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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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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.
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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.

Mentions: Embryos treated with terfenadine showed a significant increase in the variation of heart beat interval. Poincare plot, an emerging quantitative visualization tool, provides additional information about the variation between heartbeats. Typical examples for both control (Fig. 4A) and terfenadine-treated embryos with bradycardia (Fig. 4B) were shown. In control embryo, the plot had concentrated data points arranged from 0.3 to 0.6 sec, clustering around 0.48 sec. In contract, the plot of terfenadine-treated embryos was dispersed, spreading from 0.3 to 1 sec. Although there was a cluster around 0.48 seconds, some of data points were diverged from the cluster, making the standard deviation large. Variation of heart beat interval was significantly increased from 0.0615 ± 0.0107 in control embryos to 0.1857 ± 0.0627 in terfenadine treated embryos (p < 0.01), illustrating the increased variation of heartbeat by terfenadine in zebrafish embryos.


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

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

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

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

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.
Mentions: Embryos treated with terfenadine showed a significant increase in the variation of heart beat interval. Poincare plot, an emerging quantitative visualization tool, provides additional information about the variation between heartbeats. Typical examples for both control (Fig. 4A) and terfenadine-treated embryos with bradycardia (Fig. 4B) were shown. In control embryo, the plot had concentrated data points arranged from 0.3 to 0.6 sec, clustering around 0.48 sec. In contract, the plot of terfenadine-treated embryos was dispersed, spreading from 0.3 to 1 sec. Although there was a cluster around 0.48 seconds, some of data points were diverged from the cluster, making the standard deviation large. Variation of heart beat interval was significantly increased from 0.0615 ± 0.0107 in control embryos to 0.1857 ± 0.0627 in terfenadine treated embryos (p < 0.01), illustrating the increased variation of heartbeat by terfenadine in zebrafish embryos.

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