Regular heartbeat rhythm at the heartbeat initiation stage is essential for normal cardiogenesis at low temperature

Watanabe-Asaka, Tomomi; Sekiya, Yoshio; Wada, Hironori; Yasuda, Tomoyuki; Okubo, Ikuya; Oda, Shoji; Mikami, Hiroshi

doi:10.1186/1471-213x-14-12

Cited by 13 publications

(21 citation statements)

References 40 publications

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“…S4) can be applied as physical stressor in ectothermic medaka and zebrafish embryos, which regulate cardiac output in response to temperature primarily through adaption of heart rate rather than stroke volume 43,44 . Tight control of heart rate by temperature can be leveraged as readout of acute and chronic adaptive stress responses, temperature-sensitive strains 45 and of susceptibility to arrhythmia in strains with different genetic backgrounds. In summary, our detailed analysis stresses the importance to rigorously control temperature and normalize to stage-dependent changes of beat rates to ensure experimental standardization.…”

Section: Discussionmentioning

confidence: 99%

“…In human, the antihistamine terfenadine can induce adverse prolongation of QT interval resulting in increased risk for torsades de pointes tachycardia. Detailed studies have demonstrated QT prolongation resulting in AV block in zebrafish 46 and medaka 45 . Probing effects of terfenadine on zebrafish and medaka embryonic heart rate, distinct time points of induction and time courses of AV block were uncovered by our assay (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Automated high-throughput heartbeat quantification in medaka and zebrafish embryos under physiological conditions

Gierten

Pylatiuk

Hammouda

et al. 2020

Sci Rep

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Accurate quantification of heartbeats in fish models is an important readout to study cardiovascular biology, disease states and pharmacology. However, dependence on anaesthesia, laborious sample orientation or requirement for fluorescent reporters have hampered the use of high-throughput heartbeat analysis. To overcome these limitations, we established an efficient screening assay employing automated label-free heart rate determination of randomly oriented, non-anesthetized medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos in microtiter plates. Automatically acquired bright-field data feeds into an easy-to-use HeartBeat software with graphical user interface for automated quantification of heart rate and rhythm. Sensitivity of the assay was demonstrated by profiling heart rates during entire embryonic development. Our analysis revealed rapid adaption of heart rates to temperature changes, which has implications for standardization of experimental layout. The assay allows scoring of multiple embryos per well enabling a throughput of >500 embryos per 96well plate. In a proof of principle screen for compound testing, we captured concentration-dependent effects of nifedipine and terfenadine over time. Our novel assay permits large-scale applications ranging from phenotypic screening, interrogation of gene functions to cardiovascular drug development. Quantification of heartbeats is an important readout to study physiology and disease states of the heart. The resting heart rate (beats per minute, bpm) is a strong predictive risk factor of overall mortality and associated with a growing catalogue of genetic variants and environmental-sensitive alleles 1,2. However, geneticists are facing an enormous challenge to establish causality for associated candidate variants. Small fish models provide efficient means of functional assessment in the context of a vertebrate. In fish models heart rate and rhythm quantification is employed for analysing inherited or acquired arrhythmia 3 , for validation of genetic variants 4 , phenotypic drug discovery and safety pipelines 5 or for toxicological studies 6. Moreover, the recent establishment of a vertebrate panel of isogenic strains in medaka fish enables to study variations of quantitative traits such as heart rate in genome-wide association studies 7. However, there is a lack of efficient screening workflows that allow large-scale quantitative scoring of cardiac phenotypes. Therefore, novel and efficient protocols were needed encompassing sample preparation, automated imaging and image analysis. The fish species medaka (Oryzias latipes) and zebrafish (Danio rerio) emerged as major vertebrate models for phenotypic screening, follow-up functional studies, reverse genetics and for studying human pathologies in mechanistic details 8,9. Medaka and zebrafish have tractable diploid genomes, feature a short generation time (2-3 months), external embryonic development and established protocols for transgenesis and manipulation of gene function including CRISPR/Cas-based approac...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Automated high-throughput heartbeat quantification in medaka and zebrafish embryos under physiological conditions

Gierten

Pylatiuk

Hammouda

et al. 2020

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Tight control of heart rate by temperature can be leveraged as readout of acute and chronic adaptive stress responses, temperature-sensitive strains 52 and of susceptibility to arrhythmia in strains with different genetic backgrounds. In summary, our detailed analysis stresses the importance to rigorously control temperature and normalize to stage-dependent changes of beat rates to ensure experimental standardization.…”

Section: Discussionmentioning

confidence: 99%

“…In human, the antihistamine terfenadine can induce adverse prolongation of QT interval resulting in increased risk for torsades de pointes tachycardia. Detailed studies have demonstrated QT prolongation resulting in AV block in zebrafish 53 and medaka 52 . Probing effects of terfenadine on zebrafish and medaka embryonic heart rate, distinct time points of induction and time courses of AV block were uncovered by our assay (Figure 7).…”

Section: Discussionmentioning

confidence: 99%

Automated high-throughput heart rate measurement in medaka and zebrafish embryos under physiological conditions

Gierten

Pylatiuk

Hammouda

et al. 2019

Preprint

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Rationale: Accurate and efficient quantification of heartbeats in small fish models is an important readout to study cardiovascular biology, disease states and pharmacology at large scale. However, dependence on anesthesia, laborious sample orientation or requirement for fluorescent reporters have hampered the establishment of high-throughput heartbeat analysis.Objective: To overcome these limitations, we aimed to develop a high-throughput assay with automated heart rate scoring in medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos under physiological conditions designed for genetic screens and drug discovery and validation. Methods and Results:We established an efficient screening assay employing automated label-free heart rate determination of randomly oriented, non-anesthetized specimen in microtiter plates. Automatically acquired bright-field data feeds into an easy-to-use HeartBeat software, a MATLAB algorithm with graphical user interface developed for automated quantification of heart rate and rhythm. Sensitivity of the assay and algorithm was demonstrated by profiling heart rates during entire embryonic development. Our analysis pipeline revealed acute temperature changes triggering rapid adaption of heart rates, which has implications for standardization of experimental layout. The approach is scalable and allows scoring of multiple embryos per well resulting in a throughput of >500 embryos per 96-well plate. In a proof of principle screen for compound testing, our assay captured concentration-dependent effects of nifedipine and terfenadine over time. Conclusion:A novel workflow and HeartBeat software provide efficient means for reliable and direct quantification of heart rate and rhythm of small fish in a physiological environment. Importantly, confounding factors such as anesthetics or laborious mounting are eliminated. We provide detailed profiles of embryonic heart rate dynamics in medaka and zebrafish as reference for future assay development. Ease of sample handling, automated imaging, physiological conditions and softwareassisted analysis now facilitate various large-scale applications ranging from phenotypic screening, interrogation of gene functions to cardiovascular drug development pipelines.

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“…5F ). We then averaged the beat-by-beat heart rates during collection for 3 min to generate steady-state heart rates 40 .…”

Section: Methodsmentioning

confidence: 99%

In vivo 3D analysis of systemic effects after local heavy-ion beam irradiation in an animal model

Nagata

Hashimoto

Watanabe-Asaka

et al. 2016

Sci Rep

Self Cite

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Radiotherapy is widely used in cancer treatment. In addition to inducing effects in the irradiated area, irradiation may induce effects on tissues close to and distant from the irradiated area. Japanese medaka, Oryzias latipes, is a small teleost fish and a model organism for evaluating the environmental effects of radiation. In this study, we applied low-energy carbon-ion (26.7 MeV/u) irradiation to adult medaka to a depth of approximately 2.2 mm from the body surface using an irradiation system at the National Institutes for Quantum and Radiological Science and Technology. We histologically evaluated the systemic alterations induced by irradiation using serial sections of the whole body, and conducted a heart rate analysis. Tissues from the irradiated side showed signs of serious injury that corresponded with the radiation dose. A 3D reconstruction analysis of the kidney sections showed reductions in the kidney volume and blood cell mass along the irradiated area, reflecting the precise localization of the injuries caused by carbon-beam irradiation. Capillary aneurysms were observed in the gill in both ventrally and dorsally irradiated fish, suggesting systemic irradiation effects. The present study provides an in vivo model for further investigation of the effects of irradiation beyond the locally irradiated area.

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Regular heartbeat rhythm at the heartbeat initiation stage is essential for normal cardiogenesis at low temperature

Cited by 13 publications

References 40 publications

Automated high-throughput heartbeat quantification in medaka and zebrafish embryos under physiological conditions

Automated high-throughput heartbeat quantification in medaka and zebrafish embryos under physiological conditions

Automated high-throughput heart rate measurement in medaka and zebrafish embryos under physiological conditions

In vivo 3D analysis of systemic effects after local heavy-ion beam irradiation in an animal model

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