Mar 04, 2026

Imaging cardiac arrhythmia in genetic disease models in zebrafish embryos V.1

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvm1j75v3p/v1
  • Ioana Gabor1,
  • Lynn Kreutzer1,
  • Mandy Lim1,
  • Ericka Peloso1,
  • Mar Roca Cugat1,
  • Melanie Schmid1
  • 1FHML, Maastricht University
  • UM BBS 2023-26
Mar Roca Cugat
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvm1j75v3p/v1
Protocol Citation: Ioana Gabor, Lynn Kreutzer, Mandy Lim, Ericka Peloso, Mar Roca Cugat, Melanie Schmid 2026. Imaging cardiac arrhythmia in genetic disease models in zebrafish embryos. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm1j75v3p/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol. It will be tested.
Created: March 03, 2026
Last Modified: March 04, 2026
Protocol  Integer ID: 245412
Keywords: zebrafish, cardiac arrhyhmia, genetic disease model, zebrafish cardiac electrophysiology, zebrafish embryos in biomedical research, cardiac arrhythmia in genetic disease model, zebrafish embryo, genetic mechanisms of cardiac development, cardiac development, zebrafish ortholog, imaging cardiac arrhythmia, vertebrate biology, direct predictors of clinical arrhythmia, arrhythmia research, impulse generation in the human heart, vertebrate biology with human, many arrhythmia, clinical arrhythmia, differences in the heart structure, heart structure, studying genetic mechanism, vertebrate, human heart, human gene, effective vertebrate system, functional heart, inbred laboratory mice, receptor, embryo, many gene, similar organ system, including many gene, laboratory mice, ion channel
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Abstract
In biomedical research, zebrafish are broadly used because they share conserved vertebrate biology with humans while allowing some unique experimental advantages. As vertebrates, zebrafish possess similar organ systems, including a functional heart, brain, liver, and kidney, and other pathways, ion channels, receptors, and signalling cascades. Their genetical diversity also reflect humans better than the highly inbred laboratory mice. In addition to that, their embryos develop externally, are optically transparent and can survive days without effective circulation, allowing direct and non-invasive live imaging of cardiac development. (Bedell VM 2025)

Approximately 70% of human genes have at least one zebrafish ortholog, including many genes involved in cardiac development and arrhythmogenic disorders, being the focus of this study. Importantly for arrhythmia research, zebrafish cardiac electrophysiology relies on similar ionic currents that define impulse generation in the human heart. (Verkerk AO 2012) Thus, many arrhythmia-inducing drugs and neuroactive compounds produce similar effects in zebrafish and humans, making zebrafish a biologically relevant comparative model. (Gauvrit S 2022)

Despite these strengths, zebrafish should not be interpreted as direct predictors of clinical arrhythmias. Differences in the heart structure, in drug metabolism, developmental timing, and regenerative capacity must also be considered when translating findings. Overall, zebrafish provide a powerful and cost-effective vertebrate system for studying genetic mechanisms of cardiac development, rhythm, and early disease phenotypes, particularly for discovery and screening approaches.
Image Attribution
Created in BioRender
Materials

ABC
NameDescriptionSupplier/Reference
Zeiss Axiophot fluorescence microscopeMicroscope equipped for fluorescence microscopyZeiss GmbH, Oberkochen, Germany.
Bresser MikroCam II, 20MP 1”Camera used for video recordingBresser, Hoogeveen, Netherlands
20x objectiveObjective used for microscopyZeiss GmbH, Oberkochen, Germany
Custom 3D-printed mouldFor mounting, creating indentations fitting 72 hpf embryos. Contains 2% agarose in E3 mediumDepartment of Clinical Genetics, Maastricht University, Maastricht, Netherlands.
Static breeding tank with outer and inner tanksWith a sloped bottom, and perforated grate
MUSCLEMOTIONImageJ macro to measure in vitro or in vivo contraction or similar motionSala L et al., Versatile open software to quantify cardiomyocyte and cardiac muscle contraction in vitro and in vivo, bioRxiv, 2017, doi: 10.1101/160754.
Fiji (ImageJ distribution)Image processing package, a distribution of ImageJ2, bundling a lot of plugins which facilitate scientific image analysisSchindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., ... Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676–682. doi:10.1038/nmeth.2019
PythonProgramming language for general use, often used for data science, data visualisation, and machine learningG. van Rossum, Python tutorial, Technical Report CS-R9526, Centrum voor Wiskunde en Informatica (CWI), Amsterdam, 1995.
Data processing scriptsInternally developed data processing scripts I.C. Gabor, L.D.C.E. Kreutzer, M.M.H. Lim, E.R. Peloso, M. Roca Cugat, M. Schmid. https://github.com/Peiprjs/BBS3004_scripts

Other materials:
- Net or transfer pipette
- Phenanthrene (Phe) (3e99% purity)
- Terfenadine
- Dimethyl sulfoxide (DMSO) (3e99% purity)
- E3 Medium
- 6-well plates
- Micropipettes and sterile tips
- Zebrafish embryos, 3 days post-fertilization
- MS-222 (tricaine, stock solution, neutral pH)
- 2% agarose in E3 medium
- Disposable plastic pipettes
- Dissection needles
Safety warnings
During zebrafish manipulations in arrhythmia studies, appropriate health and safety measures must be followed to protect both researchers and animal welfare. Tricaine methanesulfonate (MS-222), an anaesthetic, can cause skin, eye, and respiratory irritation, it should be prepared and handled in a well-ventilated area or fume hood while wearing a lab coat, gloves, and safety glasses. Solutions must be disposed of according to institutional chemical waste procedures. For dimethyl sulfoxide (DMSO), that we’ll use as a solvent for cardiac drugs, can easily penetrate the skin and carry dissolved compounds with it, so direct contact should be avoided and gloves changed regularly. Lastly, the electrical equipment used for cardiac recordings (e.g., ECG) should be inspected at some time points to prevent electrical hazards, especially in wet working environments.

All experiments must follow institutional animal care and biosafety guidelines, with careful monitoring of anaesthesia and recovery. Spill kits, eye wash stations, and safety data sheets should be readily accessible in the laboratory.

Phenanthrene and Terfenadine must be handled with gloves and safety glasses. Swallowing and inhalation of their dust are harmful and should be avoided. The substances should be disposed of to an approved waste disposal plant, as they are toxic to aquatic life with long-lasting effects.
Spawning
1d
Assemble the breeding tank with the inner tank securely placed inside the outer tank.
Select healthy adult males and females for breeding.
Transfer the selected fish into the breeding tank in the late afternoon.
Maintain the standard 14-hour light / 10-hour dark cycle Overnight at 24 °C .

Observe the tank the following morning after lights turn on.
Confirm spawning by checking for eggs that have passed through the grate into the lower compartment.
Remove the adult fish from the breeding tank and return them to their home tanks.
Collect the eggs from the lower compartment using a pipette or fine-mesh net.
Transfer the collected eggs to embryo medium as described in the next section of the protocol.
Label and record the spawning event.
Consider a spawning successful if at least 100 fertilized eggs are present.
Record crossings without eggs or with unfertilized/degenerating eggs as unsuccessful.
Drug treatment - Sensitivity Analysis
2d
Dissolve phenanthrene in Dimethyl Sulfoxide (DMSO) to produce a 10 millimolar (mM) solution.
Vortex.

Dilute the the phenantrene stock solution 2*10^5 x in E3 medium to reach a concentration of 50 nanomolar (nM) . The final volume necessary of this solution is 20 mL. This concentration can be obtained in two steps:
Start with performing a 1000 x dilution, this can be done in 10 mL by pipetting 10 µL of Stock Phe and 9990 µL of medium. Vortex.
Perform a 200 x dilution by pipetting 100 µL of Stock Phe and 19900 µL of medium. Vortex.
Serially dilute the solution 3 times 10 x with E3 medium to obtain the concentrations 50 nanomolar (nM) , 5 nanomolar (nM) , 0.5 nanomolar (nM) , and 0.05 nanomolar (nM)

Dissolve terfenadine in DMSO to prepare 0.5 mL of 2 millimolar (mM) stock solution of 100mM. Vortex.

Dissolve the terfenadine stock solutions further in E3 medium to reach the final concentrations of 4, 6, 8, and 10 µM and mix gently with the pipette.

Final concentration (μM)Stock solution volume (mL)E3 volume (mL)Final volume (mL)
40.0419.9620 mL
60.0619.9420 mL
80.0819.9220 mL
10 0.119.920 mL
Table 1. Pipetting scheme.

Prepare the two DMSO in E3 controls. One should have a concentration of 2*10^5 x and the other 2*10^2 x .

Label three 6-well plates (two wells per condition (8) plus two wells per control (2)).
Add 6mL of the appropriate solution from step 2 to each well.

Transfer 20 embryos per well using a pipette.
Expose for 48:00:00 in total at Room temperature on a 14:10 h light-dark cycle.
Replace exposure medium daily.
2d
Embryo Immobilization (Will be checked shortly)
Prepare a 4 g/L stock solution of MS-222 in E3 medium. Adjust the pH to 7.0 using 1M NaOH, as MS-222 is acidic and unbuffered solution may harm embryos.
For use, dilute the stock solution to a final concentration of 50 mg/L in E3 medium for anaesthesia of 3 dpf embryos.
Prepare a 2% agarose solution by dissolving 2g agarose in 100 mL E3 medium.
Heat gently while stirring until the solution becomes completely clear. Avoid boiling.
Maintain melted agarose at 38-40 °C until use.
Add a thin layer of warm 2% agarose to each well of a 6-well plate, covering the bottom evenly.
Place the custom 3D-printed mould into the liquid agarose.
Gently press the mould into the agarose to create embryo-shaped indentations for 3 dpf.
Allow the agarose to solidify completely at room temperature.
Carefully remove the mould without damaging the imprints.
Pre-screen embryos under a stereomicroscope to select healthy individuals.
Expose embryos to drug-containing medium for the required pre-treatment duration.
Transfer embryos into 50 mg/L MS-222 solution for 5 minutes.
Confirm proper anaesthesia before embedding: reduced opercular (gill) movement, slowed but detectable heart rate, no escape response upon gentle tail touch.
Using a disposable plastic pipette, gently transfer anaesthetized embryos into pre-warmed 2% agarose.
Quickly position the embryo into a pre-formed mould indentation (20 embryos per well).
Align embryos laterally: the head must be oriented to the left, body straight, and the tail flat and not curved.
Use a dissection needle to adjust positioning if necessary.
Allow agarose to solidify for 10-15 minutes at room temperature.
Once set, add E3 medium to prevent drying.
Microscopy set up
Custom mould containing the 3 dpf embryos is placed on Zeiss Axiophot fluorescence microscope and visualized with a 20x objective.
Video recording
Recordings will be done at ambient temperature (21-22 °C).
60 FPS for 10-30 seconds using Bresser MikroCam II, 20 MP 1” camera. The absolute minimum frame rate is 16 fps, and the recommended minimum frame rate is 48 fps.
Convert the videos to an ImageJ-readable format using the bash/convertToAVI.sh script.
Video analysis
Start Fiji and run the MUSCLEMOTION. Use the settings from Appendix 2.
Once the analysis of MUSCLEMOTION is finished and the log file of the last measurement is shown, the results must be checked by looking at the JPEG files that are generated in the results folder. ‘Contraction.jpg’ shows the reference frame, and ‘Comparison calculated and measured speed.jpg’ can be used to check if MUSCLEMOTION worked within boundaries.
Data analysis and statistics
Open ‘Overview-results.txt’ and ‘Contraction.txt’ files in Python using the python/data_analysis.py script.
Normalize all means of the different concentrations to the mean of the baseline of that specific sample.
Calculate mean and standard error of the mean (SEM) of the different samples.
Perform exploratory data analysis through the use of graphs and calculate the speed of each contraction.
Run a One-Sample Chi-Square Test on the speed of contraction and inter-contraction period, to see whether the variance is significant, which could indicate an arrhythmia.
APPENDIX

Appendix 1: Timeline of the experiment

Protocol references
- Bedell VM 2025
- Verkerk AO 2012
- Gauvrit S 2022
- Sala L et al., Versatile open software to quantify cardiomyocyte and cardiac muscle contraction in vitro and in vivo, bioRxiv, 2017, doi: 10.1101/160754
- Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., ... Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676–682. doi:10.1038/nmeth.2019
- G. van Rossum, Python tutorial, Technical Report CS-R9526, Centrum voor Wiskunde en Informatica (CWI), Amsterdam, 1995.
- Bedell VM, Dubey P, Lee HB, Bailey DS, Anderson JL, Jamieson-Lucy A, Xiao R, Leonard EV, Falk MJ, Pack MA, Mullins M, Farber SA, Eckenhoff RG, Ekker SC. Zebrafishology, study design guidelines for rigorous and reproducible data using zebrafish. Commun Biol. 2025;8:739.
- Verkerk AO, Remme CA. Zebrafish: a novel research tool for cardiac (patho)electrophysiology and ion channel disorders. Front Physiol. 2012 Jul 10;3:255
- Gauvrit S, Bossaer J, Lee J, Collins MM. Modeling human cardiac arrhythmias: insights from zebrafish. J Cardiovasc Dev Dis. 2022 Jan 5;9(1):13
- Zhang, Y., Huang, L., Zuo, Z., Chen, Y.,  Wang, C. (2013). Phenanthrene exposure causes cardiac arrhythmia in embryonic zebrafish via perturbing calcium handling. Aquatic toxicology (Amsterdam, Netherlands), 142-143, 26–32. https://doi.org/10.1016/j.aquatox.2013.07.014
- Gu, G., Na, Y., Chung, H., Seok, S. H.,  Lee, H. Y. (2017). Zebrafish Larvae Model of Dilated Cardiomyopathy Induced by Terfenadine. Korean circulation journal, 47(6), 960–969. https://doi.org/10.4070/kcj.2017.0080
- Aleström, P., D'Angelo, L., Midtlyng, P. J., Schorderet, D. F., Schulte-Merker, S., Sohm, F.,  Warner, S. (2020). Zebrafish: housing and husbandry recommendations. Laboratory Animals, 54(3 Suppl), 213–224. https://doi.org/10.1177/0023677219869037