P.326Characterization of the ion channels involved in the increase of sarcolemmal calcium permeability in the dystrophin-deficient DMDmdx rat model

Abstract

Duchenne duscular Dystrophy (DMD) is a lethal muscle disease due to genetic mutations leading to the lack of dystrophin expression. Even if the cascade of events leading to muscle necrosis and fibrosis observed in the disease remains partly unexplained, an increase of the sarcolemmal permeability to Ca2+ (SPCa) and a consequent intracellular Ca2+ overload seem to be crucial events. Although much evidence has accumulated to designate channels belonging to the TRP family, the exact nature of the channels involved in the SPCa increase remains subject to debate. This may be because most of the studies have been done in the mdx mouse model that lacks significant clinical phenotype. We recently generated the dystrophin-deficient DMDmdx rat model, which mimics the human disease at both the muscular and the cardiac levels. Herein, we show that skeletal muscle fibers and cardiomyocytes isolated from DMDmdx rats and loaded with the Fura-2 Ca2+ probe exhibit higher cytosolic Ca2+ concentration than in wild-type rats. Additionally, we found, using the Mn2+ quenching technique, that SPCa is increased in DMDmdx rat skeletal muscle fibres. We performed immunofluorescence and western-blot analysis to determine wether SPCa increase could be related to changes in the expression and/or in the localization of six TPR channels: TRPV2, TRPC1, TRPC3 and TRPML1-3. TRPV2 is sensitive to stretch and has been shown to accumulate at the sarcolemma of dystrophic fibres. TRPC1 and TRPC3 are involved in SPCa regulation in muscle cells differentiation. TRPML are lysosomal Ca2+ channels involved in plasma membrane repair process. Our results show that TRPV2, TRPC3, TRPML1 and TRPML2 channels accumulate at the sarcolemma and are overexpressed in striated muscle cells from DMDmdx rats, leading to suggest an involvement of these channels in the SPCa increase measured in the dystrophic muscle fibers. The complete results of our investigations will be presented as well as our current perspectives.