Characterisation and implication of TRPV1 cationic channel in physiopathology of skeletal muscle

Le canal cationique TRPV1 (Transient Receptor Potential Vanilloid 1) est activé par la capsaïcine, une acidose, de fortes températures ainsi que par les anesthésiques volatils (AV) dans les neurones sensoriels. Dans le muscle squelettique, TRPV1 est impliqué dans le métabolisme énergétique et l'exercice d'endurance. Grâce à des techniques d'immunomarquage et d'imagerie calcique, la première partie de la thèse vise à caractériser TRPV1 en tant que canal de fuite fonctionnel du réticulum sarcoplasmique (RS) dans les cellules musculaires squelettiques isolées de FDB (Flexor Digitorum Brevis) de souris. Par la suite, nous nous sommes intéressés à son rôle physiopathologique dans le muscle strié squelettique. Ainsi, dans une seconde partie nous supposons une implication de TRPV1 dans les crises d'hyperthermie maligne (HM) chez l'homme. Cette pathologie musculaire correspond à une crise de métabolisme exacerbé du muscle strié squelettique menant à une brusque montée en température chez le patient (>42°C) endormi au moyen d'AV. Dans cette deuxième étude nous démontrons, à travers une approche combinant imagerie calcique et outils pharmacologiques spécifiques du canal, que TRPV1 est activé lors de l'exposition des cellules musculaires à l'isoflurane. TRPV1 est donc une cible des AV dans la cellule musculaire. Puis, des variants de TRPV1 (T612M et N394del) de patients susceptibles à l'HM ont été découvertes. Nous avons pu montrer, suite à la transfection in vivo de ces variants dans des souris déficientes en TRPV1 et grâce à la mesure de flux calciques intracellulaires, que les variants humains de TRPV1 rendent ces canaux plus sensibles aux anesthésiques volatils que le canal TRPV1 humain sauvage. La troisième partie de la thèse a pour but de déterminer le rôle de TRPV1 dans le muscle squelettique en conditions physiologiques par des études fonctionnelles (fonction locomotrice, consommation d'oxygène) sur animal entier. Les résultats préliminaires de cette étude tendent à montrer que l'entraînement physique est moins efficace sur la fonction musculaire des souris déficientes en TRPV1. En conclusion, l'ensemble de ces résultats révèlent pour la première fois que TRPV1 est un canal calcique de fuite fonctionnel du RS pouvant faire le lien entre le déclenchement de l'HM au cours des anesthésies et la présence des RyR1 mutés dans le muscle squelettiqueTRPV1 (Transient Receptor Potential Vanilloid 1) cation channel is activated by capsaicine, acidosis, high temperature and by volatile anaesthetics (VA) in sensory neurons. In skeletal muscle, TRPV1 appears to be implied in exercice endurance and energy metabolism. The present work aims first to characterize the functionality of this channel using immnostaining and calcium imaging. We report that TRPV1 is functionally expressed in isolated mouse skeletal muscle cells of FDB (Flexor Digitorum Brevis). These experiments point out that TRPV1 acts as a SR calcium leak channel. In contrast to earlier reports, our analysis shows that TRPV1 is only located to the sarcoplasmic reticulum (SR) membrane. Subsequently, we have studied its physiological role in skeletal muscle. Thus, in a second part, we suppose that TRPV1 could be involved in malignant hyperthermia (MH) crisis in human. MH is a muscular pathology linked to an abrupt increase in body temperature (> 42°C) in patients. MH crisis is a severe and feared complication of anesthesia. Nevertheless, any studies have demonstrated that RyR1 mutants are activated by VA. If the triggering agents of MH are known, their targets remain to be determined. By combining calcium imaging and pharmacological agents, our data first demonstrate that TRPV1 is activated by isoflurane in skeletal muscle cells. TRPV1 is so a target of volatile anaesthetics in skeletal muscle. Afterwards, TRPV1 mutants (T612M and N394del), obtained from susceptibles MH patients, were discovered. In the second part of the work, using in vivo transfection of TRPV1 mutants in TRPV1-/- mice and intracellular calcium measurements we have been able to demonstrate that human TRPV1 mutants are more sensitive to VA than human wild type TRPV1. The last part of the work investigates the physiological role of TRPV1 in skeletal muscle, using a functional exploration (locomotor function, oxygen consumption) in TRPV1-/- mice. Preliminary data point out that training seems to be less effective on skeletal muscle function of TRPV1-/- mice. To conclude, these results indicate for the first time that TRPV1 is a functional SR calcium leak channel and that TRPV1 may be the missing link between MH induction and RyR1 mutants in skeletal muscle during anesthesi

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

Characterization of Functional TRPV1 Channels in the Sarcoplasmic Reticulum of Mouse Skeletal Muscle

<div><p>TRPV1 represents a non-selective cation channel activated by capsaicin, acidosis and high temperature. In the central nervous system where TRPV1 is highly expressed, its physiological role in nociception is clearly identified. In skeletal muscle, TRPV1 appears implicated in energy metabolism and exercise endurance. However, how as a Ca²⁺ channel, it contributes to intracellular calcium concentration ([Ca²⁺]_i) maintenance and muscle contraction remains unknown. Here, as in rats, we report that TRPV1 is functionally expressed in mouse skeletal muscle. In contrast to earlier reports, our analysis show TRPV1 presence only at the sarcoplasmic reticulum (SR) membrane (preferably at the longitudinal part) in the proximity of SERCA1 pumps. Using intracellular Ca²⁺ imaging, we directly accessed to the channel functionality in intact FDB mouse fibers. Capsaicin and resiniferatoxin, both agonists as well as high temperature (45°C) elicited an increase in [Ca²⁺]_i. TRPV1-inhibition by capsazepine resulted in a strong inhibition of TRPV1-mediated functional responses and abolished channel activation. Blocking the SR release (with ryanodine or dantrolene) led to a reduced capsaicin-induced Ca²⁺ elevation suggesting that TRPV1 may participate to a secondary SR Ca²⁺ liberation of greater amplitude. In conclusion, our experiments point out that TRPV1 is a functional SR Ca²⁺ leak channel and may crosstalk with RyR1 in adult mouse muscle fibers.</p> </div

Na/Ca exchange in the atrium: Role in sinoatrial node pacemaking and excitation-contraction coupling

International audienceNa/Ca exchange is the dominant calcium (Ca) efflux mechanism in cardiac myocytes. Although our knowledge of exchanger function (NCX1 in the heart) was originally established using biochemical and electrophysiological tools such as cardiac sarcolemmal vesicles and the giant patch technique [1-4], many advances in our understanding of the physiological/pathophysiological roles of NCX1 in the heart have been obtained using a suite of genetically modified mice. Early mouse studies focused on modification of expression levels of NCX1 in the ventricles, with transgenic overexpressors, global NCX1 knockout (KO) mice (which were embryonic lethal if homozygous), and finally ventricular-specific NCX1 KO [5-12]. We found, to our surprise, that ventricular cardiomyocytes lacking NCX1 can survive and function by engaging a clever set of adaptations to minimize Ca entry, while maintaining contractile function through an increase in excitation-contraction (EC) coupling gain [5,6,13]. Having studied ventricular NCX1 ablation in detail, we more recently focused on elucidating the role of NCX1 in the atria through altering NCX1 expression. Using a novel atrial-specific NCX1 KO mouse, we found unexpected changes in atrial cell morphology and calcium handling, together with dramatic alterations in the function of sinoatrial node (SAN) pacemaker activity. In this review, we will discuss these findings and their implications for cardiac disease

Schematic drawing of the possible TRPV1 contribution in the maintenance of Ca²⁺ muscle homeostasis.

<p>(1) External stimuli, such as capsaicin, resiniferatoxin or high temperature, activate TRPV1 channels located at the longitudinal SR. (2) TRPV1 channels releases Ca²⁺ from SR to cytoplasm. (3) This low increase in [Ca²⁺]_c activates RyR channels (4) that in return release Ca²⁺ into cytoplasm increasing [Ca²⁺]_c (5). In permanence, SERCA1 pumps continuously back Ca²⁺ from cytoplasm to SR and could mask TRPV1 activation in resting condition.</p

Calcium release induced by pharmacological activation of TRPV1 in FDB isolated fibers.

<p>(A) The traces show representative curve obtained after stimulation of single fibers with capsaicin alone (100 µM; black line) or in the presence of capsazepine (light grey line) or dantrolene (dark grey line). B; C: changes in fluorescent ratio F/F0 (peak– resting) induced by drugs as indicated in table above the graphs. Summary data showing fluorescent changes in cells induced by agonists, B: with or without antagonist pretreatment or else after SR depletion and C: with or without RyR inhibitors pretreatment. Capsazepine (CPZ, 100 µM), cyclopiazonic acid (CPA, 25 µM), dantrolene (DAN, 25 µM) and ryanodine (Ry, 80 µM) were added 20 min prior to agonist treatment. Resiniferatoxin and thapsigargin were used at 10 µM and 1 µM respectively. Results are expressed as means ± S.E.M. of the indicated number of experiments from at least 4 independent fibers preparations. T-tests were performed by paired samples: *p<0.05, **p<0.002 and ***p<0.0002.</p

Localization of TRPV1, RyR1 and SERCA1 in mouse FDB fibers.

<p>A: Schematic organization of the sarcoplasmic reticulum (SR) in a skeletal muscle fiber. Confocal images of double immunofluorescence labeling of TRPV1 (B) and RyR1 (C) or TRPV1 (F) and SERCA1 (G). E, I: average intensity profiles from the dotted rectangle region in the next corresponding images. In isolated fibers, RyR1 shows localization in the junctional part of the SR (C) while SERCA1 appears in the longitudinal part of the SR (G). In the merged images, TRPV1 does not colocalize with RyR1 (D) while colocalization with SERCA1 staining is visible in H. Results are from at least 4 independent fibers preparations (n>10).</p

Expression of TRPV1 and SERCA1 in skeletal muscle.

<p>Left panel: Western blot analysis of total protein extracts (25 µg) with anti-SERCA1 (top panel) and anti-TRPV1 antibodies (low panel) from the following tissues: flexus digitorum brevis/interosseal (FDB/IO), soleus, extensor digitorum longus (EDL), red and white gastrocnemius (R Ga and W Ga) and brain. Right panel: Localization of TRPV1 in sarcotubular membrane fractions (R1, fraction enriched in light SR; R2, fraction enriched in longitudinal SR; R3, fraction containing a mixture of longitudinal SR and terminal cisternae; R4, fraction enriched in terminal cisternae): 30 µg of protein from each fraction was separated on SDS/PAGE.</p