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

Abstract

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...

Fast in vivo multiphoton light-sheet microscopy with optimal pulse frequency

“…This result suggests that water absorption might be involved and could induce a local heating of the specimen when the laser is on. Since the HBR is known to linearly depend on the temperature in the zebrafish embryo [35,36], S L can be related to a change in local temperature. We measured an HBR variation of ∼0.29 Hz/°C ( Fig.…”

Balancing signal and photoperturbation in multiphoton light-sheet microscopy by optimizing laser pulse frequency

“…This result suggests that water absorption might be involved and could induce a local heating of the specimen when the laser is on. Since the HBR is known to linearly depend on the temperature in the zebrafish embryo [32,33], S L can be related to a change in local temperature. We measured an HBR variation of ~0.29 Hz/°C (Fig.…”

Is laser repetition rate important for two-photon light sheet microscopy?

“…The increase in heart rate is due to the heating generated by laser illumination, since, upon switching to bright field illumination, the heart rate reverts back to its baseline value. Zebrafish heart rate has previously been established as a measure of laser-induced photo-injury in both single-photon [10,11] and two-photon light sheet microscopy [5], increasing (decreasing) linearly with a rise (fall) of temperature [14].…”