Development of a rapid and economic in vivo electrocardiogram platform for cardiovascular drug assay and electrophysiology research in adult zebrafish

Figure 1. Illustration showing the system setup and the three-needle electrode placement during real-time adult zebrafish ECG recording. The zebrafish is anesthetized and immobilized on the immersed sponge. Two of the electrodes are positioned on micromanipulators. The electrode above the zebrafish’s heart is penetrated through the dermis with a micromanipulator. In this way, the penetration depth of the electrode probe can be measured by reading from the scale on the micromanipulator. ECG kit: The signals from the electrodes are amplified, filtered and converted to digital signals in this black box. (A) Scheme of ECG recording system. (B) Photograph of the experimental setup and positions of the recording probes. (C) Standard location of the pectoral recording probe in in vivo system as indicated by a cross in the field of view under a dissecting microscope.

Figure 2. An ECG recording of adult zebrafish with the three-needle electrodes. Regular and distinct P waves, QRS complexes, and T waves can be identified. (A) Automatic identification of each heart cycle. The zebrafish raw ECG signal was similar to that in human ECG. The signal includes distinct P waves, QRS complexes, and T waves that can be easily identified. (B) Measurement of ECG intervals based on the trace.

Figure 3. Effects of anesthetics: MS-222 alone and MS-222/isoflurane combination and prolonged sedation with combined formula on heart rate. (A) MS-222 reduced the heart rate to 108 ± 16 bpm, whereas the combined formula maintained a normal heart rate of 148 ± 15 bpm. After 10 minutes, the combined-formula group had an average heart rate of 131 ± 19 bpm. Data are presented as the mean ± SD. n = 9 in the MS-222-only group; n = 10 in the combined formula group. (B) The combined-formula group maintained a normal heart rate of 148 ± 14 bpm. After 10 minutes, the combined-formula group had an average heart rate 131 ± 9 bpm. Paired t-tests showed no significant difference between one minute and 5 minutes and significant differences between the 1- to 10-minute, 20-minute and 30-minute measurements of the combined-formula anesthetic. Data are presented as the mean ± SD. n = 10; *indicates p < 0.05, **indicates p < 0.001 as determined by paired t-tests.

Figure 4. Variation of heart rate during real-time recording of adult zebrafish and the response to isoproterenol. Real-time ECG wave-form changes before and after isoproterenol treatment. (A) Raw signal showing normal ECG before isoproterenol injection. (B) Extended anesthesia by MS-222 alone to induce bradycardia. (C) After isoproterenol treatment, heart rate increased. (D) The heart rates were significant reduced after 5-min sedation. Heart rate increased by 1.25 times after 5 μl of 10 μM isoproterenol treatment (n = 6). **Indicates p < 0.001 as determined by paired t-test. (E) Dose-response curve for the effect of isoproterenol on heart rate of zebrafish heart. The fold change of heart rate in response to log of increasing concentration of isoproterenol (0.5, 1, 5, 7.5 and 10 μM, respectively).

Figure 5. Real-time ECG recording of adult zebrafish and the response to verapamil treatment. (A) Simultaneous recording of ECG during retro-orbital injection of verapamil. (B) Raw signal showing normal ECG before verapamil injection. (C) Bradycardia was observed after verapamil was injected. (D) The heart rates were significantly reduced after verapamil injection (n = 7). **Indicates p < 0.001 as determined by paired t-test.

Figure 6. Adult zebrafish ECG features and drug response to amiodarone. ECG waveforms before and after amiodarone treatment are shown. (A) Real-time ECG signal before treatment. (B) Heart rate was reduced after amiodarone treatment. (C) Heart rate, QRS interval, QT interval, PR interval and QTc interval before and after amiodarone treatment. Data are presented as the mean ± SD, n = 12. *Indicates p < 0.05, **Indicates p < 0.001 as determined by paired t-test.

Figure 7. Quinidine reduced heart rate and prolonged QTc interval in adult zebrafish heart. (A) Heart rate, QT interval, PR interval and QTc interval before and after 250 μM quinidine treatment. (B) Control QT interval before injection of quinidine. (C) QT interval was prolonged after quinidine was injected. Data are presented as the mean ± SD, n = 8. *Indicates p < 0.05, **Indicates p < 0.001 as determined by paired t-test.

Figure 8. Arrhythmia was induced after veratridine treatment in adult zebrafish. (A) Raw signal showing normal ECG before veratridine injection. (B) Significant first-degree AV block (prolonged PR interval) was observed within 5 minutes after veratridine (100 μM) was injected. (C) Heart rate, PR interval, QT interval and QTc interval before and after veratridine treatment. Data are presented as the mean ± SD, n = 10. **Indicates p < 0.001 as determined by paired t-test.