Effect of cyclopiazonic acid on heat-induced Ca ²⁺ release in R2509C-Hom primary myocytes. (A) Averaged fluorescence images of Cal-520–loaded R2509C-Hom myocytes in the presence of 20 μM CPA before (left) and during heating (middle). Images captured for 1.2 s were averaged. The image on right indicates the differences in fluorescence intensity between 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 R2509C-Hom + 20 μM CPA. Each line represents an individual primary myocyte. The pink vertical bar indicates the period of a heat pulse. ΔT = 11 ± 2°C; T₀ = 24 ± 1°C. (C) Time-dependent analyses of Cal-520 in R2509C-Hom + 20 μM CPA. The fluorescence intensity of Cal-520 before (0–1 s as F₀), during (14–15 s), and after (19–20 s) the heating in R2509C-Hom + 20 μM CPA. Thin lines represent individual cells. Thick line and squares represent averages. Data were analyzed by repeated measures one-way ANOVA with Tukey’s multiple comparisons tests. (D) Minimum values in the relative fluorescence intensity of Cal-520 during heating [(F_min − F₀) / F₀]. Dotted horizontal line indicates the baseline. R2509C-Hom: n = 66, N = 4; R2509C-Hom + 20 μM CPA: n = 30, N = 3. Values are 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.