Skip to main content
. 2020 Sep 10;21(18):6610. doi: 10.3390/ijms21186610

Figure 6.

Figure 6

Effect of the L-type Ca2+ channel blocker nifedipine on heart Ca2+ levels measured with Twitch and TN-XXL biosensors. (A) Ca2+ kinetic parameters were measured in 3 dpf zebrafish embryos expressing Twitch-4 in the heart, before and after 1 h incubation with 1, 10, and 100 µM nifedipine. Different embryos were used for each nifedipine concentration. A paired Student’s t-test was used (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (B) Average of the systolic and diastolic ratio values obtained in 3 dpf embryos expressing Twitch-1, Twitch-2, and TN-XXL, before and after 1 h incubation with 1, 10, and 100 µM nifedipine. Data are shown as the mean ± S.E.M., n = 3 to 6 embryos for each concentration, from one or two independent experiments. A paired Student’s t-test was used (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (C) HR and fractional area change in uninjected embryos incubated with 5 and 20 µM nifedipine compared to control. These embryos were not incubated with PAB. Data are shown as the mean ± S.E.M., n = 3 to 4 embryos for each group, from one independent experiment. An unpaired Student’s t-test was used to compare each treatment with the control (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (D) A representative embryo in which treatment with 100 µM nifedipine (bottom image and graph) decreased the Ca2+ levels and completely abrogated the contractions in the atrium. Regions-of-interest (ROI) were manually drawn on the atrium (red line) and ventricle (white line). The effect of nifedipine on the average ratio was more pronounced with the high affinity biosensors Twitch-1 and Twitch-2B than with the lower affinity Twitch-4 or TN-XXL, even at 1 and 10 µM nifedipine (Figure 6B and Table S7). At Ca2+ levels comparable to the Kd, the fractions of Ca2+-bound and free biosensor are most sensitive to Ca2+. Thus, despite Twitch-4 generally providing a more robust reading of Ca2+ (larger ΔR/Rdiastole, less smoothing), in some cases the higher affinity GECIs may be advantageous.