The study introduces Zebra II, an advanced system for prolonged ECG acquisition and analysis in zebrafish. This system features an independent perfusion system and multiple-point electrodes for handling multiple fish in controlled environments. Acute effects of Amiodarone were assessed and analyzed with wild-type zebrafish.
Zebrafish and their mutant lines have been extensively used in biomedical investigations, cardiovascular studies, and drug screening. In the current study, the commercial version of the novel system, Zebra II, is presented. The protocol demonstrates electrocardiogram (ECG) acquisition and analysis from multiple zebrafish within controllable working environments. The device is composed of an external and independent perfusion system, a 4-point electrode, temperature sensors, and an embedded electronic system. In previous studies, the device prototype underwent validation against the established iWORX system through several tests, demonstrating similar data quality and ECG response to drug interventions. Following this, the study delved into examining the impact of anesthetic drugs and temperature fluctuations on zebrafish ECG, necessitating instant data evaluation. Thanks to the apparatus's capacity for consistent delivery of anesthetics and drugs, it was possible to extend ECG data collection up to 1 h, markedly longer than the 5 min duration supported by current systems. This paper introduces a pioneering, cloud-based, automated analysis utilizing data from four zebrafish, offering an efficient method for conducting combination experiments and significantly reducing time and effort. The system proved effective in capturing and analyzing ECG, especially in detecting drug-induced arrhythmias in wild-type zebrafish. Additionally, the capability to gather data across multiple channels facilitated the execution of randomized controlled trials with zebrafish models. The developed ECG system overcomes existing limitations, showing the potential to greatly expedite drug discovery and cardiovascular research involving zebrafish.
Cardiovascular diseases (CVDs) are the leading cause of death worldwide. According to the 2024 AHA Update Report, around 2552 people die every day of CVDs in the U.S., and the average direct and indirect cost of CVD was estimated at $378.0 billion in 2017-20181. Atrial fibrillation (AF) is one of the most common cardiac arrhythmias in the U.S.2 and is associated with an unfavorable prognosis, increasing the risk of stroke and death in both CVDs and non-cardiovascular disease scenarios, leading to an increased risk of adverse outcomes3. This type of arrhythmia is distinguished by its rapid and irregular electrical activity in the atria, which usually results in a fast and irregular ventricular rhythm4. Further, given the recent events of COVID-19, there has been an increased number of studies and evidence that show an increased appearance of arrhythmia in relation to COVID-19, mostly to sinus bradycardia5. On the opposite side of AF, sinus bradycardia presents a decreased and slower heart rhythm, with a heart rate lower than 60 beats per minute (bpm). This diagnosis requires an electrocardiogram (ECG) showing a normal sinus rhythm at a rate lower than 60 bpm.
The zebrafish, Danio rerio, has proven to be an ideal model for cardiovascular and drug development studies because of its homology to humans in both morphology, physiology, and genetics6,7. Despite only having two discernible chambers compared to the four-chambered heart in humans, the zebrafish possesses a similar contractile structure with an analogous conduction system8,9. Hence, the zebrafish model is suitable for researching cardiac arrhythmias and exploring the association between related genetic pathways and electrophysiological characteristics through ECG analysis. Regarding the sensor design for ECG recording in zebrafish, conventional needle electrodes are commonly used10. Other studies presented alternatives for conventional needles by designing a different type of needle made of different materials11, these incorporated materials such as tungsten filament, stainless steel, and silver wire to assess the quality of the recorded data. Together with the portable ECG kit, they sought to establish a universal platform for both research and educational labs.
The needle system was also deployed in other studies11,12,13,14 for carrying out research on biological and/or drug-induced effects, in which it showed encouraging outcomes. However, to obtain optimal signals, it was necessary to carefully insert the needles through the zebrafish's dermis. This invasive procedure can cause injury to the fish's heart due to the sharpness of the needles, thus possibly changing signal morphology13. Furthermore, accurately placing the electrode on the small heart without causing harm to ensure clear ECG capture presents a significant learning curve. Consequently, various substitute probe systems have been introduced, such as microelectrode arrays (MEA) and 3D-printed sensors. Both our team and other researchers have successfully utilized MEA for data acquisition, achieving data that offer a good signal-to-noise ratio (SNR) along with high spatial and temporal resolution15,16,17. For example, we introduced an MEA that envelops the heart of the fish, allowing the capture of site-specific ECG signals15,16. In contrast, another team developed a different kind of MEA based on a flexible printed circuit board (FPCB) crafted from polyimide film aimed at recording multiple electroencephalograms (EEGs) for epilepsy research18. While MEAs enable the recording of numerous signals, they are limited by the capacity to assess only one fish at a time due to the restricted number of electrode channels. In response to this limitation, we have recently showcased an advanced system capable of simultaneous ECG recordings from multiple fish19. This system employs two electrodes, each 125 µm in thickness and made of polyimide, with gold-sputtered electrodes positioned at the bottom of their housing to facilitate data acquisition. However, the inclusion of a water circulation pump introduced noise, affecting the signal-to-noise ratio (SNR) quality of the ECG signals. Additionally, the use of bulky and costly data collection tools, which required a wired connection to a computer, was necessitated. On the market, systems like iWORX offer improved portability through a compact amplifier, yet they face unresolved issues: (i) their recording time is too short (3-5 min) for experiments needing longer durations, such as those involving acute drug effects; (ii) the requirement for anesthetizing the animals, which can stress them and skew innate cardiac electrophysiological data; (iii) manual, sequential measurements hinder research with large numbers of fish; and (iv) the need for offline, labor-intensive ECG data processing. Importantly, no high-throughput systems incorporating microelectronics for analyzing mutant phenotypes have yet emerged. Therefore, the creation of high-throughput systems that enable extended ECG recording is crucial for uncovering links between arrhythmic phenotypes and mutant genotypes, identifying multiple arrhythmic phenotypes associated with single mutant genotypes, and testing the effectiveness of cardiac drugs using the zebrafish model. Consequently, we introduced, in 2022, the groundbreaking prototype of the Zebra II system. This novel system is equipped to perform extended ECG recordings from several fish at once. To achieve this, an in-house electronic system was crafted, utilizing the Internet of Things (IoT) technology for wireless data transmission and enabling data analysis through a mobile application. The inclusion of IoT technology not only bolstered the system's flexibility and portability but also facilitated remote collaborations for research on zebrafish models. In a prior study, we validated the system's efficacy through extensive testing, showcasing its ability to 1) acquire ECG data simultaneously from four fish; 2) perform continuous ECG recordings for up to 1 h, which significantly exceeds the capacity of existing systems that only capture a few minutes; 3) minimize the confounding effects of anesthesia by employing a 50% reduced concentration of Tricaine. Furthermore, the system demonstrated a strong capability for lengthy ECG recordings across various experiments, such as those involving sodium-induced sinoatrial (SA) block, temperature-induced heart rate changes, and drug-induced arrhythmias in both Tg(SCN5A-D1275N) mutant and wildtype fish under controlled experimental conditions. The system's adoption of multiple electrode channels for extended ECG recording further enables the execution of randomized controlled trials, allowing for two fish per experimental group to be studied under identical conditions20. In this paper, we introduce the upgraded version of the system, featuring a 4-point electrode. This enhancement enables users to capture ECG readings from various locations on the fish, significantly reducing the chance of obtaining low-quality ECG signals. Moreover, this iteration builds upon and refines the majority of the functionalities outlined in previous research19. This evolved ECG system shows great promise in addressing existing limitations, thereby substantially facilitating the analysis of arrhythmic phenotypes and the screening of drugs in zebrafish. The innovative addition of the 4-point electrode was rigorously tested through a brief study on drug-induced arrhythmia in wild-type zebrafish, further validating its effectiveness.
The current steps were modified and improved according to this new device version from a previous study made by our team20. Here, a labeled computer-aided design (CAD) of the device is also included.
As highlighted in the protocol’s troubleshooting guide, the device necessitates the user to execute certain critical steps with diligence and precision, as these steps crucially affect the ECG signal’s quality. Consistent with findings from our prior study…
The authors have nothing to disclose.
We are grateful for the support from the NIH SBIR Phase II #R44OD024874 to H.C. and M.P.H.L. The authors would also like to acknowledge the financial support provided by the Consejo Nacional de Humanidades, Ciencias y Tecnologias (CONAHCYT) in Mexico through the fellowship titled Beca CONAHCYT Para Estudios de Doctorado en el Extranjero.
Amiodarone Hydrochloride | Sigma-Aldrich | PHR1164-1G | Amiodarone powder |
Benchtop pH/mV Meter | Sper Scientific | 10500308 | |
Ethyle3-Aminobenzoate, Methanesulfonic Acid Salt, 98%, ACROS Organics | Fisher Scientific | AC118002500 | Tricaine powder |
Isotemp Hot Plate Stirrer | Fisher Scientific | SP88854200 | |
Zebra II System | Sensoriis | N/A | Commercial Zebrafish ECG Recording System |
Zebrafish ECG System | iWorx | ZS-200 | Commercial Zebrafish ECG Recording System |
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