Calcium handling in zebrafish ventricular myocytes

Abstract

The zebrafish is an important model for the study of vertebrate cardiac development with a rich array of genetic mutations and biological reagents for functional interrogation. The similarity of the zebrafish (Danio rerio) cardiac action potential with that of humans further enhances the relevance of this model. In spite of this, little is known about excitation-contraction coupling in the zebrafish heart. To address this issue, adult zebrafish cardiomyocytes were isolated by enzymatic perfusion of the cannulated ventricle and were subjected to amphotericin-perforated patch-clamp technique, confocal calcium imaging, and/or measurements of cell shortening. Simultaneous recordings of the voltage dependence of the L-type calcium current (I(Ca,L)) amplitude and cell shortening showed a typical bell-shaped current-voltage (I-V) relationship for I(Ca,L) with a maximum at +10 mV, whereas calcium transients and cell shortening showed a monophasic increase with membrane depolarization that reached a plateau at membrane potentials above +20 mV. Values of I(Ca,L) were 53, 100, and 17% of maximum at -20, +10, and +40 mV, while the corresponding calcium transient amplitudes were 64, 92, and 98% and cell shortening values were 62, 95, and 96% of maximum, respectively, suggesting that I(Ca,L) is the major contributor to the activation of contraction at voltages below +10 mV, whereas the contribution of reverse-mode Na/Ca exchange becomes increasingly more important at membrane potentials above +10 mV. Comparison of the recovery of I(Ca,L) from acute and steady-state inactivation showed that reduction of I(Ca,L) upon elevation of the stimulation frequency is primarily due to calcium-dependent I(Ca,L) inactivation. In conclusion, we demonstrate that a large yield of healthy atrial and ventricular myocytes can be obtained by enzymatic perfusion of the cannulated zebrafish heart. Moreover, zebrafish ventricular myocytes differed from that of large mammals by having larger I(Ca,L) density and a monophasically increasing contraction-voltage relationship, suggesting that caution should be taken upon extrapolation of the functional impact of mutations on calcium handling and contraction in zebrafish cardiomyocytes.

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

“…Furthermore, the rapid development of genetically encoded reporters that can be localized to specific organelles within the cell make optical mapping a robust tool for understanding mechanisms (17). Yet, the basic mechanisms of zebrafish electrical activity and calcium handling remain under investigation (3,8,35).…”

“…Calcium currents in ventricular zebrafish myocytes are also larger and significant Ca 2ϩ entry through reverse-mode NCX activity occurs at positive potentials (35). The importance of the L-type calcium channel and NCX in zebrafish cardiology is also apparent in the island beat (L-type calcium channel) (29) mutant and in the tremblor (NCX) (9, 20) mutant zebrafish.…”

Optical mapping of the electrical activity of isolated adult zebrafish hearts: acute effects of temperature

“…More recent reports suggest that caution should be taken when modeling human excitation-contraction coupling in the ZF, due to some dissimilarities in calcium handling at positive membrane potentials (47), owing to the important role of the sodium-calcium exchanger (NCX). In general, ZF appear to rely primarily on transsarcolemmal calcium flux for the activation of contraction (4,47), whereas mammals rely heavily on calcium release from the sarcoplasmic reticulum (SR) (which can be triggered by I Ca ) (3).…”

Optical Mapping in the Developing Zebrafish Heart

“…Several rhythm mutants were found in the original screens, including slo mo, which has bradycardia [83], tremblor, which irregular heart beats [7], breakdance, which exhibits 2:1 block [7], and hiphop, which has 3:1 block [7]. Zebrafish were soon being used as a model to understand the basic mechanisms of cardiac electrophysiology [8,18,39,41,51,88]. Investigators began to model long QT syndrome (LQTS) and drug effects on repolarization [2,28,43,62,87].…”