Abstract
The cardiac action potential (AP) and the consequent cytosolic Ca2+ transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time) of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar) to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf). We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 and 102 h post fertilization (hpf), the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function.
Highlights
The cardiac action potential (AP) arises through the interaction of a large number of membrane proteins, and is an essential indicator of cardiac function
We found that the as-delivered spinning disk unit lacked sufficient sensitivity to image dim Archaerhodopsin 3 (Arch)(D95N) fluorescence in zebrafish heart
HIGH SENSITIVITY IMAGING WITH A MODIFIED SPINNING DISK CONFOCAL MICROSCOPE Spinning disk confocal fluorescence images of Arch(D95N) fluorescence in human embryonic kidney (HEK) cells were contaminated by significant background autofluorescence produced by the dichroic mirror (Figure 1C)
Summary
The cardiac action potential (AP) arises through the interaction of a large number of membrane proteins, and is an essential indicator of cardiac function. The action potential depolarization causes voltage-gated Ca2+ channels to open. Voltage-sensitive dyes (VSDs) have been used to study AP waveforms from excised animal hearts since the 1970’s (Salama and Morad, 1976; Entcheva and Bien, 2006; Panáková et al, 2010). Due to dye-mediated phototoxicity, optical recordings with VSDs typically do not extend beyond 1 min, and preparations are not stable for repeated imaging. Dye-mediated phototoxicity is most acute for high-magnification single-cell imaging, due to the high illumination intensity needed to produce sufficient fluorescence signal from a small field of view. The difficulty of targeting dyes to specific cell types presents a challenge for cellular-resolution voltage imaging in vivo
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