Zebrafish in Cardiotoxicity Screening

Drug‑induced cardiotoxicity remains a persistent and costly challenge in drug development, responsible for around 10–14 % of post‑market drug withdrawals. This has intensified the demand for more predictive and ethical preclinical models. In response, regulatory agencies are firmly embracing New Approach Methodologies (NAMs). In a landmark announcement in April 2025, the U.S. Food and Drug Administration unveiled its plan to phase out animal testing requirements for monoclonal antibodies and other drugs, encouraging developers to submit safety data derived from NAMs and signaling a clear paradigm shift toward modernized testing frameworks. 

Early Detection of Cardiotoxic Effects in Preclinical Drug Evaluation

Cardiotoxicity remains one of the most critical safety concerns in drug development, contributing to late-stage clinical trial failures and post-marketing withdrawals. Drugs such as certain chemotherapeutics, antiarrhythmics, and antimicrobials have been linked to cardiac damage, ranging from subtle electrophysiological alterations to severe arrhythmias and heart failure. Detecting these risks early is crucial to reducing attrition rates, safeguarding patient health, and avoiding costly setbacks.

Traditional preclinical cardiotoxicity screening often relies on rodent or large mammalian models, but these present limitations in terms of physiological relevance and throughput. Zebrafish have emerged as an attractive alternative. Adult zebrafish electrocardiograms (ECGs) display P–QRS–T complexes with measurable QT intervals, and the larval heart is fully functional by 96 hours post-fertilization (hpf), enabling rapid in vivo readouts soon after dosing, including those affecting rhythm, morphology, and contractility, at early developmental stages. 

More than 20 years ago, Milan et al. had already demonstrated in a phenotype-based screening that zebrafish repeatedly mirror pharmacology seen in patients. They tested 23 drugs with known human repolarization liability; 22 produced repolarization-related toxicity in zebrafish, underscoring conserved effect direction and supporting their use as an early in vivo model before mammalian work.

Recently, Dyballa et al. tested 92 cardiotoxic compounds and compared them with data acquired from human induced pluripotent stem cell cardiomyocytes. Zebrafish offered a more reliable prediction of cardiotoxicity. The prediction was similar to meta-studies performed with dogs, the standard regulatory preclinical model for predicting cardiotoxic liabilities before clinical phases.

Current Methods for Cardiotoxicity Screening in Preclinical Research

Current preclinical cardiotoxicity models fall into two broad categories: in vitro assays and in vivo animal testing. In vitro assays, such as hERG channel inhibition tests or cardiomyocyte cultures, provide mechanistic insights into electrophysiological effects but lack the systemic context of a living organism. They cannot replicate the complexity of drug absorption, distribution, metabolism, and excretion (ADME) processes, which are often key to understanding a compound’s cardiac impact.

In vivo mammalian models, such as mice, rats, and dogs, are the gold standard for regulatory toxicology, but their predictive accuracy is not absolute. Differences in heart rate, repolarization mechanisms, and ion channel profiles between species can lead to discrepancies in translational relevance. For example, rat hearts beat significantly faster, exceeding 300 beats/min, than human hearts in healthy individuals (70 beats/min). Rodents also rely on different potassium currents for repolarization, which can mask or exaggerate drug effects observed in human patients.

Advanced imaging and biomarker analyses are employed in both preclinical and clinical phases. While these tools are valuable, they often detect damage after it has occurred, making them less suited for preventive screening. Moreover, invasive techniques like endomyocardial biopsy, although definitive, are impractical for routine early-stage testing.

Against this backdrop, zebrafish models offer a unique middle ground, retaining the integrative complexity of a whole organism while allowing higher throughput and lower costs compared to mammalian studies.

Advantages of Using Zebrafish Models in Cardiotoxicity Assays

The zebrafish cardiotoxicity model offers several advantages for cardiotoxicity studies that address the limitations of other systems. 

Optical transparency 

Their optical transparency enables real-time, non-invasive observation of cardiac morphology, heartbeat, and blood flow. This makes it possible to measure parameters such as heart rate variability, stroke volume, and ejection fraction with high accuracy.

Electrophysiology

Zebrafish electrophysiology closely mirrors that of humans. Zebrafish heart rate is between 120 and 180 beats/min. Their cardiac action potentials exhibit a plateau phase mediated by similar calcium and potassium currents, making them more representative of human repolarization than rodents. This similarity is particularly relevant for identifying drugs that prolong the QT interval or disrupt ion channel function, effects that can lead to life-threatening arrhythmias. 

Multiple compounds with known human cardiotoxic profiles reproduce concordant phenotypes in zebrafish. Haloperidol, widely reported to block hERG and prolong QTc in patients, elicited QTc prolongation and arrhythmias in zebrafish larvae, according to Cornet et al. In the same dataset, several human QT-prolonging drugs drove bradycardia in zebrafish, mirroring clinical associations between repolarization delay and rate slowing. These concordances, measured via standardized video-ECG analysis using our ZeCardio® platform, confirm zebrafish predictive value. 

Genetic manipulation

Zebrafish allow easy genetic manipulation to create transgenic lines expressing fluorescent markers in specific cardiac tissues. These reporter lines facilitate high-content imaging and can be used to study both structural and functional effects of drug exposure.

High-throughput screening

Zebrafish are highly amenable to high-throughput phenotypic screening. Hundreds of embryos can be housed and dosed in multi-well plates, enabling parallel testing of compound libraries. The ZeGlobalTox platform, for instance, integrates cardiotoxicity assessment with neuro- and hepatotoxicity evaluation in the same larva, optimizing resources and aligning with the 3Rs principle (Replacement, Reduction, Refinement) in animal research.

Quick development and small size 

Their rapid development and small size reduce both the time and cost of experiments. After 96 hpf, the zebrafish heart development is already completed, and its heartbeat is stabilized. Effects can be detected within hours after drug exposure, accelerating decision-making during lead optimization. According to the European Directive 2010/63/EU, zebrafish are not considered animals until they have reached the stage capable of independent feeding, which typically occurs around 5 days post-fertilization (dpf). This window of opportunity allows in vivo cardiotoxicity assays without the ethical and regulatory implications associated with animal models. 

Improving Drug Safety with Zebrafish in Cardiotoxicity Studies

Zebrafish are increasingly integrated into preclinical safety pipelines to enhance early detection of cardiac risks. By bridging the gap between in vitro assays and mammalian testing, they help filter out hazardous candidates before they advance to expensive and ethically demanding stages. The combination of high predictive value, scalability, and physiological relevance positions zebrafish as a complementary model that strengthens the robustness of cardiotoxicity assessments.

We have developed CardioTox for cardiotoxicity assessment in zebrafish. Transgenic zebrafish embryos expressing green fluorescent protein (GFP) in cardiomyocytes are incubated with the No Observable Effect Concentration (NOEC) of the molecule of interest for 4 hours. At the endpoint, every larvae heart is video-imaged for 1 min and analyzed with ZeCardio unique software of analysis (ZeClinics-developed cardiovascular software). After 120 hpf, we analyze beat frequency (ventricle and atrium), arrhythmias, QTc interval, and ejection fraction. 

CardioTox shows predictability levels referred to clinical data above 85% and a comparable predictability to that reported in dogs. We can assess simultaneously neurotoxicity and hepatotoxicity through our ZeGlobalTox service. 

If you're interested in learning more about how zebrafish cardiotoxicity testing can fit into your preclinical pipeline, contact us!

References

Cornet C, Calzolari S, Miñana‑Prieto R, Dyballa S, van Doornmalen E, Rutjes H, Savy T, D’Amico D, Terriente J. ZeGlobalTox: an innovative approach to address organ drug toxicity using zebrafish. Int J Mol Sci. 2017 Apr 19;18(4):864. doi:10.3390/ijms18040864

Dyballa S, Miñana R, Rubio-Brotons M, Cornet C, Pederzani T, Escaramis G, Garcia-Serna R, Mestres J, Terriente J. Comparison of Zebrafish Larvae and hiPSC Cardiomyocytes for Predicting Drug-Induced Cardiotoxicity in Humans. Toxicol Sci. 2019 Oct 1;171(2):283-295. doi: 10.1093/toxsci/kfz165.

Kreutzer FP, Meinecke A, Schmidt K, Fiedler J, Thum T. Alternative strategies in cardiac preclinical research and new clinical trial formats. Cardiovasc Res. 2022 Feb 21;118(3):746–762. doi:10.1093/cvr/cvab075.

Leong IU, Skinner JR, Shelling AN, Love DR. Zebrafish as a model for long QT syndrome: the evidence and the means of manipulating zebrafish gene expression. Acta Physiol (Oxf). 2010 Jul 1;199(3):257–76. doi:10.1111/j.1748-1716.2010.02111.x.

Lin Z, Wei X, Wei Y, Miao Z, Ye H, Wu M, Liu X, Cai L, Yu C. Zebrafish as a rapid model system for early cardiotoxicity assessment of drugs. J Holist Integr Pharm. 2024;5(3):223–34. doi:10.1016/j.jhip.2024.09.002.

Milan DJ, Peterson TA, Ruskin JN, Peterson RT, MacRae CA. Drugs that induce repolarization abnormalities cause bradycardia in zebrafish. Circulation. 2003 Mar 18;107(10):1355-8. doi:10.1161/01.cir.0000061912.88753.87.

Seal S, Spjuth O, Hosseini‑Gerami L, García‑Ortegón M, Singh S, Bender A, Carpenter AE. Insights into Drug Cardiotoxicity from Biological and Chemical Data: The First Public Classifiers for FDA Drug‑Induced Cardiotoxicity Rank. J Chem Inf Model. 2024;64(4):1172‑1186. doi:10.1021/acs.jcim.3c01834.

US Food and Drug Administration. FDA announces plan to phase out animal testing requirement for monoclonal antibodies and other drugs [press release]. Silver Spring (MD): US FDA; 2025 Apr 10 [cited 2025 Aug 12]. Available from: https://www.fda.gov/news-events/press-announcements/fda-announces-plan-phase-out-animal-testing-requirement-monoclonal-antibodies-and-other-drugs

By Miriam

Miriam is a Human Biologist with a strong background in neuropharmacology and a passion for bridging science and innovation. After earning a master’s degree in the Pharmaceutical and Biotech Industry, she completed her PhD in Biomedicine at Pompeu Fabra University (Barcelona), where her research focused on the behavioral analysis of animal models for neurophenotypical characterization. Following her doctoral studies, Miriam transitioned into the healthcare marketing and communication sector, where she played a key role in developing impactful marketing strategies and educational campaigns for leading pharmaceutical brands. She now leverages her scientific expertise, strategic thinking, and creative communication skills in her current role at ZeClinics.