Weakness and fatigability, typical features of Duchenne muscular dystrophy, are aggravated in mdx mice by a chronic exercise on horizontal treadmill (30 min running at 12 m/min twice a week). This protocol leads to a significant decrease in limb force vs. non exercised mdx mice by grip test; preliminary results by torque recordings confirm this functional impairment. Parallel ex vivo studies show that the exercise increases the resistance to eccentric contraction in C57BL/10 wildtype (wt) extensor digitorum longus (EDL) muscles, while such an adaptation is not observed in mdx ones, which remain weaker than controls. In order to understand the molecular mechanisms underlying exercise susceptibility of mdx mice we investigated by quantitive realtime PCR 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 gastrocnemius muscle of mdx and wt mice. Basal expression of peroxisome proliferator receptor γ coactivator 1α (PGC1α) and sirtuin1 is higher in mdx vs. wt at both ages. Exercise increases PGC1α expression in wt, while in mdx mice T12 exercise down-regulates PGC1α, sirtuin1, PPARγ and the autophagy marker BNIP3. 16 week-old mdx mice show a basal overexpression of the slow phenotype genes; T12 exercise fully contrasts this basal adaptation and the high expression of follistatin and myogenin, being ineffective in wt mice. Damage and inflammation related genes, such as NADPHoxidase, TGFβ and TNFα are overexpressed in mdx muscle in all conditions. In parallel the anti-inflammatory adiponectin is lower in T12 exercised mdx muscle. Then a chronic exercise with minor adaptive effects in wt muscle, contrasts compensatory changes in the benign mdx phenotype leading to a disequilibrium between protective and damaging signals and disclosing potential drug targets (Supported by NLDDP and Italian MIUR-PRIN project n. 20108YB5W3_004).

Angiotensin II (ANG II) plays a role in muscle wasting and remodeling; however, little evidence shows its direct effects on specific muscle functions. We presently investigated the acute in vitro effects of ANG II on resting ionic conductance and calcium homeostasis of mouse extensor digitorum longus (EDL) muscle fibers, based on previous findings that in vivo inhibition of ANG II counteracts the impairment of macroscopic ClC-1 chloride channel conductance (gCl) in the mdx mouse model of muscular dystrophy. By means of intracellular microelectrode recordings we found that ANG II reduced gCl in the nanomolar range and in a concentration-dependent manner (EC50 = 0.06 μM) meanwhile increasing potassium conductance (gK). Both effects were inhibited by the ANG II receptors type 1 (AT1)-receptor antagonist losartan and the protein kinase C inhibitor chelerythrine; no antagonism was observed with the AT2 antagonist PD123,319. The scavenger of reactive oxygen species (ROS) N-acetyl cysteine and the NADPH-oxidase (NOX) inhibitor apocynin also antagonized ANG II effects on resting ionic conductances; the ANG II-dependent gK increase was blocked by iberiotoxin, an inhibitor of calcium-activated potassium channels. ANG II also lowered the threshold for myofiber and muscle contraction. Both ANG II and the AT1 agonist L162,313 increased the intracellular calcium transients, measured by fura-2, with a two-step pattern. These latter effects were not observed in the presence of losartan and of the phospholipase C inhibitor U73122 and the in absence of extracellular calcium, disclosing a Gq-mediated calcium entry mechanism. The data show for the first time that the AT1-mediated ANG II pathway, also involving NOX and ROS, directly modulates ion channels and calcium homeostasis in adult myofibers.

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.

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.

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 research

Oxidative stress, caused by reactive oxygen species (ROS), has been implicated on disease progression and chronic inflammation in Duchenne Muscular Dystrophy (DMD). NADPH oxidase 2 (NOX2) is currently considered to be a major source of ROS and it is over-expressed in skeletal muscle and heart of mdx mice, the most widely used model for DMD. Its activation in mdx myofibers via stretch-sensitive pathways has also been shown (Whitehead et al., 2010; Khairallah et al., 2012). Consequently, drugs able to reduce ROS production by inhibition of NOX-2 are potential treatment for muscular dystrophy. In line with this view, we have recently shown that enalapril, by inhibiting the production of angiotensin II (Ang II), one of the main endogenous activator of NOX, reduces the signs of oxidative stress and the percentage of p65-NF-kB positive nuclei in the mdx muscles. In this frame we also observed, in the myofibers of mdx mice treated with enalapril, a dose-dependent restoration of macroscopic chloride conductance (gCl), a sensitive biomarker of inflammation in skeletal muscle (Cozzoli et al., 2011). The aim of the present study was to investigate the involvement of NOX-2 dependent-ROS production in relation to the aberrant mechano-transduction occurring in dystrophic muscle. RT-PCR experiments confirmed a higher expression of β-tubulin and NOX2 (gp91phox) mRNA in gastrocnemius (GC) muscle of mdx mice. Interestingly, this increased expression was maintained in GC muscles of mdx mice that underwent a standard chronic (1-2 months) exercise protocol on treadmill. Then, we tested the effect of a chronic treatment with apocynin (38 mg/kg in drinking water/day for 5-9 weeks), a natural compound able to directly inhibit NOX-2, on exercised mdx mice (De Luca et al., 2003). Treatment started at 4-5 weeks of age and the outcome was evaluated by a multidisciplinary approach on pathology-related in vivo and ex vivo endpoints. In vivo, apocynin significantly increased mouse strength, with normalized forelimb force values of 6.4 ± 0.16 (n=9) vs. 5.6 ± 0.19 (n=10; p<0.05) of untreated mice, but did not improve exercise performance. Furthermore, no effect was observed on plasma creatine kinase and lactate dehydrogenase. However, the treatment with apocynin counteracted the exercise-induced impairment of total membrane conductance (gm), which is mainly sustained by the reduction of gCl, in extensor digitorum longus (EDL) muscle fibers, gm being 2536 ±105 µS/cm2 (n =37) vs. 1886 ± 92 µS/cm2 (n = 42, p <0.0001) of untreated mdx myofibers. Then the recovery score for this parameter, considering the value of 2607 ± 23 µS/cm2 (n =19) of wild-type C57BL10 myofibers, was 90% in apocynin-treated myofibers. This latter effect prompted us to investigate the possibility that the channels underlying macroscopic gCl could be target of the redox-dependent NOX actions in skeletal muscle. Parallel experiments on EDL muscle fibers of C57BL10 mouse showed that Ang II decreases gCl in a concentration-dependent manner (IC50 = 60nM) and this effect was fully contrasted by the prior incubation of apocynin (10 µM) or with a known anti-oxidant N-acetyl cysteine (5 mM). Markers of oxidative stress and inflammation, RT-PCR, histo-morphology are currently under evaluation. However, this preliminary data support the hypothesis that pharmacological targeting of NOX-2, providing protection from cross-talk between ROS production and inflammation, may represent a valuable approach in DMD. (Supported by DPP/NL and MIUR-PRIN n° 20108YB5W3). Whitehead et al. (2010). PLoS One, 5(12):e15354. Khairallah et al. (2012). Sci Signal.5(236):ra56. Cozzoli et al. (2011). Pharmacol Res. 64(5):482-92. De Luca et al. (2003) J Pharmacol Exp Ther.304(1):453-63.