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

Implication des récepteurs à l'inositol 1,4,5-triphosphate dans les libérations de calcium du reticulum sarcoplasmique dans des cellules musculaires squelettiques déficientes en dystrophine ou exprimant la mini-dystrophine

La dystrophie musculaire de Duchenne (DMD) est une maladie génétique récessive, caractérisée par l'absence de la dystrophine et des altérations de l'homéostasie calcique. Ce travail a été entrepris dans le but de comparer la participation du calcium stocké dans le RS à la dérégulation de l'homéostasie calcique entre les myotubes déficients en dystrophine (SolC1) et les myotubes exprimant la mini-dystrophine (SolD). Deux voies de libération de calcium ont été examinées, le couplage excitation-libération de Ca2+ et les évènements élémentaires de libération de Ca2+ au repos. Les résultats révèlent que dans les cellules SolC1, les libérations de Ca2+ sont plus importantes, à la fois au repos et en dépolarisation, que dans les myotubes SolD. De plus, dans les myotubes SolC1 la voie IP3 semble être plus impliquée dans ces libérations. Il semble désormais important d'étudier plus précisément le mécanisme d'interaction de la mini-dystrophine avec les canaux de libération de Ca2+.Alterations of Ca2+ homeostasis are involved in Duchenne muscular dystrophy characterized by a lack of the dystrophin protein. The aim of this study is to characterize Ca2+ release events in a dystrophin deficient cell line (SolC1), in a dystrophin forced-expression cell line (SolD). Using confocal microscopy, measurements of Ca2+ signals were performed in myotubes loaded with the Fluo-4. At rest, localized quantal Ca2+ release events (sparks) and widespread calcium releases evoked by high KCl solution were recorded. SolC1 myotubes showed a higher sparks density than SolD myotubes. Perfusion of 2-APB (inhibitor of IP3 receptor) decreased sparks density in SolC1 and SolD myotubes while ryanodine decreased sparks density only in SolD myotubes. Myotubes incubated with 2-APB showed a faster relaxation kinetic of calcium transients than the control myotubes. These data suggest that IP3 could play a substantial role in Ca2+ release from SR in dystrophic cells.POITIERS-BU Sciences (861942102) / SudocSudocFranceF

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

International audienceEvidence 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

Lack of CFTR in skeletal muscle predisposes to muscle wasting and diaphragm muscle pump failure in cystic fibrosis mice

Cystic fibrosis (CF) patients often have reduced mass and strength of skeletal muscles, including the diaphragm, the primary muscle of respiration. Here we show that lack of the CF transmembrane conductance regulator (CFTR) plays an intrinsic role in skeletal muscle atrophy and dysfunction. In normal murine and human skeletal muscle, CFTR is expressed and colocalized with sarcoplasmic reticulum-associated proteins. CFTR–deficient myotubes exhibit augmented levels of intracellular calcium after KCl-induced depolarization, and exposure to an inflammatory milieu induces excessive NF-kB translocation and cytokine/chemokine gene upregulation. To determine the effects of an inflammatory environment in vivo, sustained pulmonary infection with Pseudomonas aeruginosa was produced, and under these conditions diaphragmatic force-generating capacity is selectively reduced in Cftr 2/2 mice. This is associated with exaggerated pro-inflammatory cytokine expression as well as upregulation of the E3 ubiquitin ligases (MuRF1 and atrogin-1) involved in muscle atrophy. We conclude that an intrinsic alteration of function is linked to the absence of CFTR from skeletal muscle, leading to dysregulated calcium homeostasis, augmented inflammatory/atrophic gene expression signatures, and increased diaphragmatic weakness during pulmonary infection. These findings reveal a previously unrecognized role for CFTR in skeletal muscle function that may have major implications for the pathogenesis of cachexia and respiratory muscle pum

Quantification and inhibition of SR–mediated calcium release.

<p>(A) Group mean data for intracellular Ca²⁺ responses in <i>Cftr^+/+</i> myotubes, either without (control) or with (CFTRinh) pre-treatment by CFTR-inh172. (B) Group mean data for intracellular Ca²⁺ responses in <i>Cftr^+/+</i> versus <i>Cftr</i>^−/− myotubes. (C) Effects of inhibiting RyR or IP3R function in <i>Cftr^+/+</i> (open bars) and <i>Cftr</i>^−/− (filled bars) myotubes. For all experiments, N = minimum of 30 myotubes per group. * p<0.05 versus control, † p<0.05 for ryanodine versus 2-APB and U73122.</p

Abnormal calcium responses in CFTR–deficient muscle cells.

<p>(A) Negative immunostaining for CFTR in <i>Cftr</i>^−/− myotubes. (B) Representative Ca²⁺ responses in <i>Cftr^+/+</i> myotubes, either without (control) or with (CFTR-inh) pre-treatment by CFTR-inh172. (C) Representative Ca²⁺ responses in <i>Cftr^+/+</i> versus <i>Cftr</i>^−/− myotubes. (D) Lack of effect of CFTR-inh172 on the area under the curve or peak amplitude of Ca²⁺ responses in <i>Cftr</i>^−/− myotubes.</p

Effects of CFTR status on diaphragmatic contractile function.

<p>(A) Response of intact diaphragmatic muscle fibers to KCl-induced depolarization in <i>Cftr^+/+</i> versus <i>Cftr</i>^−/− groups, under basal conditions (N = 6–7 mice per group) or with CFTR- inh172 (N = 4–5 mice per group) added to the perfusate (100 µM). (B,C): Effects of CFTR deficiency on relaxation and contraction times of the diaphragm during electrical stimulation (white bars: <i>Cftr^+/+</i>; red bars: <i>Cftr</i>^−/−). (D) Differential effects of <i>Pseudomonas</i> lung infection on diaphragmatic force capacity in <i>Cftr^+/+</i> and <i>Cftr</i>^−/− mice (N = 5 mice per group). * p<0.05 for <i>Cftr^+/+</i> versus <i>Cftr</i>^−/−.</p

Hyperinflammatory phenotype of <i>Cftr^−/−</i> skeletal muscle in vivo.

<p>(A) Cytokine mRNA expression levels in <i>Cftr^+/+</i> versus <i>Cftr</i>^−/− diaphragms for uninfected (CTL) and <i>Pseudomonas</i>-infected (day 2) mice. N = 4–5 mice per group; * p<0.05 for control versus infected mice, † p<0.05 for <i>Cftr^+/+</i> versus <i>Cftr</i>^−/−. (B) Real-time PCR quantification of the E3 ubiquitin ligases MuRF-1 and atrogin-1 in diaphragms of control and infected mice. * p<0.05 for <i>Cftr^+/+</i> versus <i>Cftr</i>^−/−.</p