Figure 5

tnnt2a^+/RK94del mutation causes intracellular calcium dysregulation in zebrafish embryos. (A) DNA construct and sensor dynamics of GCaMP6f (left panel). GCaMP6f was placed under the control of the myl7 promoter to restrict its expression to the heart, where the Gal4FF-UAS system amplifies its expression. GCaMP6f consists of a circularly permutated enhanced green fluorescence protein (cpEGFP) fused to calmodulin (CaM) and the M13 peptide. Upon the increase of intracellular calcium (Ca²⁺), CaM binds to M13, causing increased brightness of cpEGFP. This is visualized using a high-speed epifluorescence microscope, where movies of 5 dpf non-contracting GCaMP6f embryonic hearts were recorded (left panel). (B) Schematic representation of Ca²⁺ transient parameters examined from the recorded GCaMP6f signals. (C–G) Bar graphs of the different Ca²⁺ transient parameters for tnnt2a^+/+ and tnnt2a^+/RK94del, including upstroke time, recovery time, signal amplitude, diastolic Ca²⁺ levels, and peak Ca²⁺ levels. (H) Frequency of Ca²⁺ transients tnnt2a^+/+ and tnnt2a+/RK94del. AU: arbitrary unit. Statistics: mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, tnnt2a^+/+ n = 34, tnnt2a^+/RK94del n = 19, unpaired Students t-test. A: atrium, V: ventricle.