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 96-well 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.
Highlights
Accurate quantification of heartbeats in fish models is an important readout to study cardiovascular biology, disease states and pharmacology
Challenges of current methods to capture heart rate in high-throughput include: (i) immobilisation of dechorionated embryos or hatchlings, which in the majority of cases is achieved by anaesthesia with tricaine leading to severe adverse cardiovascular effects[29], (ii) imaging of larvae in standardized orientations, involving labour-intensive manual mounting on dedicated sample carriers or embedding in low-melting agarose, (iii) fluorescent reporter readouts, which preclude studies of specific genetic backgrounds and (iv) lack of user-friendly software for efficient analysis of multiple hearts simultaneously
We implemented a rapid workflow for automated imaging of medaka and zebrafish embryos and a new HeartBeat software for extraction of heart rate and rhythm
Summary
Accurate quantification of heartbeats in fish models is an important readout to study cardiovascular biology, disease states and pharmacology. 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. Challenges of current methods to capture heart rate in high-throughput include: (i) immobilisation of dechorionated embryos or hatchlings, which in the majority of cases is achieved by anaesthesia with tricaine leading to severe adverse cardiovascular effects[29], (ii) imaging of larvae in standardized orientations, involving labour-intensive manual mounting on dedicated sample carriers or embedding in low-melting agarose, (iii) fluorescent reporter readouts, which preclude studies of specific genetic backgrounds and (iv) lack of user-friendly software for efficient analysis of multiple hearts simultaneously. This demonstrates the applicability to preclinical drug screens and toxicological tests using fish models
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
