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

Background: Zebrafish (Daniorerio), due to its optical accessibility and similarity to human,has emerged as model organism for cardiac research. Although various methodshave been developed to assess cardiac functions in zebrafish embryos, therelacks a method to assess heartbeat regularity in blood vessels. Heartbeatregularity is an important parameter for cardiac function and is associatedwith cardiotoxicity in human being. Using stereomicroscope and digital videocamera, we have developed a simple, noninvasive method to measure the heartrate and heartbeat regularity in peripheral blood vessels. Anesthetized embryoswere mounted laterally in agarose on a slide and the caudal blood circulationof zebrafish embryo was video-recorded under stereomicroscope and the data wasanalyzed by custom-made software. The heart rate was determined by digitalmotion analysis and power spectral analysis through extraction of frequencycharacteristics of the cardiac rhythm. The heartbeat regularity, defined as therhythmicity index, was determined by short-time Fourier Transform analysis.

Results: The heart rate measured by this noninvasive methodin zebrafish embryos at 52 hour post-fertilization was similar to thatdetermined by direct visual counting of ventricle beating (p > 0.05). In addition, themethod was validated by a known cardiotoxic drug, terfenadine, which affectsheartbeat regularity in humans and induces bradycardia and atrioventricularblockage in zebrafish. A significant decrease in heart rate was found by our methodin 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 intervalvariability (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 frequencyanalysis. This method is capable of measuring the heart rate and heartbeatregularity simultaneously via the analysis of caudal blood flow in zebrafishembryos. With the advantages of rapid sample preparation procedures, automaticimage analysis and data analysis, this method can potentially be applied tocardiotoxicity screening assay.