Figure 3. Ca²⁺ release kinetics in muscle fibers of two patients and expression of recombinant CASQ1^WT and CASQ1^MUT proteins.

A. Ca²⁺ release kinetics in muscle fibers of two patients. Dose-response curves of permeabilized muscle fibers from control healthy subjects (black dashed line, average of 48 fibers from 4 subjects), patient I:1 of family 2 (continuous red line, average of 15 fibers) and from the sporadic case (continuous blue line, average of 21 fibers) exposed to increasing concentrations of caffeine (from 0.1 to 20 mM). Curves are interpolated by a sigmoidal Hill equation: $\mathrm{Y}=\mathrm{B}+(\text{Amax}-\mathrm{B})/(1+10^({(\text{LogEC}50-\mathrm{X})}^{\ast }\mathrm{n}))$. The parameter logEC50 is significantly greater (p<0.01) in the patient I:1 of family 2 (0.697+/− 0.062) and in the sporadic case (0.863+/− 0.101) than in controls (0.343 +/− 0.006). B. CASQ1^WT-GFP and CASQ1^MUT-GFP were expressed in COS-7 cells. Living cells were imaged by confocal laser scan microscopy. Scale bars = 7 μm. C. Rat myotubes expressing recombinant CASQ1^WT and CASQ1^MUT were immunostained with antibodies against CASQ1 and RyR1. Scale bar = 2 μm. D. EM analysis of CASQ1^WT and CASQ1^MUT in mouse FDB fibers. Fibers expressing CASQ1^MUT show the presence of vacuoles filled with electron-dense material (asterisks). These vacuoles were not present in fibers expressing CASQ1^WT. Higher magnification shows enlarged vesicles derived from the SR terminal cisternae: arrows point to RyR-feet between an enlarged SR vesicle and a T-tubule. M, mitochondrion; TT, transverse tubule; SR, sarcoplasmic reticulum. Scale bars in left, middle and right panels = 0.5, 1 and 0.1 μm, respectively.