In vivo electrical cardiography is the only diagnostic tool for electrical phenotyping in live zebrafish in cross-sectional and longitudinal studies. Our approach is practical, minimally invasive, and cost effective because we repurpose a data acquisition system for mammals. We emphasize the strategy of real-time ECG interpretation for early validation of data quality.
Because human and zebrafish ECGs are similar, in vivo electrocardiography facilitates the use of zebrafish in modeling human cardiac physiology as well as high throughput drug screening for cardiotoxicities, such as QT prolongation. Correct lead positioning is the key to success. Because the normal zebrafish T wave is the smallest, prioritize optimizing its amplitude over those of the larger P and R waves.
Study normal and pathological ECG patterns to interpret your ECG findings yourselves. Do not rely solely on automatic interpretation of ECG analysis software. On the day of the experiment, transport the zebrafish from the aquarium to the laboratory.
To set up the in vivo ECG recording system, connect the essential pieces of equipment and insert the three color-coded, stainless steel electrodes of the ECG lead into the three color-matched access portals of the amplifier. For induction of level 4 anesthesia, immerse an adult zebrafish in a dish containing a solution of anesthetics at the lowest pre-determined concentrations approved by the institutional animal care and use committee. Once the zebrafish has maintained level 4 anesthesia for three seconds, use a pair of blunt forceps to immediately transfer the fish onto a damp sponge with a slit, ventral surface up, for placement of the three ECG electrodes.
Gently insert the positive electrode in the ventral midline at the level of the bulbus arteriosus one to two millimeters above an imaginary line connecting the two lower edges of the operculums. Position the negative electrode caudally and 0.5 to one millimeter left laterally to the positive electrode at a distance greater than the maximal apicobasal length of the adult zebrafish ventricle, then position the reference electrode caudally near the anal region. For ECG recording, start the system and open the ECG data acquisition program.
Select a desired setting from the drop-down menus for range, low pass, and high pass. Press Start to initiate continuous gap-free ECG recording at a sampling rate of one kilohertz. To optimize lead positioning for maximal signal-to-noise ratio, press Stop to stop the recording and review the trace soon after the very first recording attempt for each heart.
If the ECG is expected to be normal, confirm that all ECG waveforms are distinct and readily visible and that the P wave, net QRS complex, and T wave are all positive. The single most critical step is lead positioning to maximize the signal-to-noise ratio. Apply our four validating criteria after the very first ECG recording attempt for each fish to obtain corrective feedback.
If a normal ECG is expected, reposition the electrodes as necessary until all of these four validating criteria are satisfied. If a normal T wave is expected, but the T wave is too small, reposition the electrodes to maximize the T wave amplitude. Resume the ECG recording after optimizing the lead positioning, saving the ECG sweeps for subsequent analysis.
At the end of the ECG recording session, carefully remove the electrodes without injuring the fish. In survival studies, transfer the fish to fresh, oxygenated fish water free of tricaine. Please note that, in this video, to facilitate readers'viewing of the fish recovery from anesthesia, oxygenation is discontinued.
To facilitate recovery from the anesthesia in survival studies, use a Pasteur pipette to vigorously squirt water over the gills until the fish resumes regular gill movement or swimming, then monitor the fish for full recovery from anesthesia before returning the fish to the aquarium. The fish is considered fully recovered from anesthesia when it can swim upright for at least five seconds. To define the analysis settings, open the ECG data analysis program and open the ECG file of interest to display the full ECG trace.
Use the mouse to drag out a section of interest in the ECG trace to analyze. From the ECG Analysis menu, select ECG Settings to open a dialogue box to predefine various parameter settings for software automatic analysis. Analyze the heart rhythm and rate and determine whether the heart rhythm is sinus or not, regular or irregular.
To determine the heart rate, ensure that the software correctly identifies all of the P and R waves because the atrial and ventricular rate is based on the PP and RR interval, respectively. Correct any auto-identification mistakes by moving the misplaced cursors to the appropriate P and R waves. To calculate intervals and wave durations, open ECG analysis and averaging view to concatenate several consecutive cardiac cycles into a single average signal.
Ensure that the software correctly identifies the start and end of the P wave, the QRS complex, and the T wave, then move the misplaced cursors to the appropriate positions to correct any auto-identification mistakes. To export the ECG measurements, select Table View to review all of the ECG measurements and copy and paste the measurements of interest into a spreadsheet for subsequent analysis. To export an ECG trace, use the magnifier to highlight a section of interest in the ECG sweep and copy and paste the sweep into the desired document.
In contract to the human heart, which has two atria and two ventricles, the zebrafish heart has only one atrium and one ventricle. Despite its apparent anatomical simplicity, the zebrafish heart shares several ECG features with the human heart. Proper lead placement requires aligning the lead with the presumed cardiac main axis.
When the electrodes are inserted too superficially in the dermis, the lead is considered indirect-like and the voltage signals are small. When the electrodes are inserted to the appropriate one-millimeter depth into the pectoralis musculature, the lead becomes semi-direct and the voltage signals increase. If the electrodes are inserted deeper than one millimeter into the ventricle, however, the lead becomes direct and the voltage signals increase even further, with the R wave amplitude increasing by four-fold compared to when the electrodes are inserted at the appropriate depth.
The ECG trace from too-deeply inserted electrodes also reveals new signs of injury to the ventricular myocardium, such as a new ST depression and a new T wave inversion. If the positive and negative electrodes are switched in their placement, unusual inversions of all of the ECG waveforms are observed. An inappropriate level of sedation depth can also impair the quality of the in vivo ECG recording.
Remember to anesthetize deeply enough to immobilize the fish without killing it. Position the electrodes to maximize the signal-to-noise ratio and avoid over-reliance on automatic software analysis. Following this minimally invasive survival procedure, it is possible to perform other procedures such as partial ablation, resection of the heart, or serial ECG investigations in drug responses or longitudinal studies.
Systemic toxicities are potential complications of any anesthetic, including tricaine. These toxicities can be reduced by capitalizing on the synergy of multiple anesthetics to lower the dose of individual agents.
