Fig. 2

In vivo recording of voltage and Ca²⁺ dynamics in the embryonic zebrafish heart. (A) Recordings were performed using a custom-built upright widefield microscope equipped with a 20x objective, LED excitation light, an emission splitter unit and a high-speed fluorescence camera. Embryos were embedded in 0.3% agarose and placed in a temperature-controlled water bath. Temperature was regulated using a heated glass plate and a temperature sensor. A schematic depiction of the focal plane through the embryonic heart is visible in the right bottom frame. (B) Using the high-speed epifluorescence microscope, movies of 3 dpf non-contracting chimeric VSFP-butterfly CY embryonic hearts were recorded. Signal analysis demonstrated an increase in mCitrine (top panel) and decrease in mCerulean (bottom panel) every consecutive excitation cycle, starting in the atrium and followed by the ventricle. From these signals, a ratiometric signal could be calculated to study action potential (AP) configuration in both chambers. The selected atrial or ventricular regions are indicated by square boxes superimposed on the fluorescence images. (C) Movies of 3 dpf non-contracting GCaMP6f embryonic hearts were recorded and signal analysis demonstrated a clear oscillatory Ca²⁺ signal, starting in the atrium and followed by the ventricle. (D-E) Line plots of both chimeric VSFP-butterfly CY and GCaMP6f signals illustrating electrical impulse (D) and Ca²⁺ propagation (E) throughout the heart. Background-corrected fluorescence intensities were averaged across the width of the myocardial wall and along a trajectory defined by the course of the wall. Signal heat maps of trajectory against time depict depolarizations of the membrane (D) or increases in intracellular Ca²⁺ (E). The chimeric VSFP-butterfly CY line plot (D) clearly shows that APs originate in the atrium, have a small delay at the atrioventricular canal (AVC) and then rapidly spread throughout the ventricle. White insets represent the spatial AP traces, visible is the longer AP duration in the ventricle as compared to the atrium, as well as the regularity of the activation intervals. Analysis of GCaMP6f signal intensity versus time (E) also shows a clear activation pattern from atrium to ventricle. White insets represent the spatial Ca²⁺ transients, showing a faster speed of intracellular Ca²⁺ release in the atrium versus ventricle, similar speed in Ca²⁺ reuptake/clearance between chambers and nice regularity of activation intervals. A: atrium; cpEGFP: circularly permutated enhanced green fluorescent protein; V: ventricle.