Gene expression in mdx mouse muscle in relation to age and exercise: aberrant mechanical-metabolic coupling and implications for pre-clinical studies in duchenne muscular dystrophy

Weakness and fatigability are typical features of Duchenne muscular dystrophy patients and are aggravated in dystrophic mdx mice by chronic treadmill exercise. Mechanical activity modulates gene expression and muscle plasticity. Here, we investigated the outcome of 4 (T4, 8 weeks of age) and 12 (T12, 16 weeks of age) weeks of either exercise or cage-based activity on a large set of genes in the gastrocnemius muscle of mdx and wild-type (WT) mice using quantitative real-time PCR. Basal expression of the exercise-sensitive genes peroxisome-proliferator receptor γ coactivator 1α (Pgc-1α) and Sirtuin1 (Sirt1) was higher in mdx versus WT mice at both ages. Exercise increased Pgc-1α expression in WT mice; Pgc-1α was downregulated by T12 exercise in mdx muscles, along with Sirt1, Pparγ and the autophagy marker Bnip3. Sixteen weeks old mdx mice showed a basal overexpression of the slow Mhc1 isoform and Serca2; T12 exercise fully contrasted this basal adaptation as well as the high expression of follistatin and myogenin. Conversely, T12 exercise was ineffective in WT mice. Damage-related genes such as gp91-phox (NADPH-oxidase2), Tgfβ, Tnfα and c-Src tyrosine kinase were overexpressed in mdx muscles and not affected by exercise. Likewise, the anti-inflammatory adiponectin was lower in T12-exercised mdx muscles. Chronic exercise with minor adaptive effects in WT muscles leads to maladaptation in mdx muscles with a disequilibrium between protective and damaging signals. Increased understanding of the pathways involved in the altered mechanical-metabolic coupling may help guide appropriate physical therapies while better addressing pharmacological interventions in translational researchWeakness and fatigability are typical features of Duchenne muscular dystrophy patients and are aggravated in dystrophic mdx mice by chronic treadmill exercise. Mechanical activity modulates gene expression and muscle plasticity. Here, we investigated the outcome of 4 (T4, 8 weeks of age) and 12 (T12, 16 weeks of age) weeks of either exercise or cage-based activity on a large set of genes in the gastrocnemius muscle of mdx and wildtype (WT) mice using quantitative real-time PCR. Basal expression of the exercise-sensitive genes peroxisome-proliferator receptor g coactivator 1α (Pgc-1α) and Sirtuin1 (Sirt1) was higher in mdx versus WT mice at both ages. Exercise increased Pgc-1α expression in WT mice; Pgc-1α was downregulted by T12 exercise in mdx muscles, along with Sirt1, Pparγ and the autophagy marker Bnip3. Sixteen weeks old mdx mice showed a basal over expression of the slowMhc1 isoform and Serca2; T12 exercise fully contrasted this basal adaptation as well as the high expression of follistatin and myogenin. Conversely, T12 exercise was ineffective in WT mice. Damage-related genes such as gp91-phox (NADPH-oxidase2), Tgfβ, Tnfα and c-Src tyrosine kinase were overexpressed in mdx muscles and not affected by exercise. Likewise, the anti-inflammatory adiponectin was lower in T12-exercised mdx muscles. Chronic exercise with minor adaptive effects in WT muscles leads to maladaptation in mdx muscles with a disequilibrium between protective and damaging signals. Increased understanding of the pathways involved in the altered mechanical-metabolic coupling may help guide appropriate physical therapies while better addressing pharmacological interventions in translational research

A long-term treatment with taurine prevents cardiac dysfunction in mdx mice

Taurine is an amino acid abundantly present in heart and skeletal muscle. Duchenne muscular dystrophy (DMD) is a genetic disorder in which the absence of dystrophin leads to skeletal muscle wasting and heart failure. An altered taurine metabolism has been described in dystrophic animals and short-term taurine administration exerts promising amelioration of early muscular alterations in the mdx mouse model of DMD. To reinforce the therapeutic and nutraceutical taurine potential in DMD, we evaluated the effects of a long-term treatment on cardiac and skeletal muscle function of mdx mice in a later disease stage. Taurine was administered in drinking water (1 g/kg/day) to wt and mdx mice for 6 months, starting at 6 months of age. Ultrasonography evaluation of heart and hind limb was performed, in parallel with in vivo and ex vivo functional tests and biochemical, histological and gene expression analyses. 12-month-old mdx mice showed a significant worsening of left ventricular function parameters (shortening fraction, ejection fraction, stroke volume), which were significantly counteracted by the taurine treatment. In parallel, histologic signs of damage were reduced by taurine along with the expression of proinflammatory myocardial IL-6. Interestingly, no effects were observed on hind limb volume and percentage of vascularization or on in vivo and ex vivo muscle functional parameters, suggesting a tissue-specific action of taurine in relation to the disease phase. A trend toward increase in taurine was found in heart and quadriceps from treated animals, paralleled by a slight decrease in mdx mice plasma. Our study provides evidences that taurine can prevent late heart dysfunction in mdx mice, further corroborating the interest on this amino acid toward clinical trials

Contractile efficiency of dystrophic mdx mouse muscle: In vivo and ex vivo assessment of adaptation to exercise of functional end points

Progressive weakness is a typical feature of Duchenne muscular dystrophy (DMD) patients and is exacerbated in the benign mdx mouse model by in vivo treadmill exercise. We hypothesized a different threshold for functional adaptation of mdx muscles in response to the duration of the exercise protocol. In vivo weakness was confirmed by grip strength after 4, 8 and 12 weeks of exercise in mdx mice. Torque measurements revealed that exercise-related weakness in mdx mice correlated with the duration of the protocol, while wild-type (wt) mice were stronger. Twitch and tetanic forces of isolated diaphragm and extensor digitorum longus (EDL) muscles, were lower in mdx compared to wt mice. In mdx, both muscle types exhibited greater weakness after a single exercise bout, but only in EDL after a long exercise protocol. As opposite to wt muscles, mdx EDL ones did not show any exercise-induced adaptations against eccentric contraction force drop. qRT-PCR analysis confirmed the maladaptation of genes involved in metabolic and structural remodeling, while damage-related genes remained significantly upregulated and angiogenesis impaired. Phosphorylated AMP kinase level increased only in exercised wt muscle. The severe histopathology and the high levels of muscular TGF-β1 and of plasma matrix metalloproteinase-9 confirmed the persistence of muscle damage in mdx mice. Then, dystrophic muscles showed a partial degree of functional adaptation to chronic exercise, although not sufficient to overcome weakness nor signs of damage. The improved understanding of the complex mechanisms underlying maladaptation of dystrophic muscle paves the way to a better managment of DMD patients

Ultrasonography validation for early alteration of diaphragm echodensity and function in the mdx mouse model of Duchenne muscular dystrophy.

The mdx mouse model of Duchenne muscular dystrophy is characterized by functional and structural alterations of the diaphragm since early stages of pathology, closely resembling patients' condition. In recent years, ultrasonography has been proposed as a useful longitudinal non-invasive technique to assess mdx diaphragm dysfunction and evaluate drug efficacy over time. To date, only a few preclinical studies have been conducted. Therefore, an independent validation of this method by different laboratories is needed to increase results reliability and reduce biases. Here, we performed diaphragm ultrasonography in 3- and 6-month-old mdx mice, the preferred age-window for pharmacology studies. The alteration of diaphragm function over time was measured as diaphragm ultrasound movement amplitude. At the same time points, a first-time assessment of diaphragm echodensity was performed, as an experimental index of progressive loss of contractile tissue. A parallel evaluation of other in vivo and ex vivo dystrophy-relevant readouts was carried out. Both 3- and 6-month-old mdx mice showed a significant decrease in diaphragm amplitude compared to wild type (wt) mice. This index was well-correlated either with in vivo running performance or ex vivo isometric tetanic force of isolated diaphragm. In addition, diaphragms from 6-month-old dystrophic mice were also highly susceptible to eccentric contraction ex vivo. Importantly, we disclosed an age-dependent increase in echodensity in mdx mice not observed in wt animals, which was independent from abdominal wall thickness. This was accompanied by a notable increase of pro-fibrotic TGF-β1 levels in the mdx diaphragm and of non-muscle tissue amount in diaphragm sections stained by hematoxylin & eosin. Our findings corroborate the usefulness of diaphragm ultrasonography in preclinical drug studies as a powerful tool to monitor mdx pathology progression since early stages

GLPG0492, a novel selective androgen receptor modulator, improves muscle performance in the exercised-mdx mouse model of muscular dystrophy

Anabolic drugs may counteract muscle wasting and dysfunction in Duchenne muscular dystrophy (DMD); however, steroids have unwanted side effects. We focused on GLPG0492, a new non-steroidal selective androgen receptor modulator that is currently under development for musculo-skeletal diseases such as sarcopenia and cachexia. GLPG0492 was tested in the exercised mdx mouse model of DMD in a 4-week trial at a single high dose (30mg/kg, 6 day/week s.c.), and the results were compared with those from the administration of α-methylprednisolone (PDN; 1mg/kg, i.p.) and nandrolone (NAND, 5mg/kg, s.c.). This assessment was followed by a 12-week dose-dependence study (0.3-30mg/kg s.c.). The outcomes were evaluated in vivo and ex vivo on functional, histological and biochemical parameters. Similar to PDN and NAND, GLPG0492 significantly increased mouse strength. In acute exhaustion tests, a surrogate of the 6-min walking test used in DMD patients, GLPG0492 preserved running performance, whereas vehicle- or comparator-treated animals showed a significant increase in fatigue (30-50%). Ex vivo, all drugs resulted in a modest but significant increase of diaphragm force. In parallel, a decrease in the non-muscle area and markers of fibrosis was observed in GLPG0492- and NAND-treated mice. The drugs exerted minor effects on limb muscles; however, electrophysiological biomarkers were ameliorated in extensor digitorum longus muscle. The longer dose-dependence study confirmed the effect on mdx mouse strength and resistance to fatigue and demonstrated the efficacy of lower drug doses on in vivo and ex vivo functional parameters. These results support the interest of further studies of GLPG0492 as a potential treatment for DMD.Anabolic drugs may counteract muscle wasting and dysfunction in Duchenne muscular dystrophy (DMD); however, steroids have unwanted side effects. We focused on GLPG0492, a new non-steroidal selective androgen receptor modulator that is currently under development for musculo-skeletal diseases such as sarcopenia and cachexia. GLPG0492 was tested in the exercised mdx mouse model of DMD in a 4-week trial at a single high dose (30 mg/kg, 6 day/week s.c.), and the results were compared with those from the administration of α-methylprednisolone (PDN; 1 mg/kg, i.p.) and nandrolone (NAND, 5 mg/kg, s.c.). This assessment was followed by a 12-week dose-dependence study (0.3-30 mg/kg s.c.). The outcomes were evaluated in vivo and ex vivo on functional, histological and biochemical parameters. Similar to PDN and NAND, GLPG0492 significantly increased mouse strength. In acute exhaustion tests, a surrogate of the 6-min walking test used in DMD patients, GLPG0492 preserved running performance, whereas vehicle- or comparator-treated animals showed a significant increase in fatigue (30-50%). Ex vivo, all drugs resulted in a modest but significant increase of diaphragm force. In parallel, a decrease in the non-muscle area and markers of fibrosis was observed in GLPG0492- and NAND-treated mice. The drugs exerted minor effects on limb muscles; however, electrophysiological biomarkers were ameliorated in extensor digitorum longus muscle. The longer dose-dependence study confirmed the effect on mdx mouse strength and resistance to fatigue and demonstrated the efficacy of lower drug doses on in vivo and ex vivo functional parameters. These results support the interest of further studies of GLPG0492 as a potential treatment for DMD. © 2013 Elsevier Ltd. All rights reserved

Effect of a long-term treatment with metformin in dystrophic mdx mice: A reconsideration of its potential clinical interest in Duchenne muscular dystrophy

The pharmacological stimulation of AMP-activated protein kinase (AMPK) via metabolic enhancers has been proposed as potential therapeutic strategy for Duchenne muscular dystrophy (DMD). Metformin, a widely-prescribed anti-hyperglycemic drug which activates AMPK via mitochondrial respiratory chain, has been recently tested in DMD patients in synergy with nitric oxide (NO)-precursors, with encouraging results. However, preclinical data supporting the use of metformin in DMD are still poor, and its actions on skeletal muscle appear controversial. Therefore, we investigated the effects of a long-term treatment with metformin (200 mg/kg/day in drinking water, for 20 weeks) in the exercised mdx mouse model, characterized by a severe mechanical-metabolic maladaptation. Metformin significantly ameliorated histopathology in mdx gastrocnemius muscle, in parallel reducing TGF-β1 with a recovery score (r.s) of 106%; this was accompanied by a decreased plasma matrix-metalloproteinase-9 (r.s. 43%). In addition, metformin significantly increased mdx diaphragm twitch and tetanic tension ex vivo (r.s. 44% and 36%, respectively), in spite of minor effects on in vivo weakness. However, no clear protective actions on dystrophic muscle metabolism were observed, as shown by the poor metformin effect on AMPK activation measured by western blot, on the expression of mechanical-metabolic response genes analyzed by qPCR, and by the lack of fast-to-slow fiber-type-shift assessed by SDH staining in tibialis anterior muscle. Similar results were obtained in the milder phenotype of sedentary mdx mice. The lack of metabolic effects could be, at least partly, due to metformin inability to increase low mdx muscle levels of L-arginine, L-citrulline and taurine, found by HPLC. Our findings encourage to explore alternative, metabolism-independent mechanisms of action to differently repurpose metformin in DMD, supporting its therapeutic combination with NO-sources

Assessment of resveratrol, apocynin and taurine on mechanical-metabolic uncoupling and oxidative stress in a mouse model of Duchenne muscular dystrophy: a comparison with the gold standard, α-methyl prednisolone.

Antioxidants have a great potential as adjuvant therapeutics in patients with Duchenne muscular dystrophy, although systematic comparisons at pre-clinical level are limited. The present study is a head-to-head assessment, in the exercised mdx mouse model of DMD, of natural compounds, resveratrol and apocynin, and of the amino acid taurine, in comparison with the gold standard α-methyl prednisolone (PDN). The rationale was to target the overproduction of reactive oxygen species (ROS) via disease-related pathways that are worsened by mechanical-metabolic impairment such as inflammation and over-activity of NADPH oxidase (NOX) (taurine and apocynin, respectively) or the failing ROS detoxification mechanisms via sirtuin-1 (SIRT1)-peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) (resveratrol). Resveratrol (100mg/kg i.p. 5days/week), apocynin (38mg/kg/day per os), taurine (1g/kg/day per os), and PDN (1mg/kg i.p., 5days/week) were administered for 4-5 weeks to mdx mice in parallel with a standard protocol of treadmill exercise and the outcome was evaluated with a multidisciplinary approach in vivo and ex vivo on pathology-related end-points and biomarkers of oxidative stress. Resveratrol≥taurine>apocynin enhanced in vivo mouse force similarly to PDN. All the compounds reduced the production of superoxide anion, assessed by dihydroethidium staining, with apocynin being as effective as PDN, and ameliorated electrophysiological biomarkers of oxidative stress. Resveratrol also significantly reduced plasma levels of creatine kinase and lactate dehydrogenase. Force of isolated muscles was little ameliorated. However, the three compounds improved histopathology of gastrocnemius muscle more than PDN. Taurine>apocynin>PDN significantly decreased activated NF-kB positive myofibers. Thus, compounds targeting NOX-ROS or SIRT1/PGC-1α pathways differently modulate clinically relevant DMD-related endpoints according to their mechanism of action. With the caution needed in translational research, the results show that the parallel assessment can help the identification of best adjuvant therapies