Abstract
BackgroundZebrafish (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.ResultsThe 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.ConclusionThe 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.
Highlights
Zebrafish (Danio rerio), due to its optical accessibility and similarity to human, has emerged as model organism for cardiac research
We described a simple, non-invasive methodology by video-recording under stereomicroscope and analyzing the video records with our cuctom made software, which integrated digital motion analysis [20] and power spectral analysis, to determine the heart rate
The results demonstrated that our method is able to assess the cardiac physiology, in term of heart rate and rhythmicity, in zebrafish embryos
Summary
Zebrafish (Danio rerio), due to its optical accessibility and similarity to human, has emerged as model organism for cardiac research. There are several methods to assess cardiac functions in zebrafish embryos, including the simplest stopwatch counting [9], micro pressure system [10], Laser Doppler microscope technique [11] and electrocardiogram [12]. All these techniques are labor-intensive, timeconsuming and requires skillful operator to perform the experiments, and limits their applications for largescale studies [11], such as drug toxicity evaluation. Schwerte and his co-workers had developed a non-invasive method to assess heart rate variability in zebrafish embryos [22]. I.e. disturbance of heartbeat regularity [23], is associated with cardiotoxicity effect of drugs and sudden cardiac death [24]
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