Figure 4.
Ca
2+
transients by electrical stimulation or caffeine and KCl application in WT and R2509C-RYR1 primary myocytes. (A) Left: Typical time course of Ca2+ transients induced by platinum field electrode stimulation (electrical stimulation; ES) in WT, R2509C-Het (Het), and R2509C-Hom (Hom) myocytes measured using Cal-520. The fluorescence intensity F was normalized to the initial value of F (F0). Right: Graph showing peak amplitudes (Fmax/F0) of ES in WT (n = 176, N = 3), R2509C-Het (Het, n = 384, N = 4), and R2509C-Hom (Hom, n = 194, N = 3) primary myocytes. The horizontal dotted line indicates F0. Values are mean ± SD. (B) K+-induced depolarization in WT, R2509C-Het (Het) and R2509C-Hom (Hom) myocytes. KCl (7–60 mM) was applied at the time points indicated by the grey horizontal bar. Values are mean ± SEM (n = 36–107, N = 3, see Table 2). (C) Left: The magnitude of the peak Ca2+ transients was plotted against KCl concentrations and fitted to the dose–response curve in WT (black), R2509C-Het (red), and R2509C-Hom (blue). Right: Normalized for maximal peak amplitudes (Fmax/F0) to KCl. To eliminate run-down, only one dose was administered to the myocytes and the Ca2+ response was analyzed. (D) Ca2+ transients induced by caffeine in WT, R2509C-Het (Het), and R2509C-Hom (Hom) myocytes measured using Cal-520. Caffeine (0.1–20 mM) was applied at the time points indicated by the grey horizontal bar. Values are mean ± SEM (n = 20–104, N = 3, see Table 2). (E) Left: The magnitude of the peak Ca2+ transients was plotted against caffeine concentrations and fitted to the dose–response curve in WT (black), R2509C-Het (red), and R2509C-Hom (blue). Right: Normalized for maximal peak amplitudes (Fmax/F0) to caffeine. Curves are fittings based on the Hill equation: where R, C, k, and n' represent the normalized response, either KCl or caffeine concentration, EC50 and Hill coefficient. Bottom and Top represent the maximum and minimum responses. Statistical significance was determined using a one-way ANOVA with Tukey’s test. Cells on days 2–4 after differentiation were analyzed. “n” indicates the number of myocytes, and “N” the number of animals.