In vivo calcium and voltage mapping of the zebrafish heart

Abstract

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Research Foundation Flanders (FWO) Purpose Inherited cardiac arrhythmias are characterized by a disturbance in the normal electrical functioning of the heart due to a dysfunction of cardiac ion channels, accessory proteins and/or structural components. This complex process is difficult to model in vitro, while in vivo mouse models differ substantially from humans with regards to cardiac electrophysiology. Thus, there is a need for alternative disease models in cardiac arrhythmia research. The zebrafish heart shows remarkable similarity to humans in its electrocardiogram and action potential (AP) morphology. Due to the optical translucency of zebrafish larvae, it is possible visualize the cardiac calcium and voltage dynamics across their entire hearts in vivo by use of genetically encoded calcium and voltage indicators (GECI and GEVI). We combined the fast kinetics of a next generation voltage reporter (Ace2N-mNeon) with a spectrally compatible calcium sensor (R-GECO) to create a sensitive high throughput method for in vivo characterization of the cardiac AP. Methods Ace2N-mNeon and R-GECO were expressed under control of a myocardial specific promoter via the Tol2kit system. The effect of sensor expression on normal physiology was assessed by patch clamp experiments. Fluorescence recordings were obtained with a Leica SP8 DLS microscope and a custom-built light sheet microscope. Our zebrafish sensor line was validated by experiments with quinidine drug exposure, as well as a novel model for long QT syndrome (LQTS) with a knock-in (KI) mutation in the cacna1c gene. Results Sensor expression did not significantly alter cardiac electrophysiology. The tg(myl7:Ace2N-mNeon/R-GECO) line demonstrated alterations in the heart rate, depolarization and repolarization induced by quinidine exposure. We observed a significantly prolonged ventricular repolarization in the LQTS KI line. Three-dimensional optical maps of AP characteristics across the entire zebrafish heart exhibited the physiological characteristics of the zebrafish cardiac electrophysiology, with activation initiated at the atrial venous pole and propagated towards the ventricular outflow tract, prolonged repolarization in the ventricles compared to the atria and slowed depolarization in the atrio-ventricular canal. Conclusion By expressing Ace2N-mNeon and R-GECO in the zebrafish heart, we were able to accurately detect drug- and mutation induced alterations in the cardiac AP, as well as AP characteristics across the entire heart, without compromising normal cardiac electrophysiology. Our transgenic line will provide a promising assay for future studies of cardiac arrhythmia.