Stéphane Sebille

Regulation of capacitative calcium entries by α1-syntrophin: association of TRPC1 with dystrophin complex and the PDZ domain of α1-syntrophin

Calcium mishandling in Duchenne dystrophic muscle suggested that dystrophin, a membrane‐associated cytoskeleton protein, might regulate calcium signaling cascade such as calcium influx pathway. It was previously shown that abnormal calcium entries involve uncontrolled stretch‐activated currents and store‐operated Ca2+ currents supported by TRPC1 channels. Moreover, our recent work demonstrated that reintroduction of minidystrophin in dystrophic myotubes restores normal capacitative calcium entries (CCEs). However, until now, no molecular link between the dystrophin complex and calcium entry channels has been described. This study is the first to show by coimmunoprecipitation assays the molecular association of TRPC1 with dystrophin and α1‐syntrophin in muscle cells. TRPC1 was also associated with α1‐syntrophin in dystrophic muscle cells independently of dystrophin. Furthermore, glutathione S‐transferase (GST) pull‐down assays showed that TRPC1 binds to the α1‐syntrophin PDZ domain. Transfected recombinant α1‐syntrophin formed a complex with TRPC1 channels and restored normal CCEs in dystrophic muscle cells. We suggest that normal regulation of CCEs in skeletal muscle depends on the association between TRPC1 channels and α1‐syntrophin that may anchor the store‐operated channels to the dystrophin‐associated protein complex (DAPC). The loss of this molecular association could participate in the calcium alterations observed in dystrophic muscle cells. This study provides a new model for the regulation of calcium influx by interaction with the scaffold of the DAPC in muscle cells.—Vandebrouck, A., Sabourin, J., Rivet, J., Balghi, H., Sebille, S., Kitzis, A., Raymond, G., Cognard, C., Bourmeyster, N., Constantin, B. Regulation of capacitative calcium entries by α1‐syntrophin: association of TRPC1 with dystrophin complex and the PDZ domain of α1‐syntrophin. FASEB J. 21, 608–617 (2007)

New insights in the regulation of calcium transfers by muscle dystrophin-based cytoskeleton: implications in DMD

Calcium mishandling in Duchenne muscular dystrophy (DMD) suggested that dystrophin, a membrane-associated cytoskeleton protein, may regulate calcium-signalling cascades such as calcium entries. Calcium overload in human DMD myotubes is dependent on their contractile activity suggesting the involvement of channels being activated during contraction and/or calcium release. Forced expression of mini-dystrophin in dystrophin-deficient myotubes, reactivates appropriate sarcolemmal expression of dystrophin-associated proteins and restores normal calcium handling in the cytosol. Furthermore, the recombinant mini-dystrophin reduced the store-operated calcium influx across the sarcolemma, and the mitochondrial calcium uptake during this influx. A slow component of calcium release dependent on IP3R, as well as the production of IP3, were also reduced to normal levels by expression of mini-dystrophin. Our studies provide a new model for the convergent regulation of transmembrane calcium influx and IP3-dependent calcium release by the dystrophin-based cytoskeleton (DBC). We also suggest molecular association of such channels with DBC which may provide the scaffold for assembling a multiprotein-signalling complex that modulates the channel activity. This suggests that the loss of this molecular association could participate in the alteration of calcium homeostasis observed in DMD muscle cells.

Non-acidic activation of pain-related Acid-Sensing Ion Channel 3 by lipids

Extracellular pH variations are seen as the principal endogenous signal that triggers activation of Acid‐Sensing Ion Channels (ASICs), which are basically considered as proton sensors, and are involved in various processes associated with tissue acidification. Here, we show that human painful inflammatory exudates, displaying non‐acidic pH, induce a slow constitutive activation of human ASIC3 channels. This effect is largely driven by lipids, and we identify lysophosphatidylcholine (LPC) and arachidonic acid (AA) as endogenous activators of ASIC3 in the absence of any extracellular acidification. The combination of LPC and AA evokes robust depolarizing current in DRG neurons at physiological pH 7.4, increases nociceptive C‐fiber firing, and induces pain behavior in rats, effects that are all prevented by ASIC3 blockers. Lipid‐induced pain is also significantly reduced in ASIC3 knockout mice. These findings open new perspectives on the roles of ASIC3 in the absence of tissue pH variation, as well as on the contribution of those channels to lipid‐mediated signaling.

Mini-dystrophin Expression Down-regulates Overactivation of G Protein–mediated IP3 Signaling Pathway in Dystrophin-deficient Muscle Cells

We present here evidence for the enhancement of an inositol 1,4,5-trisphosphate (IP3) mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(−)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, we demonstrated that calcium rise, induced by the perifusion of a solution containing a high potassium concentration, was higher in SolC1(−) than in SolD(+) myotubes. The analysis of amplitude and kinetics of the calcium increase in SolC1(−) and in SolD(+) myotubes during the exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) suggested the presence of two mechanisms of SR calcium release: (1) a fast SR calcium release that depended on ryanodine receptors and (2) a slow SR calcium release mediated by IP3 receptors. Detection analyses of mRNAs (reverse transcriptase [RT]-PCR) and proteins (Western blot and immunolocalization) demonstrated the presence of the three known isoforms of IP3 receptors in both SolC1(−) and SolD(+) myotubes. Furthermore, analysis of the kinetics of the rise in calcium revealed that the slow IP3-dependent release may be increased in the SolC1(−) as compared to the SolD(+), suggesting an inhibitory effect of mini-dystrophin in this signaling pathway. Upon incubation with pertussis toxin (PTX), an inhibitory effect similar to that of the IP3R inhibitor (2-APB) was observed on K+-evoked calcium release. This result suggests the involvement of a Gi protein upstream of the IP3 pathway in these stimulation conditions. A hypothetical model is depicted in which both Gi protein and IP3 production could be involved in K+-evoked calcium release as well as a possible interaction with mini-dystrophin. Our findings demonstrate the existence of a potential relationship between mini-dystrophin and SR calcium release as well as a regulatory role of mini-dystrophin on intracellular signaling.

Negative modulation of inositol 1,4,5-trisphosphate type 1 receptor expression prevents dystrophin-deficient muscle cells death.

Evidence for a modulatory effect of cyclosporin A (CsA) on calcium signaling and cell survival in dystrophin-deficient cells is presented. Our previous works strongly supported the hypothesis of an overactivation of Ca(2+) release via inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) in dystrophin-deficient cells, both during membrane depolarization and at rest, through spontaneous Ca(2+) release events. Forced expression of mini-dystrophin in these cells contributed, during stimulation and in resting condition, to the recovery of a controlled calcium homeostasis. In the present work, we demonstrate that CsA exposure displayed a dual-modulator effect on calcium signaling in dystrophin-deficient cells. Short-time incubation induced a decrease of IP3-dependent calcium release, leading to patterns of release similar to those observed in myotubes expressing mini-dystrophin, whereas long-time incubation reduced the expression of the type I of IP3 receptors (IP3R-1) RNA levels. Moreover, both IP3R-1 knockdown and blockade through 2-aminoethoxydiphenyle borate or CsA induced improved survival of dystrophin-deficient myotubes, demonstrating the cell death dependence on the IP3-dependent calcium signaling as well as the protective effect of CsA. Inhibition of the IP3 pathway could be a very interesting approach for reducing the natural cell death of dystrophin-deficient cells in development.

Mini-dystrophin expression down-regulates IP3-mediated calcium release events in resting dystrophin-deficient muscle cells.

We present here evidence for the enhancement, at rest, of an inositol 1,4,5-trisphosphate (IP3)–mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(−)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, the number of sites discharging calcium (release site density [RSD]) was quantified and found more elevated in SolC1(−) than in SolD(+) myotubes. Variations of membrane potential had no significant effect on this difference, and higher resting [Ca2+]i in SolC1(−) (Marchand, E., B. Constantin, H. Balghi, M.C. Claudepierre, A. Cantereau, C. Magaud, A. Mouzou, G. Raymond, S. Braun, and C. Cognard. 2004. Exp. Cell Res. 297:363–379) cannot explain alone higher RSD. The exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) and phospholipase C inhibitor (U73122) significantly reduced RSD in both cell types but with a stronger effect in dystrophin-deficient SolC1(−) myotubes. Immunocytochemistry allowed us to localize ryanodine receptors (RyRs) as well as IP3 receptors (IP3Rs), IP3R-1 and IP3R-2 isoforms, indicating the presence of both RyRs-dependent and IP3-dependent release systems in both cells. We previously reported evidence for the enhancement, through a Gi protein, of the IP3-mediated calcium signaling pathway in SolC1(−) as compared to SolD(+) myotubes during a high K+ stimulation (Balghi, H., S. Sebille, B. Constantin, S. Patri, V. Thoreau, L. Mondin, E. Mok, A. Kitzis, G. Raymond, and C. Cognard. 2006. J. Gen. Physiol. 127:171–182). Here we show that, at rest, these regulation mechanisms are also involved in the modulation of calcium release activities. The enhancement of resting release activity may participate in the calcium overload observed in dystrophin-deficient myotubes, and our findings support the hypothesis of the regulatory role of mini-dystrophin on intracellular signaling.

Stretch-activated TRPV2 channels: Role in mediating cardiopathies

Transient receptor potential vanilloid type 2, TRPV2, is a calcium-permeable cation channel belonging to the TRPV channel family. Although this channel has been first characterized as a noxious heat sensor, its mechanosensor property recently gained importance in various physiological functions. TRPV2 has been described as a stretch-mediated channel and a regulator of calcium homeostasis in several cell types and has been shown to be involved in the stretch-dependent responses in cardiomyocytes. Hence, several studies in the last years support the idea that TRPV2 play a key role in the function and structure of the heart, being involved in the cardiac compensatory mechanisms in response to pathologic or exercise-induced stress. We present here an overview of the current literature and concepts of TRPV2 channels involvement (i) in the mechanical coupling mechanisms in heart and (ii) in the mechanisms that lead to cardiomyopathies. All these studies lead us to think that TRPV2 may also be an important cardiac drug target based on its major physiological roles in heart.

Axial stretch-dependent cation entry in dystrophic cardiomyopathy: Involvement of several TRPs channels

In Duchenne muscular dystrophy (DMD), deficiency of the cytoskeletal protein dystrophin leads to well-described defects in skeletal muscle but also to dilated cardiomyopathy (DCM). In cardiac cells, the subsarcolemmal localization of dystrophin is thought to protect the membrane from mechanical stress. The dystrophin deficiency leads to membrane instability and a high stress-induced Ca(2+) influx due to dysregulation of sarcolemmal channels such as stretch-activated channels (SACs). In this work divalent cation entry has been explored in isolated ventricular Wild Type (WT) and mdx cardiomyocytes in two different conditions: at rest and during the application of an axial stretch. At rest, our results suggest that activation of TRPV2 channels participates to a constitutive basal cation entry in mdx cardiomyocytes.Using microcarbon fibres technique, an axial stretchwas applied to mimic effects of physiological conditions of ventricular filling and study on cation influx bythe Mn(2+)-quenching techniquedemonstrated a high stretch-dependentcationic influx in dystrophic cells, partially due to SACs. Involvement of TRPs channels in this excessive Ca(2+) influx has been investigated using specific modulators and demonstratedboth sarcolemmal localization and an abnormal activity of TRPV2 channels. In conclusion, TRPV2 channels are demonstrated here to play a key role in cation influx and dysregulation in dystrophin deficient cardiomyocytes, enhanced in stretching conditions.

Effets du décocté de Biophytum petersianum (Oxalidaceae) sur la libération du calcium du réticulum sarcoplasmique des cellules musculaires squelettiques

RésuméLe décocté de la plante entière de Biophytum petersianum (Oxalidaceae) est utilisé dans la médecine traditionnelle au Togo pour traiter l’hypertension artérielle. Le présent travail évalue les effets du décocté de la plante entière de Biophytum petersianum sur la libération du calcium stocké dans le réticulum sarcoplasmique des cellules musculaires squelettiques. Les résultats ont montré qu’en présence du décocté, la libération du calcium du réticulum sarcoplasmique induite par la caféine est diminuée. Les récepteurs à l’inositol triphosphate et la pompe calcique du réticulum sarcoplasmique connus pour intervenir dans la libération du calcium du réticulum sarcoplasmique n’ont pas été affectés par le décocté de Biophytum petersianum. Ces résultats ont montré que le décocté possède un ou des composé(s) actif(s) capable(s) d’inhiber la libération du calcium du réticulum sarcoplasmique, ce qui justifie son utilisation dans la médicine traditionnelle pour traiter l’hypertension artérielle.AbstractThe decoction of the whole plant of Biophytum petersianum (Oxalidaceae) was used in traditional medicine to treat hypertension in Togo. The present work was performed to investigate the decoction of the whole plant of Biophytum petersianum for possible interference with sarcoplasmic reticulum calcium mobilising in skeletal muscle cells. The results showed that the decoction has a significant antagonistic effect on caffeine-induced calcium release from sarcoplasmic reticulum. The inositol 1,4,5-triphosphate receptor and the sarcoplasmic reticulum calcium pump, known to be involved in the regulation of internal free calcium cycling were not affected by the Biophytum petersianum decoction. The results reported here show that the BP decoction possess active component capable of inhibiting calcium release from the sarcoplasmic reticulum and this might explain its use as antihypertensive in traditional medicine.

Structural, Contractile and Electrophysiological Adaptations of Cardiomyocytes to Chronic Exercise

Cardiac beneficial effects of chronic exercise is well admitted. These effects mainly studied at the organ and organism integrated levels find their origin in cardiomyocyte adaptation. This chapter try to highlight the main trends of the data related to the different parameters subject to such adaptations. This is addressed through cardiomyocytes size and structure, calcium and contractile properties, and finally electrophysiological alterations induced by training as they transpire from the literature. Despite the clarifications needed to decipher healthy cardiomyocyte remodeling, this overview clearly show that cardiac cell plasticity ensure the cardiac adaptation to exercise training and offers an interesting mean of action to counteract physiological disturbances induced by cardiac pathologies.