CRFR2 agonist, Urocortin 2 (30µg/kg) both administered by 6 subcutaneous injections over 2 weeks completely rescued Mdx diaphragm muscle force loss compared to saline treatment. Mdx mice have pronounced respiratory muscle dysfunction and impaired normoxic ventilation suggestive of hypoventilation. Enhanced hypoxic chemosensitivity and respiratory muscle tolerance of hypoxic stress are suggestive of adaptation to chronic hypoxaemia in young adult life. Hypoxia, stress and immune factors are implicated in diaphragm dysfunction in the Mdx mouse model of DMD.The MR1‐61 antibody was gifted by Chugai Pharmaceuticals, Tokyo, Japan.

Respiratory Control in the mdx Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a genetic disease caused by defects in the dystrophin gene resulting in loss of the structural protein dystrophin. Patients have reduced diaphragm functional capacity due to progressive muscle weakness. Respiratory morbidity in DMD is further characterised by hypoxaemic periods due to hypoventilation. DMD patients die prematurely due to respiratory and cardiac failure. In this study, we examined respiratory function in young adult male mdx (dystrophin deficient) mice (C57BL/10ScSn-Dmd(mdx)/J; n = 10) and in wild-type controls (WT; C57BL/10ScSnJ; n = 11). Breathing was assessed in unrestrained, unanaesthetised animals by whole-body plethysmography. Ventilatory parameters were recorded during air breathing and during exposure to acute hypoxia (F(i)O(2) = 0.1, 20 min). Data for the two groups of animals were compared using Student's t tests. During normoxic breathing, mdx mice had reduced breathing frequency (p = 0.011), tidal volume (p = 0.093) and minute ventilation (p = 0.033) compared to WT. Hypoxia increased minute ventilation in WT and mdx animals. Mdx mice had a significantly increased ventilatory response to hypoxia which manifest as an elevated % change from baseline for minute ventilation (p = 0.0015) compared to WT. We conclude that mdx mice have impaired normoxic ventilation suggestive of hypoventilation. Furthermore, mdx mice have an enhanced hypoxic ventilatory response compared to WT animals which we speculate may be secondary to chronic hypoxaemia. Our results indicate that a significant respiratory phenotype is evident as early as 8 weeks in the mdx mouse model of DMD.

The extraocular muscle stem cell niche is resistant to ageing and disease.

Specific muscles are spared in many degenerative myopathies. Most notably, the extraocular muscles (EOMs) do not show clinical signs of late stage myopathies including the accumulation of fibrosis and fat. It has been proposed that an altered stem cell niche underlies the resistance of EOMs in these pathologies, however, to date, no reports have provided a detailed characterization of the EOM stem cell niche. PW1/Peg3 is expressed in progenitor cells in all adult tissues including satellite cells and a subset of interstitial non-satellite cell progenitors in muscle. These PW1-positive interstitial cells (PICs) include a fibroadipogenic progenitor population (FAP) that give rise to fat and fibrosis in late stage myopathies. PICs/FAPs are mobilized following injury and FAPs exert a promyogenic role upon myoblasts in vitro but require the presence of a minimal population of satellite cells in vivo. We and others recently described that FAPs express promyogenic factors while satellite cells express antimyogenic factors suggesting that PICs/FAPs act as support niche cells in skeletal muscle through paracrine interactions. We analyzed the EOM stem cell niche in young adult and aged wild-type mice and found that the balance between PICs and satellite cells within the EOM stem cell niche is maintained throughout life. Moreover, in the adult mdx mouse model for Duchenne muscular dystrophy (DMD), the EOM stem cell niche is unperturbed compared to normal mice, in contrast to Tibialis Anterior (TA) muscle, which displays signs of ongoing degeneration/regeneration. Regenerating mdx TA shows increased levels of both PICs and satellite cells, comparable to normal unaffected EOMs. We propose that the increase in PICs that we observe in normal EOMs contributes to preserving the integrity of the myofibers and satellite cells. Our data suggest that molecular cues regulating muscle regeneration are intrinsic properties of EOMs.

Dystropathology Increases Energy Expenditure and Protein Turnover in the Mdx Mouse Model of Duchenne Muscular Dystrophy

The skeletal muscles in Duchenne muscular dystrophy and the mdx mouse model lack functional dystrophin and undergo repeated bouts of necrosis, regeneration, and growth. These processes have a high metabolic cost. However, the consequences for whole body energy and protein metabolism, and on the dietary requirements for these macronutrients at different stages of the disease, are not well-understood. This study used juvenile (4- to 5- wk-old) and adult (12- to 14-wk-old) male dystrophic C57BL/10ScSn-mdx/J and age-matched C57BL/10ScSn/J control male mice to measure total and resting energy expenditure, food intake, spontaneous activity, body composition, whole body protein turnover, and muscle protein synthesis rates. In juvenile mdx mice that have extensive muscle damage, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were higher than in age-matched controls. Adaptations in food intake and decreased activity were insufficient to meet the increased energy and protein needs of juvenile mdx mice and resulted in stunted growth. In (non-growing) adult mdx mice with less severe dystropathology, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were also higher than in age-matched controls. Food intake was sufficient to meet their protein and energy needs, but insufficient to result in fat deposition. These data show that dystropathology impacts the protein and energy needs of mdx mice and that tailored dietary interventions are necessary to redress this imbalance. If not met, the resultant imbalance blunts growth, and may limit the benefits of therapies designed to protect and repair dystrophic muscles.

Filamentous structures in skeletal muscle: anchors for the subsarcolemmal space

In skeletal muscle fibers, intermediate filaments and actin filaments provide structural support to the myofibrils and the sarcolemma. For many years, it was poorly understood from ultrastructural observations that how these filamentous structures were kept anchored. The present study was conducted to determine the architecture of filamentous anchoring structures in the subsarcolemmal space and the intermyofibrils. The diaphragms (Dp) of adult wild type and mdx mice (mdx is a model for Duchenne muscular dystrophy) were subjected to tension applied perpendicular to the long axis of the muscle fibers, with or without treatment with 1% Triton X-100 or 0.03% saponin. These experiments were conducted to confirm the presence and integrity of the filamentous anchoring structures. Transmission electron microscopy revealed that these structures provide firm transverse connections between the sarcolemma and peripheral myofibrils. Most of the filamentous structures appeared to be inserted into subsarcolemmal densities, forming anchoring connections between the sarcolemma and peripheral myofibrils. In some cases, actin filaments were found to run longitudinally in the subsarcolemmal space to connect to the sarcolemma or in some cases to connect to the intermyofibrils as elongated thin filaments. These filamentous anchoring structures were less common in the mdx Dp. Our data suggest that the transverse and longitudinal filamentous structures form an anchoring system in the subsarcolemmal space and the intermyofibrils.

Targeting NRF2 Activation Modulates X-Ros Signaling in Dystrophic Skeletal Muscle

Oxidative stress is a critical disease modifier in the muscular dystrophies. Recently we discovered X-ROS, a pathway by which mechanical stretch activates NADPH Oxidase 2 (NoX2) dependent ROS generation (Prosser et al, 2011). Our work in dystrophic skeletal muscle (Khairallah et al, 2012) revealed X-ROS is excessive in dystrophin deficient (mdx) skeletal muscle where it contributes muscle injury susceptibility, a hallmark of the dystrophic process. Our transcriptional interrogation of the X-ROS pathway in human DMD and in mdx revealed a widespread alteration in genes associated with redox homeostasis. As Nrf2 plays an essential role in the transcriptional regulation of genes involved in redox homeostasis we hypothesized that reductions in Nrf2 activity may contribute to the enhanced X-ROS signaling by reducing redox buffering. In our first experiment, we treated adult (6 month) mdx mice with sulforaphane, an activator of Nrf2 which transcriptionally up regulates antioxidant enzyme expression thus increasing the ability of cells to scavenge ROS. Following 5 days of treatment the sentinel antioxidant enzyme, glutathione reductase, was significantly increased in the muscle. Furthermore, X-ROS was significantly abrogated in treated vs. untreated animals. To directly test the effect of decreased Nrf2 activity, Nrf2 was genetically silenced in the A/J model of dysferlinopathy; a model is notorious for having a mild functional phenotype, which has raised doubts about its relevance for dysferlinopathy. With Nrf2 silencing, 1 year old A/J mice had significant muscle specific functional deficits and altered histopathology compared to non-silenced controls and X-ROS was dramatically enhanced. We propose that strategies targeting Nrf2 activation will address generalized reduction in redox homeostasis, which is a core mechanism of dystrophic progression.

Decrease in prosaposin in the Dystrophic mdx mouse brain.

BackgroundDuchenne muscular dystrophy caused by a mutation in the X-linked dystrophin gene induces metabolic and structural disorders in the brain. A lack of dystrophin in brain structures is involved in impaired cognitive function. Prosaposin (PS), a neurotrophic factor, is abundant in the choroid plexus and various brain regions. We investigated whether PS serves as a link between dystrophin loss and gross and/or ultrastructural brain abnormalities.Methodology/Principal FindingsThe distribution of PS in the brains of juvenile and adult mdx mice was investigated by immunochemistry, Western blotting, and in situ hybridization. Immunochemistry revealed lower levels of PS in the cytoplasm of neurons of the cerebral cortex, hippocampus, cerebellum, and choroid plexus in mdx mice. Western blotting confirmed that PS levels were lower in these brain regions in both juveniles and adults. Even with low PS production in the choroids plexus, there was no significant PS decrease in cerebrospinal fluid (CSF). In situ hybridization revealed that the primary form of PS mRNA in both normal and mdx mice was Pro+9, a secretory-type PS, and the hybridization signals for Pro+9 in the above-mentioned brain regions were weaker in mdx mice than in normal mice. We also investigated mitogen-activated protein kinase signalling. Stronger activation of ERK1/2 was observed in mdx mice, ERK1/2 activity was positively correlated with PS activity, and exogenous PS18 stimulated both p-ERK1/2 and PS in SH-SY5Y cells.Conclusions/SignificanceLow levels of PS and its receptors suggest the participation of PS in some pathological changes in the brains of mdx mice.

P.20.7 Longitudinal neuromuscular responses in mdx mice challenged with or without addition of forced exercise

Duchenne muscular dystrophy (DMD) is a lethal X-linked neuromuscular disease caused by a mutation in the dystrophin gene, and the mdx mouse is the most widely used model of DMD used in preclinical therapeutic screening. Many neuromuscular diseases such as DMD are characterized by an exaggerated exercise-induced fatigue response, disproportionate to activity level, known as fatigability which is observed in both DMD patients and the mdx mouse. As such, we developed a hind limb fatigue challenge (HLFC) test utilizing postural fatigue and identified a robust, early in vivo fatigability phenotype in the mdx mouse. In the HLFC test, subjects are evaluated in a locomotor task, are then forced to stand on their hind limbs for an extended period of time, and are subsequently evaluated in the same locomotor task. Whereas other exercise paradigms such as the running wheel or treadmill can be used to elicit whole body fatigue incorporating a more robust cardiovascular component, the HLFC test isolates the fatigue to the hindlimb muscles, and we observe consistent early differences in fatigability between mdx and control animals. However, as the mdx mouse exhibits substantial muscle regeneration leading to a mild progression of the disease, the extent of muscle pathology in sedentary adult mdx mice is low and running wheel or treadmill exercise are often used to exacerbate myofibre necrosis, biomarkers indicative of muscle damage, and behavioral deficits. Therefore, the current study directly compares cohorts of mice evaluated in the HLFC longitudinally with or without exercise in between HLFC testing ages. Ultimately, our goal is to develop a behavioral battery which will reliably capture neuromuscular deficits and provide for efficient and robust preclinical testing in the mdx mouse model of DMD. The effectiveness of currently used compounds with therapeutic value in DMD patients, such as prednisone, will be screened using the newly established efficacy study platform.

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

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

T.P.12 Dietary phosphate intake and exercise-induced stress induce severe muscle necrosis in adult mdx mice

Progressive muscle degeneration, ectopic calcification in skeletal muscle, and high serum phosphate levels are characteristic features of muscular dystrophy especially in mdx mice. However, the effects of dietary phosphate intake on ectopic calcification and muscle pathology are not well studied. We previously reported that increased dietary phosphate intake caused elevated serum phosphate (Pi) levels, severe ectopic calcification and reduced muscle function in mdx mice. In contrast, lowering phosphate intake ameliorated the pathogenesis of the mice. In this study, we examined the effects of dietary phosphate intake and exercise-induced stress on muscle pathology of 90days old mdx mice (adult mdx mice). Voluntary running on activity wheel (VR) increases physical activity and negatively affects muscle pathology of the mice. We found that 10days of VR exacerbated muscle degeneration, increased macrophage infiltration and increased ectopic calcification in limb muscles of high-phosphate fed adult mdx mice. Tibialis anterior (TA) muscle, which consists mostly of muscle fibers with central nuclei and minimal necrotic areas when the mice were kept in a normal cage, was the most affected muscle by VR. Since numerous muscle fibers were embryonic heavy chain positive, high-phosphate intake did not interfere with muscle regeneration but dramatically progressed muscle degeneration. Interestingly, exercise and low-phosphate intake had relatively few areas of group degeneration and no ectopic calcification in TA muscle of mdx mice. The muscle pathologies of the exercised and non-exercised mdx mice fed low-Pi diet were similar. Therefore we conclude that high dietary phosphate intake progresses the pathogenesis of muscular dystrophy, and lowering phosphate intake could increase resistance to exercise-induced damage.

T.O.7 Blockade of Myostatin/ActRIIB signalling induces skeletal muscle fatigability and exercise intolerance

<h2>Abstract</h2> Blockade of myostatin/ActRIIB signaling has been proposed as a therapeutic strategy for Duchenne muscular dystrophy (DMD) because of its supposed stimulating effect on skeletal muscle size and function. Here we show that constitutive lack of myostatin in Mstn−/− mice resulted in increased muscle fatigability, decreased exercise performance, exercise-induced abnormal lactic acidemia and decreased mitochondrial respiration. However, Mstn−/− mice develop a fiber type disproportion towards a glycolytic muscle phenotype with less mitochondria and decreased capillary density, and these developmental abnormalities may account for the observed changes in muscle function and energy metabolism. To determine the role of myostatin/ActRIIB signaling on adult muscle physiology and metabolism, we treated adult wild type mice and mdx mice for 4months with soluble Activin IIB receptor (sActRIIB-Fc) in order to block signaling by myostatin and homologous proteins. In wild type mice, sAcRIIB-Fc strongly increased muscle size and tetanic force, whereas fatigue resistance decreased. Mice exhausted precociously during treadmill exercise and this was associated with grossly elevated serum lactate levels. Treatment of mdx mice with sActRIIB-Fc, however, resulted in largely decreased specific force. Mdx mice developed extreme exercise intolerance and exercise induced lactate acidosis. Mitochondrial enzyme activities and fiber type distribution remained unchanged, thus a primary mitochondrial deficit was unlikely the cause of the exercise failure. Importantly, capillary density of muscle strongly decreased following treatment with sActRIIB-Fc and this was associated with marked dystrophic changes. Our results suggest that myostatin optimizes muscle metabolism by adapting muscle vascularization to oxygen requirements. Blockade of myostatin/ActRIIB signaling causes a capillary-fiber mismatch especially in de- and regenerating dystrophic muscle, provoking a secondary metabolic myopathy.

Effect of rapamycin on immunity induced by vector-mediated dystrophin expression in mdx skeletal muscle

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. Therapeutic gene replacement of a dystrophin cDNA into dystrophic muscle can provide functional dystrophin protein to the tissue. However, vector-mediated gene transfer is limited by anti-vector and anti-transgene host immunity that causes rejection of the therapeutic protein. We hypothesized that rapamycin (RAPA) would diminish immunity due to vector-delivered recombinant dystrophin in the adult mdx mouse model for DMD. To test this hypothesis, we injected limb muscle of mdx mice with RAPA-containing, poly-lactic-co-glycolic acid (PLGA) microparticles prior to dystrophin gene transfer and analyzed treated tissue after 6 weeks. RAPA decreased host immunity against vector-mediated dystrophin protein, as demonstrated by decreased cellular infiltrates and decreased anti-dystrophin antibody production. The interpretation of the effect of RAPA on recombinant dystrophin expression was complex because of an effect of PLGA microparticles.