Calcium homeostasis is altered in skeletal muscle of spontaneously hypertensive rats cytofluorimetric and gene expression analysis

Hypertension is often associated with skeletal muscle pathological conditions related to function and metabolism. The mechanisms underlying the development of these pathological conditions remain undefined. Because calcium homeostasis is a biomarker of muscle function, we assessed whether it is altered in hypertensive muscles. We measured resting intracellular calcium and store-operated calcium entry (SOCE) in fast- and slow-twitch muscle fibers from normotensive Wistar-Kyoto rats and spontaneously hypertensive rats (SHRs) by cytofluorimetric technique and determined the expression of SOCE gene machinery by real-time PCR. Hypertension caused a phenotype-dependent dysregulation of calcium homeostasis; the resting intracellular calcium of extensor digitorum longus and soleus muscles of SHRs were differently altered with respect to the related muscle of normotensive animals. In addition, soleus muscles of SHR showed reduced activity of the sarcoplasmic reticulum and decreased sarcolemmal calcium permeability at rest and after SOCE activation. Accordingly, we found an alteration of the expression levels of some SOCE components, such as stromal interaction molecule 1, calcium release-activated calcium modulator 1, and transient receptor potential canonical 1. The hypertension-induced alterations of calcium homeostasis in the soleus muscle of SHRs occurred with changes of some functional outcomes as excitability and resting chloride conductance. We provide suitable targets for therapeutic interventions aimed at counterbalancing muscle performance decline in hypertension, and propose the reported calcium-dependent parameters as indexes to predict how the antihypertensive drugs could influence muscle function

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

EFFETS MYOTROPHIQUES D'UN FACTEUR NEUROTROPHIQUE, LE CNTF, SUR LES MUSCLES SQUELETTIQUES DE RAT EN CONDITIONS DE MICROGRAVITE SIMULEE

NANTES-BU Sciences (441092104) / SudocSudocFranceF

Influence of metallic mixture exposure on subcellular metal distribution in Daphnia magna

International audienceAquatic ecosystems are constantly subjected to the introduction of anthropogenic pollutants which constitute a threat for living organisms. Metals are among the major contaminants, because of their persistence and may have short and long term deleterious effects. The whole body metal concentrations give useful information on the bioavailability of these compounds, but they are inadequate to predict their potential toxicity. As only a portion of incorporated metals is potentially toxic, the knowledge of the subcellular behaviour of metals in living organisms is fundamental. In several studies, authors show that effects were linked to the metal concentrations in the cytosolic fraction and particularly to the metal bound to low molecular weight compounds (Wang et al, 1999) or to high molecular weight compounds (Perceval et al, 2006). The aim of this project was to assess on one hand the metal distribution between cytosolic and insoluble fractions and on the other hand the cytosolic metal distribution according to three molecular mass class compounds including a fraction containing metallothionein proteins

Importance of metallothioneins in the cadmium detoxification process in Daphnia magna

International audienceGood knowledge of the relationship between toxic metals and biological systems, particularly the sub-cellular fraction, could be a suitable early indicator of toxic effects. These effects and the sub-cellular behaviour of cadmium were studied with a widely used species in freshwater toxicity bioassays, Daphnia magna. In spite of this very commonplace usage in ecotoxicological studies, very few data are available on its toxicant metabolism and in particular metal homeostasis. Combining multi-tools analysis, a soluble protein was found: it is heat-stable, rich in sulfhydryl groups (differential pulse polarography), characterised by a molecular mass of approximately 6.5 kDa, with a G-75 chromatographic profile corresponding to the rabbit metallothioneins monomer, with few if any aromatic-containing amino acids, it binds metals (e.g. Cd, Cu), and its concentration increases with Cd exposure. This evidence led us to hypothesise that metallothioneins (MTs) are present in D. magna. Up to 75% of the Cd body burden with Cd exposure is bound to the MTs fraction. The increase in the Cd concentration in the surrounding medium and concomitantly in daphnids induces sub-cellular reorganisation of essential metals such as Cu and Zn. The rate of metals in the soluble cellular fraction and associated with MTs increases with the Cd body burden. Monitoring sub-cellular distribution of metals after exposure in the natural environment could be very useful for ecotoxicological assessment

Dystrophin restoration therapy improves both the reduced excitability and the force drop induced by lengthening contractions in dystrophic mdx skeletal muscle

International audienceBackgroundThe greater susceptibility to contraction-induced skeletal muscle injury (fragility) is an important dystrophic feature and tool for testing preclinic dystrophin-based therapies for Duchenne muscular dystrophy. However, how these therapies reduce the muscle fragility is not clear.MethodsTo address this question, we first determined the event(s) of the excitation-contraction cycle which is/are altered following lengthening (eccentric) contractions in the mdx muscle.ResultsWe found that the immediate force drop following lengthening contractions, a widely used measure of muscle fragility, was associated with reduced muscle excitability. Moreover, the force drop can be mimicked by an experimental reduction in muscle excitation of uninjured muscle. Furthermore, the force drop was not related to major neuromuscular transmission failure, excitation-contraction uncoupling, and myofibrillar impairment. Secondly, and importantly, the re-expression of functional truncated dystrophin in the muscle of mdx mice using an exon skipping strategy partially prevented the reductions in both force drop and muscle excitability following lengthening contractions.ConclusionWe demonstrated for the first time that (i) the increased susceptibility to contraction-induced muscle injury in mdx mice is mainly attributable to reduced muscle excitability; (ii) dystrophin-based therapy improves fragility of the dystrophic skeletal muscle by preventing reduction in muscle excitability

Evaluation des risques environnementaux concernant 6 substances pharmaceutiques : les procédures de l`évaluation des risques environnementaux sont-elles suffisantes pour protéger l`écosystème aquatique ?

[Departement_IRSTEA]MA [TR1_IRSTEA]QSA / EXPERInternational audienceIn this study, acute and chronic data of 6 human pharmaceuticals (Carbamazepine, Clofibric acid, Diclofenac, Ofloxacin, Propranolol and Sulphamethoxazole) have been collected, using our own experimental data and literature data. From this data collection, the 2-tiered European draft guideline on the environmental risk assessment of such substances was tested. Firstly, measured environmental concentrations (MEC) in effluents from France and from published data obtained within effluents and surface waters in Germany were compared to the predicted environmental concentrations (PEC) respectively in both countries. Secondly, in a similar manner, using the compiled toxicity data on aquatic organisms, predicted no effect concentrations (PNEC) derived from chronic data were compared to PNEC derived from acute data. Globally, results demonstrated that (a) all environmental concentrations (predicted or measured) for each considered pharmaceutical exceeded the 10 ng/L cut-off value, which requires the second tier assessment based on ecotoxicity data, (b) the 6 pharmaceuticals showed a relatively limited acute toxicity and (c) Carbamazepine and Propranolol were inaccurately identified as having negligible risks under the current European draft procedure. Such results should help to provide further sound considerations for discussing the actual procedure on pharmaceuticals, especially on the need of appropriate ecotoxicity tests

Effets des processus avancés d'oxydation (AOPs) sur la toxicité d'un mélange de produits pharmaceutiques

[Departement_IRSTEA]MA [TR1_IRSTEA]QSA / EXPERInternational audienceThe possibility of applying main AOP techniques, namely ozonation, H2O2/UV photolysis and TiO2 photocatalysis to provide a significant reduction of toxicity of pharmaceutical mixtures has been evaluated. For the preparation of the mixture six pharmaceuticals were chosen among those found at highest concentrations in Sewage Treatment Plant effluents, namely carbamazepine, clofibric acid, diclofenac, sulfamethoxazole, ofloxacin and propranolol. The blue-green alga Synechococcus leopoliensis and the rotifer Brachyonus calyciflorus were utilised to assess the toxicity of the mixtures after AOP treatments. All the toxicity tests were performed using chronic standardized bioassays. The best results were obtained with ozonation. With this type of treatment a complete removal of mixture toxicity on S. leopolensis was obtained even after the shortest time of application (1 min). The ozonation treatment leads also to removal of all the pharmaceutical mixture toxicity on B. calyciflorus, by applying the oxidizing agent for at least for 2 minutes

Mechanical Overloading Increases Maximal Force and Reduces Fragility in Hind Limb Skeletal Muscle from Mdx Mouse

International audienceThere is fear that mechanical overloading (OVL; ie, high-force contractions) accelerates Duchenne muscular dystrophy. Herein, we determined whether short-term OVL combined with wheel running, short-term OVL combined with irradiation, and long-term OVL are detrimental for hind limb mdx mouse muscle, a murine model of Duchene muscular dystrophy exhibiting milder dystrophic features. OVL was induced by the surgical ablation of the synergic muscles of the plantaris muscle, a fast muscle susceptible to contraction-induced muscle damage in mdx mice. We found that short-term OVL combined with wheel and long-term OVL did not worsen the deficit in specific maximal force (ie, absolute maximal force normalized to muscle size) and histological markers of muscle damage (percentage of regenerating fibers and fibrosis) in mdx mice. Moreover, long-term OVL did not increase the alteration in calcium homeostasis and did not deplete muscle cell progenitors expressing Pax 7 in mdx mice. Irradiation before short-term OVL, which is believed to inhibit muscle regeneration, was not more detrimental to mdx than control mice. Interestingly, short-term OVL combined with wheel and tong-term OVL markedly improved the susceptibility to contraction-induced damage, increased absolute maximal force, induced hypertrophy, and promoted a slower, more oxidative phenotype. Together, these findings indicate that OVL is beneficial to mdx muscle, and muscle regeneration does not mask the potentially detrimental effect of OVL

Extensive characterisation of a new rat model of Duchenne muscular dystrophy.

Extensive characterisation of a new rat model of Duchenne muscular dystrophy.. Congress of the ESVP, ECVP and EST