Fig. 6. Effect of acute pressure-overload on ROS, Ca²⁺ sparks, CaM-RyR2 interaction, and hypertrophic signaling in WT and V3599K cardiomyocytes.

a Timing of acute pressure overload by air compression (+150 mmHg) to cardiomyocytes. Top: Ca²⁺ transient; bottom: sarcomere shortening. b Representative images of DCFHDA fluorescence and the summarized data. N = 30–51 cells from 3 to 4 hearts. c Representative images of Ca²⁺ sparks and the summarized data. N = 19–28 cells from 3 to 4 hearts. d Representative images of endogenous CaM, co-localized with RyR2, and the summarized data of the Z-line bound CaM and nuclear CaM. The immuno-fluorescence signal of the Z-line bound CaM was divided by that of RyR2, normalized to control (baseline of WT), and expressed as a ratio. The immuno-fluorescence signal of the nuclear CaM was divided by that of DAPI for nuclear staining, normalized to control (baseline of WT), and expressed as a ratio. N = 23–39 cells from 3 hearts. Values for individual mice are plotted together with mean ± SEM. Parentheses indicate the number of mice. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA with post-hoc Tukey’s multiple comparison test).