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

Chan PK, Lin CC, Cheng SH - BMC Biotechnol. (2009)

Bottom Line: 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.This method is capable of measuring the heart rate and heartbeat regularity simultaneously via the analysis of caudal blood flow in zebrafish embryos.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, HKSAR, PR China.

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.

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Related in: MedlinePlus

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).
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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).

Mentions: Videos of caudal circulation in terfenadine-treated embryos were analyzed with our program. Direct observation of caudal circulation in terfenadine-treated embryos showed that movement of blood cells in dorsal aorta was pulsatile as seen in control embryos. An example of waveform of dynamic pixels (Fig. 3A) of a terfenadine-treated embryo with severe effect and its corresponding power spectrum (Fig. 3B) were shown. Although the waveform of dynamic pixels also exhibited oscillatory change of blood cells velocity along the time being analyzed, the fbasic was hard to identify in the power spectrum (Fig. 3B). In general, the fbasic of terfenadine-treated embryos were just a peak a little bit higher than other frequency components and was less dominant in the power spectra, as compared with the power spectra of control embryos (Fig. 1B).


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

Chan PK, Lin CC, Cheng SH - BMC Biotechnol. (2009)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664803&req=5

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).
Mentions: Videos of caudal circulation in terfenadine-treated embryos were analyzed with our program. Direct observation of caudal circulation in terfenadine-treated embryos showed that movement of blood cells in dorsal aorta was pulsatile as seen in control embryos. An example of waveform of dynamic pixels (Fig. 3A) of a terfenadine-treated embryo with severe effect and its corresponding power spectrum (Fig. 3B) were shown. Although the waveform of dynamic pixels also exhibited oscillatory change of blood cells velocity along the time being analyzed, the fbasic was hard to identify in the power spectrum (Fig. 3B). In general, the fbasic of terfenadine-treated embryos were just a peak a little bit higher than other frequency components and was less dominant in the power spectra, as compared with the power spectra of control embryos (Fig. 1B).

Bottom Line: 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.This method is capable of measuring the heart rate and heartbeat regularity simultaneously via the analysis of caudal blood flow in zebrafish embryos.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, HKSAR, PR China.

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.

Show MeSH
Related in: MedlinePlus