Key result
Mini-dystrophin expression in dystrophin-deficient muscle cells down-regulates the overactivation of the G protein-mediated IP3 signaling pathway during potassium-evoked calcium release.
Why the study?
Does mini-dystrophin expression reduce overactivation of the IP3-mediated calcium signaling pathway in dystrophin-deficient muscle cells?
Does mini-dystrophin expression reduce overactivation of the IP3-mediated calcium signaling pathway in dystrophin-deficient muscle cells?
Mini-dystrophin expression down-regulates the overactivation of the G protein-mediated IP3 calcium signaling pathway in dystrophin-deficient muscle cells, highlighting its regulatory role in intracellular signaling.
Hypothesis-generating in dystrophin-deficient models; leaves open clinical translation of mini-dystrophin gene therapy.
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.
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Balghi et al. (2006) studied Duchenne muscular dystrophy (in vitro model). Mini-dystrophin expression vs. Dystrophin-deficient cells (SolC1(-)) was evaluated on Intracellular calcium increase (amplitude and kinetics) upon depolarization with high potassium solution. Mini-dystrophin expression in dystrophin-deficient muscle cells down-regulates the overactivation of the G protein-mediated IP3 signaling pathway during potassium-evoked calcium release.
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