Evaluation of the dystrophin carboxy-terminal domain for micro-dystrophin gene therapy in cardiac and skeletal muscles in the DMDmdx rat model

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the gene encoding dystrophin. Gene therapy using micro-dystrophin (MD) transgenes and recombinant adeno-associated virus (rAAV) vectors hold great promise. To overcome the limited packaging capacity of rAAV vectors, most MD do not include dystrophin carboxy-terminal (CT) domain. Yet, the CT domain is known to recruit α1- and β1-syntrophins and α-dystrobrevin, a part of the dystrophin-associated protein complex (DAPC), which is a signaling and structural mediator of muscle cells. In this study, we explored the impact of inclusion of the dystrophin CT domain on ΔR4-23/ΔCT MD (MD1), in DMDmdx rats, which allows for relevant evaluations at muscular and cardiac levels. We showed by LC-MS/MS that MD1 expression is sufficient to restore the interactions at a physiological level of most DAPC partners in skeletal and cardiac muscles, and that inclusion of the CT domain increases the recruitment of some DAPC partners at supra-physiological levels. In parallel, we demonstrated that inclusion of the CT domain does not improve MD1 therapeutic efficacy on DMD muscle and cardiac pathologies. Our work highlights new evidences of the therapeutic potential of MD1 and strengthens the relevance of this candidate for gene therapy of DMD

Analyse fonctionnelle, cellulaire et moléculaire des effets du stress oxydatif sur l'homéostasie calcique du muscle dystrophique

La myopathie de Duchenne (DMD) est une maladie neuromusculaire affectant environ 1 garçon sur 3500. Elle est due à des mutations dans le gène codant pour la dystrophine, protéine de 420 kDa identifiée en 1987, localisée sous la membrane de la fibre musculaire. Une perturbation de l homéostasie calcique est l un des mécanismes impliqués dans le processus physiopathologique de cette maladie. Cependant, l absence de la dystrophine induit également la délocalisation de l enzyme NO-synthase provoquant une diminution de la libération de NO, ce qui pourrait être à l origine de l accumulation de radicaux libres, inducteurs potentiels de la nécrose musculaire. Le but de ce travail a donc été d étudier le rôle du stress oxydatif dans le processus dystrophique musculaire du diaphragme de souris mdx, modèle animal de la DMD, et tout particulièrement sur les mécanismes participant à la régulation du Ca2+ intracellulaire. Ainsi, les effets potentiels d oxydants tels que le peroxyde d hydrogène (H2O2) et l acide hypochlorique (HOCl) ont été analysés sur les structures impliquées dans la fonction musculaire telles que les myofilaments et le réticulum sarcoplasmique, organite impliqué dans la régulation du Ca2+ et apparaissant aussi comme des cibles privilégiées de l oxydation cellulaire. Les résultats obtenus sur le modèle de fibres perméabilisées au triton X-100 montrent que seul HOCl induit une augmentation de la sensibilité au Ca2+ des protéines contractiles. L analyse des contractures induites par l activation des récepteurs à la ryanodine par la caféine sur le modèle de fibres perméabilisées à la saponine montre que H2O2 et HOCl induisent une diminution des contractures développées traduisant une diminution de la quantité de Ca2+ libérée, ces effets étant significativement plus importants pour le muscle dystrophique. Lors d approches conduites sur des vésicules de réticulum sarcoplasmique, la sensibilité accrue des muscles dystrophiques à l action des oxydants a été également mise en évidence. Ainsi, dans ces cellules squelettiques de diaphragme où la dystrophine est absente, nos résultats montrent que les oxydants peuvent altérer la fonction contractile par une action au niveau des mécanismes cellulaires impliqués dans le contrôle du Ca2+, suggérant à nouveau l implication d un stress oxydatif dans le processus dystrophique et son association étroite avec la perturbation de l homéostasie calcique. Aucun traitement curatif de la myopathie de Duchenne est actuellement disponible, seule la prescription de stéroïdes comme traitement palliatif permet de ralentir la maladie mais comporte de nombreux effets secondaires. La suite de l étude a donc été consacrée à l analyse des effets bénéfiques potentiels de traitements pharmacologiques. Les souris mdx traitées à base de créatine ou de L-arginine montrent une récupération de certaines fonctions motrices associée à une amélioration de la fonction de charge en Ca2+ du réticulum sarcoplasmique. Ces résultats démontrent l importance de cet organite dans le contrôle du Ca2+ du muscle dystrophique. Les protéines impliquées dans la régulation du Ca2+ apparaissent donc comme des cibles privilégiées dans le cadre de la mise en place de nouveaux traitements de la DMDDuchenne muscular dystrophy (DMD) is characterized by necrosis and progressive muscle weakness. In DMD patients and mdx mouse model, dystrophin deficiency lead to altered total Ca2+ content but also to an oxidative stress. This study examine the direct effect of oxidant on the contractile apparatus and sarcoplasmic reticulum (SR) function in diaphragm from mdx mice. Exposing triton skinned fibres to H2O2 or HOCl induced a decrease of tension. Data from saponin skinned fibres and SR vesicles show that oxidant slowing-down the SR Ca2+ uptake. Furthermore, mdx muscle was more affected by H2O2, suggesting that oxidant could been implicated in alteration of Ca2+ homeostasis via disruption of SR function. A second part of this study investigated the potential benefits of pharmacological tools in reversing the Ca2+ sequestration function of SR. Creatine or L-arginine treated mdx mice show an improvement of motor capacity, of cellular contractile function and particularly of the SR Ca2+ uptake. Taken together, these data show the importance of SR function in Ca2+ regulation in dystrophic muscle.NANTES-BU Sciences (441092104) / SudocSudocFranceF

Rôle des mécanismes intracellulaires de régulation du calcium dans la dystrophie musculaire de Duchenne (approche physiologique, pharmacologique et moléculaire)

Le but de ce travail est d'étudier les fonctions du réticulum sarcoplasmique (RS) des muscles EDL et soléaire de souris mdx. Les résultats montrent, dans les deux types de muscles, un ralentissement de la charge en Ca2+ sans modification de la capacité maximale de stockage. Une altération des mécanismes de charge pourrait impliquer la Ca2+-ATPase et/ou la présence d'une fuite passive de Ca2+. Les résultats issus d'approches physiopharmacologiques, biochimiques et moléculaires indiquent, dans l'EDL mdx, une diminution de la sensibilité à un inhibiteur de la Ca2+-ATPase du RS, associée à l'expression de son isoforme lente SERCA2a. Dans le soléaire mdx seule une importante fuite de Ca2+ du RS a été observée, impliquant les canaux calciques : récepteurs à la ryanodine et à l'inositol triphosphate. Ces résultats soulignent le rôle majeur du RS dans la perturbation de l'homéostasie calcique des cellules musculaires dystrophiques impliquant différents mécanismes selon le type musculaire.NANTES-BU Sciences (441092104) / SudocSudocFranceF

Cyclopiazonic acid-induced changes in the contraction and Ca 2+ transient of frog fast-twitch skeletal muscle

International audienceThe effects of cyclopiazonic acid (CPA) were investigated on isolated skeletal muscle fibers of frog semitendinosus muscle. CPA (0.5-10 microM) enhanced isometric twitch but produced little change in resting tension. At higher concentrations (10-50 microM), CPA depressed twitch and induced sustained contracture without affecting resting and action potentials. In Triton-skinned fibers, CPA had no significant effect on myofibrillar Ca2+ sensitivity but decreased maximal activated force at concentrations > 5 microM. In intact cells loaded with the Ca2+ fluorescence indicator indo 1, CPA (2 microM) induced an increase in Ca(2+)-transient amplitude (10 +/- 2.5%), which was associated with an increase in time to peak and in the time constant of decay. Consequently, peak force was increased by 35 +/- 4%, and both time to peak and the time constant of relaxation were prolonged. It is concluded that CPA effects, at a concentration of up to 2 microM, were associated with specific inhibition of sarcoplasmic reticulum Ca(2+)-adenosinetriphosphatase in intact skeletal muscle and that inhibition of the pump directly affected the handling of intracellular Ca2+ and force production

The Contractile Phenotype of Skeletal Muscle in TRPV1 Knockout Mice Is Gender-Specific and Exercise-Dependent

International audienceThe transient receptor potential vanilloid 1 (TRPV1) belongs to the transient receptor potential superfamily of sensory receptors. TRPV1 is a non-selective cation channel permeable to Ca 2+ that is capable of detecting noxious heat temperature and acidosis. In skeletal muscles, TRPV1 operates as a reticular Ca 2+-leak channel and several TRPV1 mutations have been associated with two muscle disorders: malignant hyperthermia (MH) and exertional heat stroke (EHS). Although TRPV1 −/− mice have been available since the 2000s, TRPV1's role in muscle physiology has not been thoroughly studied. Therefore, the focus of this work was to characterize the contractile phenotype of skeletal muscles of TRPV1-deficient mice at rest and after four weeks of exercise. As MS and EHS have a higher incidence in men than in women, we also investigated sex-related phenotype differences. Our results indicated that, without exercise, TRPV1 −/− mice improved in vivo muscle strength with an impairment of skeletal muscle in vitro twitch features, i.e., delayed contraction and relaxation. Additionally, exercise appeared detrimental to TRPV1 −/− slow-twitch muscles, especially in female animals

Soluble Milk Protein Supplementation with Moderate Physical Activity Improves Locomotion Function in Aging Rats

International audienceAging is associated with a loss of muscle mass and functional capacity. Present study was designed to compare the impact of specific dairy proteins on muscular function with or without a low-intensity physical activity program on a treadmill in an aged rat model. We investigated the effects of nutritional supplementation, five days a week over a 2-month period with a slow digestible protein, casein or fast digestible proteins, whey or soluble milk protein, on strength and locomotor parameters in sedentary or active aged Wistar RjHan rats (17-19 months of age). An extensive gait analysis was performed before and after protein supplementation. After two months of protein administration and activity program, muscle force was evaluated using a grip test, spontaneous activity using an open-field and muscular mass by specific muscle sampling. When aged rats were supplemented with proteins without exercise, only minor effects of different diets on muscle mass and locomotion were observed: higher muscle mass in the casein group and improvement of stride frequencies with soluble milk protein. By contrast, supplementation with soluble milk protein just after physical activity was more effective at improving overall skeletal muscle function in old rats compared to casein. For active old rats supplemented with soluble milk protein, an increase in locomotor activity in the open field and an enhancement of static and dynamic gait parameters compared to active groups supplemented with casein or whey were observed without any differences in muscle mass and forelimb strength. These results suggest that consumption of soluble milk protein as a bolus immediately after a low intensity physical activity may be a suitable nutritional intervention to prevent decline in locomotion in aged rats and strengthen the interest to analyze the longitudinal aspect of locomotion in aged rodents

Skeletal muscle relaxant effect of a standardized extract of Valeriana officinalis L. after acute administration in mice

Valeriana officinalis L. root extracts are traditionally taken for their sedative and anxiolytic properties and are also used for muscle relaxation. Relaxant effects were clearly observed on smooth muscle whereas data on effects on skeletal muscle are scarce and inconsistent. The aim of this study was to assess whether a standardized extract (SE) of V. officinalis had myorelaxant effects by decreasing skeletal muscle strength and/or neuromuscular tone in mice. Mice received an acute dose of V. officinalis SE (2 or 5 g/kg per os) or tetrazepam (10 mg/kg ip), a standard myorelaxant drug. Thirty minutes later, the maximal muscle strength was measured using a grip test, while global skeletal muscle function (endurance and neuromuscular tone) was assessed in a wire hanging test. Compared to tetrazepam, both doses of V. officinalis SE induced a pronounced decrease in skeletal muscle strength without any significant effects on endurance and neuromuscular tone. This study provides clear evidence that the extract of V. officinalis tested has a relaxant effect on skeletal muscle. By decreasing skeletal muscle strength without impacting endurance and neuromuscular tone, V. officinalis SE could induce less undesirable side effects than standard myorelaxant agents, and be particularly useful for avoiding falls in the elderly. Keywords: Valeriana officinalis, Skeletal muscle relaxant, Strength, Hydroethanolic root extract, Acute treatment, Mous

Characterization of brain dystrophins absence and impact in dystrophin-deficient Dmdmdx rat model

International audienceCharacterization of brain dystrophins absence and impact in dystrophin-deficient Dmdmdx rat mode

Similar total amount of protein ingested during study in sedentary or active old rats.

<p>For each sedentary (<i>A</i>) and active (<i>B</i>) rats, the weekly protein intake was calculated, normalized to body weight and summarized to compare the total amount of protein ingested during the 2 months of the supplementation with casein, whey or soluble milk protein. With or without a low physical activity, the total amount of protein intake during the 2 months was not significantly different between specific proteins. Values are means ± SEM.</p

Protein supplementation without food deprivation induced a significant and enduring increase in total protein intake.

<p>For sedentary (<i>A</i>) and active (<i>B</i>) old rats, supplementation significantly increased the total protein intake (<i>i</i>.<i>e</i> food and specific proteins) over the course of the study period. For each analysed time point, there was an equal amount of total protein ingested, regardless of the specific protein being tested. Values are means ± SEM. W0 to W8: weeks of protocol with W0 being the week prior to physical activity and/or protein supplementation. * p<0.05 <i>vs</i>. W0.</p