Medicine
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Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice
Chapters
Summary July 5th, 2021
This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.
Transcript
Measuring intrinsic heart rate is an important indicator of cardiac health. This technique accurately measures this rate while excluding the majority of confounding influences on the sinoatrial node. MEA recording of intrinsic heart rate captures accurate firing rate data similar to single-cell recordings without extensive electrophysiology training.
Individuals using this technique may struggle to obtain healthy tissue preparations in early experiments, so practice the dissection and optimize the buffers and data collection settings before beginning the real data collection. Demonstrating the procedure will be Doctors Man Si and Praveen Kumar, post-doctoral researchers in my laboratory. Begin by holding the skin of the euthanized mouse with a hemostat and use surgical scissors to make a transverse incision in the skin just beneath the bottom of the ribcage from the left costal arch to the right costal arch.
Use surgical scissors to cut open the peritoneum and carefully separate the liver from the diaphragm without nicking the liver to prevent excessive bleeding. Incise the diaphragm along the thorax to expose the thoracic cavity. Use the surgical scissors to cut the lateral walls of the ribcage from the edges of the costal arches up to the clavicles to expose the heart, then use a 23 gauge syringe needle to pin the ribcage over the shoulder.
Use a transfer pipette to drop warm heparinized complete Tyrode's solution on the heart to keep it moist. Hold the lungs with extra fine Graefe forceps and sever the trachea with surgical scissors to remove the lungs. For removing the heart, hold the apex of the heart with extra fine Graefe forceps and cut the aorta and inferior vena cava with surgical scissors.
Transfer the heart to a Petri dish containing cured silicone elastomer and use a transfer pipette for bathing the heart with two to three milliliters of warm heparinized complete Tyrode's solution. Attach the apex of the heart to the dish with a dissection pin. Hold the inferior vena cava with Dumont 2 laminectomy forceps.
Insert a 22 gauge syringe needle through the inferior and superior vena cava to locate their position in the right atrium, which also identifies the approximate position of the sinoatrial node. Hold the right atrial appendage with Dumont 2 laminectomy forceps, and put a dissection pin through the right atrial appendage to hold it in place. Repeat the same procedure for the left atrial appendage, then remove the syringe needle that spans the vena cavae.
Use Castroviejo scissors to remove the apex of the heart by making a transverse incision across the ventricles for releasing the blood from the heart, then wash the heart by adding warm heparinized complete Tyrode's solution. Use Castroviejo scissors to cut along the atrioventricular septum until the atria are separated from the ventricles and keep the incision closer to the ventricle. Cut along the interarterial septum to remove the left atrium.
Place the dissection pins in the periphery of the right atrium to make it lay flat. Remove any remaining fat, vessels, or tissue from the atrium using the Castroviejo scissors and locate the sinoatrial node in the right atrium. Add Tyrode's solution to the input solution model and turn on the flow of carbogen gas to oxygenate the Tyrode's solution.
Set the peristaltic pump to 25 RPM, which gives a flow rate of two milliliters per minute. After starting the pump, ensure that the buffer is not leaking from the system and set the temperature controller to 37 degrees Celsius. Use Dumont 55 forceps to transfer the dissected tissue from the dissecting Petri dish onto the microelectrode array grid.
Gently position the tissue with a soft paintbrush to overlay the sinoatrial node region of the electrode grid. Then place the mesh over the tissue using the bone forceps or any curved forceps. Using the bone forceps, position the harp anchor on the mesh to hold everything in place.
Arrange the microelectrode array dish on the connector plate. Carefully place the perfusion cap on the microelectrode array dish without disturbing the harp slice anchor and secure the perfusion gap with tape. Turn on the amplifier and set up a workflow for the recording in the software.
Select beat_recording. moflo template. Open it and set the number of traces, trace duration, trace interval, input voltage, sampling rate, and other recording parameters according to the desired recording conditions.
Click the record and play button to start the recording and acquire data for 10 traces of one-minute duration with two minutes of intervals between the traces. Pause the pump and switch the pump inflow tubing from the normal recording solution to Tyrode's solution containing the desired drug of choice. Restart the pump and resume the recording.
Once the drug-infused Tyrode's solution has reached the tissue, record 10 traces in the same manner as done previously for the baseline recordings. Take a final picture of the positioning of the tissue on the microelectrode array. Open the saved recorded data file in the beat frequency analysis template of the analysis software.
Click on the play and allow the entire recording to run for data visualization and then assign appropriate analysis parameters. Select the channels to be included in the analysis and set the desired amplitude maxima or amplitude minima threshold values for automated waveform peak identification. Click on the play icon again to rerun the dataset and confirm that the analysis parameters are appropriate for a spike extraction.
For analysis, identify the three most stable consecutive traces that exhibit a stable beating rate for each trace across most channels during the baseline period of the experiment and another three consecutive stable traces during the drug exposure period. Click the play and record icon to start the analysis. The data was collected from a 45-day-old male wild-type black Swiss mouse for the sinoatrial node beat frequency measurement.
The waveforms with different shapes and amplitudes were observed in different channels and all the channels showed identical interspike intervals and firing frequencies. However, the degree of tissue contact with the electrode may also influence the waveform characteristics such as amplitude. From the 10 recorded traces, the three consecutive channels with stable beat frequency and interspike interval were chosen for further analysis.
The bad extracted spike patterns should be absent, but if present are either influenced by noise or unstable. The waveforms that correspond to individual heartbeats reflect intrinsic cardiac pacemaking activity. The microelectrode array system allows easy application of drug agents to analyze the pharmacological effects.
The intrinsic firing rate of selected three traces across all 64 channels was found to be approximately 320 beats per minute in the sampling data. The introduction of 4-aminopyrimidine increased the interspike intervals as expected, which decreased the beat frequency from 320 to 210 beats per minute. To collect reliable data for analysis, it is important to confirm that tissue is healthy during recording by verifying that traces are stable and meet the standard criteria.
MEAs can be used to record cardiac activity in other regions of the heart, allowing detailed region-specific characterization that is amenable to study the effects of genetic and pharmacological manipulation.
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