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

Abstract

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

“…Our previous works strongly supported the hypothesis of an overactivation of Ca 2ϩ release in dystrophin-deficient cells, both during membrane depolarization (4) and, at rest, through spontaneous Ca 2ϩ release events (5). Previous studies from other groups have provided evidence for at least two phases in the calcium rise observed in cultured skeletal muscle cells exposed to a high potassium solution (27,40).…”

Altered Gene Expression Pathways in Duchenne Muscular Dystrophy

“…We recently found that both expression and localization of IP 3 Rs are different in normal and dystrophic human skeletal muscle and cell lines (Cárdenas et al 2010). On the other hand, experiments performed using two types of myotubes originated from the same Sol8 cell line -dystrophin deficient myotubes, SolC1(-), and myotubes transfected to express the minidystrophin, SolD(+) -show that Ca 2+ rise evoked by potassium depolarization was higher in SolC1(-) than in SolD(+) myotubes (Balghi et al 2006a). Analysis of the kinetics of the Ca 2+ rise, reveals that the slow IP 3 -dependent release may be increased in the SolC1(-) as compared to the SolD(+), suggesting an inhibitory effect of mini-dystrophin on IP 3 R-dependent K + -evoked Ca 2+ release (Balghi et al 2006a).…”

“…Analysis of the kinetics of the Ca 2+ rise, reveals that the slow IP 3 -dependent release may be increased in the SolC1(-) as compared to the SolD(+), suggesting an inhibitory effect of mini-dystrophin on IP 3 R-dependent K + -evoked Ca 2+ release (Balghi et al 2006a). Moreover, it has been described that IP 3 production after membrane depolarization is significantly elevated in dystrophin-deficient myotubes and that the presence of minidystrophin under the membrane leads to reduced IP 3 production (Balghi et al 2006a). In fact, we have recently demonstrated, using normal (RCMH) and dystrophic (RCDMD) human skeletal muscle cell lines, that IP 3 dependent, slow Ca 2+ transients evoked by electrical stimulation are faster in dystrophic cells, compared to normal myotubes (Cárdenas et al 2010).…”

“…On the other hand, experiments performed using two types of myotubes originated from the same Sol8 cell line -dystrophin deficient myotubes, SolC1(-), and myotubes transfected to express the minidystrophin, SolD(+) -show that Ca 2+ rise evoked by potassium depolarization was higher in SolC1(-) than in SolD(+) myotubes (Balghi et al 2006a). Analysis of the kinetics of the Ca 2+ rise, reveals that the slow IP 3 -dependent release may be increased in the SolC1(-) as compared to the SolD(+), suggesting an inhibitory effect of mini-dystrophin on IP 3 R-dependent K + -evoked Ca 2+ release (Balghi et al 2006a). Moreover, it has been described that IP 3 production after membrane depolarization is significantly elevated in dystrophin-deficient myotubes and that the presence of minidystrophin under the membrane leads to reduced IP 3 production (Balghi et al 2006a).…”

“…In dystrophic skeletal muscle, it has been suggested that an alteration of Ca 2+ homeostasis occurs and might be responsible for muscle degeneration (Turner et al 1988;Turner et al 1991). Several studies indicate that IP 3 pathways could be involved in the DMD pathophysiology (Liberona et al 1998;Balghi et al 2006a;Balghi et al 2006b). We recently found that both expression and localization of IP 3 Rs are different in normal and dystrophic human skeletal muscle and cell lines (Cárdenas et al 2010).…”