Mapping Calcium Dynamics in the Heart of Zebrafish Embryos with Ratiometric Genetically Encoded Calcium Indicators

Abstract

Zebrafish embryos have been proposed as a cost-effective vertebrate model to study heart function. Many fluorescent genetically encoded Ca2+ indicators (GECIs) have been developed, but those with ratiometric readout seem more appropriate to image a moving organ such as the heart. Four ratiometric GECIs based on troponin C, TN-XXL, Twitch-1, Twitch-2B, and Twitch-4 were expressed transiently in the heart of zebrafish embryos. Their emission ratio reported the Ca2+ levels in both the atrium and the ventricle. We measured several kinetic parameters of the Ca2+ transients: systolic and diastolic ratio, the amplitude of the systolic Ca2+ rise, the heart rate, as well as the rise and decay times and slopes. The systolic ratio change decreased in cells expressing high biosensor concentration, possibly caused by Ca2+ buffering. The GECIs were able to report the effect of nifedipine and propranolol on the heart, which resulted in changes in heart rate, diastolic and systolic Ca2+ levels, and Ca2+ kinetics. As a result, Twitch-1 and Twitch-4 (Kd 0.25 and 2.8 µM, respectively) seem the most promising GECIs for generating transgenic zebrafish lines, which could be used for modeling heart disorders, for drug screening, and for cardiotoxicity assessment during drug development.

Cardioluminescence in transgenic zebrafish embryos: a Ca2+imaging tool to study drug effects and pathological modeling

“…Ca 2+ levels also decreased by the β-adrenergic blocker propranolol. Using fluorescent biosensors, we and others have reported a decrease in HR and Ca 2+ levels induced by propranolol [19,24]. The treatment of 3 dpf zebrafish embryos with the antihistamine drug terfenadine for 24 hours has been reported to induce heart failure [40,41], involving arrhythmia and systolic disfunction, but potential changes in cytosolic Ca 2+ were not investigated.…”

“…This requires disrupting cardiac mechanical function with morpholino oligomers against myosin II [22] or with inhibitors like para-amino blebbistatin [23] to avoid motion imaging artifacts. We have previously used ratiometric biosensors, which require special optical components to acquire two emission images simultaneously [24], to correct these artifacts. Although fluorescent biosensors provide good temporal resolution of the Ca 2+ transients, the continuous illumination may cause phototoxicity and photobleaching of the biosensor, limiting the duration of an imaging experiment to a few seconds, depending on the instrumentation.…”

Mechanisms of TTNtv-Related Dilated Cardiomyopathy: Insights from Zebrafish Models

“…Accurate methods for cardiac electrophysiological assessment are currently still missing from the adult zebrafish phenotyping toolbox. Although parameters such as calcium transient characteristics and conduction velocity have been successfully studied in larval zebrafish using zebrafish using transgenic [ 82 , 83 , 84 ] and optogenetic lines [ 85 , 86 ] or the application of calcium or voltage dyes [ 87 , 88 , 89 ], these have not yet been adapted for use in adult zebrafish. Recently, progress has been made using ex vivo adult heart preparations [ 90 , 91 ]; however, in vivo methods are yet to be developed.…”

Optical Mapping in hiPSC-CM and Zebrafish to Resolve Cardiac Arrhythmias

“…The above techniques have been used frequently in the past decade to characterise ICAs both in hiPSC-CM and zebrafish hearts. They offer a valid alternative or are used complementary to the conventional electrophysiological patch clamping techniques [85,[99][100][101][102][103]. So far, characterising ICAs in hiPSC-CM solely via optical mapping is not yet an established practice.…”

“…ECG recordings or (high-speed) light microscopy videos (i.e., monitoring heart rate) of non-transparent adult zebrafish have been utilised for the study of LQTS [54,108,109], SQTS [69] and BrS [110]. Most recent development and implementation of state-of-the-art fluorescence microscopy techniques and GEFIs enable reliable in vivo optical mapping in the whole heart of zebrafish larvae [85,101,103]. Table 2.…”