Heat-induced Ca ²⁺ release in WT and R2509C-RYR1 primary myocytes. (A) Averaged fluorescence images of Cal-520–loaded WT myocytes before (left) and during heating (middle). Images captured for 1.2 s were averaged. Image on the right indicates the differences in fluorescence intensity between the left and middle images (ΔF/F₀). A closed red circle indicates the position of focused infrared (IR) laser beam for micro-heating. (B) Time-course of fluorescence intensity of Cal-520 in WT myocytes. Each line represents an individual primary myocyte. The pink vertical bar indicates the period of a heat pulse. (C) Same as in A for R2509C-Het myocytes. (D) Same as in B for R2509C-Het myocytes. (E) Same as in A for R2509C-Hom myocytes. (F) Same as in B for R2509C-Hom myocytes. (G–I) Time-dependent analyses in WT (G), R2509C-Het (H), and R2509C-Hom (I) myocytes. The Ca²⁺ levels before (F₀), during (14–15 s) and after (19–20 s) heating were analyzed. Thin lines represent individual cells, and thick line and squares represent averages. Data are analyzed by repeated measures one-way ANOVA with Tukey's multiple comparisons tests. (J) Basal fluorescence intensities of Cal-520 in WT, R2509C-Het, and R2509C-Hom myocytes before heating (F₀). (K) Maximum changes in the relative fluorescence intensity of Cal-520 during heating (ΔF_max/F₀). Dotted horizontal line indicates the baseline. WT: n = 14, N = 3; R2509C-Het: n = 72, N = 4; R2509C-Hom: n = 66, N = 4. Myocytes from WT mice were analyzed 3 d after the onset of differentiation and those from R2509C-Het and R2509C-Hom 2–4 d after the onset of differentiation. Values are shown as mean ± SD. Statistical significance was determined using a one-way ANOVA with Tukey’s test. ΔT = 11 ± 2°C; T₀ = 24 ± 1°C. “n” is the number of myocytes and “N” is the number of animals.