Name: microelectrode array recording of sinoatrial node firing rate to identify intrinsic cardiac pacemaking defects in mice
Uploaded: 2021-07-05T15:00:00
Description: Watch this Scientific Journal Video about Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice at JoVE.com

This work was funded by the National Institutes of Health, grant numbers R01NS100954 and R01NS099188.

All experimental procedures described here have been carried out in accordance with the guidelines of the National Institutes of Health (NIH), as approved by the Institutional Animal Care and Use Committee (IACUC) at Southern Methodist University.
1. Coating the multielectrode array (MEA) for recording
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Practicing and mastering the SAN dissection process is imperative since the tissue is fragile and healthy tissue is necessary for a successful recording. During the SAN dissection, correct orientation is essential to obtain the proper region of tissue. However, the original orientation of the heart can be easily lost during the dissection process, which complicates this endeavor. Therefore, to ensure the proper left-right orientation, the atria should be visually inspected. Typically, the right atrium tends to be more tr...

The heart is a complex organ governed by both cardiac-intrinsic and extrinsic influences such as those that originate in the brain. The sinoatrial node (SAN) is a defined region in the heart that houses the pacemaker cells (also referred to as sinoatrial cells, or SA cells) responsible for the initiation and perpetuation of the mammalian heartbeat1,2. The intrinsic heart rate is the rate driven by the pacemaker cells without influence by other cardiac or neuro-humoral influences, but traditional measures of heart rate in humans and live animals, such as electrocardiograms, reflect both the pacemaker and neural influences on the heart. The most notable neural influence on SA cells is from the autonomic nervous system, which constantly modulates firing patterns to meet the physiological requirements of the body3. Supporting this idea, both sympathetic and parasympathetic projections can be found near the SAN4. The intrinsic cardiac nervous system (ICNS) is another important neural influence where ganglionated plexi, specifically in the right atria, innervate and regulate the activity of the SAN5,6.
Understanding pacemaking deficits is clinically important, as dysfunction can underlie many cardiac disorders, as well as contribute to the risk of other complications. Sick sinus syndrome (SSS) is a category of diseases characterized by dysfunction of the sinoatrial node which impedes proper pacemaking7,8. SSS can present with sinus bradycardia, sinus pauses, sinus arrest, sinoatrial exit block, and alternating bradyarrythmias and tachyarrhythmias9 and can lead to complications including increased risk of embolic stroke and sudden death8,10. Those with Brugada syndrome, a cardiac disorder marked by ventricular fibrillation with an increased risk of sudden cardiac death, are at greater risk for arrhythmogenic events if they also have comorbid SAN dysfunction11,12. Sinoatrial dysfunction may also have physiological consequences beyond the heart. For example, SSS has been observed to trigger seizures in a patient due to cerebral hypoperfusion13.
To identify sinoatrial pacemaking deficits, intrinsic heart rates need to be determined by measuring the activity of the SAN without the influence of the autonomic nervous system or humoral factors. Clinically, this can be approximated by pharmacological autonomic blockade14, but this same technique can also be applied in mammalian models to study intrinsic cardiac function15,16. While this approach blocks a large portion of contributing neural influences and allows for in vivo cardiac examination, it does not completely eliminate all extrinsic influences on the heart. Another research technique used to study intrinsic cardiac function in animal models is isolated heart recordings using Langendorff-perfused hearts, which typically involve measurements using electrograms, pacing, or epicardial multielectrode arrays17,18,19,20. While this technique is more specific to cardiac function since it involves removing the heart from the body, the measurements may still be influenced by mechano-electric autoregulatory mechanisms that could influence intrinsic heart rate measurements21. The isolated heart recordings may also still be influenced by autonomic regulation through the ICNS5,6,22,23. Furthermore, maintaining a physiologically relevant temperature of the heart, which is critical for cardiac function measurements, can be difficult in isolated heart approaches20. A more direct method to study SAN function is to specifically isolate SAN tissue and measure its activity. This can be accomplished through SAN strips (isolated SAN tissue) or isolated SAN pacemaker cells24,25. Both require a high degree of technical training, as the SAN is a very small and highly defined region, and cell isolation poses an even greater challenge as dissociation can damage the overall health of the cell if not performed correctly. Furthermore, these techniques require expert electrophysiological skills in order to successfully record from the tissue or cells using individual recording microelectrodes.
In this protocol, we describe a technique to record the SAN in vitro by using a microelectrode array (MEA) to obtain intrinsic heart rate measurements. This approach has the advantage of making highly specific electrophysiological recordings accessible to researchers lacking intensive electrophysiological skillsets. MEAs have previously been used to study cardiomyocyte function in primary cardiomyocyte cultures26,27,28,29,30,31,32, cardiac sheets33,34,35,36,37,38,39, and tissue whole mounts40,41,42,43,44,45,46,47. Previous work has also been done to examine field potentials in SAN tissue41,42. Here, we provide a methodology to use the MEA to record and analyze murine intrinsic SAN firing rates. We also describe how this technique can be used to test pharmacological effects of drugs on SAN intrinsic firing rates by providing a sample experiment showing the effects of 4-aminopyridine (4-AP), a voltage-gated K+ channel blocker. Using defined anatomical landmarks, we can accurately record the SAN without having to perform the extensive tissue dissections or cell isolations required in other methods. While the MEA can be cost-prohibitive, the recordings provide highly specific and reliable measures of pacemaking that can be used in a vast array of clinical and physiological research applications.

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			<td>4-Aminopyridine</td>
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			<td>22 gauge syringe needle</td>
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			<td>23 gauge syringe needle</td>
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			<td>1000 mL Pyrex Bottle</td>
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			<td>208337-100G</td>
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			<td>MED64 Head Amplifier</td>
			<td>MED64</td>
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			<td>MED64</td>
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			<td>MED64</td>
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			<td>MED64</td>
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			<td>Parafilm</td>
			<td>Genesee Scientific</td>
			<td>PM-996</td>
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			<td>Peristaltic Pump</td>
			<td>Gilson</td>
			<td>F155009</td>
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			<td>Peristaltic Pump Tubing</td>
			<td>Fisher Scientific</td>
			<td>14-171-298</td>
			<td>1/8&#39;&#39; Interior Diameter</td>
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			<td>P3143</td>
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			<td>Sigma-Aldrich</td>
			<td>P9333-500G</td>
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			<td>Sigma-Aldrich</td>
			<td>P5655-500G</td>
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			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S6297</td>
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			<td>Sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S671-3</td>
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			<td>Sylgruard Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td></td>
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		<tr>
			<td>Sodium Phosphate Monobasic</td>
			<td>Sigma</td>
			<td>S6566</td>
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			<td>Sodium tetraborate</td>
			<td>Sigma</td>
			<td>S9640</td>
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			<td>Surgical Scissors</td>
			<td>Fine Science Tools</td>
			<td>14074-09</td>
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			<td>Transfer Pipets (3mL graduated)</td>
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			<td>225</td>
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microelectrode array recording of sinoatrial node firing rate to identify intrinsic cardiac pacemaking defects in mice

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

After allowing the tissue to acclimate in the dish for 15 min, 10 one-min traces are recorded. Our current protocol records activity for over an hour, but we have recorded stable firing patterns for &#8805;4 h in unpublished data not shown here. If an experimental preparation is good for data collection, each recording channel should exhibit consistent and evenly spaced recurring waveforms (i.e., spikes) of uniform shape for a given channel (Figure 11D). These waveforms correspond to individ...

Watch this Scientific Journal Video about Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice at JoVE.com

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

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.

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or ...

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