Construction and use of a zebrafish heart voltage and calcium optical mapping system, with integrated electrocardiogram and programmable electrical stimulation

Abstract

Zebrafish are increasingly being used as a model of vertebrate cardiology due to mammalian-like cardiac properties in many respects. The size and fecundity of zebrafish make them suitable for large-scale genetic and pharmacological screening. In larger mammalian hearts, optical mapping is often used to investigate the interplay between voltage and calcium dynamics and to investigate their respective roles in arrhythmogenesis. This report outlines the construction of an optical mapping system for use with zebrafish hearts, using the voltage-sensitive dye RH 237 and the calcium indicator dye Rhod-2 using two industrial-level CCD cameras. With the use of economical cameras and a common 532-nm diode laser for excitation, the rate dependence of voltage and calcium dynamics within the atrial and ventricular compartments can be simultaneously determined. At 140 beats/min, the atrial action potential duration was 36 ms and the transient duration was 53 ms. With the use of a programmable electrical stimulator, a shallow rate dependence of 3 and 4 ms per 100 beats/min was observed, respectively. In the ventricle the action potential duration was 109 ms and the transient duration was 124 ms, with a steeper rate dependence of 12 and 16 ms per 100 beats/min. Synchronous electrocardiograms and optical mapping recordings were recorded, in which the P-wave aligns with the atrial voltage peak and R-wave aligns with the ventricular peak. A simple optical pathway and imaging chamber are detailed along with schematics for the in-house construction of the electrocardiogram amplifier and electrical stimulator. Laboratory procedures necessary for zebrafish heart isolation, cannulation, and loading are also presented.

Zebrafish heart as a model to study the integrative autonomic control of pacemaker function

“…In the present study, the origin of myocyte electrical activity was located within the SAR, in agreement with previous functional (24,34,46,49,51,60,61) and anatomical (57,60) data. The mean time for propagation of atrial excitation, as well as the atrioventricular delay, corresponded with the delay calculated from ECG-derived data and with values previously reported in other optical imaging studies of the zebrafish heart (34,35). The observed location of the initiation and spread of excitation thus conformed with the recognized pattern of normal heart activation in this species, and established a functional baseline for mapping the effects of VSN stimulation.…”

Atrium‐specific ion channels in the zebrafish—A role of IKACh in atrial repolarization

“…In preliminary experiments, we found that constant perfusion of the hearts during recordings resulted in increased signal noise, so we proceeded without perfusion of the hearts during the experiments. Lin et al showed that passive oxygenation through diffusion maintains the heart rate for hours after isolation, likely because the mature zebrafish heart is 3‐4 cell layers thick, which aids diffusion.…”

Breakthroughs in modern cancer therapy and elusive cardiotoxicity: Critical research‐practice gaps, challenges, and insights

“…The key morphological, functional, mechanobiological, electrophysiological, metabolic, and molecular profiles, as well as many cardiac events, overlap with those in humans, including the kinome profiles, where most kinase inhibitors interact with the kinases . Moreover, the cardiac electrophysiological properties of the hearts of zebrafish larvae and adults resemble those of humans in many aspects . It is of particular importance to evaluate the pharmacological response of cardiac function in an animal model relevant to the human heart for an appropriate assessment of the safety (drug‐induced cardiotoxicity) and efficacy of drugs that target cardiovascular diseases (cardiopharmacology).…”

“…423,424 Moreover, the cardiac electrophysiological properties of the hearts of zebrafish larvae and adults resemble those of humans in many aspects. 413,[425][426][427][428] It is of particular importance to evaluate the pharmacological response of cardiac function in an animal model relevant to the human heart for an appropriate assessment of the safety (drug-induced cardiotoxicity) and efficacy of drugs that target cardiovascular diseases (cardiopharmacology). Many human cardiovascular drugs have identical effects on zebrafish physiology, and numerous human cardiovascular disorders have been recapitulated in zebrafish genetic models.…”

“…417,429 Importantly, many drugs that cause QT prolongation in humans consistently cause bradycardia and AV block in zebrafish, 413 suggesting that zebrafish are a rational, predictive model for cardiac safety liability of chemicals/drugs and also a valued, mechanistic model of human cardiovascular diseases. The overall physiology, genetics, and cardiovascular pharmacology similarities strongly support the use of zebrafish as the most reasonable approach for modeling human cardiovascular diseases or events in vivo, 420,428,[430][431][432][433] monitoring drug-induced in vivo cardiotoxicity, 413,417,418,[434][435][436] and in vivo cardiovascular drug discovery. 429 Furthermore, zebrafish are a diurnal vertebrate; 437 thus, the zebrafish CTS is expected to be compatible with the human counterpart for studies of chronobiology and chronopharmacology.…”