Noninvasive technique for measurement of heartbeat regularity in zebrafish (Danio rerio) embryos

Abstract

Background: Zebrafish (Danio rerio), due to its optical accessibility and similarity to human, has emerged as model organism for cardiac research. Although various methods have been developed to assess cardiac functions in zebrafish embryos, there lacks a method to assess heartbeat regularity in blood vessels. Heartbeat regularity is an important parameter for cardiac function and is associated with cardiotoxicity in human being. Using stereomicroscope and digital video camera, we have developed a simple, noninvasive method to measure the heart rate and heartbeat regularity in peripheral blood vessels. Anesthetized embryos were mounted laterally in agarose on a slide and the caudal blood circulation of zebrafish embryo was video-recorded under stereomicroscope and the data was analyzed by custom-made software. The heart rate was determined by digital motion analysis and power spectral analysis through extraction of frequency characteristics of the cardiac rhythm. The heartbeat regularity, defined as the rhythmicity index, was determined by short-time Fourier Transform analysis.

Results: The heart rate measured by this noninvasive method in zebrafish embryos at 52 hour post-fertilization was similar to that determined by direct visual counting of ventricle beating (p > 0.05). In addition, the method was validated by a known cardiotoxic drug, terfenadine, which affects heartbeat regularity in humans and induces bradycardia and atrioventricular blockage in zebrafish. A significant decrease in heart rate was found by our method in treated embryos (p < 0.01). Moreover, there was a significant increase of the rhythmicity index (p < 0.01), which was supported by an increase in beat-to-beat interval variability (p < 0.01) of treated embryos as shown by Poincare plot.

Conclusion: The data support and validate this rapid, simple, noninvasive method, which includes video image analysis and frequency analysis. This method is capable of measuring the heart rate and heartbeat regularity simultaneously via the analysis of caudal blood flow in zebrafish embryos. With the advantages of rapid sample preparation procedures, automatic image analysis and data analysis, this method can potentially be applied to cardiotoxicity screening assay.

Figure 1

A typical example of the signal of dynamic pixels (A) and its corresponding power spectrum (B) obtained from the caudal circulation of a 52-hpf control embryo. f_basic: first frequency component

Figure 2

(A) Heart rate determined from visual examination of heart beats from 52 hpf zebrafish embryos is corresponding to the one determined from power spectral analysis. (B) Linear correlation between the heart beats analyzed by power spectrum and visual counting was found.

Figure 3

A typical example of the signal of dynamic pixels (A) and its corresponding power spectrum (B) obtained from the caudal circulation of terfenadine-treated embryo at 52 hpf. (C) The heart beat frequency of control and terfenadine-treated embryos with bradycardia analyzed by power spectrum. A significant decrease in heart beat frequency was found in terfenadine-treated embryos (p < 0.01).

Figure 4

Poincare plot of time interval of heart beat from (A) control and (B) terfenadine-treated embryos at 52 hpf. X-axis is the time interval of heart beat at a particular time while Y-axis is the time interval of next heart beat. The shape and size of cluster illustrates the increase of heart beat variability in terfenadine-treated embryos.

Figure 5

Short-time Fourier Transform (STFT) analysis. The figure illustrates the STFT analysis of signal of dynamic pixels in every 0.5 sec along time. The time interval for each analysis is 1 sec. Intensity of green colour is corresponding to the power value while the red colour indicates the f_basic. (A) Control embryo showed consistent f_basic during the examination period. In contrast, the f_basic varied time by time in terfenadine-treated embryos, no matter in embryo with (B) decreased heart rate or (C) normal heart rate.

Figure 6

Effect of terfenadine on heart rate and heart rate variability. Bradycardia was induced by terfenadine treatment (A). Rhythmicity index was increased by terfenadine treatment (B), suggesting induction of irregularity of heart rate by terfenadine.

Figure 7

Diagram showing the caudal region of embryo to be analyzed (A) and the result image of subtraction between 2 consecutive video frames (B) with yellow pixels representing the dynamic pixels. Schematic diagram illustrated the parameters being analyzed in the signal of dynamic pixels (C). a_max: maximum value of dynamic pixels; a_min: minimum value of dyannmic pixels; heart beat interval: the time for a complete heart beat between 2 toughs; Scale Bar: 50 μm; Right to anterior (head).