Function In Mdx Mice Research Articles

ACE2 Is Augmented in Dystrophic Skeletal Muscle and Plays a Role in Decreasing Associated Fibrosis

Duchenne muscular dystrophy (DMD) is the most common inherited neuromuscular disease and is characterized by absence of the cytoskeletal protein dystrophin, muscle wasting, and fibrosis. We previously demonstrated that systemic infusion or oral administration of angiotensin-(1-7) (Ang-(1-7)), a peptide with opposing effects to angiotensin II, normalized skeletal muscle architecture, decreased local fibrosis, and improved muscle function in mdx mice, a dystrophic model for DMD. In this study, we investigated the presence, activity, and localization of ACE2, the enzyme responsible for Ang-(1-7) production, in wild type (wt) and mdx skeletal muscle and in a model of induced chronic damage in wt mice. All dystrophic muscles studied showed higher ACE2 activity than wt muscle. Immunolocalization studies indicated that ACE2 was localized mainly at the sarcolemma and, to a lesser extent, associated with interstitial cells. Similar results were observed in the model of chronic damage in the tibialis anterior (TA) muscle. Furthermore, we evaluated the effect of ACE2 overexpression in mdx TA muscle using an adenovirus containing human ACE2 sequence and showed that expression of ACE2 reduced the fibrosis associated with TA dystrophic muscles. Moreover, we observed fewer inflammatory cells infiltrating the mdx muscle. Finally, mdx gastrocnemius muscles from mice infused with Ang-(1-7), which decreases fibrosis, contain less ACE2 associated with the muscle. This is the first evidence supporting ACE2 as an important therapeutic target to improve the dystrophic skeletal muscle phenotype.

Dietary quercetin enrichment improves respiratory function in mdx mice (884.17)

Duchenne muscular dystrophy (DMD) is caused by a dystrophin deficiency and results in progressive muscle injury culminating in death due to respiratory or cardiac failure. We have established that gene transfer of the exercise mimetic, PGC‐1α, will decrease disease severity and improve muscle function in skeletal muscle taken from mdx mice, the mouse model of DMD. While this approach is effective and repeatable, at present it is not readily translatable. Hence, the purpose of this project was to determine the extent to which oral delivery of quercetin, a PGC‐1α activator, would improve respiratory function in mdx mice. To address this purpose baseline respiratory function was measured in vivo in two month old C57 and mdx mice. After measurement, the mdx group was randomly assigned into groups receiving a control diet (mdx) or diet enriched with 0.2% quercetin (mdx‐Q). Respiratory function was measured again at four and six months of age. By six months of age we found that a 16% (p<0.05) increase in respiratory frequency and a 12% (p<0.05) increase in tidal volume caused a near 30% (p<0.05) increase in minute ventilation in mdx‐Q compared to mdx. Further, peak inspiratory flow was increased 20% (p<0.05) and peak expiratory flow was increased 26% (p<0.05) in mdx‐Q compared to mdx. These data suggest strongly that dietary enrichment of the orally available PGC‐1α activator, quercetin, attenuated muscle injury and improved respiratory function in a mouse model of Duchenne muscular dystrophy.Grant Funding Source: Supported by the Duchenne Alliance and its member foundations

Post-natal induction of PGC-1α protects against severe muscle dystrophy independently of utrophin

BackgroundDuchenne muscle dystrophy (DMD) afflicts 1 million boys in the US and has few effective treatments. Constitutive transgenic expression of the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α improves skeletal muscle function in the murine “mdx” model of DMD, but how this occurs, or whether it can occur post-natally, is not known. The leading mechanistic hypotheses for the benefits conferred by PGC-1α include the induction of utrophin, a dystrophin homolog, and/or induction and stabilization of the neuromuscular junction.MethodsThe effects of transgenic overexpression of PGC-1β, a homolog of PGC-1α in mdx mice was examined using different assays of skeletal muscle structure and function. To formally test the hypothesis that PGC-1α confers benefit in mdx mice by induction of utrophin and stabilization of neuromuscular junction, PGC-1α transgenic animals were crossed with the dystrophin utrophin double knock out (mdx/utrn-/-) mice, a more severe dystrophic model. Finally, we also examined the effect of post-natal induction of skeletal muscle-specific PGC-1α overexpression on muscle structure and function in mdx mice.ResultsWe show here that PGC-1β does not induce utrophin or other neuromuscular genes when transgenically expressed in mouse skeletal muscle. Surprisingly, however, PGC-1β transgenesis protects as efficaciously as PGC-1α against muscle degeneration in dystrophin-deficient (mdx) mice, suggesting that alternate mechanisms of protection exist. When PGC-1α is overexpressed in mdx/utrn-/- mice, we find that PGC-1α dramatically ameliorates muscle damage even in the absence of utrophin. Finally, we also used inducible skeletal muscle-specific PGC-1α overexpression to show that PGC-1α can protect against dystrophy even if activated post-natally, a more plausible therapeutic option.ConclusionsThese data demonstrate that PGC-1α can improve muscle dystrophy post-natally, highlighting its therapeutic potential. The data also show that PGC-1α is equally protective in the more severely affected mdx/utrn-/- mice, which more closely recapitulates the aggressive progression of muscle damage seen in DMD patients. The data also identify PGC-1β as a novel potential target, equally efficacious in protecting against muscle dystrophy. Finally, the data also show that PGC-1α and PGC-1β protect against dystrophy independently of utrophin or of induction of the neuromuscular junction, indicating the existence of other mechanisms.

Resveratrol improves muscle function but not oxidative capacity in young mdx mice

Patients with Duchenne muscular dystrophy (DMD) have reduced muscle function due to chronic muscle damage, inflammation, oxidative stress, and reduced oxidative capacity. Resveratrol reduces inflammation and oxidative stress, and increases oxidative capacity in other disease models. The purpose of this study was to determine the effects of resveratrol on muscle function, muscle pathology, and oxidative capacity in young mdx mice. For this, 4- to 5-week-old male mdx mice were randomized into control or resveratrol-treated groups and given resveratrol (100 mg/kg body mass) or an equal volume of water by gavage every other day for 8 weeks. Muscle function was assessed pre- and post-treatment. Central nucleation, total immune cell infiltrate, oxidative stress, and oxidative capacity were measured post-treatment. Resveratrol mediated substantial improvements in rotarod performance and in-situ peak tension by 53% and 17%, respectively, and slight improvements in central nucleation and oxidative stress. Resveratrol did not affect total immune cell infiltrate at 12 weeks of age, and had no effect on oxidative capacity. Resveratrol improves muscle function in mdx mice despite small changes in muscle pathology. The likely mechanism is a resveratrol-mediated reduction in immune cell infiltrate at the early stages of this disease, as previously reported by our laboratory.

Nifedipine Treatment Improves Muscle Function in Mdx Mice

Duchenne muscular dystrophy (DMD) is a common genetic disorder characterized by chronic inflammation and severe muscle wasting caused by the absence of dystrophin. In mdx muscle fibers, we have shown that basal ATP release was increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, ATP release is blocked by nifedipine leading us to study the potential therapeutic effect of nifedipine in mdx mice and its relation with extracellular ATP signaling. Six-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1mg/Kg for 1-week. This treatment significantly reduced extracellular ATP levels in adult mdx flexor digitorum brevis (FDB) fibers and lowered the intracellular resting Ca2+ concentration ([Ca2+]r) measured in vivo in the vastus lateralis. Acute treatment of mdx FDB fibers with apyrase, reduced [Ca2+]r to a similar extent as was seen in vastus lateralis after nifedipine treatment. Physiologically, nifedipine treatment increased muscle strength assessed by both the inverted grip-hanging test and forced swimming test, and decreased serum CK in mdx mice. Exploring mechanisms, nifedipine treatment reduced mRNA levels of iNOS, gp91phox, p47phox and Bax in mdx diaphragm muscles. We hypothesize that nifedipine, reduces basal ATP release thereby decreasing purinergic receptor activation, which in turn reduces [Ca2+]r and muscle destruction in mdx skeletal muscles. Funding: FONDECYT 1110467, Proyecto Anillo ACT1111 and AFM14562 (EJ, MC), NIH AR43140, AR052534 (PDA), U-INICIA VID 2011, U-INICIA 02/12M (MC).

P.11.3 Long term treatment with naproxcinod significantly improves skeletal and cardiac function in mdx mouse model of dystrophy

There is evidence that nitric oxide (NO) plays a critical role in skeletal muscle. In Duchenne muscular dystrophy (DMD) patients and the mdx mouse model of DMD, dystrophin deficiency causes a decrease and mislocalization of muscle-specific neuronal nitric oxide synthase (nNOSμ), leading to a variety of functional impairments such as muscle ischemia and compromised myogenesis. Previous studies have shown that NO donation associated with anti-inflammatory action showed beneficial effects in dystrophic mouse models. In this study, we have investigated the effects of naproxcinod, an NO donating naproxen, on skeletal and cardiac muscle function in mdx mice. 4-week-old mdx mice were orally treated for 9 months with three different doses of naproxcinod (10, 21 and 41 mg/kg/day) compared with 0.9 mg/kg of prednisolone. Functional and behavioral parameters using a grip strength meter and open field digiscan were measured at 3, 6, and 9 months of treatment using SOPs developed by TREAT-NMD. Additionally, in vitro EDL force contraction, optical imaging of inflammation, echocardiography and blood pressure were evaluated at the 9 months prior to sacrifice. Naproxcinod treatment at 10 and 21 mg/kg resulted in significant improvements in hindlimb grip strength as well as approximately a 25–30% decrease in inflammation in fore and hind limbs measured by in vivo optical imaging in mdx mice. Naproxcinod induced significant improvements in heart function as evidenced by ameliorated fraction shortening and ejection fraction measured using echocardiography along with improvements in systolic blood pressure. Moreover, the long term detrimental effects of prednisolone typically observed in mdx skeletal and heart function were not observed at the effective doses of naproxcinod. In conclusion, naproxcinod seems to have significant potential as a safe therapeutic option for the treatment of muscular dystrophies.

Inhibition of iPLA2β and of stretch‐activated channels by doxorubicin alters dystrophic muscle function

Chronic elevation in intracellular Ca(2+) concentration participates in death of skeletal muscle from mdx mice, a model for Duchenne muscular dystrophy (DMD). Candidate pathways mediating this Ca(2+) overload involve store-operated channels (SOCs) and stretch-activated channels (SACs), which are modulated by the Ca(2+) -independent form of PL A2 (iPLA2 ). We investigated the effect of doxorubicin (Dox), a chemotherapeutic agent reported to inhibit iPLA2 in other systems, on the activity of this enzyme and on the consequences on Ca(2+) handling and muscle function in mdx mice. Effects of Dox on iPLA2 activity, reactive oxygen species production and on Ca(2+) influx were investigated in C2C12 and mdx myotubes. The mechanism of Dox-mediated iPLA2 inhibition was evaluated using purified 6x histidine-tagged enzyme. Aequorin technology was used to assess Ca(2+) concentrations underneath the plasma membrane. Isolated muscles were exposed to fatigue protocols and eccentric contractions to evaluate the effects of Dox on muscle function. Dox at 1-30 μM inhibited iPLA2 activity in cells and in the purified enzyme. Dox also inhibited SAC- but not SOC-mediated Ca(2+) influx in myotubes. Stimulated elevations of Ca(2+) concentrations below the plasmalemma were also blocked. Exposure of excised muscle to Dox was not deleterious to force production and promoted recovery from eccentric contractions. Dox showed efficacy against targets known to play a role in the pathology of DMD, namely iPLA2 and SAC. The potent SAC inhibitory effect of Dox is a novel finding that can explain partly the cardiomyopathy seen in chronic anthracycline treatment.

Cardiac Characterization ofmdxMice Using High-Resolution Doppler Echocardiography

Objectives Duchenne muscular dystrophy is an X-linked neuromuscular disorder. The heart is traditionally involved, leading to heart failure. The mdx mouse is a natural animal model of the disease. We conducted a prospective study to analyze left ventricular (LV) function in mdx mice at different ages using high-resolution Doppler echocardiography. Methods Echocardiography was performed with a 30-MHz cardiac probe. Wild-type and mdx mice were scanned at 10 and 12 months. We measured the interventricular septal wall thickness, posterior wall thickness, and LV diameter in systole and diastole. The LV shortening fraction, LV ejection fraction, and LV mass were then calculated. Results At 10 months, the shortening fractions in mdx and wild-type mice were relatively close (29% ± 5% versus 25% ± 4%). We found a significant difference in the posterior wall thickness change (40% ± 12% in mdx versus 28% ± 10% in wild-type; P = .048). The LV mass/body weight ratio was higher in mdx than wild-type mice (3.67 ± 0.25 versus 3.39 ± 0.26; P = .05). At 12 months, the LV mass was elevated in mdx mice compared to wild-type (152 ±16 versus 135 ± 3.7 mg; P = .04). The diastolic posterior wall thickness change was decreased in mdx mice at 12 months compared to wild-type (21% ± 7% versus 33% ± 4%; P = .01). The LV ejection fraction was not statistically different between mdx and wild-type mice (50% ± 6% versus 54% ± 2%). Conclusions Ten-month-old mdx mice had a significantly higher posterior wall thickness than wild-type mice, but it was not significant at 12 months. In 12-month-old mdx mice, the posterior wall thickness change was significantly lower, and the LV mass was significantly higher. These findings indicate the role of LV function in the early stages of Duchenne muscular dystrophy.

Cardiac Characterization of mdx Mice Using High-Resolution Doppler Echocardiography

Duchenne muscular dystrophy is an X-linked neuromuscular disorder. The heart is traditionally involved, leading to heart failure. The mdx mouse is a natural animal model of the disease. We conducted a prospective study to analyze left ventricular (LV) function in mdx mice at different ages using high-resolution Doppler echocardiography. Echocardiography was performed with a 30-MHz cardiac probe. Wild-type and mdx mice were scanned at 10 and 12 months. We measured the interventricular septal wall thickness, posterior wall thickness, and LV diameter in systole and diastole. The LV shortening fraction, LV ejection fraction, and LV mass were then calculated. At 10 months, the shortening fractions in mdx and wild-type mice were relatively close (29% ± 5% versus 25% ± 4%). We found a significant difference in the posterior wall thickness change (40% ± 12% in mdx versus 28% ± 10% in wild-type; P = .048). The LV mass/body weight ratio was higher in mdx than wild-type mice (3.67 ± 0.25 versus 3.39 ± 0.26; P = .05). At 12 months, the LV mass was elevated in mdx mice compared to wild-type (152 ±16 versus 135 ± 3.7 mg; P = .04). The diastolic posterior wall thickness change was decreased in mdx mice at 12 months compared to wild-type (21% ± 7% versus 33% ± 4%; P = .01). The LV ejection fraction was not statistically different between mdx and wild-type mice (50% ± 6% versus 54% ± 2%). Ten-month-old mdx mice had a significantly higher posterior wall thickness than wild-type mice, but it was not significant at 12 months. In 12-month-old mdx mice, the posterior wall thickness change was significantly lower, and the LV mass was significantly higher. These findings indicate the role of LV function in the early stages of Duchenne muscular dystrophy.

Voluntary Physical Activity Protects from Susceptibility to Skeletal Muscle Contraction–Induced Injury But Worsens Heart Function in mdx Mice

It is well known that inactivity/activity influences skeletal muscle physiological characteristics. However, the effects of inactivity/activity on muscle weakness and increased susceptibility to muscle contraction-induced injury have not been extensively studied in mdx mice, a murine model of Duchenne muscular dystrophy with dystrophin deficiency. In the present study, we demonstrate that inactivity (ie, leg immobilization) worsened the muscle weakness and the susceptibility to contraction-induced injury in mdx mice. Inactivity also mimicked these two dystrophic features in wild-type mice. In contrast, we demonstrate that these parameters can be improved by activity (ie, voluntary wheel running) in mdx mice. Biochemical analyses indicate that the changes induced by inactivity/activity were not related to fiber-type transition but were associated with altered expression of different genes involved in fiber growth (GDF8), structure (Actg1), and calcium homeostasis (Stim1 and Jph1). However, activity reduced left ventricular function (ie, ejection and shortening fractions) in mdx, but not C57, mice. Altogether, our study suggests that muscle weakness and susceptibility to contraction-induced injury in dystrophic muscle could be attributable, at least in part, to inactivity. It also suggests that activity exerts a beneficial effect on dystrophic skeletal muscle but not on the heart.

Beta-blockers, left and right ventricular function, and in-vivo calcium influx in muscular dystrophy cardiomyopathy.

Beta-blockers are used to treat acquired heart failure in adults, though their role in early muscular dystrophy cardiomyopathy is unclear. We treated 2 different dystrophic mouse models which have an associated cardiomyopathy (mdx: model for Duchenne Muscular Dystrophy, and Sgcd-/-: model for limb girdle muscular dystrophy type 2F) and wild type controls (C57 Bl10) with the beta blocker metoprolol or placebo for 8 weeks at an early stage in the development of the cardiomyopathy. Left and right ventricular function was assessed with cardiac magnetic resonance imaging (MRI) and in-vivo myocardial calcium influx with manganese enhanced MRI. In the mdx mice at baseline there was reduced stroke volume, cardiac index, and end-diastolic volume with preserved left ventricular ejection fraction. These abnormalities were no longer evident after treatment with beta-blockers. Right ventricular ejection fraction was reduced and right ventricular end-systolic volume increased in the mdx mice. With metoprolol there was an increase in right ventricular end-diastolic and end-systolic volumes. Left and right ventricular function was normal in the Sgcd-/- mice. Metroprolol had no significant effects on left and right ventricular function in these mice, though heart/body weight ratios increased after treatment. In-vivo myocardial calcium influx with MEMRI was significantly elevated in both models, though metoprolol had no significant effects on either. In conclusion, metoprolol treatment at an early stage in the development of cardiomyopathy has deleterious effects on right ventricular function in mdx mice and in both models no effect on increased in-vivo calcium influx. This suggests that clinical trials need to carefully monitor not just left ventricular function but also right ventricular function and other aspects of myocardial metabolism.

Loss of cIAP1 attenuates soleus muscle pathology and improves diaphragm function in mdx mice

The cellular inhibitor of apoptosis 1 (cIAP1) protein is an essential regulator of canonical and noncanonical nuclear factor κB (NF-κB) signaling pathways. NF-κB signaling is known to play important roles in myogenesis and degenerative muscle disorders such as Duchenne muscular dystrophy (DMD), but the involvement of cIAP1 in muscle disease has not been studied directly. Here, we asked whether the loss of cIAP1 would influence the pathology of skeletal muscle in the mdx mouse model of DMD. Double-mutant cIAP1(-/-);mdx mice exhibited reduced muscle damage and decreased fiber centronucleation in the soleus, compared with single-mutant cIAP1(+/+);mdx mice. This improvement in pathology was associated with a reduction in muscle infiltration by macrophages and diminished expression of inflammatory cytokines such as IL-6 and tumor necrosis factor-α. Furthermore, the cIAP1(-/-);mdx mice exhibited reduced serum creatine kinase, and improved exercise endurance associated with improved exercise resilience by the diaphragm. Mechanistically, the loss of cIAP1 was sufficient to drive constitutive activation of the noncanonical NF-κB pathway, which led to increased myoblast fusion in vitro and in vivo. Collectively, these results show that the loss of cIAP1 protects skeletal muscle from the degenerative pathology resulting from systemic loss of dystrophin.

Combined Effect of AMPK/PPAR Agonists and Exercise Training in mdx Mice Functional Performance

The present investigation was undertaken to test whether exercise training (ET) associated with AMPK/PPAR agonists (EM) would improve skeletal muscle function in mdx mice. These drugs have the potential to improve oxidative metabolism. This is of particular interest because oxidative muscle fibers are less affected in the course of the disease than glycolitic counterparts. Therefore, a cohort of 34 male congenic C57Bl/10J mdx mice included in this study was randomly assigned into four groups: vehicle solution (V), EM [AICAR (AMPK agonist, 50 mg/Kg-1.day-1, ip) and GW 1516 (PPARδ agonist, 2.5 mg/Kg-1.day-1, gavage)], ET (voluntary running on activity wheel) and EM+ET. Functional performance (grip meter and rotarod), aerobic capacity (running test), muscle histopathology, serum creatine kinase (CK), levels of ubiquitined proteins, oxidative metabolism protein expression (AMPK, PPAR, myoglobin and SCD) and intracellular calcium handling (DHPR, SERCA and NCX) protein expression were analyzed. Treatments started when the animals were two months old and were maintained for one month. A significant functional improvement (p<0.05) was observed in animals submitted to the combination of ET and EM. CK levels were decreased and the expression of proteins related to oxidative metabolism was increased in this group. There were no differences among the groups in the intracellular calcium handling protein expression. To our knowledge, this is the first study that tested the association of ET with EM in an experimental model of muscular dystrophy. Our results suggest that the association of ET and EM should be further tested as a potential therapeutic approach in muscular dystrophies.

Exercise Training Improves Plantar Flexor Muscle Function in mdx Mice

We tested the hypothesis that low-intensity exercise in mdx mice improves plantar flexor muscle contractile function, resistance to fatigue, and mitochondrial adaptations without exacerbating muscular dystrophy. We subjected mdx mice to 12 wk of voluntary low-resistance wheel running (Run, n = 17) or normal cage activities (sedentary (Sed), n = 16) followed by in vivo analyses for plantar flexor torque generation and fatigue resistance or running capacity on a treadmill. Gastrocnemius muscles were further evaluated for exercise-induced mitochondrial adaptations and fiber type distribution and central nuclei. t-tests were used to determine differences between the Sed and Run groups. Plantar flexor submaximal isometric torques and maximal isometric torque at multiple ankle joint angles and resistance to fatigue were greater in Run compared with Sed mdx mice (P G 0.05). Citrate synthase and A-hydroxyacyl-CoA dehydrogenase enzyme activities and cytochrome c oxidase IV protein expression in gastrocnemius muscles were greater in Run than in Sed mdx mice(P e 0.04), along with a trend of fiber type transformation from Type IIb to Type IIx fibers. Exercise training in mdx mice did not elevate serum creatine kinase levels but led to a significant reduction of centrally nucleated myofibers. Voluntary low-resistance wheel running in mdx mice can result in skeletal muscle adaptation, leading to improved contractile function and reduced fatigability,with no indication that exercise was detrimental. This study supports the need for further investigation of low-intensity exercise as an early therapeutic intervention in ambulatory boys with Duchenne muscular dystrophy.

T.P.4 Treatment with oral metformin improves muscle function in mdx mice

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by an absence of dystrophin. DMD is caused by out of frame mutations in the dystrophin gene and is characterised by muscle inflammation, necrosis and eventual loss of function of cardiac and skeletal muscle. Slow, oxidative fibres appear to be relatively protected compared to fast, glycolytic fibres. Evidence from transgenic animals suggests that driving muscle into a more aerobic phenotype by upregulating PPAR gamma coactivator 1-alpha (PGC1α) may be beneficial in mdx mice, the murine genetic homologue of DMD. PGC1α is a downstream target of AMP-activated protein kinase (AMPK). Metformin (N,N-dimethylbiguanide) hydrochloride is an AMPK agonist and a commonly prescribed antidiabetic drug used as a first-line treatment for type II diabetes. We hypothesised that metformin would improve skeletal muscle function in mdx mice. Mdx mice were treated for 10weeks from 14weeks of age with metformin at 2mg/ml in their drinking water. Age-matched controls received drinking water alone and all mice were weighed twice a week. The effect of treatment on muscle function was assessed using a standardised in vivo exercise test on the Tibialis Anterior (TA) muscle in situ. The test comprised a warm up, measurement of the force–frequency relationship and a series of eccentric contractions with a 10% stretch. Mice were then euthanised and their muscles’ collected for sectioning and histological analysis. Treatment with metformin at 2mg/ml in drinking water reduced the magnitude and speed of the force drop associated with 10 eccentric contractions compared to untreated controls, thereby improving the muscle’s resistance to contraction induced injury. There was no significant effect of metformin treatment on body weight. Further work is ongoing to assess the effect of metformin treatment on muscle histopathology, free radical content, fibre type distribution, and downstream targets (by western-blotting).

T.P.52 Pilot study of flavocoxid in ambulant DMD patients

Abstract Muscle degeneration in Duchenne muscular dystrophy (DMD) is exacerbated by the endogenous inflammatory response and increased oxidative stress. A key role is played by nuclear factor (NF)-kappaB. We previously showed that flavocoxid, a flavonoid with antioxidant and anti-inflammatory properties, ameliorates muscle pathology and function in mdx mice. This effect seemed to be mediated by the inhibition of NF-kappaB, tumor necrosis factor-α, cyclooxygenase-2/5-lipoxygenase and MAPKs expression in muscle. Moreover, flavocoxid has been shown to decrement serum levels of IL-1β and TNF-α in in vivo studies. Primary end-point of this pilot study was to evaluate safety and tolerability of flavocoxid administered daily per os for one year in ambulant DMD patients. We also evaluated function, muscle strength and quality of life. The effects of flavocoxid on selected biomarkers was also assessed. We recruited 20 patients and herein present preliminary data on 15 patients who completed 9 months of treatment. We did not report any treatment-related adverse event and clinically meaningful change in laboratory findings. Serum expression analysis of inflammatory cytokines showed a significant reduction of TNF-α ( p

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.

Metabolic remodeling agents show beneficial effects in the dystrophin- deficient mdx mouse model

BackgroundDuchenne muscular dystrophy is a genetic disease involving a severe muscle wasting that is characterized by cycles of muscle degeneration/regeneration and culminates in early death in affected boys. Mitochondria are presumed to be involved in the regulation of myoblast proliferation/differentiation; enhancing mitochondrial activity with exercise mimetics (AMPK and PPAR-delta agonists) increases muscle function and inhibits muscle wasting in healthy mice. We therefore asked whether metabolic remodeling agents that increase mitochondrial activity would improve muscle function in mdx mice.MethodsTwelve-week-old mdx mice were treated with two different metabolic remodeling agents (GW501516 and AICAR), separately or in combination, for 4 weeks. Extensive systematic behavioral, functional, histological, biochemical, and molecular tests were conducted to assess the drug(s)' effects.ResultsWe found a gain in body and muscle weight in all treated mice. Histologic examination showed a decrease in muscle inflammation and in the number of fibers with central nuclei and an increase in fibers with peripheral nuclei, with significantly fewer activated satellite cells and regenerating fibers. Together with an inhibition of FoXO1 signaling, these results indicated that the treatments reduced ongoing muscle damage.ConclusionsThe three treatments produced significant improvements in disease phenotype, including an increase in overall behavioral activity and significant gains in forelimb and hind limb strength. Our findings suggest that triggering mitochondrial activity with exercise mimetics improves muscle function in dystrophin-deficient mdx mice.

Doxycycline ameliorates the dystrophic phenotype of skeletal and cardiac muscles in mdx mice

We examined whether doxycycline, an antibiotic member of the tetracycline family, improves the histopathology and muscle function in mdx mice, the experimental model of DMD. Doxycycline was administered for 36 days (starting on postnatal day 0) and for 9 months (starting at 8 months of age) in drinking water. Histopathological, biochemical (creatine kinase), and functional (forelimb muscle grip strength) parameters were evaluated in limb, diaphragm, and cardiac muscle. Doxycycline significantly minimized the dystrophic phenotype of skeletal and cardiac muscles and improved forelimb muscle strength. The drug protected muscle fibers against myonecrosis and reduced inflammation. Furthermore, it slowed the progression of myocardial fibrosis. This study provides evidence that doxycycline may be a potential therapeutic agent for DMD.

Resveratrol affects inflammation and muscle function in mdx mice

Duchenne Muscular Dystrophy (DMD) is a genetic disease characterized by chronic inflammation and muscle degeneration/regeneration. Resveratrol is a compound that has anti inflammatory properties. However its effect on inflammation and muscle function in DMD is unknown. The purpose of this study was to investigate the effect of resveratrol on inflammation and muscle function in the mdx mouse model of DMD. A ten day dosage trial and an eight week treatment trial were performed. Ten days of resveratrol supplementation (100mg/kg) significantly increased Sirt1 gene expression 1.57 ± 0.11 fold (p&lt;0.01), IL‐6 gene expression 3.52 ± 0.023 fold (p&lt;0.05), and did not affect TNF alpha gene expression in the gastrocnemius. This dose increased tibialis anterior H2O2 content 80.6% (p&lt;0.05). Eight weeks of resveratrol supplementation (100 mg/kg) increased peak tension of the tricep surae muscle complex by 9.5% (p&lt;0.05). There was no effect on time to peak tension or muscle fatigue. Our data suggests a positive effect of resveratrol on inflammation and muscle function in the mdx mouse. This project was funded by The Center for Alternative Medicine at The University of South Carolina School of Medicine.