Analysis of ryanodine receptor 1 (RyR1) and voltage-gated Ca2+ channel (VGCC) alpha1S subunit (Cav1.1) pre-mRNA splicing and correlation with intracellular calcium signals in myotonic dystrophy type 1 (DM1) and in myotonic dystrophy type 2 (DM2) myotubes.

Introduction. The pathogenesis of myotonic dystrophy type 1 (DM1) and type 2 (DM2) has been related to nuclear accumulation of RNAs containing expanded CUG and CCUG sequences, respectively, which sequester RNA-binding proteins, thus affecting in trans the alternative splicing of several genes. The evidence of increased intracellular Ca2+ ([Ca2+]i) levels documented in DM1 myotubes suggested that an altered Ca2+ homeostasis may contribute to the muscle pathology in myotonic dystrophy. Accordingly, aberrant splicing of ryanodine receptor 1 (RyR1) and sarcoplasmatic/endoplasmatic Ca2+-ATPase were found in DM1 muscle tissues. However, data regarding RyR1 expression and intracellular Ca2+ handling in DM2 muscle tissues have not been reported. Objectives. To determine whether the expression of RyR1 and voltage-gated Ca2+ channel (VGCC) alpha1s subunit (Cav1.1) genes in myotubes from DM1 and DM2 patients are associated with alteration of [Ca2+]i transients. Methods. The mRNA processing of RyR1 and Cav1.1 in myotubes from healthy controls, DM1 and DM2 patients was studied by RT-PCR. [Ca2+]i transients induced by VGCC or RyR1 activation through stimulation with 100mM KCl or 20mM caffeine were studied by confocal Ca2+ imaging. Results. Abnormal RyR1 splicing was found in DM1 but not in DM2 myotubes. On the other hand, Cav1.1 mRNA processing was normal in both muscle cultures. In DM1 myotubes the mean amplitude of [Ca2+]i transients induced by KCl was greater than controls (6.6±1.4 vs. 5.7±1.9 ΔF/F, respectively, n=53, P<0.05), whereas in DM2 it was lower (3.7±1.4 ΔF/F, P<0.01). In both DM1 and DM2 myotubes Ca2+ released through RyRs after caffeine stimulation was lower than controls (-25 and -30%, respectively, P<0.01). Conclusion. Our data suggest that [Ca2+]i transients induced by KCl and caffeine are altered in both myotonic dystrophies, but only in DM1 these alterations are related with RyR1 aberrant splicing.